Poster Walks (PW):

8:00 PM
PL-02-1 — Advancing surgical guidance: From (hybrid) molecule to man and beyond (#598)

N. S. van den Berg1

1 Stanford University, Department of Otolaryngology, Head and Neck Surgery, Stanford, United States of America

Content

Surgery, often combined with (neo-adjuvant) chemo-, hormonal- or radiotherapy, is considered the main pillar in cancer management. However, during the surgical procedure it is not always clear what exactly has to be removed. My PhD research at the Leiden University Medical Center (dept. Radiology)- Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital (dept. Urology & dept. Head and Neck Surgery and Oncology) aimed to use interventional molecular imaging technologies to provide the surgeon with additional guidance during surgery as such to improve outcome. We focused on the clinical evaluation and validation of the hybrid, radioactive and fluorescent, tracer indocyanine green (ICG)-technetium 99m-nanocolloid to identify and excise lymph nodes that are in direct contact with the primary tumor (so-called sentinel nodes) in e.g. patients with head-and-neck or urological cancer. Using the hybrid tracer, we created a hybrid approach wherein diagnostic imaging findings could be directly translated into the operation room. Here, for diagnostic imaging the radioactive signature of the tracer could be used to non-invasively identify the target lesions (sentinel nodes). In addition, during surgery, the fluorescent signature of the hybrid tracer allowed visual identification of the sentinel nodes. In comparison to the conventional approach of radiocolloid and blue dye a definitive value of the hybrid tracer was found with more sentinel nodes identified during surgery. This value seemed biggest when sentinel nodes are located near the injection site and/or at locations of complex anatomy (e.g. parotid gland, presacral). Secondly, in collaboration with industrial partners, optimization of radioactive- and fluorescence imaging devices was investigated. Subsequently, in clinical first-in-human pilot studies we found that we were able to improve intraoperative lesion detection and resection. For example, using an optimized fluorescence camera, compared to its predecessor, we could visually more sentinel nodes. Even more so, the use of these optimized fluorescence camera’s resulted in a shift where by the surgeon now can perform real-time fluorescence-guided sentinel node excision rather than using fluorescence imaging for confirmation only.

8:30 PM
PL-02-2 — Changes of Paradigm in Biomedical Ultrasound (#574)

M. Tanter1

1 Inserm, Institut Langevin (ESPCI Paris, CNRS, Inserm, PSL University), Paris, France

Content

In the last twenty years, the introduction of plane or diverging wave transmissions rather than line by line scanning focused beams broke the resolution limits of ultrasound imaging. By using such large field of view transmissions, the frame rate reaches the theoretical limit of physics dictated by the ultrasound speed and an ultrasonic map can be provided typically in tens of micro-seconds (several thousands of frames per second). Interestingly, this leap in frame rate is not only a technological breakthrough but it permits the advent of completely new ultrasound imaging modes, including shear wave elastography1,2, electromechanical wave imaging, ultrafast Doppler, ultrafast contrast imaging, and even functional ultrasound imaging of brain activity (fUltrasound) introducing Ultrasound as an emerging full-fledged neuroimaging modality.

At ultrafast frame rates, it becomes possible to track in real time the transient vibrations – known as shear waves – propagating through organs. Such "human body seismology" provides quantitative maps of local tissue stiffness whose added value for diagnosis has been recently demonstrated in many fields of radiology (breast, prostate and liver cancer, cardiovascular imaging, ...).

For blood flow imaging, ultrafast Doppler permits high-precision characterization of complex vascular and cardiac flows. It also gives ultrasound the ability to detect very subtle blood flow in very small vessels. In the brain, such ultrasensitive Doppler paves the way for fUltrasound (functional ultrasound imaging) of brain activity with unprecedented spatial and temporal resolution compared to fMRI (figure 1).

It provides the first modality for imaging of the whole brain activity working on awake and freely moving animals with unprecedented resolutions 3,4,5.

Finally, we recently demonstrated that it can be combined with 3 µm diameter microbubbles injections in order to provide a first in vivo and non-invasive imaging modality at microscopic scales deep into organs (figure 2) combined with contrast agents by localizing the position of millions of microbubbles at ultrafast frame rates.

This ultrasound localization microscopy technique solves for the first time the problem of in vivo imaging at microscopic scale the whole brain vasculature 6. Beyond fundamental neuroscience or stroke diagnosis, it will certainly provide new insights in the understanding of tumor angiogenesis.

All these new features of ultrafast Ultrasound could be combined in the future with PET/CT acquisitions for unique hybrid and simultaneous imaging of anatomy, metabolism, hemodynamics and functional activity as recently demonstrated7.
 

References

  1. M. Tanter et al, Ultrasound in Medicine and Biology, 34(9), 2008
  2. M.E. Fernandez-Sanchez et al, Nature, July 2015
  3. Mace et al., Nature Methods, Jun. 2011
  4. Osmanski et al, Nature Comm., Oct. 2014
  5. L.A. Sieu et al, Nature Methods, Jul. 2015
  6. C.Errico et al, Nature, Dec. 2015
  7. J. Provost et al, Nature Biomedical Engineering, Feb. 2018

 

 

 

Acknowledgement

This work was supported by a research grant from the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013) / ERC Advanced grant agreement n° 339244-FUSIMAGINE and , by France Life Imaging and by the Inserm Technology Research Accelerator in Biomedical Ultrasound.

Ultrafast Ultrasound becomes a full fledged neuroimaging and microscopic modality

A & B.) 3D Functional Ultrasound imaging of the rat brain neuronal activity during a visual stimulus (from Gesnik et al Neuroimage 2015)

C) Superresolution Ultrasound based on ultrasound localization microscopy leads to non invasive deep  in vivo microscopy of the vasculature up to the capillary level (from Errico et al Nature 2015)

Hybrid PET/CT and Ultrafast Ultrasound imaging of a growing tumor in mice
Ultrafast Ultrasound can be implemented simultaneously with PET/CT for an hybrid modality providing simultaneously anatomy, metabolism, hemodynamics and functional activity (from Provost et al, Nature BME 2018) : Images of mice tumor at different growth stages with vascularization provided by Ultrasound, Metabolism provided by PET, and Anatomy by CT.

4:00 PM
emptyVal-1 — Introductory Talk by Adrian Rodriguez-Contreras - New York, USA

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

4:20 PM
PS-17-2 — Monitoring neural lineage differentiation of stem cell grafts: An in vivo bioluminescence imaging and light sheet microscopy study (#229)

S. Vogel1, C. Schäfer1, M. Aswendt2, M. K. Schwarz3, 4, C. Niedworok3, A. Minassian1, K. Folz-Donahue5, C. Kukat5, M. Ehrlich6, 7, H. Zaehres7, M. Hoehn1, 8

1 Max Planck Institute, In-vivo-NMR Laboratory, Cologne, Germany
2 University Hospital Cologne, Department of Neurology, Cologne, Germany
3 University of Bonn, Department of Epileptology, Bonn, Germany
4 Life and Brain GmbH, Bonn, Germany
5 Max Planck Institute for Biology of Ageing, FACS & Imaging Core Facility, Cologne, Germany
6 University Hospital Münster, Institute of Neuropathology, Muenster, Germany
7 Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology, Muenster, Germany
8 Leiden University Medical Center, Department of Radiology, Leiden, Netherlands

Introduction

The potential of neural stem/progenitor cells (NSC/NPC) to differentiate into mature neural cells and thus to replace or repair brain tissue is one of the most promising novel therapies to treat neurodegenerative diseases and brain injuries. Here, we present a molecular imaging approach based on long-term bioluminescence imaging (BLI) complemented by light sheet fluorescence microscopy (LSFM) to evaluate the integration and differentiation capacities of engrafted NPCs and to monitor NPC differentiation longitudinally in vivo.

Methods

The neural progenitor cell line fNSC-iPSC-NPC [1] was lentivirally transduced to express the BLI reporter Luc2 and the fluorescence reporter EGFP under constitutive (EF1) or cell specific (MAP2 for neurons, GFAP for astroglia or PLP for oligodendrocytes) promoters. All genetically modified cell lines were sorted for EGFP expression and were characterized via immunofluorescence, Western Blot and qPCR. To monitor differentiation of these cell lines in vivo, 1.5x105 cells were engrafted into the cortex of male nude mice and photon emission (PE) of the cell graft is acquired over 12 weeks [2]. The fate of the engrafted cells is characterized by LSFM [3] for spatial dynamics (migration or integration), and validated via histology and electrophysiology.

Results/Discussion

In vitro, all transgenic cell lines show an upregulation of the imaging reporter Luc2 and EGFP in line with the upregulation of lineage specific neural markers over time. Under in vivo conditions, all cell lines show a PE increase within the first six weeks post engraftment (Fig 1). Interestingly, NPCs show a massive astroglia (GFAP) upregulation, followed by slower neuronal (MAP2) differentiation and weak oligodendrocyte (PLP) differentiation. Migration from the graft location (Fig. 2A) to distal brain regions is detected by LSFM (Fig. 2B, C). Ongoing experiments assess the excitability of engrafted MAP2-specific cells via patch clamp electrophysiology. The potential of the grafted cells to develop efferent projections and to transmit signals is another significant event for the reconstruction of neuronal networks and thus the recovery of connectivity after neurodegeneration.

Conclusions

A comprehensive understanding of the differentiation processes of engrafted NPCs and their interaction with host cells is a crucial factor for the translation of stem cell approaches into the clinic. Here, we have determined the in vivo time profile of differentiation, distinct for all three neural lineages. The combination with LSFM allows the characterization of the spatial dynamics, assessing migration capacity and projections into the host brain. This approach unravels for the first time the temporal and spatial fate of stem cells in the brain under true in vivo conditions.

References

1. Hargus G, Ehrlich M, Arauzo-Bravo MJ, et al. (2014) Origin-dependent neural cell identities in differentiated human iPSCs in vitro and after transplantation into the mouse brain. Cell reports 8:1697-1703.

2. Aswendt M, Adamczak J, Couillard-Despres S, Hoehn M (2013) Boosting bioluminescence neuroimaging: an optimized protocol for brain studies. PloS one 8:e55662.

3. Schwarz MK, Scherbarth A, Sprengel R, Engelhardt J, Theer P, Giese G (2015) Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains. PloS one 10:e0124650.

Acknowledgement

We thank Fabian Distler, Marieke Nill and Melanie Nelles for technical support. This work was financially supported by grants from the EU-FP7 programs TargetBraIn (HEALTH-F2-2012-279017) and BrainPath (PIAPP-GA-2013-612360) and by a grant from the German Research Foundation DFG (AS-464/1-1).

Development of the lineage-dependent in vivo bioluminescence of the engrafted NPCs over time.
Signal intensities were normalized to 7 days post injection (dpi) and plotted as fold change. All data are presented as mean plus standard deviation. Number of animals per group: EF1 (n=10), MAP2 (n=18), PLP (n=11) and GFAP (n=9).

Migration capacities of engrafted cells detected by LSFM.
Presentation of the stem cell graft in the mouse brain cortex on EGFP fluorescence of brain slices (A). Migration of cells (↑) distant to the primary graft (▲), detected on LSFM at 12 weeks post implantation in sagittal (B) and axial (C) orientation.

4:30 PM
PS-17-3 — The contractile brain: actomyosin-dependent rapid remodeling of brain tissue and its control by cannabinoids (#452)

J. Ferrier1, 2, 3, A. Ricobaraza2, 3, C. Demene4, 5, 6, M. Humbert-Claude2, 3, A. Ugarte7, J. Oyarzabal7, M. Tanter4, 5, 6, Z. Lenkei1, 2, 3

1 INSERM U894, Center of Psychiatry and Neurosciences, Paris, France
2 CNRS, UMR8249, Paris, France
3 Brain Plasticity Unit, ESPCI Paris, Paris, France
4 Institut Langevin, ESPCI Paris, Paris, France
5 CNRS, UMR 7587, Paris, France
6 INSERM U979, Wave Physics for Medicine Lab, Paris, France
7 Center for Applied Medical Research (CIMA), University of Navarra, Small Molecule Discovery Platform, Pamplona, Spain

Introduction

From a mechanical point of view, the adult brain is currently considered as a soft solid without autonomous movements. Although local volume changes of cerebral tissue have already been reported following neuronal activity or acute drug treatment, the ability of brain tissue for active movement is currently unknown. As previously reported at the microscopic level in neurons [1], we hypothesized that cannabinoid treatment could induce large-scale tissue motions through actomyosin contraction.

Methods

Here we applied ultrafast ultrasound imaging [2] over complete coronal sections of the living adult rat brain. Anesthesia was maintained using 1.5% isoflurane. Ultrasound plane wave compounding (8 angles, PRF=500) was used to insonify the region of interest every 12 s during 2 hours. Each 0.5 s burst was then filtered using a spatiotemporal clutter for parallel measurements of local cerebral blood volume (through highly-resolved ultrafast Doppler imaging) and localized microscopic brain deformations (along the vertical axis), precisely synchronized with the cardiac cycle. Effect of cannabinoid on brain tissue strain was assessed by administrating CP 55,940 (0.7 mg/kg) with or without Blebbistatin (1.5 mg/kg, a myosin II inhibitor) pre-treatment.

Results/Discussion

We report that brain regions may dilate and contract due to contractility induced by non-muscle myosin II at the minute time-scale, independently of local blood flow changes. Blebbistatin administration resulted in highly-reproducible strain change characterized by well-localized and symmetrical contraction and dilation of hippocampal and neocortical areas, respectively. Inversely, activation of type-1 cannabinoid receptors, brain targets of marijuana and of endocannabinoids, a major central neurotransmitter system, resulted in symmetrical tissue deformations in brain areas rich in CB1R, starting between 7 and 15 minutes following CP 55,940 administration and manifested in the contraction of hippocampal and dilation of neocortical areas along the vertical axis. These deformations were abolished by blebbistatin pre-treatment and were not correlated with changes in cerebral blood volume.

Conclusions

Taken together, the above results suggest that adult brain tissue, traditionally considered lacking active movement, is capable of large-scale (i.e. muscle-like) actomyosin contractility. Significant actomyosin contractility is already present at steady-state and this tissue contractility is further enhanced by activation of CB1R cannabinoid receptors. Our study indicates a novel physiological role for a major brain neurotransmitter system and also introduces a novel and powerful experimental tool for measurement of brain tissue mechanics over several spatio-temporal scales.

References

[1] Roland AB, Ricobaraza A, Carrel D, Jordan BM, Rico F, Simon A, Humbert-Claude M, Ferrier J, McFadden MH, Scheuring S, Lenkei Z. Cannabinoid-induced actomyosin contractility shapes neuronal morphology and growth. Elife. 2014 Sep 15;3:e03159.

[2] Tanter, M. & Fink, M. Ultrafast imaging in biomedical ultrasound. Ultrasonics (2014). doi:10.1109/TUFFC.2014.6689779.

Acknowledgement

The research leading to these results has received funding from the European program FUSIMICE of the Human Brain Project and by two research grants : to Z.L. from the French Agence Nationale de la Recherche (ANR-09-MNPS-004-01) and to M.T. from the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013) / ERC Advanced grant agreement n° 339244-FUSIMAGINE. A.U. and J.O. also thank the Foundation for Applied Medical Research (FIMA), University of Navarra, for financial support.

4:40 PM
PS-17-4 — In vivo Diffusion Tensor Imaging reveals White Matter Abnormalities after Surgical Menopause in a Mouse Model of Alzheimer’s Disease (#345)

F. Kara1, Z. Sarwari1, G. Yadav1, A. Langbeen2, R. Voncken1, C. Anckaerts1, J. Hamaide1, P. Ponsaerts3, J. Daans3, P. Bols2, S. Roßner4, A. Van der Linden1, M. Verhoye1

1 Antwerp University-, Bio-imaging Lab-Member of INMIND consortium, Antwerp, Belgium
2 Antwerp University, Laboratory of Veterinary Physiology and Biochemistry, Antwerp, Belgium
3 Antwerp University, Experimental Cell Transplantation Group, Antwerp, Belgium
4 Leipzig University, Paul Flechsig Institute for Brain Research, Leipzig, Germany

Introduction

The decline in estrogen (E2) level at menopause has been proposed as a gender-specific risk factor for Alzheimer’s disease (AD). Depletion of E2 may affect the brain integrity through white matter degeneration [1]. However, little is known about how and when white matter alterations occur in late postmenopausal women. To address these questions we employed in vivo diffusion tensor imaging  (DTI) to investigate white matter abnormalities after surgical menopause (i.e. ovariectomization) in a mouse  model of AD (i.e. Tg2576 mice [2]). 

Methods

Tg2576/ wild-type mice were sham operated (n= 12/12) or ovariectomized (OVX) (n= 12/12) at 3.5 months of age. When the animals were 18 months of age, DTI was performed at 9.4T (Bruker) (TR 7500 ms; TE 23.5 ms; 30 coronal slices at (0.214x0.214x 0.2) mm3; 60 diffusion gradient directions; diffusion gradient duration & separation, 4 & 12 ms; b 800 s/mm2 ). The diffusion metrics (fractional anisotropy (FA), mean, axial and radial diffusivity (MD, AD, RD, respectively) ) were extracted for corpus callosum (CC), external capsule (EC), optic tract (OT), cerebral peduncle (CP) and fimbriae (Fmb). Histology was performed to quantify Aβ plaques [3]. Luteinizing hormone (LH) level was assessed with ELISA [4]. One-way & two-way ANOVA and two sample student T tests were used for statistical testing.

Results/Discussion

TG-OVX exhibited significantly reduced MD compared to WT-OVX (Fig. 1). These results may imply that changes in white matter architecture after OVX were only visible between TG-OVX and WT-OVX groups. We observed a significant decrease in FA values in TG mice compared to WT mice in AC region. Our results may imply an increased white mater pathology related with Aβ (Fig. 2) and gliosis  present in TG mice (Fig. 2). In Fmb, TG-OVX depicted lower axial diffusivity compared to TG-Sham. This result may imply that low E2 when coupled with AD pathology accelerates manifestation of axonal damage. Interestingly, TG-OVX depicted lower axial diffusivity compared to WT-OVX in sCC suggesting low E2 might be associated with axonal integrity. Quantitative Aβ plaque analysis of white matter regions depicted that ovariectomy did not modulate the levels of Aβ in the white matter of  TG mice brain at old age. High LH levels indicates that the OVX operation was successful (Fig 2). 

Conclusions

Overall our results suggest that white matter architecture involving axonal and myelin integrity is sensitive to AD pathology and post-menopause related depletion of ovarian hormones. Apparent resilience of some white matter regions against low E2 and AD pathology may indicate presence of local compensation mechanisms which need to be investigated in future studies. Overall our results suggest that alterations in the white matter architecture can be one of the underlying reasons of higher incidence of  AD in postmenopausal women.

References

[1]Klosinski, L.P., et al., EBioMedicine, 2015. 2(12): p. 1888-1904; [2] Hsiao, K., et al., Science, 1996. 274(5284): p. 99-103;[3] Shah, D., et al., Alzheimers Dement, 2016. 12(9): p. 964-976;[4]Steyn, F.J., et al., Endocrinology, 2013. 154(12): p. 4939-45.

Acknowledgement

Acknowledgement: Scientific Research Flanders (FWO) (grant agreement G.0D76.14, G.0587.14.),postdoctoral FWO number (no 12S4815N),INMiND(278850) and Molecular Imaging of Brain Pathophysiology (BRAINPATH).

Figure 1.

Mean diffusivity alterations in white matter brain regions (i.e. EC, AC, Fmb and sCC) of 18-month-old mice. Statistics for genotype (G) and interaction (genotype x operation) : *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 in two-way ANOVA; interaction: post hoc one-way ANOVA with Bonferroni correction: #P ≤ 0.0125. EC, external capsule; AC, anterior commissure; Fmb, fimbriae; sCC, splenium corpus callosum.

Figure 2.

Percent (%) plaque area in OVX and sham operated Tg2576 mice in CC (A) and EC regions (B). Plasma LH levels of mice after OVX or sham operation (between 8-14 months of age) (C). Data are expressed as mean ± SD. ***P ≤ 0.001 (student T test). Abbreviation: TG, transgenic; WT, wild-type; OVX, ovariectomized; sham, sham-operated; CC, corpus callosum; EC, external capsule; LH, luteinizing hormone.

4:50 PM
PS-17-5 — Brain state defines glymphatic washout in the rat brain (#350)

M. Segeroth1, L. Wachsmuth1, F. Albers1, C. Faber1

1 Department of Clinical Radiology, Translational Research Imaging Center, University Hospital Muenster, Muenster, Germany

Introduction

The Glymphatic System has been identified (1) as a brain-wide paravascular pathway controlling the flux of CSF in the brain. CSF flow elutes soluble proteins and waste, and is intensified during sleep or anesthesia (2). To assess whether the depth of anesthesia and thus possibly also the sleep state affects waste clearance, we investigated contrast agent distribution and water diffusivity by measuring the ADC by DWI  in the rat brain under two different anesthetic protocols. To mimic slow wave sleep, we used Isoflurane, while sedation with higher brain activity was induced by Medetomidine.

Methods

A modified standard venous catheter (26G) was implanted into the Cisterna Magna of 15 rats under Isoflurane. MRI at 9.4T was performed under Isoflurane (1.3-1.5%) (n=8) or Medetomidine (n=7) anesthesia. Apparent diffusion coefficient (ADC) maps were acquired in 10/15 rats and in 7 rats without catheter (TR/TE:4250/42.8 ms, duration 4 ms, separation 30 ms, b=10, 200, 400, 600, 800, 1000, 1200, 1400, 1500). Then contrast agent (Gadovist 21 mM) was administered via the catheter (50 min continuous infusion at 1.6 µl/min) and at least 40 T1 weighted 3D FLASH scans (TR/TE:15/ 3.8 ms, FA 15°) were acquired during 6 hours. ROI analysis was performed in CSF and brain parenchyma. In 3/7 Medetomidine anesthetized rats the brain state was simultaneously monitored by optical calcium recordings.

Results/Discussion

Optical calcium recordings revealed slow oscillations or burst suppression-like brain state, resembling slow wave sleep, under Isoflurane. More brain activity, resembling awake state, was observed under Medetomidine (Fig. 1). The ADC did not significantly change over time under Isoflurane (-1.2±3.4% ADC reduction from baseline) but decreased when anesthesia was switched to Medetomidine (-5.1±4.7%) (Fig. 1). T1w scans during and after contrast agent application showed a higher signal increase in subarachnoidal space under Medetomidine (572±148% maximal change from baseline) compared to Isoflurane (303±210%) (Fig. 2), indicating higher contrast agent concentration. Also in brain parenchyma signal increase was higher under Medetomidine (70±18%) as compared to Isoflurane (43±21%). These data support the hypothesis that different brain states, induced by different anesthetics, have impact on glymphatic waste clearance.

Conclusions

Altered glympahtic clearance under different anesthetic regimens has been reported previously (3), similar to our observations. Our additional DWI data are in line with the notion that lower diffusivity in the awake-like state under Medetomidine may be due to swelling activated neurons and a reduced extracellular water pool (4,5). Together DWI and contrast enhanced MRI data allow for the conclusion that the higher signal increase under Medetomidine indicates lower CSF efflux and thus less efficient glymphatic clearance in an awake-like brain state.

References

(1) Jeffrey J. Iliff, et al., Sci Transl Med. 2012 August 15; 4(147): 147ra111. doi:10.1126/scitranslmed.3003748 

 

(2) Lulu Xie, et al., SCIENCE VOL 342 18 OCTOBER 2013 

 

(3) Helene Benveniste, et al., Anesthesiology 2017; XXX:00-00

 

(4) Philippe Coulon, et al., The Journal of Experimental Biology 211, 630-641 doi:10.1242/jeb.008565

 

(5) Yoshifumi Abe, et al., PLoS Biol 15(4): e2001494. https://doi.org/10.1371/ journal.pbio.2001494

Effect of the different anesthetic protocols on brain state and the apparent diffusion coefficient

(A) Calcium trace under Isoflurane shows burst suppression-like brain state. (B) Calcium trace under Medetomidine shows higher brain activity similar to an awake-like state. (C) ADC reduction in a cortical ROI (relative to baseline under Isoflurane), acquired after 40 min ether with or without switching anesthesia to Medetomidine.

 

Contrast enhanced MRI after contrast administration into Cisterna Magna

(A, B) T1w MR images illustrating ROI analysis in subarachnoidal space (A) and brainstem parenchyma (B), reveal higher signal increase under Medetomidine compared to Isoflurane. (C, D) Averaged time signal curves represented as mean±confidential interval.

5:00 PM
PS-17-6 — Acquisition and reversal learning in the Morris water maze lead to different patterns of functional connectivity alterations in the mouse brain (#105)

D. Shah1, 2, M. Verhoye1, A. Van der Linden1, R. D'Hooge2

1 Bio-Imaging Lab, Biomedical Sciences, Wilrijk, Belgium
2 Laboratory of Biological Psychology, Psychology, Leuven, Belgium

Introduction

Learning processes induce structural and functional changes in the brain at the synaptic level. Previous studies have established resting-state functional MRI (rsfMRI) as a tool to investigate the effect of synaptic modulations on brain functional connectivity (FC) in mice (1, 2). RsfMRI could thus allow exploring spatiotemporal changes of brain FC after learning processes non-invasively and in the entire brain. This study assessed for the first time the effects of spatial learning in the Morris water maze on FC in mice.

Methods

We conjecture that acquisition and reversal training engage different brain regions (3) and thus lead to different patterns of FC alterations in the brain. Mice (C57BL/6, female, N=12/training) were either subjected to Morris water maze training during 2 days of acquisition learning (i.e. learning the location of the hidden platform), 10 days of acquisition learning, or 5 days of reversal learning (i.e. after changing the platform location). As controls, separate groups of mice which did not perform spatial memory training were added for each training protocol: cage controls (N=10/training) and swim controls (N=10/training). After the behaviour protocols all mice were sedated using a combination of medetomidine and isoflurane and subjected to rsfMRI scans acquired on a 9.4 T MRI scanner.

Results/Discussion

Already after 2 days of acquisition training, FC increases were observed in the hippocampus and cingulate-, motor- and visual cortex, each of which play a role in learning processes and spatial memory (Figure 1). With longer periods of training a higher number of brain regions presented increased FC. Moreover, different patterns of FC alterations were observed at different training phases, i.e. more involvement of the hippocampus, cingulate and caudate putamen during acquisition learning and of the visual cortex during reversal learning (Figures 1 and 2). This shift in FC patterns correlated to the strategy the mice applied to find the location of the hidden platform (Figure 2). FC between the hippocampus, cingulate-, motor- and visual cortex demonstrated an overall positive correlation with the use of spatial strategies and a negative correlation with the use of non-spatial strategies for each training.

Conclusions

This study showed that specific learning processes recruit a different pattern of FC changes in the mouse brain, which is correlated to the strategy that is applied for successful task performance. These results show how rsfMRI can detect the brain’s remarkable plasticity and provide more insight into the relation between brain function and animal behaviour. This opens doors for rigorous investigations of how different types of learning processes affect FC in the healthy brain and how this is modulated by disease processes.

References

1) Shah D et al., NeuroImage, 109:151-159, 2) Shah D., et al, Brain Structure and Function 221(6):3067-79. 3) D'Hooge R, De Deyn PP. Brain research reviews. 2001;36:60-90.

Acknowledgement

This research was supported by BRAINPATH (grant nr 612360) within IAPP, by FLAG-ERA JTC 2015, and by IWT Flanders (grant nr 13160)

Figure 1: Spatial learning increases FC in the mouse brain

Figure 2: FC and spatial strategies differ between acquisition and reversal learning

5:10 PM
PS-17-7 — Resting state fMRI alterations during epileptogenesis in relation to seizure burden in the KASE rat model of temporal lobe epilepsy (#184)

E. Jonckers1, D. Bertoglio2, 3, A. Idrish3, J. Verhaeghe2, M. Verhoye1, S. Dedeurwaerdere3, 4

1 Bio-Imaging Lab, University of Antwerp - Biomedical Sciences, Wilrijk, Belgium
2 Molecular Imaging Center Antwerp, University of Antwerp - Medical Sciences, Wilrijk, Belgium
3 Department of Translational Neurosciences, University of Antwerp - Biomedical Sciences, Wilrijk, Belgium
4 UCB Pharma, Braine l-Alleud, Belgium

Introduction

Epileptogenesis in acquired epilepsies is initiated by an epileptogenic event (e.g. brain trauma or infection) and is characterized by an initial latent period during which the development and extension of tissue capable of generating spontaneous seizures occurs. This then leads to the development of an epileptic condition with abnormal neuronal activity in the brain (1, 2). We hypothesize that resting state fMRI (rsfMRI) functional connectivity (FC) can reveal brain network disruption and abnormalities as a candidate mechanism in epileptogenesis and seizure generation.

Methods

Status Epileticus was induced by kainic acid (KASE) in 12 Male Wistar-Han (Charles River) of 7.5 weeks. These, and 11 controls, were measured on a 9.4T Biospec MRI scanner (Brüker) during epileptogenesis (2w post KASE) and after establishment of epilepsy (4w post KASE) (3). Rats were anesthetized with medetomidine (Pfizer; 0.05 mg/kg + 0.1 mg/kg/h, SC) and monitored during the MRI scans. 45 min post-bolus, 300 GE-EPI (TE/TR= 16/2000ms) images were acquired during 5 min (in plane resolution (0.23 x 0.23) mm2). FC calculation was performed for regions with previously reported increased inflammation in this model (3) (See Fig 1). Another cohort of KASE animals was videoEEG monitored during 12w enabling correlations between FC and the number of spontaneous recurrent seizures (SRS).

Results/Discussion

The rats were video monitored to verify no seizures occurred before w2 and all the animals experienced at least 2 seizures before w4. Statistical comparison of the correlation matrices including Cingulate-, Entorhinal-, Insular-, Motor-, Piriform- and Sensory Cortex, Hippocampus, Hypothalamus, Striatum, and Thalamus showed significantly decreased FC in the KASE animals 2w post injection, while no significant differences were found at 4w post injection (Fig 1). Interestingly, the BOLD FC of the hippocampus with the rest of the brain at 4 weeks post injection positively correlated with the SRS frequency at the same time point (Fig 2).

Conclusions

RsfMRI shows a disruption of neuronal networks predisposing to altered neuronal synchrony in KASE rats suggesting that the technique might reveal brain network reorganization during epileptogenesis. Interestingly, when the epileptic condition is established, known to be characterized by abnormal neuronal activity in the brain, the level of FC is higher in animals with a higher seizure burden (2).

References

(1) Lillis, K.P., et al. 2015. (2) Pitkanen, A., et. al. 2014. (3) Bertoglio, D., et al. 2017.

Acknowledgement

E.J. D.B and S.D. are supported by Research Foundation Flanders funding (post-doctoral fellowship FWO, 12R1917 N; PhD fellowship FWO, 11W2516N/11W2518N; Projects 1.5.110.14N, 1.5.144.12N) S.D. is supported by ERA-NET NEURON G.A009.13N, and by Queen Elisabeth Medical Foundation for Neurosciences. A.V.D.L. is supported by the European Union’s Seventh Framework Programme (278850; INMiND) and BRAINPATH (FP7-PEOPLE-2013-IAPP-612360)

Figure 1
Left: FC Matrix in KASE and control animals 2 weeks post seizure induction (* indicate significant differences). Warmer colors indicate higher FC. Right: Mean FC over the different regions in the matrix compared for both groups and time points § p < 0.1 * p < 0.05, ** p < 0.01) . 

Figure 2
Correlation analysis between mean FC of Hippocampus with the rest of the investigated regions and seizure burden (#SRS/day).

5:20 PM
PS-17-8 — DREADD-MRI reveals functional connectivity changes upon inactivation of the right dorsomedial prefrontal cortex in mice. (#388)

L. Peeters1, R. Hinz1, S. Missault1, M. Verhoye1, A. Van der Linden1, G. A. Keliris1

1 Bio-Imaging Lab - University of Antwerp, Department of Biomedical Sciences, Wilrijk, Belgium

Introduction

Chemogenetics allow researchers to probe the brain’s neural circuitry by controlling the activity of specific neurons. Recently, a new inhibitory Kappa Opioid Receptor (KOR) DREADD, selectively activated by the inert ligand Salvinorin B (SalB), was developed 1. Here, we unilaterally targeted the dorsomedial prefrontal cortex (dmPFC), a major node of the attention network, and used pharmacological MRI to assess the spatial distribution and time evolution of the DREADD effects, and resting state fMRI (rsfMRI) to measure DREADD-induced alterations in FC within and across networks.

Methods

During a stereotactic surgery, mice received either (n=12), AAV-CaMKII-HA-KORD-IRES-mCitrine, or a control virus (n=9), AAV-CaMKIIa-EGFP, in the right dmPFC. Imaging procedures were performed on anesthetized mice (medetomidine: 0.05mg/kg bolus, 0.01mg/kg infusion with 0.3% isoflurane) using a 9.4T Biospec. Two separate pharmacological fMRI (phMRI) scans were performed in different scanning sessions using two concentrations of SalB (low: 3mg/kg, high: 6mg/kg). PhMRI scans (Two-shot GE-EPI: TE=20ms, TR=15000ms, matrix=64x64, FOV=(20x20)mm2, 16 slices) were acquired starting 10 min before to 50 min after s.c. SalB administration (duration 1h). RsfMRI scans (GE-EPI: TE=16ms, TR=2000ms, matrix=128x64, FOV=(20x20)mm2, 16 slices) were acquired 10 min after s.c. injection of SalB (3mg/kg) or DMSO.

Results/Discussion

PhMRI showed decreased BOLD signals after administration of SalB (3mg/kg and 6mg/kg) in the KORD treated mice. The changes were more pronounced at 10-20min after SalB injection and gradually diminished back to baseline levels. The low SalB dose was selected for rsfMRI scans during the 10–20min post-injection time window. Upon dmPFC inactivation, ROI-based rsfMRI analysis revealed significantly decreased FC between various regions of the attention network and within the right attention network (Fig 1). Furthermore, seed based analysis of the right visual cortex revealed significantly decreased FC in the visual network (Fig 2).

Conclusions

Right dmPFC inactivation using KORD reduces the BOLD signal in a wide network of areas. Moreover, the results demonstrated altered FC in the targeted attentional network. Interestingly, FC alterations could also be observed in a distinct sensory network, i.e. the visual network. Our study demonstrates that KORD based DREADD-fMRI is a promising tool to assess large-scale network FC and activity upon neural activity modulation in selected neural populations.

References

1Vardy et al., 2015

Acknowledgement

This research was supported by University Research Fund of University of Antwerp (FFB150340) and by Research Foundation Flanders (G048917N).

Functional correlation between regions of the attention network.

(A) FC matrix shows correlations between regions of the attention network upon injection of DMSO (lower) or SalB (upper). Colorbar indicates functional Pearson’s correlation values; stars indicate significant FC differences between the groups. *p<0.05 (B+C) Graphs show Z scores (transformed correlation values) in the attention network of both hemispheres upon DMSO or SalB injection. **p<0.001.

Seed-based analysis of right visual cortex.
(A) Statistical FC map for a seed in the right visual cortex upon DMSO injection and SalB injection (uncorrected p<0.001, cluster size k≥10). (B+C) Graphs show the mean T-values ± SEM and the total cluster size of all significantly correlated clusters (minimal cluster size = 10) with the seed region. ** p<0.001, * p<0.05.

1:30 PM
emptyVal-1 — Introductory Lecture by René Botnar - London, UK

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

1:50 PM
PS-04-2 — Antibiotic treatment with Minocycline affects vascular elastin remodeling distally to the site of injury in a murine model of atherosclerosis (#109)

B. Lavin1, A. Phinikaridou1, M. E. Andia2, M. Potter3, R. M. Botnar1, 2

1 King's College London, School of Biomedical Engineering Imaging Sciences, London, United Kingdom
2 Pontificia Universidad Católica de Chile, Radiology department, School of Medicine,, Santiago, Chile
3 King's College London, GKT School of Medicine, London, United Kingdom

Introduction

Vascular interventions aim to treat focal stenosis however, they may trigger systemic responses that accelerate lesions elsewhere. The antibiotic agent minocycline has demonstrated substantial effect on matrix remodeling1-3 and late gadolinium enhancement (LGE) MRI using an elastin-specific contrast agent (Gd-ESMA) can assess matrix remodelling in cardiovascular diseases4-6. We study the merits of Gd-ESMA to assess the impact of aortic injury and minocycline treatment on the plaques located distally to the site of injury in vivo, and whether the response to injury could be driven by monocytes.

Methods

Study design is summarized in Fig.1A. Animal surgical model: Aortic injury was performed as described7. In vivo MRI: A 3T MR scanner (Achieva, Philips Healthcare, NL) equipped with a 23mm single-loop microscopy surface coil were used. Images were acquired 2h after intravenous administration of Gd-ESMA (0.2mmol/kg). Acquisition parameters are summarised in Fig.1B. Histology: Atherosclerotic plaques in the brachiocephalic artery (BCA) were analyzed using Verhoeff Van Gieson elastic stain and tropoelastin immunohistochemistry (IHC). FACS: Monocyte count was performed using B220, F4/80, CD155 and Ly6C antibodies.

Results/Discussion

LGE-MRI images of the BCA (Fig 1C) showed greater enhancement (Fig 1D) and higher R1 relaxation rate (Fig 1E) in the HFD-injury group compared to all other groups, suggesting that vascular injury accelerates atherosclerosis in distal locations compared to HFD alone. Mice treated with minocycline showed decreased Gd-ESMA uptake compared to the untreated groups (Fig 1E-1D). Histology showed increased elastin fibers in the HFD and HFD-injury groups but not in minocycline-treated mice (Fig 1C).

Flow cytometry of monocytes (Fig 2A) showed blood monocytosis in HFD and HFD-injured mice, but not in the minocycline-treated group (Fig 2B). HFD-injured mice showed increased monocyte recruitment to the BCA compared to other groups (Fig 2D). In blood and BCA, a shift from Ly6Chigh to Ly6Clow subtypes was observed in the minocycline-treated mice (Fig 2C-2E). Positive correlation was detected between vessel wall enhancement, R1 and Ly6Chigh monocytes (Fig 2F-2H) measured in the aorta and the BCA.

Conclusions

We demonstrate that focal vascular injury accelerates atherosclerosis in distal vessel segments through a systemic response driven by monocytes. Minocycline treatment alters elastin remodeling and promotes a shift from Ly6Chigh inflammatory to Ly6Clow reparative monocytes retarding intimal thickening distally to the site of injury.

References

1Ohshima, S. JACC. 2010. 2Shahzad, K. Atherosclerosis. 2011. 3Phinikaridou A. J Am Heart Assoc. 2013. 4Makowski, M.R. Nature Medicine. 2011. 5Botnar, R.M. Circ Cardiovasc Imaging. 2014. 6Protti, A. J Am Heart Assoc. 2015.7Lavin, B. Circ Cardiovasc Imaging. 2015.

Acknowledgement

The British Heart Foundation (RG/12/1/29262).

Figure 1
(A) Study design. (B) MRI acquisition parameters. (C) 1st row: Fused LGE-MRI/MRA images of the BCA vessel wall; 2nd row: Verhoeff Van Gieson elastic stain of the BCA showing a thicker fibrous cap in the injured groups (arrow); 3rd row: Tropoelastin IHC showing fiber deposition (asterisk) in the HFD and HFD-injury groups. (D) LGE-MRI area and (E) relaxation rate (R1) quantification measured by MRI.

Figure 2
(A) Flow cytometry monocyte gating strategy. (B) Quantification of monocytes (C) and percentage of Ly6Chigh (orange) and Ly6Clow (blue) monocytes in blood. (D) Quantification of monocytes (E) and percentage of Ly6Chigh (orange) and Ly6Clow (blue) monocytes in BCA. LGE-MRI (F), R1 (G) and Ly6Chigh (H) Pearson correlations between aorta and BCA. BCA: Brachiocephalic artery.

2:00 PM
PS-04-3 — Molecular imaging of tropoelastin in plaque progression and instability (#47)

A. Phinikaridou1, S. Lacerda2, B. Lavin1, M. E. Andia3, R. M. Botnar1

1 King's College London, Biomedical Engineering, London, United Kingdom
2 Centre de Biophysique Moléculaire, CNRS, Orléans, France
3 Pontificia Universidad Católica de Chile, Radiology Department, School of Medicine, Santiago, Chile

Introduction

Elastolysis and ineffective elastogenesis favor the accumulation of tropoelastin, rather than cross-linked elastin, in atherosclerotic plaques and has been associated with lesion progression and destabilization [1, 2]. We have developed a tropoelastin-binding gadolinium-based MRI contrast agent (Gd-TESMA) and demonstrated the feasibility of molecular imaging of tropoelastin in mice and rabbits.

Methods

Atherosclerotic ApoE-/- mice and New Zealand rabbits were used. Gd-TESMA was synthesized and characterized prior to in vivo use [3]. 3 Tesla MRI: Angiography, delayed-enhanced (DE) MRI and T1 mapping of the brachiocephalic artery (BCA) in mice was performed at 4, 8, and 12 weeks of high-fat-diet and 30min post-injection of Gd-TESMA. Rabbits were imaged two times before and one time after triggering for plaque rupture [4]. Pre-triggered, rabbits were scanned with an elastin-binding contrast agent (Gd-ESMA)(Lantheus Medical Imaging) and then with Gd-TESMA. T1-black-blood (BB), DE-MRI and T1 mapping images were acquired for plaque characterization. Post-triggered images were used to classify plaques in ruptured and stable. Elastin and tropoelastin stainings were used for validation.

Results/Discussion

DE-MRI (Fig. 1A1-E1 & A2-E2) and R1 maps (Fig. 1A3-E3), post-injection of Gd-TESMA, showed increased vascular enhancement and R1 relaxation rate of the BCA with disease progression and regression after statin-treated treatment (Fig.1E1-3). Histology verified the deposition of tropoelastin fibres (Fig. 1A4-E4, A5-E5, A6-E6). A scrambled probe showed less vascular enhancement compared with the non-scrambled probe (Fig. 1F1-F3). Pre-trigger images of a stable (Fig. 2A-E) and rupture-prone plaques (Fig. 2G-K) detect the lesions. DE-MRI showed vascular enhancement and reduction of T1 relaxation time post-injection of both agents. Post-trigger T1BB images showed thrombus only attached to the ruptured lesion (Fig. 2F, L). Rupture-prone plaques had higher R1 relaxation rate post-injection of Gd-TESMA compared with stable plaques and that allowed their detection with high sensitivity and specificity. Conversely, uptake of Gd-ESMA was similar between the two groups (Fig. 2M-N)

Conclusions

Molecular imaging of tropoelastin allows monitoring of lesion progression and detection of rupture-prone plaque.   

References

1.  Krettek, A., et al. ATVB, 2003.

2.  Makowski, M., et al., Nat Med, 2011.

3.  Phinikaridou, A., et al., ISMRM proceedings, 2016.

4.  Phinikaridou, A., et al., Radiol. 2014.

Acknowledgement

British Heart Foundation

Figure 1: MRI of tropoelastin monitors atherosclerosis progression in mice.

Figure 2: MRI of tropoelastin allows detection of rupture-prone rabbit plaques.

2:10 PM
PS-04-4 — Targeting malondialdehyde-acetaldehyde epitopes with a human antibody fragment detects clinically relevant atherothrombotic lesions and allows non-invasive PET/MR imaging in experimental models (#119)

M. L. Senders1, 2, X. Que3, Y. S. Cho4, 5, C. Yeang5, H. Groenen1, F. Fay1, A. E. Meerwaldt1, C. Calcagno1, S. Green5, P. Miu5, M. E. Lobatto6, T. Reiner7, Z. A. Fayad1, J. L. Witztum3, W. J. M. Mulder1, S. Tsimikas5, C. Pérez-Medina1

1 Icahn School of Medicine at Mount Sinai, Translational and Molecular Imaging Institute, New York, New York, United States of America
2 Academic Medical Center, Department of Medical Biochemistry, Amsterdam, Netherlands
3 University of California San Diego, Division of Endocrinology and Metabolism, Department of Medicine, La Jolla, California, United States of America
4 Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
5 University of California San Diego, Division of Cardiovascular Diseases, Sulpizio Cardiovascular Center, Department of Medicine, La Jolla, California, United States of America
6 Academic Medical Center, Department of Radiology, Amsterdam, Netherlands
7 Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, New York, United States of America

Introduction

Oxidation-specific epitopes (OSE) are well defined danger-associated molecular patterns that activate inflammatory pathways leading to initiation and progression of atherosclerosis1,2, the major underlying cause of cardiovascular disease. Despite its tremendous socioeconomic impact, clinical characterization of atherosclerosis remains a challenge, as it mainly relies on the detection of the degree of stenosis3. Here we present a novel positron emission tomography (PET) probe that allows non-invasive imaging of OSE-rich atherosclerotic lesions.

Methods

The human monoclonal antigen-binding fragment (Fab) LA25 was identified and characterized after multiple rounds of screening against the oxidation-specific malondialdehyde-acetaldehyde (MAA) epitope. Pharmacokinetics, biodistribution and plaque specificity studies were performed in Apoe-/- mice with Zirconium-89 (89Zr)-labeled LA25. In rabbits, 89Zr-LA25 was used in combination with an integrated clinical PET/magnetic resonance (PET/MR) system. 18F-fluorodeoxyglucose (18F-FDG)-PET and dynamic contrast-enhanced MR imaging (DCE-MRI) were used to evaluate vessel wall inflammation and plaque neovascularization, respectively. Extensive ex vivo validation was carried out by a combination of gamma counting, near-infrared fluorescence, autoradiography, immunohistochemistry, and immunofluorescence.

Results/Discussion

LA25 bound specifically to MAA epitopes in advanced and ruptured human atherosclerotic plaques with accompanying thrombi and in debris from distal protection devices. In Apoe-/- mice, 89Zr-LA25 accumulation in the aorta was significantly higher than for 89Zr-LA24, a non-targeted Fab that was used as chemical control (Fig. 1B-C). Analysis of aortic root sections revealed extensive co-localization of 89Zr-LA25 radioactivity to macrophage-rich areas (Fig. 1E). PET/MR imaging 24 hours after injection of 89Zr-LA25 showed increased uptake in the abdominal aorta of atherosclerotic rabbits compared to non-atherosclerotic controls, confirmed by ex vivo gamma counting (P=0.02) and autoradiography. 18F-FDG-PET, DCE-MRI, and NIRF signals were also significantly higher in atherosclerotic rabbit aortas compared to controls (Fig. 2). Enhanced liver uptake was also noted in atherosclerotic animals, confirmed by the presence of MAA epitopes by immunostaining.

Conclusions

The human Fab antibody LA25 targeting the malondialdehyde-acetaldehyde epitope detects clinically relevant atherothrombotic lesions and allows non-invasive PET imaging of atherosclerotic plaques in rabbits. Ultimately, this radiotracer could serve as a marker to evaluate and inform therapeutic interventions.

 

References

1. Miller YI, Choi SH, Wiesner P et al. Oxidation-specific epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate immunity. Circ Res 2011;108:235-48.

2. Binder CJ, Papac-Milicevic N and Witztum JL. Innate sensing of oxidation-specific epitopes in health and disease. Nat Rev Immunol 2016;16:485-97.

3. Dweck MR, Aikawa E, Newby DE, Tarkin JM, Rudd JH, Narula J and Fayad ZA. Noninvasive Molecular Imaging of Disease Activity in Atherosclerosis. Circ Res 2016;119:330-40.

Acknowledgement

Fondation Leducq, R01-HL119828, R01-HL078610, R01-HL106579, R01 HL128550, R01 HL136098, P01 HL136275, R35 HL135737, P01-HL055798, (ST and/or JLW), R01 EB009638 (ZAF), P01-HL131478 and R01-HL125703 (WJMM), and AHA 16SDG31390007 (CPM). MLS is supported by AHA 17PRE33660729 and the Foundation “De Drie Lichten” in The Netherlands.

 

Figure 1. 89Zr-LA25 evaluation in Apoe-/- mice
Blood time-activity curve (A), aortic accumulation (B-C), and radioactivity distribution in selected tissues 4 hours post injection (D) of 89Zr-LA25 or control Fab 89Zr-LA24 in Apoe-/- mice. Immunofluorescence and autoradiography on aortic sections from Apoe-/- mice after 89Zr-LA25 injection showing radioactivity deposition on macrophage-rich (CD68) areas (E).

Figure 2. Phenotyping of rabbit atherosclerotic plaques by PET/MRI.
Representative coronal aortic fused PET/MR imaging 24 hours p.i. of 89Zr-LA25 (A), autoradiographs and gamma counting (whole aortas) 28 hours p.i. of 89Zr-LA25 (B), MR T2-weighted imaging (C), 18F-FDG PET/MRI (D), DCE-MRI (E), and DiD-rHDL near-infrared fluorescence imaging (F), in healthy control (white) and atherosclerotic abdominal aortas (black).

2:20 PM
PS-04-5 — Platelet-targeted microbubbles for diagnostic and theranostic approach of thrombosis using in vivo molecular ultrasound imaigng: Treatment without bleeding complications (#325)

X. Wang1, 2, Y. Gkanatsas1, K. Peter1, 2

1 Baker Heart and Diabetes Institute, Atherothrombosis and Vascular Biology, Melbourne, VIC, Australia
2 Monash Univeristy, Department of Medicine, Melbourne, VIC, Australia

Introduction

Most acute cases of myocardial infarction and stroke are caused by atherothrombosis, when platelet adhesion, activation and aggregation, lead to thrombus formation and vessel occlusion. Glycoprotein (GP) IIb/IIIa complex, is the most abundant receptor expressed on the platelet surface, responsible for adhesion and aggregation. We have developed conformation-specific single-chain antibodies (scFv) that bind specifically to ligand-induced binding sites (LIBS) on activated GPIIb/IIIa.

Methods

For diagnostic imaging, MB were conjugated to either a single-chain antibody (scFv) specific for activated GPIIb/IIIa (LIBS-MB), or a non-specific scFv (control-MB). For theranostic approach, we conjugated thrombolytic drugs, such as single chain urokinase plasminogen activator (scuPA), to form targeted theranostic MBs (TT-MB). In a ferric chloride induced carotid artery thrombi mouse model, imaging was performed before and after MB injection.

Results/Discussion

LIBS-MBs strongly adhered to immobilized activated platelets and micro-thrombi under flow. A significant increase in decibel (dB) was observed after LIBS-MB but not after control-MB injection (9.55 ± 1.7 versus 1.46 ± 1.3 dB; p<0.01). For theranostic approach, TT-MB significantly reduced thrombus size after 45 min, while no significant difference was noted in the MB that were targeted but without urokinase (37 ± 6 vs. 97 ± 4, mean % change ± SEM, normalized to baseline thrombus size, p<0.001). The same degree of efficient thrombolysis was only achievable using a high dose of urokinase (NS). The targeting and thus clot-enrichment effect of TT-MBs results in a highly potent fibrinolysis that could only be matched using high doses of non-targeted urokinase. However, the latter is associated with a highly prolonged bleeding time (79 ± 7 vs. 1079 ± 261, sec ± SEM, p<0.001). In contrast, TT-MB does not prolong bleeding time (NS).

Conclusions

Our LIBS-MBs specifically bind to activated platelets in vitro and allow real-time molecular imaging of acute arterial thrombosis in vivo. TT-MBs conjugated with recombinant urokinase represent a novel and unique theranostic approach to simultaneously diagnose thrombosis, as well as to treat and monitor the success or failure of thrombolysis. This unique, non-invasive and cost effective technology holds promise for major progress towards rapid diagnosis and bleeding-free, potent therapy of the vast number of patients suffering from thrombotic diseases.

2:30 PM
PS-04-6 — Nanobody-facilitated multiparametric PET/MRI phenotyping of atherosclerosis (#81)

M. L. Senders1, 2, S. Hernot3, G. Carlucci4, 5, J. C. van de Voort2, F. Fay2, 6, C. Calcagno2, J. Tang2, A. Alaarg2, Y. Zhao2, S. Ishino2, A. Palmisano2, G. Boeykens2, A. E. Meerwaldt2, B. L. Sanchez-Gaytan2, S. Baxter2, L. Zendman2, M. E. Lobatto2, 7, N. A. Karakatsanis2, P. M. Robson2, A. Broisat8, G. Raes9, 10, J. S. Lewis5, 11, 12, S. Tsimikas13, T. Reiner5, 11, Z. A. Fayad2, N. Devoogdt3, W. J. M. Mulder1, 2, C. Pérez-Medina2

1 Icahn School of Medicine at Mount Sinai, Translational and Molecular Imaging Institute, New York, United States of America
2 Academic Medical Center, Department of Medical Biochemistry, Amsterdam, Netherlands
3 Vrije Universiteit Brussel, In vivo Cellular and Molecular Imaging laboratory, Brussels, Belgium
4 New York University, Bernard and Irene Schwarz Center for Biomedical Imaging, New York, United States of America
5 Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, United States of America
6 York College of The City University of New York, Department of Chemistry, New York, United States of America
7 Academic Medical Center, Department of Radiology, Amsterdam, Netherlands
8 INSERM UMR S 1039, Bioclinic Radiopharmaceutics Laboratory, Grenoble, France
9 Vrije Universiteit Brussel, Research Group of Cellular and Molecular Immunology, Brussels, Belgium
10 VIB Inflammation Research Center, Laboratory of Myeloid Cell Immunology, Ghent, Belgium
11 Weill Cornell Medical College, Department of Radiology, New York, United States of America
12 Memorial Sloan Kettering Cancer Center, Molecular Pharmacology Program, New York, United States of America
13 UCSD, Division of Cardiovascular Diseases, San Diego, United States of America

Introduction

Non-invasive characterization of atherosclerosis, the pathophysiological process of stroke and myocardial infarction1, remains a challenge in clinical practice. The limitations of current diagnostic methods demonstrate that, in addition to atherosclerotic plaque morphology and composition, disease activity needs to be evaluated2. Here, we aimed to combine target-specific nanobody-positron emission tomography (PET) imaging information with functional and anatomical magnetic resonance imaging (MRI) readouts to develop an integrative multiparametric atherosclerotic plaque phenotyping procedure.

Methods

We screened three nanobody radiotracers targeted to different biomarkers of atherosclerosis progression, namely vascular cell adhesion molecule 1 (VCAM-1), lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), and macrophage mannose receptor (MMR). The nanobodies, initially radiolabeled with Copper-64 (64Cu), were extensively evaluated in Apoe-/- mice and atherosclerotic rabbits using a combination of in vivo PET/MRI readouts and ex vivo radioactivity counting, autoradiography and histological analyses.

Results/Discussion

The three nanobody radiotracers accumulated in atherosclerotic plaques and displayed short circulation times due to fast renal clearance. The MMR nanobody was selected for labeling with Gallium-68 (68Ga), a short-lived radioisotope with high clinical relevance, and used in an ensuing atherosclerosis progression PET/MRI study. Macrophage burden was longitudinally studied by 68Ga-MMR-PET, plaque burden by T2-weighted MRI (T2W-MRI), and neovascularization by dynamic contrast enhanced MRI (DCE-MRI). Additionally, inflammation and microcalcifications were evaluated by 18F-fluorodeoxyglucose and 18F-sodium fluoride PET, respectively. Using our multiparametric approach, we were able to detect an increase in macrophage and plaque burden, neovascularization, inflammation and microcalcifications as disease progressed, and correlated with histopathological features.

Conclusions

We have evaluated nanobody-based radiotracers in rabbits and developed an integrative PET/MR imaging protocol that allows non-invasive assessment of different processes relevant to atherosclerosis progression. This multimodal imaging approach would not only have a potential impact on future anti-atherosclerosis clinical drug trials, but is immediately relevant on a preclinical level, both for a better understanding of atherosclerosis biology and the development and evaluation of new drugs, noninvasively and longitudinally in animals.

References

1. Hansson GK. Inflammation, Atherosclerosis, and Coronary Artery Disease. N Engl J Med. 2005;352(16):1685-1695. 

2. Dweck MR, Aikawa E, Newby DE, Tarkin JM, Rudd JHF, Narula J, Fayad ZA. Noninvasive Molecular Imaging of Disease Activity in Atherosclerosis. Circ Res. 2016;119(2):330-340.

 

Acknowledgement

This work was supported by the National Institutes of Health grants R01 EB009638, P01 HL131478 (Z.A.F.), R01 HL125703, R01 HL118440 (W.J.M.M.), P30 CA008748, the American Heart Association 16SDG31390007 (C.P.M.), 17PRE33660729 (M.L.S.), the Netherlands Organization for Scientific Research NWO Vidi (W.J.M.M) and the “De Drie Lichten” Foundation in The Netherlands (M.L.S.). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) for financial support (T.R.).

Figure 1. Nanobody-radiotracer screening in mice.

Radioactivity distribution at 3 h p.i. of 64Cu-nanobodies. B) Autoradiography (AR) and C) radioactivity concentration in aortas at 3 h p.i. D) PET/CT images 1 h p.i. of 64Cu-VCAM (left) and 64Cu-MMR (right). E) Aortic root sections showing H&E staining (top left), AR (top right), CD31 (endothelial cells, bottom left) and CD68 (macrophages, bottom right) immunostaining. All in Apoe-/- mice.

Figure 2. PET/MRI evaluation of atherosclerosis progression.

A) Coronal aortic PET/MR images for 18F-FDG (left), 68Ga-MMR (middle) and 18F-NaF (right), and B) T2W-MRI (left) and DCE-MRI (right) images from healthy and atherosclerotic rabbits (4 or 8 months on high-fat diet, HFD). C) Cardiac PET/MR images and aorta-to-heart ratios in rabbits with atherosclerosis (8HFD). D) Aortic sections from rabbits stained with H&E and RAM-11 (macrophages). * P < 0.05.

2:40 PM
PS-04-7 — Quantitative Intravascular Fluorescence-Ultrasound Imaging In Vivo (#58)

D. Bozhko1, V. Ntziachristos1

1 Technische Universität München, CBI, Munich, Bavaria, Germany

Introduction

The need to identify and quantify the vulnerability state of the atherosclerotic plaques led to the development of multi-modality intravascular imaging systems with molecular sensitivity [1,2]. While intravascular ultrasound (IVUS) imaging and optical coherence tomography (OCT) reveal information related to arterial structures and stents, they fail to assess the biological features of vascular disease. To enable a co-registration of molecular and morphological aspects of arterial disease in vivo a hybrid near-infrared fluorescence - intravascular ultrasound (NIRF-IVUS) imaging was introduced.

Methods

Fully integrated NIRF-IVUS catheter was engineered to accurately co-register biological and morphological readings in vivo. A correction algorithm utilizing IVUS information was developed to account for the distance-related fluorescence attenuation due to through-blood imaging. NIRF-IVUS was validated in various clinically-relevant scenarios including a model of angioplasty-induced vascular injury and a model of fibrin deposition on coronary artery stents in pigs.

Results/Discussion

Co-registration of NIRF and IVUS signals, and a NIRF attenuation blood correction model, were verified to be physically accurate in vitro. We discovered that distance-correction model calibrated empirically on ex vivo data overestimates the degree of light attenuation that occurs in vivo. Therefore, we calibrated our model based on in vivo measurements. Next, concurrent in vivo intravascular imaging was performed through flowing blood. The addition of ICG-enhanced NIRF assessment improved the detection of angioplasty-induced endothelial damage compared to standalone IVUS.  NIRF detection of coronary stent fibrin illuminated stent pathobiology that was concealed on standalone IVUS. Fluorescence reflectance imaging and microscopy of resected tissues corroborated the in vivo findings.

Conclusions

Integrated NIRF-IVUS enables simultaneous co-registered through-blood imaging of disease related morphological and biological alterations in coronary and peripheral arteries in vivo. Clinical translation of NIRF-IVUS may significantly enhance knowledge of arterial pathobiology, leading to improvements in clinical diagnosis and prognosis, and help guide the development of new therapeutic approaches for arterial diseases. 

References

1. Bourantas C V., Jaffer FA, Gijsen FJH, Soest G van, Madden SP, Courtney BK, Fard AM, Tenekecioglu E, Zeng Y, Steen AFW van der, Emelianov S, Muller J, Stone PH, Marcu L, Tearney GJ, Serruys PW. Hybrid intravascular imaging: recent advances, technical considerations, and current applications in the study of plaque pathophysiology. Eur Heart J 2016;ehw097.

2. Ma T, Zhou B, Hsiai TK, Shung KK. A Review of Intravascular Ultrasound-based Multimodal Intravascular Imaging: The Synergistic Approach to Characterizing Vulnerable Plaques. Ultrason Imaging 2016;38:314–331.

Schematic of the NIRF-IVUS imaging system for intravascular through-blood imaging.
The NIRF-IVUS imaging catheters (insets on top left) consist of an ultrasound transducer and NIRF optical fiber. The dual-modality imaging probe rotates and pulls back inside a transparent catheter sheath using an electro-optical rotary joint, which connects the moving catheter with the stationary back-end console (insets on bottom right).

In vivo validation of NIRF-IVUS in clinically-relevant conditions.

(a) In vivo NIRF-IVUS imaging of vascular injury with ICG in a swine iliac artery using the 9F/15MHz hybrid NIRF-IVUS catheter. (b) In vivo imaging of the coronary artery with an implanted NIR fluorescent fibrin-labeled stent.

2:50 PM
PS-04-8 — Non-invasive imaging of macrophage-rich atherosclerotic plaques using Zirconium-89 (89Zr)-labelled desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO)-coupled anti-Galectine-3 (Gal-3)-F(ab')2. (#374)

Z. Varasteh1, F. De Rose1, S. Mohanta2, Y. Li2, M. Braeuer1, B. Miritsch3, S. Nekolla1, A. Habenicht2, H. Sager3, A. Bartolazzi4, M. Schwaiger1, C. D'Alessandria1

1 Klinikum rechts der Isar-TUM, Nuclear medicine, Munich, Bavaria, Germany
2 Institute for Cardiovascular Prevention, University Hospital of Ludwig-Maximilians-University, Munich, Bavaria, Germany
3 Deutsches Herzzentrum München, Klinik für Herz und Kreislauferkrankungen-TUM, Munich, Bavaria, Germany
4 St. Andrea University Hospital, Department of Pathology, Rome, Italy

Introduction

Atherosclerosis remains the main cause of mortality in industrialized countries. Identification of the lesions prone to rupture, may lead to the application of pharmacological/mechanical strategies to prevent clinical events. High macrophage density is considered as potential marker of plaque vulnerability. Galectin-3 (Gal-3) has been reported to be expressed on activated macrophages. In the present project, our aim was to evaluate the potential of 89Zr-anti-Gal-3-F(ab')2 to selectively target infiltrated macrophages and non-invasively image atherosclerotic plaques in ApoE-KO mice using PET.

 

Methods

DFO-anti-Gal-3-F(ab')2 was labelled with 89Zr. The binding selectivity of the fluorescent and 89Zr-labelled tracer was evaluated in vitro, on M0, M1 and M2 activated macrophages. The radiotracer was injected into ApoE-KO and age-matched control mice. Animals were scanned 48 h p.i. Upon PET/CT scans, mice were sacrificed and organs were collected for radioactivity measurement. The whole length aortas were harvested free from adipose tissue for Sudan-IV staining and autoradiography. Cryosections were prepared for immunofluorescence staining (IFS).

Results/Discussion

Fluorescent and 89Zr-labelled tracer accumulated in vitro mainly in M2 activated macrophages (Figure 1). 89Zr-DFO-anti-Gal-3-F(ab')2 accumulated in vivo in the liver (7.8±1.3 %ID/g), spleen (13.7±3.2 %ID/g) and kidneys (69±7 %ID/g, 48 h p.i.). It showed only low residual blood signal (0.5±0.1 %ID/g, 48 h p.i.). Focal signals could be detected in the atherosclerotic plaques of ApoE-KO mice (Figure 2) whereas no signal was detected in the aortas extracted from control mice. 89Zr-DFO-anti-Gal-3-F(ab')2 uptake was observed in atherosclerotic plaques on autoradiography correlating well with Sudan-IV-positive areas. The 89Zr-DFO-anti-Gal-3-F(ab')2 uptake in the plaques was associated with subendothelial accumulations of Gal-3 expressing CD68-positive macrophages confirmed by IFS. No Gal-3 expression was observed in the adventitia and adipose tissue. In the plaques, the Gal-3 expression was higher in the shoulder region (Figure 2).

Conclusions

Our data suggest that 89Zr-DFO-anti-Gal-3-F(ab')2 may serve as both an imaging agent for macrophage-rich plaques and a novel therapy assessment tracer in anti-inflammatory strategies for the treatment of atherosclerosis. Small dimension atherosclerotic plaques could be efficiently targeted and visualized in vivo using 89Zr-DFO-anti-Gal-3-F(ab')2 in ApoE-KO mouse model. However, substantial uptake of 89Zr-DFO-anti-Gal-3-F(ab')2 in the liver, spleen and the kidneys may impede the interpretation of the signals originating from abdominal aorta in mice.

In vitro binding selectivity tests
A) Optical and fluorescence microscopy images of M0, M1 and M2 macrophages incubated with fluorescent tracer. B) Cell associated radioactivity of M0, M1 and M2 macrophages incubated with radiolabelled tracer.

In vivo PET/CT and ex vivo IFS
Axial, coronal and sagittal views of PET/CT images acquired in vivo 48 h p.i. of 89Zr-DFO-anti-Gal-3-F(ab')2. Note the intense focal signal in the atherosclerotic plaque of the aortic arch in an ApoE-KO mouse (white arrows). Photomicrographs of Gal-3 (green), CD68 (red) immunofluorescence staining. Overlaping domains of expression is shown in yellow. DAPI stained nuclei are shown in blue.

6:00 PM
SG-01-1 — Optimal imaging protocols, tricks and pitfalls for MRI (#608)

M. R. Makowski1

1 Charité, Radiology, Berlin, Berlin, Germany

Content

Optimal imaging protocols, tricks and pitfalls for MRI

This presentation will give an overview regarding optimal imaging protocols, tricks and pitfalls for magnetic resonance imaging with a specific focus on cardiovascular diseases. The first part of the presentation will focus on the technical aspects of imaging atherosclerotic plaque with MRI. The second part of the presentation will focus on the protocols and techniques needed for the visualization of molecular imaging probes. In the context of atherosclerosis, MRI allows the noninvasive characterization of the relative plaque composition, the integrity of the fibrous cap and the quantification of plaque burden. By multiparametric imaging protocols in combination with MR probes, contrast between plaque structures, such as the necrotic core and the fibrous cap, can be visualized. Most MRI probes are based on paramagnetic complexes, for instance paramagnetic gadolinium (Gd) based substances or iron-oxide nano-particles. Molecular Gd-based probes are a combination of a chelated Gd with a target specific component. Gd-based probes cause a positive signal effect, as a result of the shortening of the T1 relaxation time. Iron oxide based nanoparticles on the other hand cause a strong negative signal effect, as a result of the shortening of the T2/T2* relaxation time. The advantage of MRI is the detection and visualization of molecular probes with a high spatial and temporal resolution, compared to modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT). MRI also allow the generation of anatomical images and enables the native characterization of atherosclerotic plaque components with a high soft tissue contrast based on local tissue properties. Additionally, MRI is a radiation free technique, therefore imaging can be repeated multiple times without associated risks. On the other hand, MRI is a relatively time-consuming technique, compared to e.g. CT and the sensitivity for the detection of molecular probes is lower compared to nuclear techniques like PET and SPECT. 

6:15 PM
SG-01-2 — A new picture of atherosclerosis – can Optical Imaging do more? (#576)

M. Wildgruber1

1 Westfälische Wilhelms Universität Münster, Translational Research Imaging Center, Münster, Germany

Content

As atherosclerosis in generally perceived as an inflammatory disease, capturing immune and inflammation related biology is an important task for the molecular imaging community.

Optical Imaging has the advantage to cover a wide range of contrasts and synthesis of optical reporters targeting specific cellular or molecular aspects is chemically easier compared to the synthesis of Magnetic Resonance Agents or radiotracers. The limited penetration depth however remains the major hurdle of optical technologies. Diffuse attenuation and scattering of the light in complex tissue decreases signal intensity, spatial resolution and makes quantification of fluorescence cumbersome. Similarly, clinical translation of optical technologies for imaging cardiovascular disease is limited.

However, recent approaches such a hybridized optical imaging as well as photoacoustic imaging overcome several of the traditional drawbacks, improve spatial resolution and quantification and enable first attempts of clinical translation. Invasive optical technologies that bring the light source and detector close to the target via catheter devices are promising tools to move optical molecular imaging towards clinical applications.

6:30 PM
SG-01-3 — Pre-clinical nuclear imaging of atherosclerosis. (#582)

Z. Varasteh1

1 Klinikum rechts der Isar-TUM, Nuclear medicine, Munich, Germany

Content

Atherosclerosis is one of the most actively investigated fields in medical imaging. Nuclear imaging of plaque instability targets a wide range of pathophysiologic processes involved in atherosclerosis;

The accumulation of LDL or its oxidized derivatives is the first step of atherosclerosis which was adapted as target for imaging of atherosclerotic plaques in early studies (1, 2).

Endothelial activation is the sliding door for inflammation. The expression of VCAM-1 or selectins on the activated endothelium has been selected for plaque imaging and some radiotracers were utilized to this aim (3, 4).

Activated macrophages are the most extensively investigated imaging targets for assessing plaque vulnerability and [18F]-FDG is the most widely used imaging probe to this aim. The close correlation between [18F]-FDG uptake and macrophage accumulation has been reported both in animal models and human studies. However, high tracer uptake into myocytes of the heart in addition to lack of the specificity of [18F]-FDG to inflammatory cells limits its utility. Therefore, more specific tracers for detection of inflammatory activity in the vessel walls need to be pursued. Many surface receptors expressed on activated macrophages, including somatostatin receptor SSTR2, translocator protein TSPO, mannose receptor MR and chemokine receptor CXCR4, were utilized for plaque imaging (5, 6, 7, 8).

Several proteases released by macrophages, e.g. matrix metalloproteinase MMP, which are responsible to loosen the extracellular space so that inflammatory as well as smooth muscle cells can easily migrate through the space, were also targeted to image prone to rupture atherosclerotic plaques (9).

Increase of the plaque size as well as active and prolonged inflammation in vulnerable plaques leads to hypoxic conditions in the local tissue. [18F]-FMISO has been utilised to image hypoxia for plaque visualization (10).

Neoangiogenesis is another hallmark of vulnerable plaque. Radiotracers targeting integrin αvβ3 and VEGF receptors have been evaluated for plaque imaging (11, 12).

Apoptosis, which occurs in macrophages and other cell types as a result of inflammation, has also been adapted for atherosclerotic plaque imaging (13).

It has been proposed that by imaging calcification as a final process in inflammation we may be able to identify early calcific deposits and hence rupture-prone plagues (14).

In spite of acquired promising results, there are still considerable limitations in nuclear imaging of atherosclerosis. The main challenge to plaque imaging is that the accumulation of an imaging agent and hence signal is not enough in the target lesion because most of the lesions are very small and easily affected by the partial volume effect. Therefore, it may be necessary to employ a partial volume correction to improve the quantitative assessment of the plaque. Ex vivo confirmation of the tracer accumulation in the lesions is essential for tracer characterization. In addition, comparison studies to [18F]-FDG are likely required to determine the added value of the new radiotracer for plaque imaging.

Altogether, the range of targets reflects the complexity of the disease process, and definition of the optimal target for identification of vulnerability remains unclear.

 

 

 

 

References

1. Shaw PX, Hörkkö S, Tsimikas S, Chang MK, Palinski W, Silverman GJ, et al. Human-derived anti-oxidized LDL autoantibody blocks uptake of oxidized LDL by macrophages and localizes to atherosclerotic lesions in vivo. Arterioscler Thromb Vasc Biol. 2001;21:1333–1339.

2. Tekabe Y, Li Q, Rosario R, Sedlar M, Majewski S, Hudson BI, et al. Development of receptor for advanced glycation end products-directed imaging of atherosclerotic plaque in a murine model of spontaneous atherosclerosis. Circ Cardiovasc Imaging. 2008;1:212–219.

3. Nahrendorf M, Keliher E, Panizzi P, Zhang H, Hembrador S, Figueiredo JL, et al. 18F-4V for PET-CT imaging of VCAM-1 expression in atherosclerosis. JACC Cardiovasc Imaging. 2009;2:1213–1222.

4. Li X, Bauer W, Israel I, Kreissl MC, Weirather J, Richter D, et al. Targeting P-selectin by gallium-68-labeled fucoidan positron emission tomography for noninvasive characterization of vulnerable plaques: correlation with in vivo 17.6T MRI. Arterioscler Thromb Vasc Biol. 2014;34:1661–1667.

5. Rinne P, Hellberg S, Kiugel M, Virta J, Li XG, Kakela M, et al. Comparison of Somatostatin Receptor 2-Targeting PET Tracers in the Detection of Mouse Atherosclerotic Plaques. Mol Imaging Biol. 2016;18(1):99-108.

6. Hellberg S, Silvola JMU, Kiugel M, Liljenback H, Savisto N, Li XG, et al. 18-kDa translocator protein ligand 18F-FEMPA: Biodistribution and uptake into atherosclerotic plaques in mice. J Nucl Cardiol. 2017;24(3):862-71.

7. Tahara N, Mukherjee J, de Haas HJ, Petrov AD, Tawakol A, Haider N, et al. 2-deoxy-2-[18F]fluoro-D-mannose positron emission tomography imaging in atherosclerosis. Nat Med. 2014;20(2):215-9.

8. Hyafil F, Pelisek J, Laitinen I, Schottelius M, Mohring M, Doring Y, et al. Imaging the Cytokine Receptor CXCR4 in Atherosclerotic Plaques with the Radiotracer (68)Ga-Pentixafor for PET. J Nucl Med. 2017;58(3):499-506.

9. Schäfers M, Riemann B, Kopka K, Breyholz HJ, Wagner S, Schäfers KP, et al. Scintigraphic imaging of matrix metalloproteinase activity in the arterial wall in vivo. Circulation. 2004;109:2554–2559.

10. Mateo J, Izquierdo-Garcia D, Badimon JJ, Fayad ZA, Fuster V. Noninvasive assessment of hypoxia in rabbit advanced atherosclerosis using (1)(8)F-fluoromisonidazole positron emission tomographic imaging. Circ Cardiovasc Imaging. 2014;7(2):312-20.

11. Golestani R, Zeebregts CJ, Terwisscha van Scheltinga AG, Lub-de Hooge MN, van Dam GM, Glaudemans AW, et al. Feasibility of vascular endothelial growth factor imaging in human atherosclerotic plaque using (89)Zr-bevacizumab positron emission tomography. Mol Imaging. 2013;12(4):235-43.

12. Laitinen I, Saraste A, Weidl E, Poethko T, Weber AW, Nekolla SG, et al. Evaluation of alphavbeta3 integrin-targeted positron emission tomography tracer 18F-galacto-RGD for imaging of vascular inflammation in atherosclerotic mice. Circ Cardiovasc Imaging. 2009;2(4):331-8.

13. Johnson LL, Schofield L, Donahay T, Narula N, Narula J. 99mTc-annexin V imaging for in vivo detection of atherosclerotic lesions in porcine coronary arteries. J Nucl Med. 2005;46:1186–1193.

14. Irkle A, Vesey AT, Lewis DY, Skepper JN, Bird JL, Dweck MR, et al. Identifying active vascular microcalcification by (18)F-sodium fluoride positron emission tomography. Nat Commun. 2015;6:7495.

 

6:45 PM
SG-01-4 — The immune system as a target for treating complex cardiovascular disease (#613)

W. Mulder1, 2

1 Icahn School of Medicine at Mount Sinai, Department of Radiology, New York, United States of America
2 Academic Medical Center, Cardiovascular Nanomedicine, Amsterdam, Netherlands

Content

Thrombotic events in atherosclerosis are the ultimate consequence of chronic, maladaptive lipid-driven vessel wall inflammation. As atherosclerotic disease progresses, plaque monocyte-derived macrophages destabilize the vessel wall by secreting inflammatory cytokines and proteases that digest extracellular matrix, thereby weakening the protective fibrous cap and promoting thrombosis. Immunological studies have elucidated that macrophage dynamics in atherosclerosis is a complex systemic process which, after initial production of monocytes in the bone marrow, involves monocyte egress from the bone marrow and spleen, and subsequent plaque monocyte recruitment, resulting in increased macrophage accumulation. To add to the complexity, recent preclinical and clinical data describe a direct causal link between clinical cardiovascular events and the aggravation of inflammatory atherosclerosis.

In this presentation, macrophage dynamics in atherosclerosis and the role of imaging to noninvasively quantify this process systemically are elucidated. Lessons learned from the first trial focusing on inflammation (CANTOS) have redefined the cardiovascular therapeutic landscape, generating new opportunities for nanoimmunotherapy, one such — we recently developed — will be highlighted.

7:00 PM
emptyVal-1 — Open Discussion on future directions, group activities, and review article.

2:30 PM
ES-03-1 — Watching synthetic biology at work (#596)

G. G. Westmeyer1, 2

1 Technical University of Munich, Nuclear Medicine, Munich, Bavaria, Germany
2 Helmholtz Zentrum Munich, Institute for Biological and Medical Imaging, Neuherberg, Bavaria, Germany

Objectives

As indicated in the 'Topics Covered,' this educational talk will introduce fundamental principles and concepts of synthetic biology and the capabilities that arise from them for molecular imaging across different scales and organ systems - to complement the expert talks by Drs. Jerala and Witney on designer proteins and genetic circuits and translational gene reporter imaging, respectively.

We especially invite students and postdocs to join us in this educational session and the following interest group meeting so we can connect the next generation of multidisciplinary molecular imagers.

Content

Reporter genes such as GFP are indispensable for biological research, and targeted modifications of this protein have famously lead to variants with all colors of the rainbow.

As our understanding of biomolecules and fundamental cellular processes grows, the capabilities of synthetic biology expand far beyond reporter genes. Bottom-up biological engineering can construct designer DNA, RNA, proteins, protein assemblies, cellular compartments, …, to control cellular machinery and, e.g., reprogram input/output behavior of cells or install new cellular and tissue functions.

In a positive feedback loop, new biosynthetic sensors will further increase the capabilities of molecular imaging to quantify cellular parameters and reverse-engineer cell and tissue functions. Furthermore, molecular imaging will be vital to spatiotemporally control molecular interventions that can be exerted by bioengineered actuators such as optogenetic tools. 

Importantly, because of the enormous success of cellular therapies such as those based on CAR T-cells, there is a growing need to monitor - and in the future control - the function of genetically modified cells in the body of patients. These exciting biomedical advances call for the combined expertise and joint efforts of synthetic biologists and molecular imagers as we will outline in this educational session.

Relevant Publications

Kitada, T., DiAndreth, B., Teague, B., Weiss, R., 2018. Programming gene and engineered-cell therapies with synthetic biology. Science 359, eaad1067.

Huang, P.-S., Boyken, S.E., Baker, D., 2016. The coming of age of de novo protein design. Nature 537, 320–327. doi:10.1038/nature19946

Sigmund, F., Massner, C., Erdmann, P., Stelzl, A., Rolbieski, H., Fuchs, H., de Angelis, M.H., Desai, M., Bricault, S., Jasanoff, A., Ntziachristos, V., Plitzko, J., Westmeyer, G.G., 2017. Eukaryotically expressed encapsulins as orthogonal compartments for multiscale molecular imaging. bioRxiv. doi:10.1101/222083

Massner, C., Sigmund, F., Pettinger, S., Seeger, M., Hartmann, C., Ivleva N., Niessner, R., Fuchs H., Hrabé de Angelis, M., Stelzl, A., Koonakampully, N., Rolbieski, H., Wiedwald, U., Spasova, M., Wurst, W., Ntziachristos, V., Winklhofer, M., Westmeyer, G.G. Genetically controlled lysosomal entrapment of superparamagnetic ferritin for multimodal and multiscale imaging and actuation with low tissue attenuation. Advanced Functional Materials, in press

Westmeyer, G.G., Jasanoff, A., 2007. Genetically controlled MRI contrast mechanisms and their prospects in systems neuroscience research. Magnetic Resonance Imaging 25, 1004–1010. doi:10.1016/j.mri.2006.11.027

Acknowledgement

Funding by the ERC-SG ‘Magnetogenetics’, the DFG priority program SPP 1665, and the Bavarian Research Network for Molecular Biosystems is gratefully acknowledged.

Biological Engineering of cell therapies

Genetically modified therapeutic cell running a therapeutic program.

The figure is taken from: Kitada, T., DiAndreth, B., Teague, B. & Weiss, R. Programming gene and engineered-cell therapies with synthetic biology. Science 359, eaad1067 (2018). Rights for reuse were obtained from the publisher. 

3:30 PM
ES-03-2 — Synthetic biology design from new protein folds to cellular circuits (#590)

R. Jerala1

1 National institute of chemistry, Synthetic biology and immunology, Ljubljana, Slovenia

Objectives

Two areas of synthetic biology - design of transcriptional genetic circuits and designed proteins will be presented.

Content

Synthetic biology introduces engineering principles into biological or biomimetc systems, combining features of both approaches. Construction of devices for a complex cellular environment requires orthogonal building elements, which may be difficult to harvest from nature. Nucleotide sequence provides a large and easily accessible combinatorial diversity that can be used to program biological systems. Designable DNA-binding TALE domains can be used to construct an almost limitless number of artificial transcriptional regulators enabling construction of orthogonal genetic logic NOR gates. This allows construction of complex logic functions, which was demonstrated by all 16 two-input logic gates in mammalian cells. Construction of genetic bistable switches based on designable DNA binding modules required introduction of additional feedback loops and competition. An even more fundamental challenge for the synthetic biology is construction of new protein folds instead of modifying or combining the existing protein domains. We decided to use a modular engineering approach based on designed orthogonal coiled-coil building elements. This principle allows the design of completely new protein folds, composed of a single polypeptide chain of concatenated coiled-coil building elements called coiled-coil protein origami (CCPO). CCPO represents a new type of protein folds not found in the nature, where the structure is defined by the order of interacting segments defining the final topology rather than by compact hydrophobic core as natural proteins. Second generation CCPOs demonstrated that designed protein polyhedra can self-assemble bacterial or mammalian cells and may be applied to design intracellular scaffolds of molecular machines.

Relevant Publications

  1. Gradišar H, Božič S, Doles T, Vengust D, Hafner-Bratkovič I, Mertelj A, Webb B, Šali A, Klavžar S, Jerala R. (2013) Nat Chem Biol. 6:362-6.
  2. 2. Kočar V, Schreck JS, Čeru S, Gradišar H, Bašić N, Pisanski T, Doye JP, Jerala R. Nat Commun. (2016) 7:10803.
  3. Drobnak I, Gradišar H, Ljubetič A, Merljak E, Jerala, R. (2017) Modulation of Coiled-Coil Dimer Stability through Surface Residues while Preserving Pairing Specificity. J.Am.Chem.Soc.139: 8229-8236.
  4. Ljubetič, A., Lapenta, F., Gradišar, H., Drobnak, I., Aupič, J., Strmšek, Ž., Lainšček, D., Hafner-Bratkovič, I.,  Majerle, A., Krivec, N.,  Benčina, M., Pisanski, T., Ćirković Veličković, T., Round, A., Carazo, J.M., Melero, R. and Jerala, R., (2017) Design of in vivo self-assembling coiled-coil protein origami. Nat Biotech, 35:1094-1101.

Acknowledgement

Funded by the Slovenian Research Agency and ERANET Synthetic biology project Bioorigami. Contribution of the members of the Department of synthetic biology and immunology at NIC and slovenian iGEM teams to research results is acknowledged.

Example of designed antiinflammatory cellular device.

Design of coiled-coil protein origami cages.

4:30 PM
emptyVal-1 — BREAK

5:00 PM
ES-03-4 — Reporter gene imaging of CAR-engineered T-cells in patients: a path for synthetic biology into clinical practice (#589)

T. H. Witney1

1 University College London's Centre for Advanced Biomedical Imaging, Division of Medicine, London, United Kingdom

Objectives

  • To understand the role of non-invasive imaging in the tracking of therapeutic cells in the body.
  • To be able to differentiate between direct and indirect cell labelling strategies, along with their advantages and disadvantages.
  • How HSV1-tk reporter gene expression can be used to track CAR-engineered T-cells in humans, along with its inherent limitations.
  • That quantification of reporter gene tracking is non-trivial.

Content

Immunotherapy holds great potential for the treatment and management of cancer patients with advanced disease (1). Through numerous divergent mechanisms, the body’s adaptive immune system can be primed to target malignancies normally recognized as ‘self’. While the success of recent Phase III trials have validated the principle that immunotherapy can sometimes extend cancer patient survival (2), tumour cells are known to escape immune surveillance and develop resistance to immunotherapy (3). Given the variable success of immunotherapy in the clinic, there is an urgent need to design non-invasive techniques that could give early indications of response to treatment and help predict patient outcome.

Through imaging it is possible to monitor the viability, biodistribution and trafficking of therapeutic cells to the site of the tumour. Two main approaches have been undertaken for cell tracking: direct and indirect labelling methods (4, 5). Direct labelling of cells involves incubation and retention of a contrast agent by the therapeutic cells, which are injected into the subject. Whilst relatively cheap and easy to perform, these methods are hampered by potential toxicity to the therapeutic cells (6). Moreover, the contrast agent becomes diluted upon cell division, and is lost from the cells upon cell death, making image analysis difficult to interpret. In order to circumvent these issues, indirect imaging reporter strategies have been developed to successfully track these cells. These methods enable imaging over the entire lifetime of the cell, with signal maintained following cell division, and provide information regarding cell viability (5). Here, I exemplify the power of this synthetic biology strategy to track HSV1-tk reporter gene expression present in CAR-engineered T-cells in humans using 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG) positron emission tomography (Figure 1) (7).

Relevant Publications

K.V. Keu*, T.H. Witney*, S. Yaghoubi*, J. Rosenberg, A. Kurien, R. Magnusson, J. Williams, F. Habte, J.R. Wagner, S. Forman, C. Brown, M. Allen-Auerbach, J. Czernin, W. Tang, M.C. Jensen, B. Badie, S.S. Gambhir (2017). Reporter Gene Imaging of Targeted T-Cell Immunotherapy in Recurrent Glioma. Sci Transl Med 9, eeag2196.

References

1.         M. Vanneman, G. Dranoff. Nat Rev Cancer 12, 237-251 (2012).

2.         F. S. Hodi et al. N Engl J Med 363, 711-723 (2010).

3.         S. Kelderman, T. N. Schumacher, J. B. Haanen. Mol Oncol 8, 1132-1139 (2014).

4.         M. F. Kircher, S. S. Gambhir, J. Grimm. Nat Rev Clin Oncol 8, 677-688 (2011).

5.         D. M. Kurtz, S. S. Gambhir. Adv Cancer Res 124, 257-296 (2014).

6.         C. Botti, D. R. et al. Eur J Nucl Med 24, 497-504 (1997).

7.         K.V. Keu et al. Sci Transl Med 9, eeag2196 (2017).

Acknowledgement

I wish to acknowledge salary and research support from The Wellcome Trust and The Royal Society and am grateful for the donation of slides by Dr Tammy Kalber.

Figure 1. Reporter Gene Imaging of Targeted T-Cell Immunotherapy in Recurrent Glioma

Strategy for imaging engineered cytotoxic T lymphocytes (left) and for monitoring response to this novel cellular immunotherapy (right) using a highly specific in vivo PET imaging agent, 18F-FHBG.

6:15 PM
emptyVal-1 — Introductory Talk by Amir Rosenthal - Haifa, Israel

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

6:45 PM
PS-12-2 — Eigenvector centrality mapping and seed-based analysis of resting state fMRI during acute brainstem-coma recovery in the rat (#319)

P. Pais1, 4, B. Edlow2, Y. Jiang1, J. Stelzer1, M. Zou3, X. Yu1

1 Max Planck Institute - Cybernetics, High Field MRI, Tuebingen, Germany
2 Massachusetts General Hospital, Neurosciences intensive Care Unit, Boston, Massachusetts, United States of America
3 Wenzhou Medical University, Wengzhou, China
4 Graduate Training Center of Neuroscience, Tuebingen, Germany

Introduction

Despite the known association between brainstem lesions and coma, a circuit-based understanding of coma pathogenesis and mechanisms of recovery is lacking1. We recently developed a model of coma in the rat with focal injury to the brainstem, which allows investigating the neural mechanisms of coma emergence and recovery2. Resting state functional MRI (rs-fMRI) experiments along coma evolution in the rat provided evidence for an acute recovery mechanism by which subcortical arousal centers outside the brainstem reactivate the cerebral cortex.

Methods

rs-fMRI scans were acquired during the first 8 hours post-coma using a 3D EPI sequence (TE, 12.5 ms; TR, 1s; matrix size, 48x48x32; resolution, 400x400x600 µm; 925 TRs) on a 14.1 T/26 cm magnet interfaced to an Avance III console. Pre-processing was performed in AFNI3 and Lipsia4. For each rs-fMRI scan, a voxel-wise map of eigenvector values was computed (eigenvector centrality map), indicating the importance of the respective voxel within the network, followed by least squares fit regression of the eigenvector values over the temporal succession. This resulted in a certain slope, informative of the increase or decrease in connectivity at a given voxel of the brain (Fig.1). Additionally, seed-based analysis was performed by calculating the Pearson's correlation coefficient between regions.

Results/Discussion

The eigenvector centrality mapping-based whole brain functional connectivity analysis showed increases along the acute recovery from coma in thalamus, basal forebrain and basal ganglia (Fig.1). Additionally, seed-based analysis revealed higher correlations along the post-coma period between the central and reticular thalamus, striatum, globus pallidus and the nuclei in the basal forebrain (Fig.2). Interestingly, the time courses of these nuclei increased their correlation with those in cingulate and somatosensory cortex only after 4 hours post-coma (Fig.2). Concurrent electrophysiology and behavioral assessment in the rats demonstrated recovery of the neurological function during the period of study. This result provides evidence for the participation of the thalamic-basal forebrain-basal ganglia network in recovery of consciousness during acute recovery from coma, a time window that is not accessible in the clinical practice for systematic study of the human brain function.

Conclusions

The convergent results from whole brain and seed-based fMRI analysis of connectivity highly suggest a potential role for the basal forebrain-basal ganglia-thalamocortical network in the initial phase of restoration of consciousness after brainstem injury. This study further verifies the applicability of the rat brainstem coma model to investigate brain dynamics during the acute phase of coma.

References

1. Schiff, N. D. 2010; 2. P.Pais et al 2017; 3. Cox, R. W. 1996; 4. Lohmann, G. et al. 2001.

Acknowledgement

Graduate Training Center of Neuroscience Tuebingen.

Figure 1. Analysis of the whole brain rs-fMRI in rats recovering from coma
A: experimental design and general principle of the slope map. B: averaged slope maps of the comatose animals and of a control group anesthetized with 2% isoflurane. C: brain map showing the z-statistic from the 2-tailed 2-sample t-test between coma and control slope maps.

Figure 2. ROI-specific analysis of rs-fMRI
The central graph summarizes the strengthened connections during the acute recovery phase of coma. The seed-based graphs show the connectivity between specific regions at 4 different post-coma times.

6:55 PM
PS-12-3 — Radiomics for the Discrimination of Tuberculosis Lesions (#207)

P. Gordaliza1, 2, J. J. Vaquero1, 2, S. Sharpe3, M. Desco Menéndez1, 2, 4, A. Muñoz-Barrutia1, 2

1 Universidad Carlos III de Madrid, Bioingenieria e Ingenieria Aeroespacial, Leganés, Madrid, Spain
2 Instituto de Investigación Sanitaria Gregorio Marañón, Laboratorio de Imagen Médica, Madrid, Madrid, Spain
3 Public Health England, Microbiology Services Division, Porton Down, England, United Kingdom
4 Centro de Investigaciones Cardiovasculares Carlos III, Madrid, Madrid, Spain

Introduction

Tuberculosis (TB) is an infectious disease with a high incidence and mortality1. Traditionally, TB has been considered a binary disease, latent/active, due to the limited specificity of the traditional diagnostic tests2. Computer Tomography (CT) images of TB infected subjects presents specific manifestations4 and radiomics techniques can be applied for a more discriminant TB characterization5. Here, we proposed a methodology to automatically extract informative features and discriminate between five different types of lesions.

Methods

The main steps of our pipeline are (Fig.1): 1) Automatic lung segmentation and airway tree extraction6; 2) Selection of relevant volumes (i.e., TB lesions) employing Statistical Region Merging7; 3) Extraction of 26 texture features from each volume at 6 grey level quantizations (L=[8,16,32,64,128,256])8; 4) Parameter optimization of the Random Forest (RF) classifiers9 at each L employing different number of features (100-fold cross validation). Inherent data imbalance is handled employing Tomek Links10; 5) Evaluation of the classifiers performance using the F1-score to distinguish among 5 types of lesions: granulomas, conglomerations, trees in bud, consolidations and ground glass opacities. Selection of  informative features employing the Gini Importance (IG) given by each optimal RF.

Results/Discussion

Fig.2.a) shows the IG of each feature at each quantization level for the optimal RF classifier. Features become much informative for large L’s (difference var., L=8,IG=0.14; information measure of correlation 1, L=256,IG= 0.45). Fig.2.b) depicts the weighted F1 obtained in function of the number of features. At large L’s, a few number of features (6) are enough to reach a good precision (L=256,F1=0.844) close to convergence. These results indicate that using the proposed methodology, it would be possible to longitudinally characterize  the disease based on the discrimination among diverse TB manifestations. These lesions would be characterized by the computation of a small number of features which is crucial to build interpretable models.  Namely, the analysis of the feature Info. Measure Correlation 1, particularly high at the largest L’s (L=256,F1=0.45), allows to characterize complex relationships between adjacent voxels that constitute a unique signature of each type of lesion.

Conclusions

In conclusion, the proposed radiomics framework shows encouraging results in its ability to extract informative features to characterize tuberculosis. In particular, we have proved that it is possible to achieve a reasonable good discrimination of the most frequent TB lesion types and more importantly, that our model achieves an effective quantification of the changes that occur in the lung. In the future, this work will be the base for further studies on the characterization of the biological changes induced by TB infection that would lead to an improved understanding of the disease’s course.

References

 

  1. World Health Organization and others, “Global tuberculosis report 2016,” Tech. Rep., 2016.

  2. Barry 3rd, C. E., Boshoff, H., Dartois, V., Dick, T., Ehrt, S., Flynn, J.,Young, D. (2009). The spectrum of latent tuberculosis: rethinking the goals of prophylaxis. Nature Reviews. Microbiology, 7(12), 845–855. http://doi.org/10.1038/nrmicro2236

  3. Pai, M., Behr, M. A., Dowdy, D., Dheda, K., Divangahi, M., Boehme, C. C.,Raviglione, M. (2016). Tuberculosis. Nature Reviews Disease Primers, 2. http://doi.org/dx.doi.org/10.1038/nrdp.2016.76

  4. Nachiappan, A. C., Rahbar, K., Shi, X., Guy, E. S., Mortani Barbosa Jr, E., Shroff, G. S.,Hammer, M. M. (2017). Pulmonary Tuberculosis: Role of Radiology in Diagnosis and Management. RadioGraphics, 37(1), 52–72. http://doi.org/10.1148/rg.2017160032

  5. Gillies, R. J., Kinahan, P. E., & Hricak, H. (2016). Radiomics: Images Are More than Pictures, They Are Data. Radiology, 278(2), 563–577. http://doi.org/10.1148/radiol.2015151169

  6. Artaechevarria, X., Blanco, D., Pérez-Martín, D., De Biurrun, G., Montuenga, L. M., De Torres, J. P., Ortiz-De-Solorzano, C. (2010). Longitudinal study of a mouse model of chronic pulmonary inflammation using breath hold gated micro-CT. European Radiology, 20(11), 2600–2608. http://doi.org/10.1007/s00330-010-1853-0

  7. Nock, R., & Nielsen, F. (2004). Statistical Region Merging. IEEE Transactions on Pattern Analysis and Machine Intelligence, 26(11), 1452–1458.

  8. Saeys, Y., Abeel, T., & de Peer, Y. (2008). Robust Feature Selection Using Ensemble Feature Selection Techniques. In ECML PKDD (pp. 313–325). http://doi.org/10.1007/978-3-540-87481-2_21

  9. Ma, L., & Fan, S. (2017). CURE-SMOTE algorithm and hybrid algorithm for feature selection and parameter optimization based on random forests. BMC Bioinformatics, 18(1), 169. http://doi.org/10.1186/s12859-017-1578-z

  10. He, H., & Garcia, E. A. (2009). Learning from Imbalanced Data. IEEE Transactions on Knowledge and Data Engineering, 21(9), 1263–1284. http://doi.org/10.1109/TKDE.2008.239

 

Acknowledgement

The research leading to these results received funding from the Innovative Medicines Initiative (www.imi.europa.eu) Joint Undertaking under grant agreement no. 115337, whose resources comprise funding from EU FP7/2007-2013 and EFPIA companies in kind contribution. This work was partially funded by projects TEC2013-48552-C2-1-R, RTC-2015-3772-1, TEC2015-73064-EXP and TEC2016-78052-R from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC), TOPUS S2013/MIT-3024 project from the regional government of Madrid and by the Department of Health, UK. The CNIC is supported by the MEIC and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

Figure 1: Fully-automatic radiomics workflow for the extraction of informative features on the lung

Results

7:05 PM
PS-12-4 — Tumor heterogeneity as a PET-biomarker predicts overall survival of pancreatic cancer patients (#173)

E. M. M. Smeets1, J. Teuwen1, J. A. W. M. van der Laak2, M. Gotthardt1, F. Ciompi2, E. H. J. G. Aarntzen1

1 Radboud university medical center, Department of Radiology and Nuclear Medicine, Nijmegen, Netherlands
2 Radboud university medical center, Department of Pathology, Nijmegen, Netherlands

Introduction

Pancreatic ductal adenocarcinoma (PDAC) shows a 5-year survival rate of 8% [1], mostly due to the lack of effective treatment options [2]. PDAC has a remarkable fibrotic reaction [3] that impacts tumor metabolism, which can be measured on PET/CT images [4]. To date, standard PET-derived parameters, e.g. SUVmax, have not been able to provide prognostic information. In this study, we developed a logistic regression model based on FDG-PET texture features (TF) that classifies PDAC as heterogeneous or homogeneous and shows a good correlation with overall survival.

Methods

Patients with histologically proven PDAC (n=121) who underwent 18F-FDG PET/CT (Siemens Biograph mCT, Knoxville, US) were selected from the hospital system. Eighty-six EANM reconstructed scans [5] were visually labeled as ‘homogenous’ (n=40) or ‘heterogeneous’ (n=46) by two experienced Nuclear Medicine physicians.  In order to extract features, tumors were first delineated using 40% threshold of the SUVmax with manual correction. TF previously shown to be robust with respect to PET scan variability [6-8] were extracted using the PyRadiomics toolbox [9]. A logistic regression classifier was build and applied to the 35 cases held out. The training set was classified via leave-one-out cross validation. Prognostic impact was assessed by Kaplan Meier survival analysis and log-rank test.

Results/Discussion

Optimal performance of the leave-one-out cross-validation classifier in the training set yielded an accuracy of 0.73 and AUC of 0.71 in classifying PDAC as heterogeneous or homogeneous tumors. For the 121 patients the overall survival of PDAC tumors classified as heterogeneous was significantly worse than for homogeneous tumors; median overall survival 69 weeks (95%CI 64 to 91 weeks) versus median 95 weeks (95%CI 76 to 114), p= 0.0285). This is in contrast with single standard PET parameters (SUVmax, kurtosis, metabolic tumor volume), single TF (entropy) or visual labeling, which had no significant prognostic impact (see figure 1).

Conclusions

We developed an algorithm that accurately classifies PDAC as metabolic heterogeneous or homogeneous, based on a set of 18F-FDG PET derived texture features. We showed that the classification result has prognostic value, improving upon standard PET derived parameters and single texture-features. Further validation of this algorithm in an external cohort of PDAC patients warranted.

References

  1. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2016. CA Cancer J Clin, 2016. 66(1): p. 7-30.
  2. Ryan, D.P., T.S. Hong, and N. Bardeesy, Pancreatic adenocarcinoma. N Engl J Med, 2014. 371(11): p. 1039-49.
  3. Neesse, A., et al., Stromal biology and therapy in pancreatic cancer: a changing paradigm. Gut, 2015. 64(9): p. 1476-84.
  4. Heid, I., et al., Co-clinical Assessment of Tumor Cellularity in Pancreatic Cancer. Clin Cancer Res, 2017. 23(6): p. 1461-1470.
  5. Boellaard, R., et al., FDG PET and PET/CT: EANM procedure guidelines for tumour PET imaging: version 1.0. Eur J Nucl Med Mol Imaging, 2010. 37(1): p. 181-200.
  6. Yan, J., et al., Impact of Image Reconstruction Settings on Texture Features in 18F-FDG PET. J Nucl Med, 2015. 56(11): p. 1667-73.
  7. Leijenaar, R.T., et al., The effect of SUV discretization in quantitative FDG-PET Radiomics: the need for standardized methodology in tumor texture analysis. Sci Rep, 2015. 5: p. 11075.
  8. Grootjans, W., et al., The Impact of Optimal Respiratory Gating and Image Noise on Evaluation of Intratumor Heterogeneity on 18F-FDG PET Imaging of Lung Cancer. J Nucl Med, 2016. 57(11): p. 1692-169
  9. van Griethuysen, J.J.M., et al., Computational Radiomics System to Decode the Radiographic Phenotype. Cancer Res, 2017. 77(21): p. e104-e107.

Acknowledgement

This study was supported by a grant from the Radboud Oncology Fonds/Stichting Bergh in het Zadel (KUN2015-8106).

Tumor heterogeneity classification based on logistic regression has a prognostic value.
Kaplan-Meier analysis for overall survival according to of PET-derived parameters (a) SUVmax, (b) kurtosis, (c) metabolic tumor volume in cm3, (d) texture-feature derived entropy, (e) visual labeling by two Nuclear Medicine physicians, (f) texture-feature derived classifier.

7:15 PM
PS-12-5 — Dynamic PET Data Analysis Without Frames (#185)

J. L. Herraiz1, E. Fernandez-Garcia2, M. A. Morcillo3, J. M. Udías1

1 University Complutense of Madrid, Nuclear Physics Group, Madrid, Spain
2 University of Oviedo, Science Department, Oviedo, Spain
3 Research Centre for Energy, Environment and Technology - Ciemat, Dep. of Environment, Madrid, Spain

Introduction

Dynamic PET studies provide useful information of the evolution of the biodistribution of tracers in the body. Standard dynamic analysis is performed dividing the acquired data into several time frames which are reconstructed independently. The relevant parameters of a region-of-interest (ROI) are obtained fitting the activity concentration in each frame to a specific function. Nevertheless, this approach requires many image reconstructions, and the use of short frames usually produces noisy images with significant bias. In this work, we propose a novel method to address these problems.

Methods

In our method, each event of the list-mode data is weighted based on the time t they were detected, and then histogrammed into standard sinograms. Weights can be chosen to be, for instance, {1, t, t2} to create the zero, first, and second-order momentum sinograms respectively. The zero-order momentum sinogram corresponds to a single-frame. These sinograms are reconstructed normally, and as they contain all the available data they do not suffer from significant noise or bias. The dynamic parameters of interest in a ROI can be then easily derived from the resulting images using simple algebraic relations. The method has been verified with data from many different preclinical and clinical scanners.

Results/Discussion

As a first example, we used our method to determine the initial activity of a decaying 13N cylinder acquired for 5 minutes with the Biograph scanner. We compared these results with the ones obtained using 10 frames of 30 seconds. Both methods yield similar results, but the estimated uncertainty of our method is smaller. As an example of its application in preclinical studies, we performed a PatLak analysis in the myocardium region of a rat injected with 18F-FDG and acquired in the Argus PET/CT scanner. Using the last 10 out of 44 dynamic frames and an image-derived input function provided a value of Ki=0.091 min-1. We obtained the same result (Ki=0.093 min-1) with our method, but using only 2 reconstructed images, corresponding to the zero and first-order momentum of the acquired data.

Conclusions

The proposed method is a completely new approach to dynamic analysis. Instead of reconstructing multiple images and then fit their values to a particular function, we directly reconstruct the images using the weights needed for the fit. It can be applied to many different dynamic studies using the appopriate set of weights. In cases with a well-determined protocol, such as the PatLak analysis described before, it can be a very effective way to reduce the computational cost and bias in the results.

References

Walker MD et al. Bias in iterative reconstruction of low-statistics PET data: benefits of a resolution model. Phys Med Biol. 2011 Feb 21;56(4):931-49.

Patlak Analysis: http://doc.pmod.com/pxmod/pmclass.lib.pmod.models.PMpatlakV2.htm

 

Acknowledgement

This work was supported by Comunidad de Madrid (S2013/MIT-3024 TOPUS-CM), Spanish Ministry of Science and Innovation, Spanish Government (FPA2015-65035-P, RTC-2015-3772-1). This is a contribution for the Moncloa Campus of International Excellence. Grupo de Física Nuclear-UCM, Ref.:  910059. This work acknowledges support by EU's H2020 under MediNet a Networking Activity of ENSAR-2 (grant agreement 654002). J. L. Herraiz was also funded by the EU Cofund Fellowship Marie Curie Actions, 7th Frame Program.

Motivation of the method

The method is inspired in the way least-square fit of scattered data {xi,yi} is performed, in which the relevant variables of interest are not the individual values of each data point but the sum of {xi, yi,xi*yi,xi*xi}. Therefore, list-mode data do not need to be histogrammed into frames to be analyzed.

Description of the method and Patlak Plot
The proposed method incorporate the dynamic information into the sinograms and therefore it can be recovered from the reconstructed images. It eliminate the need to perform multiple image reconstructions. In many cases two or three images is enough. As an example, our equivalent method to the Patlak plot is derived. 

7:25 PM
PS-12-6 — Fast Magnetic Particle Imaging using Angular Subsampling Based Reconstruction (#332)

R. Orendorff1, E. Frenklak2, E. Y. Yu1, Y. Shi3, B. Zheng1, S. M. Conolly1, 2

1 University of California Berkeley, Bioengineering, Berkeley, California, United States of America
2 University of California Berkeley, Electrical Engineering and Computer Science, Berkeley, California, United States of America
3 Beijing Institute of Technology, School of Information and Electronics, Beijing, China

Introduction

Magnetic Particle Imaging (MPI) is a promising new modality that images only a magnetic tracer, commonly super-paramagnetic iron oxide (SPIO) nanoparticles [1, 2]. This technique requires manipulating a magnetic field gradient over space to acquire projections of the tracer concentrations in the imager (similar to pencil beam CT), taking an hour or more acquire 3D data [3]. We propose a novel and efficient field free line (FFL) MPI 3D image reconstruction method via prox-linear optimization to significant undersample of the required number of projections.

Methods

A simulation of the Berkeley 7 T/m FFL MPI scanner was used to create sample images of a coronary phantom. The required number of projections to satisfy the Nyquist sampling theorem was calculated and used to simulate a image acquisition with an SNR of 50 using 25nm diameter iron particles. Image acquisition with fewer projections was also simulated, down to 25 projections. The acquired data was then reconstructed using the filtered back-projection (FBP) and using the proposed sparse sampling algorithm.

The novel sparse sampling algorithm solves a prox-linear [5] optimization problem that minimizes a data consistency term and a sparse domain (total variation on the image). The algorithm is matrix-free by a in-house package called PyOp [4] that enables low memory use and fast reconstruction.

Results/Discussion

The coronary phantom was reconstructed using FBP and the proposed SS MPI method. 100 projections was calculated as the minimum number of projections satisfying the Nyquist sampling theorem. The SS reconstruction achieves similar results as the fully sampled image without streak artifacts in a fourth of the number of projections. Sinogram data was simulated with a 25nm iron particle (0.95mm FWHM in a 6.3 T/m FFL MPI scanner) with a 4cm by 4cm, 64x64 pixel FOV. 2D reconstruction time is 1 second; a tilted 3D version of the coronary phantom takes ~10 seconds for a 64x64x64 pixel image.

 

The results show that while FBP for images sampled with ~1/4 the number of projections leads to significant artifacts, the SS method correctly reconstructs the coronary phantom, leading to a 4x acquisition time reduction. As each projection on the Berkeley FFL MPI takes approximately 30 seconds to acquire, this reconstruction allows for 3D datasets to be taken in approximately ten minutes.

Conclusions

The SS MPI algorithm enables fast and robust reconstruction of undersampled, noisy FFL MPI data in a manner that reconstructs the underlying data more accurately than the state of the art reconstruction method. The SS method took 10 seconds to reconstruct a 3D version of the phantom, demonstrating that this algorithm is fast in practice. In addition, the novel forward model can be used to reconstruct data taken using any arbitrary scan trajectory, opening up new possibilities such as incrementally/locally updating images and constant SNR trajectories.

References

[1] P.W. Goodwill et al. IEEE Trans. Med. Imaging, 2010

[2] T. Knopp et al. Magnetic Particle Imaging, 2012

[3] J. J. Konkle et al. Biomed Tech (Berl). 2013

[4] R. Orendorff et al. WMIC 2016

[5] A. Beck et al. SIAM J. Imaging Sciences. 2009

Acknowledgement

We are grateful for funding support from the Keck Foundation Grant 009323, NIH 1R01EB019458, NIH 1R24MH106053, and a UC Discovery Grant. Ryan Orendorff would like to thank the NSF GRFP for funding support. 

Model of FFL MPI for Sparse Subsampling Algorithm
Sparse sampling (SS) reconstruction algorithm for FFL MPI. (a) Image of the Berkeley FFL MPI scanner simulated in this study. (b) The FFL MPI forward model is calculated from a generalized Radon transform that allows for arbitrary trajectories of space. The model is used in a prox-linear algorithm to reconstruct the image given some penalty on the TV norm.

Angular Sparse Subsampling (SS) Algorithm faithfully reconstructs coronary phantom

The results of the reconstruction on a simulated phantom image. 100 projections was calculated as the Nyquist rate, given a 25nm MPI tracer. The SS (25 projection) reconstruction achieves similar results as the fully sampled (100 projection) image without streak artifacts, enabling a 4x reduction in image acquisition time.

7:35 PM
PS-12-7 — Tumor Detection and Characterization in Ultrasound Data by Automated Feature Extraction and Pattern Recognition (#538)

Z. A. Magnuska1, T. Opacic1, F. Kiessling1, B. Theek1

1 Uniklinik RWTH Aachen, Experimental Molecular Imaging, Aachen, North Rhine-Westphalia, Germany

Introduction

The primary goal of radiomics is to improve precision medicine by developing classification models, which are supporting physicians in diagnosis and therapy evaluation. Until now, only a few radiomic analysis has been carried out for ultrasound (US) data [1,2]. Therefore, we developed an advanced, user-independent workflow to conduct a radiomic analysis of US images and evaluated its capability to automatically differentiate three different experimental tumor models.

Methods

US B-mode images of 3 different subcutaneous tumor models (lung cancer(n=3), ovarian cancer(n=3), SCC(n=3)) scanned at 2 positions, were retrospectively analyzed. Cascade classifiers (CCs) were trained for an automated tumor detection (Fig. 1A) [3]. The detection box was the seeding point for the following tumor segmentation, which is based on an active contour model and morphological operations [4]. From the segmented region, a total of 230 intensity-based, textural and wavelet-based features were mined (Fig. 2). Dedicated feature selection was performed to identify three linearly independent features for the final tumor classification model. The generated radiomic signature (RS) was validated with a k-NN learning algorithm, based on the leaving-one-out cross-validation scheme.

Results/Discussion

The proposed algorithm for the automated detection and segmentation of tumors, which was based on the Viola-Jones algorithm and an active contour segmentation, achieved a high detection accuracy (89% of correct tumor detections), and automated segmentations overlapped with the manual segmentations (81% ± 3% overlap) (Fig. 1B). The developed imaging biomarker extraction and selection algorithm identified the RS consisting of the following three independent features: median (intensity-based feature), correlation (textural feature) and short run emphasis (wavelet-based feature). The isolated tumor classification model assigned correctly 80% of tumors to their histological group (p=0.8 [95% CI 0.6-0.9]) (Fig. 2).

Conclusions

Radiomic analysis of US images can be performed to classify tumors, and should be more extensively evaluated for clinical translation. The developed framework might be able to standardise tumor detection and segmentation and to support clinicians in tumor recognition and characterisation.

References

[1] Andrekute K. et al. Ultrasound Med Biol 2016, 42(12): 2834-2843.

[2] Guo Y., et al. Clin Breast Cancer, 2017.

[3] Viola P. and Jones M. CVPR, 2001.

[4] Kass, M et al. Int J Comput Vision 1988, 1: 321-331.

Figure 1
Automated Tumor Detection and Segmentation

Figure 2
Workflow of Automated Tumor Differentiation

10:00 AM
EuBI-1 — EuroBioImaging: ready to start (#617)

S. Aime1, K. Sheehan-Rooney2, G. Digilio3

1 University of Turin, Torino, AL, Italy
2 EMBL, Heidelberg, Germany
3 University of Eastern Piedmont, Alessandria, Italy

Content

'Rome wasn’t built in a day’ ...and this is equally true for an European Research Infrastructure Consortium (ERIC). Euro-BioImaging (EuBI) is a distributed research infrastructure aiming at providing open access, services and training to a broad range of state-of-the-art biological and medical imaging technologies. After almost a decade of planning, and after the start of interim operation in May 2016, many European countries and EMBL are now committing to commonly launch the EuBI-ERIC, and open this Imaging Research Infrastructure to European scientists. All countries are now invited to sign the legal documents and become founding members of EuBI. The aim of the meeting is to describe where EuBI stands, what are the very next steps, what are the perspectives for EuBI service providers who adhered to the EuBI interim phase (i.e. the EuBI Nodes), and what steps have to be taken to get involved. Last but not least, industry-related matters and opportunities will be presented. Everyone who is interested in the EuBI initiative (as a service provider, or industry delegate, or as a user) is warmly invited to the meeting.

 

Meeting agenda:

- Update on the status of EuBI-ERIC set-up

- Involvement of service providers (the EuBI Nodes)

- How to join EuBI

- Showcasing of new imaging technologies

- Industry related matters and opportunities

- Q&A

9:15 AM
IND-04-1 — Comprehensively characterize the tumor microenvironment in spatial context with Imaging Mass Cytometry™ (#577)

R. Spada1, H. Jackson2

1 Fluidigm, Les Ulis, France
2 University of Zürich, Bodenmiller Lab, Zurich, Switzerland

Content

Biomarker discovery efforts in translational medicine and pharma R&D are increasingly reliant on antibody-mediated approaches for protein detection in both suspension and fixed tissue sections. The Hyperion™ Imaging System enables simultaneous interrogation of 4 to 37 protein markers using proven CyTOF® technology together with imaging capability, facilitating Imaging Mass Cytometry™ applications. The system is ideal for characterization of the tissue microenvironment across a breadth of disease research areas. This presentation will provide an overview of the Hyperion Imaging System.

Presenters:

Roberto Spada Market Development Specialist, Fluidigm

Introduction to Hyperion™ Imaging System

Hartland Jackson Post-Doctoral Fellow, Bodenmiller Lab, University of Zürich

The single cell landscape of breast cancer by Imaging Mass Cytometry™

 

6:15 PM
FS-03-1 — European Research Council - Funding opportunities and guide for applicants (#597)

O. Limaj1

1 European Research Council, Brussels, Belgium

Content

The European Research Council (ERC) is a research funding body who aims to support the best and most creative researchers and help them identify and explore new opportunities and directions in any field of research.

The ERC received a budget of over €13 billion under the European Union research programme Horizon 2020 (2014-2020). Currently, it has funded more than 6 000 projects in the domains of life sciences, physical sciences and engineering and social sciences and humanities.

This presentation aims to give an overview of the available ERC funding schemes (Starting, Consolidator, Advance and Synergy Grants), describe the evaluation process and provide step-by-step guidelines for prospective applicants.

More information can be found at: https://erc.europa.eu/

1:30 PM
emptyVal-1 — Introductory Talk by Kevin Brindle - Cambridge, UK

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

1:50 PM
PS-13-2 — SQUID based magnetic resonance imaging for the investigation of in situ and in vivo hyperpolarization techniques (#85)

K. Buckenmaier1, M. Rudolph1, 2, P. Fehling1, C. Back2, J. Bernarding3, D. Koelle2, R. Kleiner2, K. Scheffler1, M. Plaumann3

1 Max Planck Institute for Biological Cybernetics, High-Field Magnetic Resonance Center, Tuebingen, Baden-Württemberg, Germany
2 University of Tuebingen, Physikalisches Institut and Center for Collective Quantum Phenomena in LISA+, Tuebingen, Baden-Württemberg, Germany
3 Otto-von-Guericke University, Department for Biometrics and Medical Informatics, Magdeburg, Germany

Introduction

Ultralow-field (ULF) nuclear magnetic resonance (NMR) is a promising spectroscopy method allowing for, e.g., the simultaneous detection of multiple nuclei. To overcome the low signal-to-noise ratio that usually hampers a wider application, we present an alternative approach to prepolarized ULF NMR employing hyperpolarization techniques like signal amplification by reversible exchange (SABRE) or Overhauser dynamic nuclear polarization (ODNP). Both techniques allow continuous hyperpolarization of 1H as well as other MR-active nuclei.

Methods

To be able to measure 1H and 19F simultaneously, a superconducting quantum interference device (SQUID)-based ULF NMR/MRI detection unit was constructed (see fig. 1). Due to the very low intrinsic noise level, SQUIDs are superior to conventional Faraday detection coils at ultralow-fields. Additionally, the broad band characteristics of SQUIDs enable them to simultaneously detect the MR signal of different nuclei such as 13C, 19F or 1H. Since SQUIDs detect the MR signal directly, they are an ideal tool for a quantitative investigation of hyperpolarization techniques such as SABRE or ODNP.

Results/Discussion

Using SABRE we successfully hyperpolarized fluorinated pyridine derivatives and quantitatively characterized the dependency of the magnetization transfer reaction from parahydrogen, which bonds to an iridium complex as well as to the 1H and 19F nuclei of an exchangeable ligand, as a function of hyperpolarization time and magnetic field strength [1]. Spectra (see fig. 2) and images of the samples were acquired.

With ODNP we were able to measure the coupling constant of solutions containing free radicals. Enhancement factors of over 100 were reached in in situ experiments. First proof-of-principle ex vivo images of rats using ODNP enhanced, SQUID based ULF-MRI have been acquired successfully.

Conclusions

We successfully built a SQUID-based ULF NMR/MRI system to quantitatively investigate the hyperpolarization techniques SABRE and ODNP.

References

[1]    Buckenmaier et al. SQUID-based detection of ultralow-field multinuclear NMR of substances hyperpolarized using signal amplification by reversible exchange. Scientific Reports, 7:13431 (2017).

Acknowledgement

We thank Hermann Mayer, Tomasz Misztal, Rebekka Bernard and Rolf Pohmann for their support in this project.

Figure 1.
Photo and scheme of the ULF MRI system (a), and scheme of the SQUID based magnetic field detector (b).

Figure 2.
Ultralow-field 19F and 1H MR spectra of hyperpolarized 3-fluoropyridine. Substances and catalysts were dissolved in methanol and measured at 144 µT. The blue lines represent the measured spectra whereas the green lines represent simulated spectra based on high-field determined coupling constants.

2:00 PM
PS-13-3 — Deuteration of hyperpolarized 13C-labelled zymonic acid enables sensitivity-enhanced dynamic MRI of pH (#222)

C. Hundshammer1, 2, 4, S. Düwel1, 2, 4, S. S. Köcher2, 3, M. Gersch2, B. Feuerecker1, C. Scheurer2, A. Haase4, S. J. Glaser2, M. Schwaiger1, F. Schilling2

1 Technical University Munich, Klinikum rechts der Isar, Department of Nuclear Medicine, Munich, Bavaria, Germany
2 Technical University Munich, Department of Chemistry, Garching, Bavaria, Germany
3 Forschungszentrum Jülich, Institute of Energy and Climate Research (IEK–9), Jülich, North Rhine-Westphalia, Germany
4 Technical University Munich, Munich School of Bioengineering, 85748, Bavaria, Germany

Introduction

Polarization techniques like DNP1 have been developed to overcome signal limitations of MRSI, which triggered the development of hyperpolarized (HP) pH sensors2 that potentially could help to improve precision medicine.3,4 13C-zymonic acid (ZA) is the first chemical shift based HP sensor that has been applied in vivo so far.5,6

We show that deuterated ZA (ZAd) exhibits an up to 39% longer spin lattice relaxation time in vitro, while its in vivo SNR can be increased by up to 46%. For the first time, we further demonstrate that ZAd is capable to sense dynamic pH changes with spatial resolution. 

Methods

ZA/ZAd synthesis & hyperpolarization (Hypersense, Oxford Instruments) was performed according to Hundshammer et al.6 T1 decay curves were measured at a 3T PET/MR scanner (Siemens, 15° FA, 5s TR).5,6 Chemical shift imaging (CSI) was performed at the PET/MR & at a 7T small animal MR scanner (GE/Agilent). Acquisition parameters, reconstruction schemes & pH calculation procedures are given by Hundshammer et al.For dynamic pH imaging, vitamin C was either added stepwise or as dissolvable vitamin C tablet to 72mL of 80mM Tris buffered solution after 3mL ZAd (cfinal=2mM) has been added & one image was recorded. For in vivo pH imaging, same amounts of ZA & ZAd (+urea) were polarized to saturation & injected into the tail-vein of three rats (n=3) bearing subcutaneous MAT-B-III tumors.6

Results/Discussion

At 3T, the T1 of C1 & C5 of ZAd are 49±8s & 71±3s respectively & thus 14% & 39% longer than the ones of ZA. In vivo, successive static CSI images demonstrate an SNR gain by deuteration amounting to 43±16% & 46±4% for C1 & C5, respectively (Figure 1). ZA & urea signals were observed in the vena cava & in the tumors. Representative spectra indicate a pH 7.53±0.08 (mean±std) in the vena cava compared to the extracellular tumor pH 7.10±0.05. Values are in agreement with the literature.5  

Stepwise addition of vitamin C with subsequent mixing delivered solutions of uniformly decreasing pH that could accurately be measured with ZAd (Figure 2). Addition of a dissolvable vitamin C tablet steadily decreased the pH of an aqueous solution of initial pH 7.58±0.02 (neutral) to pH≈5.0-5.7 (acidic). Mainly two peak pairs of ZAwere observed, because the CSI slice covered regions of acidic and neutral pH at the same time. The pH values were calculated by weighting peak intensities with respective pH.

Conclusions

Deuteration of zymonic acid prolongs the hyperpolarized signal lifetime, which can be used to non-invasively image pH in vivo. Furthermore we show that this sensor is usable to spatially resolve dynamic pH changes on a time-scale of seconds. In the future, this could help to detect immediate pH changes for instance in cases where proton concentrations are altered by enzymatic reactions,7 in alkaline treatments of acute metabolic acidosis8 & in exercised muscle.9 Additionally, time-resolved imaging of pH could verify targeted drug delivery for localized acidification or basification.

References

1             Ardenkjaer-Larsen, J. H. et al. Increase in signal-to-noise ratio of > 10,000 times in liquid-state NMR. Proceedings of the National Academy of                             Sciences of the United States of America, 100, 10158-10163, 2003.

2             Hundshammer, C. et al. Imaging of Extracellular pH Using Hyperpolarized Molecules. Israel Journal of Chemistry, doi:10.1002/ijch.201700017, 2017.

3             Collins, F. S. et al. A New Initiative on Precision Medicine. New England Journal of Medicine, 372, 793-795, 2015.

4             Friedman, A. A. et al. Precision medicine for cancer with next-generation functional diagnostics. Nature reviews. Cancer 15, 747-756, 2015.

5             Düwel, S. et al. Imaging of pH in vivo using hyperpolarized 13C-labelled zymonic acid. Nature Communications 8, 15126, doi:10.1038/ncomms1512,                   2017.

6             Hundshammer, C. et al. Deuteration of hyperpolarized 13C-labelled zymonic acid enables sensitivity-enhanced dynamic MRI of pH. Chemphyschem,                   doi:10.1002/cphc.201700779, 2017.

7             Gallagher, F. A. et al. Imaging pH with hyperpolarized 13C. NMR in biomedicine 24, 1006-1015, doi:10.1002/nbm.1742, 2011.

8             Som, A. et al. Monodispersed calcium carbonate nanoparticles modulate local pH & inhibit tumor growth in vivo. Nanoscale 8, 12639-12647, 2016.

9             Kemp, G. et al. Lactate accumulation, proton buffering, and pH change in ischemically exercising muscle. American journal of physiology. Regulatory,                     integrative and comparative physiology 289, R895-901; author reply R904-910, 2005.

Acknowledgement

We appreciate the help of Miriam Braeuer, Anna Bartels, Birgit Blechert & Michael Michalik with animal experiments. We acknowledge support from EU Grant No. 294582 (MUMI), BMBF (FKZ 13EZ1114) & DFG (SFB 824). We thank Cambridge Isotope Laboratories Inc. who provided [1-13C]pyruvate for the synthesis of zymonic acid.

Figure 1: In vivo SNR comparison of ZA & ZAd
ZA intensity images of ZAd (A) & ZA (E) overlaid on 1H images. (B, F) pH maps. (C, G) ZAd & ZA spectra of a vena cava voxel (white squares). (DH) Spectra of a tumor voxel (red squares) show two pairs of ZAd or ZA, which indicate a vascular tumor pH~7.5 & an acidic extracellular pH≈7.1 compared to the vena cava spectra showing only one ZAd or ZA peak pair corresponding to blood pH≈7.5.

Figure 2: Spatially resolved imaging of temporal pH changes.
(A, D) Experimental setups. B) Time-resolved pH maps of a beaker filled with 72mL of 80mM Tris at pH7.7 & 3mL co-polarized ZAd (cfinal=2mM) & urea (cfinal=3mM). C) Spectra of the mean of all pixels from B). E) Time-resolved pH maps after addition of a dissolvable vitamin C tablet to a solution as in B) (white arrow). F) Representative spectra of voxels (red squares) from timestep 1 & 5 from E). 

2:10 PM
PS-13-4 — Hyperpolarized Silicon-29 Nanoparticles for In-Vivo MR Imaging (#43)

G. Kwiatkowski1, P. Wespi1, J. Steinhauser1, M. Ernst2, S. Kozerke1

1 University and ETH Zurich, Institute for Biomedical Engineering, Zurich, Switzerland
2 ETH Zurich, Labolatory of Physical Chemistry, Zurich, Switzerland

Introduction

A new class of hyperpolarized Mangetic Resonance contrast agents, characterized with a long lifetime were recently proposed which are based on micro/nanoparticles of elemental silicon. Experimental results1,2 revealed a lifetime of the hyperpolarized signal >30min, exceeding those of any other 13C based probes reported so far. The bio-compatibility of silicon and its versatile surface chemistry makes it well suited for in vivo use3. The objective of the present work was to demonstrate the imaging capability of hyperpolarized nanometer size silicon particles in an experimental in-vivo setting.

Methods

Silicon nanoparticles: Silicon nanopowder obtained commercially, with average particle size (APS) ~ 55nm was used.

Hyperpolarization of silicon: The nuclear polarization of 29Si nuclei was enhanced by dynamic nuclear polarization (DNP) exploiting endogenous defects between the crystalline silicon core and the oxidized shell as electronic spin pool.

Surface functionalization: The surface of the silicon nanoparticles was functionalized with NHS-dPEG4-(m-PEG12)3-ester4, Heparin5, Dextran6 or Lipid bilayer7.

MRI experiments: After 24h of polarization, the samples were taken out of the polarizer and transferred to a horizontal 9.4 T imaging system.

All animal experiments were performed with adherence to the Swiss Animal Protection law and were approved by the regional veterinary office.

Results/Discussion

While early reports1,8,9 of hyperpolarized silicon particles demonstrated the feasibility of imaging silicon microparticles (APS ∼2 μm), the relatively large particle size did limit  in-vivo application. The present work shows that functionalized nanoparticles (APS∼55 nm) exhibit comparable relaxation and polarization properties (T1 ~ 30min, maximum achievable polarization 10-12 %), with improved in-vivo compatibility due to their smaller size. For in-vivo imaging, 30 mg of mPEG functionalized silicon nanoparticles were dispersed in 500 μl for an i.g. injection, which corresponded to a concentration of ∼90 mM of 29Si. The injected hyperpolarized silicon could be imaged in the two test animals in vivo with an average SNR/pixel inside the gastrointestinal tract of 12±4 and 9±2. The signal level measured with hyperpolarized silicon-29 was found to be comparable to the signal obtained with a standard concentration of hyperpolarized 1-13C pyruvate (80 mM) used in our laboratory10.

Conclusions

No significant effect of surface functionalization on DNP properties of silicon nanoparticles was found. Overall, good quality images were obtained despite the small amount of material used. In the future, further gains in SNR may be achieved by enriching the nanoparticles with silicon-29. Potential effects on relaxation properties, however, remain to be studied. In the example shown in this work, i.g. injection was used in the animals. In future work we will also investigate other routes of injection to study other organ systems of interest in nanomedicine applications.

References

1.        Cassidy MC, Chan HR, Ross BD, Bhattacharya PK, Marcus CM. In vivo magnetic resonance imaging of hyperpolarized silicon particles. Nat Nanotechnol. 2013;8(5):363-368. doi:10.1038/nnano.2013.65.

2.        Kwiatkowski G, Jähnig F, Steinhauser J, Wespi P, Ernst M, Kozerke S. Nanometer size silicon particles for hyperpolarized MRI. Sci Rep. 2017;7(July):1-6. doi:10.1038/s41598-017-08709-0.

3.        Park J-H, Gu L, von Maltzahn G, Ruoslahti E, Bhatia SN, Sailor MJ. Biodegradable luminescent porous silicon nanoparticles for in vivo applications. Nat Mater. 2009;8(4):331-336. doi:10.1038/nmat2398.

4.        Aptekar JW, Cassidy MC, Johnson  a. C, et al. Hyperpolarized Long-T1 Silicon Nanoparticles for Magnetic Resonance  Imaging. 2009:1-5. doi:10.1021/nn900996p.

5.        Argyo C, Cauda V, Engelke H, Rädler J, Bein G, Bein T. Heparin-coated colloidal mesoporous silica nanoparticles efficiently bind to antithrombin as an anticoagulant drug-delivery system. Chem - A Eur J. 2012;18(2):428-432. doi:10.1002/chem.201102926.

6.        Schulz A, Woolley R, Tabarin T, McDonagh C. Dextran-coated silica nanoparticles for calcium-sensing. Analyst. 2011;136(8):1722-1727. doi:10.1039/c0an01009j.

7.        Meng H, Wang M, Liu H, et al. Use of a Lipid-Coated Mesoporous Silica Nanoparticle Platform for Synergistic Gemcitabine and Paclitaxel Delivery to Human Pancreatic Cancer in Mice. ACS Nano. 2015;9(4):3540-3557. doi:10.1021/acsnano.5b00510.

8.        Whiting N, Hu J, Shah J V., et al. Real-Time MRI-Guided Catheter Tracking Using Hyperpolarized Silicon Particles. Sci Rep. 2015;5:12842. doi:10.1038/srep12842.

9.        Whiting N, Hu J, Constantinou P, et al. Developing hyperpolarized silicon particles for advanced biomedical imaging applications. Proc SPIE. 2015;9417:941702. doi:10.1117/12.2082252.

10.      Krajewski M, Wespi P, Busch J, et al. A Multisample Dissolution Dynamic Nuclear Polarization System for Serial Injections in Small Animals. Magn Reson Med. 2017;77:904–910. doi:10.1002/mrm.26147.

Acknowledgement

We would like to thank Daniel Zindel, Laboratory of Physical Chemistry, ETH Zurich for his help with the surface modification of diamond powder. Financial support by the Swiss National Science Foundation (SNF grants 320030_153014, 200021_149707 and 200020_169879) are gratefully acknowledged.

Figure 1.
Anatomical image in coronal (A) and transvers plane (B). Corresponding silicon-29 images recorded at 5 min after sample injection (500 μl of 60 mg/ml) C,D). Overlay of the two images threshholded at 35% threshold level for transparency E,F).

2:20 PM
PS-13-5 — An in vivo metabolic imaging study of myopathy in transgenic mice using hyperpolarized [1-13C]pyruvate generated by ParaHydrogen (#424)

E. Cavallari1, C. Carrera1, M. Sorge1, S. Aime1, F. Reineri1

1 Torino University, Department of molecular biotechnology and health sciences, Torino, Italy

Introduction

Hyperpolarized [1-13C]pyruvate is an MRI probe that allows to monitor metabolism in vivo, in real time. ParaHydrogen Induced Polarization (PHIP) is a portable, cost effective technique able to generate hyperpolarized molecules in few seconds. The introduction of the Side Arm Hydrogenation (SAH) strategy offered a way to widen the field of PHIP generated systems and to make this approach competitive with the currently applied dissolution-DNP (Dynamic Nuclear Polarization) method.

Here, the first in vivo metabolic study using the PHIP-SAH hyperpolarized [1-13C]pyruvate is reported.

Methods

Biocompatible aqueous solution of HP pyruvate was obtained according to the PHIP-SAH procedure. Hydrogenation was carried out in an organic phase in order to eliminate the toxic methanol used in the previously reported proof-of-concept. Hydrolysis was obtained using a diluted aqueous base (NaOH 0.1M) and sodium ascorbate (as scavenger of paramagnetic impurities), physiological pH was reached adding Hepes (144mM). The concentration of the injected pyruvate was 50±5mM (doses of HP pyruvate: 0.3-0.35 mmol/Kg).

4-months-old male (n=3) and 6-months-old male (n=4) LmnaH222P/H222P mice and 6-months-old male WT mice (n=4) were investigated.

13C dynamic studies were performed using series of 13C-MR spectra, with small flip angle pulses and a tailored slice-selective sequence, at 1 Tesla (2M Aspect).

Results/Discussion

The hydrolysis and phase extraction steps took few tens of seconds, nevertheless, the HP observed on the 13C carboxylate signal (3.9±0.5%) appears already sufficient for carrying out in vivo studies.

The pyruvate-lactate exchange rate of the 13C hyperpolarized label was obtained, using the model free approach based on the ratio of the total area under the curve of these metabolites.

The results clearly show a marked decrease of lactate/pyruvate ratio in heart muscle of the LmnaH222P/H222P with respect to WT mice. The reduced pyruvate/lactate exchange rate is a reporter of the general cells metabolic activity and might be due either to lower activity of the transporters (MCT) or to an altered cytosolic redox state.

No difference on kidney metabolism was observed, this observation supports the view that the metabolic result obtained in the heart reports on differences in the metabolism of the cardiac tissue and not from an impairment in the distribution of HP metabolites in the blood pool.

Conclusions

This study shows that in vivo metabolic investigations based on the administration of [1-13C]pyruvate obtained from the PHIP-SAH procedure is possible, also at this relatively low magnetic field strength (1 Tesla).

By comparing Pyruvate-Lactate 13C label exchange rate in healthy and genetically modified Lmna mice, it was found that the metabolic dysfunction occurring in the cardiac muscle of the transgenic (LmnaH222P/H222P) mice can be detected well before the disease can be assessed by echographic investigations.

References

Reineri F.et al.Nat.Commun.2015;6:5858

Cavallari E.et al.J.Phys.Chem.B,2015,Vol. 119,10053-10041

Arimura, T. et al. Hum. Mol. Genet., 2005; 14, 155–169

Acknowledgement

We gratefully acknowledge AIRC (2015 TRIDEO call) for financial support.

Figure 1

Lactate/pyruvate ratios obtained from the 13C-MR spectra centered on the heart (red symbols) of mutant Lmna mice and WT mice. The L/P ratio in the heart of Lmna is significantly lower than in WT mice, already at 4 months of age and becomes more significant on 6 months old Lmna mice. When the 13C-MR spectra are centered on the kidneys (blue symbols) there is not any significant difference.

Figure 2

a) Stacked plot of 13C-MR spectra at 1T from a dynamic study on the heart of a WT mouse; spectra were acquired every 2’’ from a 12 mm thick axial slice centered on the heart. The lactate peak is easily identifiable (182 ppm) (13Cpyruvate signals 170 ppm).

b) Dynamic curves of metabolite levels in same slice, the metabolite level was obtained from the integral of the metabolite peak.

2:30 PM
PS-13-6 — Eight-Fold Resolution Improvement in Magnetic Particle Imaging with Pulsed Waveforms For Large Core Size Magnetic Nanoparticles (#478)

Z. W. Tay1, D. Hensley2, P. W. Goodwill2, B. Zheng1, S. M. Conolly1

1 University of California, Berkeley, Bioengineering, Berkeley, California, United States of America
2 Magnetic Insight, Inc, Alameda, California, United States of America

Introduction

Magnetic Particle Imaging (MPI) is an emerging tracer modality with high sensitivity [1-2] and zero ionizing radiation. Applications include stem cell tracking [3], tumor imaging [4], stroke detection / angiography [5] and gut bleed imaging [6]. One challenge is improving the spatial resolution of MPI (currently ~ 1.5 mm w/o deconvolution). While Langevin theory predicts a cubic improvement in MPI resolution with increasing magnetic nanoparticle (tracer) core size, relaxation blurring has been a barrier[7]. Here, we use pulsed waveforms to circumvent relaxation and achieve Langevin resolution.

Methods

Imagion Biosystems PrecisionMRX® SPIO nanoparticles (Imagion Biosystems, Albuquerque, NM, USA) with carboxylic acid coated outer shell and varying core diameters were used. All single cores are single crystalline magnetite (Fe3O4) as confirmed in [8]. Fig2 used VivotraxTM (Magnetic Insight, Alameda, CA, USA) for small-core size images. 1D point spread functions and resolution measurements were obtained on the Berkeley arbitrary waveform relaxometer (1D scanner) described in [9]. 2D resolution simulations were performed using custom matlab code and assuming a Debye relaxation model. 2D images were obtained on a modified version of the abovementioned relaxometer with a permanent magnet field-free-line configuration with gradient strength of 3.5 x 3.5 T/m for the x and z axes. 

Results/Discussion

Pulsed MPI uses square waveforms to wait for MPI tracer magnetization relaxation to complete before scanning the next voxel (Fig 1b). In contrast, standard MPI does not wait and causes MPI signal to blur between voxels. As a result, Pulsed MPI is able to circumvent relaxation-induced blurring that worsens with larger core sizes to uncover the cubic resolution improvement with core size predicted by the Langevin Model. Experimental 1D measurements (Fig 1c) show achieved resolution closely approaches the Langevin Model. This is verified by 2D simulations (Fig 1d) and 2D imaging data (Fig 2). Standard MPI has been limited to smaller core sizes (< 25 nm) due to increased relaxation-induced blurring at larger core sizes. With Pulsed MPI, we can unlock the potential of larger core sizes, achieving 0.6 mm resolution with our current setup. By improving gradient to 7 T/m and increasing core size from 27.4 nm to 40 nm, ~ 0.1 mm resolution should be achievable (before deconvolution).

Conclusions

While Pulsed MPI needs significant MPI hardware changes, unlocking cubic resolution gains with tracer core size can dramatically improve resolution (8-fold for 25 nm to 50 nm). Using large core sizes also gives increased sensitivity to the microenvironment for binding contrast and in vivo viscosity measurements [10]. Lastly, the resolution gains from pulsed MPI can be traded-off for lower field gradients to make clinical translation of MPI hardware easier and to enable faster scanning in situations where scan speed is limited by gradient vs. SAR and magnetostimulation safety limits [11]. 

References

  1. Gleich B, Weizenecker J. Tomographic imaging using the nonlinear response of magnetic particles. Nature. 2005 Jun 30;435(7046):1214–1217.
  2. Graeser M, Knopp T, et al. Towards Picogram Detection of Superparamagnetic Iron-Oxide Particles Using a Gradiometric Receive Coil. Sci Rep. 2017 Jul 31;7(1):6872. 
  3. Them K, Salamon J, et al. Increasing the sensitivity for stem cell monitoring in system-function based magnetic particle imaging. Phys Med Biol. 2016 May 7;61(9):3279–3290. 
  4. Yu EY, Bishop M, et al. Magnetic Particle Imaging: A Novel in vivo Imaging Platform for Cancer Detection. Nano Lett [Internet]. 2017 Feb 16; 
  5. Ludewig P, Gdaniec N, et al. Magnetic Particle Imaging for Real-Time Perfusion Imaging in Acute Stroke. ACS Nano. 2017 Oct 24;11(10):10480–10488. 
  6. Yu EY, Chandrasekharan P, et al. Magnetic Particle Imaging for Highly Sensitive, Quantitative, and Safe in Vivo Gut Bleed Detection in a Murine Model. ACS Nano [Internet]. 2017 Nov 30; Available from: http://dx.doi.org/10.1021/acsnano.7b04844 PMID: 29165995
  7. Tay ZW, Hensley D, et al. The relaxation wall: experimental limits to improving MPI spatial resolution by increasing nanoparticle core size. Biomed Phys Eng Express [Internet]. IOP Publishing; 2017 Apr 3 
  8. Vreeland E C et al. 2015 Enhanced nanoparticle size control by extending lamers mechanism Chem. Mater. 27 6059–66
  9. Tay ZW, Goodwill PW, et al. A High-Throughput, Arbitrary-Waveform, MPI Spectrometer and Relaxometer for Comprehensive Magnetic Particle Optimization and Characterization. Sci Rep. Nature Publishing Group; 2016 Sep 30;6:34180.
  10. Fidler F, Steinke M, et al. Stem Cell Vitality Assessment Using Magnetic Particle Spectroscopy. IEEE Trans Magn. ieeexplore.ieee.org; 2015 Feb;51(2):1–4.
  11. Saritas EU, Goodwill PW, et al. Magnetostimulation Limits in Magnetic Particle Imaging. IEEE Trans Med Imaging. 2013;32(9):1600–1610.

Acknowledgement

We would like to acknowledge NIH funding and the A*STAR NSS-PhD and the Siebel Scholars fellowship (ZW Tay).

Pulsed MPI Drive Waveforms achieves Cubic Resolution Improvement with Tracer Core Size
a) Photo of 2D Arbitrary Waveform Relaxometer used.  b) Square (pulsed) drive waveforms waits for tracer relaxation to complete before scanning the next voxel in contrast to standard (sine) continuous scanning.  c) Pulsed MPI achieves cubic resolution improvement with core size (Langevin model) while sine MPI stops improving at 25 nm.   d) 2D simulation shows improved resolution with Pulsed MPI

Experimental 2D MPI Images show Eight-Fold Resolution Improvement with Pulsed MPI Drive Waveforms
Pulsed MPI circumvents relaxation blurring to uncover the good resolution native to large core sizes. 0.6 mm spacing is just resolved for a 27.4 nm core tracer. This is 8-fold better than Standard MPI on the same tracer that has ~ 5.0 mm resolution. Standard MPI typically uses small core sizes with poorer native resolution but no relaxation blurring. Pulsed MPI is 5-fold better than std MPI setup.

2:40 PM
PS-13-7 — Noninvasive Detection and Quantification of Gastrointestinal Bleeding with Magnetic Particle Imaging (#346)

E. Y. Yu1, P. Chandrasekharan1, R. Berzon1, Z. W. Tay1, X. Y. Zhou1, A. P. Khandhar2, R. M. Ferguson2, S. J. Kemp2, B. Zheng1, P. W. Goodwill3, M. F. Wendland1, K. M. Krishnan2, 4, S. Behr5, J. Carter6, S. M. Conolly1, 7

1 University of California, Berkeley, Department of Bioengineering, Berkeley, California, United States of America
2 Lodespin Labs, LLC, Seattle, Washington, United States of America
3 Magnetic Insight, Inc., Alameda, California, United States of America
4 University of Washington, Department of Material Science and Engineering, Seattle, Washington, United States of America
5 University of California, San Francisco, Department of Radiology and Biomedical Imaging, San Francisco, California, United States of America
6 University of California San Francisco Medical Center, San Francisco, California, United States of America
7 University of California, Berkeley, Department of Electrical Engineering and Computer Science, Berkeley, California, United States of America

Introduction

Colon polyps are a known cause of gastrointestinal bleeding (GIB) [1]. 99mTc-RBC scintigraphy is the most sensitive imaging technique for GIB detection. However, drawbacks include radioactive dose, poor spatial resolution (~4 mm), bulky radio-pharmaceutical agent preparation and long scan times, which can delay surgical interventions. We present the use of Magnetic Particle Imaging (MPI) [2-5] to detect GIB using long-circulating superparamagnetic iron-oxide (SPIO) tracer as the vascular tracer agent in a mouse model of Familial Adenomatous Polyposis.

Methods

Five 12 week old C57BLK6/Apcmin/+ mice [6] with a genetic mutation in FAP (JAX® Laboratory) (Hct=0.21-0.30) [7] were used in this study. Three Wild-type C57BL/6 mice were used as control. SPIOs (LodeSpin-017, 5 mg Fe/kg in 100 μL) and heparin were injected through a lateral tail vein catheter for each animal. MPI was performed with a custom-built vertical bore field-free line (FFL) MPI scanner with a gradient strength of 6.3 T/m. Twenty-one dynamic projection MPI scans were acquired with respiratory gating over 130 minutes. Region-Of-Interest based compartment fitting was performed after converting the MPI signal to iron concentration for flow quantification.

Results/Discussion

MPI images of Apcmin/+ and wild type mice at 2 time points post-SPIO injection are shown in Fig. 1. MPI signal was observed to accumulate in the gut lumen of Apcmin/+ mice, whereas the wild type showed signal throughout the vasculature, typical of LS-017 (a blood pool tracer). In Fig. 2 (a), the MPI image at the first time point was digitally subtracted from all images in time course to capture positive tracer accumulation. The GIB is visualized with extraordinary contrast in the Apcmin/+ mice, whereas minimal positive accumulation is observed in the control mice.

The rate of tracer accumulation of the gut lumen was between 1 – 5 µL/min. A representative two-compartment model fitting result is shown in Fig. 2 (b). GIB was detected using MPI at a sensitivity rivaling that of 99mTc-RBC scintigraphy at clinically relevant doses, but by using a blood-pool, non-radioactive SPIO tracer [8]. Additional sensitivity improvement is possible through tracer and receive hardware optimization [9,10].

Conclusions

In this work, we have demonstrated highly sensitive detection of GI bleeding in a murine model using MPI. Although there is still a long path to clinical translation for MPI tracers and imager, MPI is a clinically translatable imaging modality with superb contrast, sensitivity, linear quantitation and safety. We believe that in the future, MPI could complement the current clinical workflow for cases of occult or obscure GI bleeding. This may significantly improve the accuracy of diagnosis and ultimately reduce cost and improve outcome.

References

[1] Carrera, V. G., Villaverde, A. F., Campos, A. C. & López, S. V. Acute lower gastrointestinal bleeding from a polyp. Ann. Gastroenterol. Hepatol. 27, 263 2014.

[2] B. Gleich and J. Weizenecker. Tomographic imaging using the nonlinear response of magnetic particles. (2005). Nature, 435(7046):1217-1217, 2005. 

[3] T. Knopp and T. M. Buzug. Magnetic Particle Imaging: An Introduction to Imaging Principles and Scanner Instrumentation. Springer, Berlin/Heidelberg, 2012. [4] Goodwill, P. W. & Conolly, S. M. The X-space formulation of the magnetic particle imaging process: 1-D signal, resolution, bandwidth, SNR, SAR, and magnetostimulation. IEEE Trans. Med. Imaging 29, 1851–1859, 2010. 

[5]  Goodwill, P. W., Konkle, J. J., Zheng, B., Saritas, E. U. & Conolly, S. M. Projection x-space magnetic particle imaging. IEEE Trans. Med. Imaging 31, 1076–1085, 2012.

[6]  Wei, H., Shang, J., Keohane, C., Wang, M., Li, Q., Ni, W., O’Neill, K. & Chintala, M. A novel approach to assess the spontaneous gastrointestinal bleeding risk of antithrombotic agents using Apc(min/+) mice. Thromb. Haemost. 111, 1121–1132, 2014.

[7] Graça, B. M., Freire, P. A., Brito, J. B., Ilharco, J. M., Carvalheiro, V. M. & Caseiro-Alves, F. Gastroenterologic and radiologic approach to obscure gastrointestinal bleeding: how, why, and when? Radiographics 30, 235–252, 2010. 

[8] Yu, E. Y., Chandrasekharan, P., Berzon, R., Tay, Z., Zhou, X. Y., Khandhar, A. P., Ferguson, R. M., Kemp, S. J., Zheng, B., Goodwill, P. W., Wendland, M. F., Krishnan, K. M., Behr, S., Carter, J., and Conolly, S. M. Magnetic Particle Imaging for Highly Sensitive, Quantitative, and Safe in vivo Gut Bleed Detection in a Murine Model. ACS Nano, 2017. 

[9] Ferguson, R. M., Minard, K. R., Khandhar, A. P. & Krishnan, K. M. Optimizing magnetite nanoparticles for mass sensitivity in magnetic particle imaging. Med. Phys. 38, 1619–1626, 2011. 

[10] Zheng, B., Goodwill, P. W., Dixit, N., Xiao, D., Zhang, W., Gunel, B., Lu, K., Scott, G. C. & Conolly, S. M. Optimal Broadband Noise Matching to Inductive Sensors: Application to Magnetic Particle Imaging. IEEE Trans. Biomed. Circuits Syst, 2017. 

Acknowledgement

The authors would like to acknowledge funding support from NIH 5R01EB019458-03, NIH 5R24MH106053-03, UC Discovery Grant 29623, W. M. Keck Foundation Grant 009323, and NSF GRFP for this work. Additionally, work at Lodespin Labs and University of Washington was supported by NIH 1R41EB013520-01 and NIH 2R42EB013520-02A1.

Dynamic projection MPI captures whole body tracer bio-distirbution.

Figure 1 Representative MPI images with CT overlay of an ApcMin/+ (left) and Wild-Type (right) mouse over time. MPI clearly captures dynamics of tracer extravasation into the gut in the ApcMin/+ mouse, whereas no tracer extravasation into the gut is seen in the Wild-Type mouse.

MPI quantitatively captures positive tracer accumulation over time.

(a) Representative subtracted MPI images of an ApcMin/+ mouse (left) and a wild-type mouse (right) over time with CT overlay. The GI bleed is visualized with extraordinary contrast in the ApcMin/+ mouse, while for the wild-type mouse, the tracer stayed in the blood pool throughout the study, hence minimal subtracted signal. (b) Two compartment model fit results for ApcMin/+ and wild-type mice.

2:50 PM
PS-13-8 — Retrospectively gated cardiac PET-MR imaging in rodents using MRI-based cardiac motion information. (#549)

W. Gsell1, 2, A. Nauerth3, C. Molinos4, C. Correcher4, A. J. Gonzalez5, S. Sven3, T. Greeb3, R. Polo4, B. Holvoet2, 6, C. M. Deroose2, 6, U. Himmelreich1, 2, M. Heidenreich3

1 University of Leuven, Imaging and Pathology, Biomedical MRI, Leuven, Belgium
2 University Of Leuven, MoSAIC, Leuven, Belgium
3 Bruker BioSpin, Preclinical Imaging MRI, Ettlingen, Germany
4 Bruker BioSpin, Preclinical Imaging NMI, Valencia, Spain
5 Institute for Instrumentation in Molecular Imaging, i3M-CSIC, Valencia, Spain
6 University of Leuven, Imaging and Pathology, Nuclear Medicine, Leuven, Belgium

Introduction

Preclinical PET inserts for MRI scanners enable simultaneous assessment of cardiac function, tissue integrity and molecular pathways. The acquisition of cardiac PET-MRI in rodents remains challenging due to the short cardiac cycle, implying the use of rapid imaging with ECG gating. Strong gradients and fast gradient switching can interfere with the ECG signal, rendering the conventional ECG gating very difficult if not achievable. The aim of this work was to test a method based on an MRI self-gating technique (IntraGate)1 to provide the gating information for retrospective PET reconstruction.

Methods

PET-MRI data were acquired on a BioSpec 70/30 MRI system equipped with a SiPM based PET insert (Bruker Biospin), using a quadrature volume coil (diameter of 86 mm). Wistar rats und B6 mice were used. Animals were not fasted to ensure good FDG uptake. Anaesthesia was induced and maintained through inhalation of 2% Isoflurane carried by 100% oxygen. 18F-FDG (46.1 ± 9.7 MBq) was administered intravenously through the tail vein. For MRI, we used a self-gated gradient echo sequence (igFLASH) with the following parameters: TE: 3,585 ms, TR: 10 ms, flip angle: 15 degrees, matrix: 128x128, FOV: 60x60 mm, 10 contiguous slices of 1.5 mm thickness, scan time: 53:20 min. Simultaneously, PET data were acquired using a 1hr static scan. Retrospective PET-MRI was achieved using IntraGate (see below).

Results/Discussion

The MRI sequence was modified to send a TTL signal of 4 ms duration at each TR loop to the PET DAQ electronics. Then, the IntraGate navigator information was used to derive the required retrospective data ordering that represents the position within the cardiac cycle (Fig.1). The list-mode PET data were rebinned accordingly to reconstruct 4-16 cardiac frames. All PET data were reconstructed using MLEM with 12 iterations, no smoothing, no PSF modeling and 0.5mm isotropic resolution.

MRI data were first evaluated providing a time-resolved cardiac movie with up to 20 frames per cardiac cycle identifying the end diastole and end systole. The same information used for MRI reconstruction was used to achieve a time synchronization of simultaneous PET/MRI. A first application of this technique has resulted in self-gated cardiac imaging, both in mice and rats. The cardiac cycle has been correctly resolved in both modalities and allows for a dynamic co-registration of the images (Fig.2).

Conclusions

We hereby propose for the first time a method enabling PET cardiac imaging solely based on motion information derived from MRI. Our preliminary results demonstrate the advantage of such technique for true synchronization of the PET and MRI cardiac acquisition, the easier animal handling (no ECG electrodes required), access to the full cardiac cycle and the possibility to retrospectively assess the quality of the gating information.

References

[1] A. Nauerth, E. Heijman, C. Diekmann. Slice refocusing signal for retrospective reconstruction of CINE cardiac MR images. Proc. Intl. Soc. Mag. Reson. Med. 14 (2006).

Figure 1

Principle of the retrospectively gated PET-MRI technique: Navigator information form the intragate MRI sequence enable to extract information about respiration and cardiac motion (red box) used to assign each image to the corresponding cardiac frame, retrospectively. PET and MRI are synchronized through a TTL signal enabling to assign each time stamp of the PET list-mode to a cardiac frame.

Figure 2

PET and MRI data fusion using 8 frames across the cardiac cycle starting from end-systole (yellow box).

6:00 PM
SG-02-1 — Imaging-guided development of nanomedicines (#612)

W. Mulder1, 2

1 Icahn School of Medicine at Mount Sinai, Department of Radiology, New York, United States of America
2 Academic Medical Center, Cardiovascular Nanomedicine, Amsterdam, Netherlands

Content

A way to overcome a drug’s side effects is by its more efficient delivery to diseased sites. This can be accomplished by nanoparticles, tiny carrier vehicles that can be loaded with drugs, known as nanomedicines. Imaging techniques, including MRI, PET and optical imaging, can monitor the drug-carrier association and help identify key parameters that determine drug-carrier compatibility. These findings can serve as drug delivery efficiency guidelines that can be applied to improve nanomedicines.

Despite nanomedicine’s promise and the field’s research activity, its potential is not being fully met and implementation in clinical care is falling behind. In part this is due to the technology’s immaturity, but – more importantly – ways to stratify patients that may benefit from nanomedicine-based therapy are nonexistent.

The ability to non-invasively evaluate nanomedicine targeting would greatly improve patient care by allowing swift adjustments in dosage and/or treatment regimen. In this talk, imaging-facilitated optimization of nanomedicine and the “companion diagnostic’ concept, the latest advances in these fields, and translational considerations will be discussed.

6:30 PM
Array — Triggered radiosensitizer release by radiosensitizer-loaded thermosensitive liposomes and hyperthermia improves efficacy of radiotherapy: an in vitro proof of concept study (#271)

H. Besse1, C. Bos1, M. Zandvliet2, C. Moonen1, R. Deckers1

1 University Medical Center Utrecht, Center of Imaging Sciences, Utrecht, Netherlands
2 University Utrecht, Department of Clinical Sciences of Companion Animals, Utrecht, Netherlands

Introduction

To increase the efficacy of radiotherapy (RT), it could be combined with drugs, radiosensitizers. This results in clinical improvement1, although it also leads to increased toxicity2.

Here, a new concept, local radiosensitizer delivery, is introduced in which radiosensitizers are released from thermosensitive liposomes (TSL) by local hyperthermia (HT) followed by RT.

In this study we investigate in vitro if triggered release of a radiosensitizer, doxorubicin (DOX), from a TSL (ThermoDox) by HT improves the efficacy of RT and if the radiosensitization effect of DOX is concentration dependent.

Methods

HT1080, human fibrosarcoma, cells were used. Cells were exposed to ThermoDox (0.02 µg/ml) or DOX (0.01, 0.02 or 0.06 µg/ml) for 1 hour in a water bath of 37°C or 43°C. 45 minutes after ThermoDox or DOX incubation, cells were irradiated by a linear accelerator (Elekta, 6MV). Subsequently cell viability was measured by clonogenic assay.

Results/Discussion

RT combined with ThermoDox at 37°C showed similar efficacy compared to RT as a single treatment, Figure 1, due to retention of DOX in the liposomes at 37°C3. ThermoDox and DOX at 43°C showed equal efficacy. However, ThermoDox at 43°C resulted in less radiosensitization at high RT doses when compared to DOX at 43°C, a difference that is not yet understood. DOX showed direct toxicity on cell survival, and proved an effective enhancement of the RT, although the enhancement of RT was only to some extend DOX concentration dependent, Figure 2.

Conclusions

ThermoDox in combination with HT improves the efficacy of RT in vitro, whereas in the absence of HT the efficacy was similar to that of only RT. This provides a first indication that in the concept of local radiosensitizer delivery, local heating of the tumor tissue will reduce systemic toxicity as well as toxicity to normal tissue in the beam path. Unfortunately, higher local concentrations of DOX will not lead to an extra sensitization boost in the tumor. In summary this study provided the first proof that local radiosensitizer delivery may improve efficacy, while reducing systemic toxicity.

References

1Pisters, ASO, 2002; 2Myrehaug, L&L, 2008; 3Needham, FD, 2013

Acknowledgement

ERC Sound Pharma – 268906 (CM)

Figure 1
Cells treated without and with DOX or ThermoDox at a concentration of 0.02 µg/ml at 37°C or 43°C in a water bath with or without RT at 6 Gy

Figure 2

Surviving fraction as function of radiation dose for different DOX concentrations. Cells were exposed for 1 hour to DOX (0.01, 0.02 or 0.06 µg/mL) before RT. Survival fraction calculated by samples treated with the corresponding DOX concentration (A) and by untreated sample (B).

6:38 PM
Array — Synthesis and radiolabelling (124I) of PEG-stabilized gold nanorods (AuNRs) as boron drug delivery candidates with potential application in Boron Neutron Capture Therapy. (#123)

K. R. Pulagam1, J. Kumar2, V. Gómez-Vallejo1, L. Liz-Marzan2, J. Llop1

1 CIC biomaGUNE, Radiochemistry and Nuclear imaging Lab, San Sebastian, Spain
2 CIC biomaGUNE, Bionanoplasmonics lab, San Sebastian, Spain

Introduction

Boron neutron capture therapy (BNCT) is widely accepted as a promising cancer treatment. One limitation of BNCT is the need to develop drugs that are able to deposit a sufficient number of 10B atoms specifically in tumor cells. Here, we report the preparation and characterization of AuNRs functionalized with Poly(ethylene glycol) methyl ether thiol and boron-based molecule cobalt bis(dicarbollide), [3,3’-Co(1,2-C2B9H11)2]-, commonly known as COSAN. The resulting functionalized AuNRs were radiolabelled with 124I either on the surface of the gold core or covalently attached to the COSAN.

Methods

The cobalt-bis(dicarbollide) anion (COSAN) was treated with tetrahydropyran and sequentially reacted with potassium thioacetate (KSAc) and iodine to achieve THP ring opening and iodination of the cluster (Figure 1). Radiolabelling was achieved by palladium-catalyzed isotopic exchange using 124I on compound [6]-. Final hydrolysis yielded the thio-compound, ready to be incorporated into AuNRs. The synthesis of AuNRs followed a reported seed-mediated method.Radiolabelled, boron-rich AuNRs were prepared using two strategies: (i) absorption of 124I- on the surface of the gold core followed by attachment of PEG-SH and COSAN derivatives; and (ii) functionalisation of CTAB-stabilized AuNRs with PEG-SH and (124I-[6]- + [4]-), to incorporate the radionuclide at the shell of the AuNRs (Figure 2).

Results/Discussion

The (protected) thiolated iodo-cobalt bis(dicarbollide) derivative [6]- could be obtained in an overall yield of 70±6%. Radiolabelling by isotopic exchange (T=100ºC, t=15 min) resulted in conversion values in the range of 75-80%. COSAN could be efficiently incorporated into AuNRs (size: 10 x 30 nm) as demonstrated by X-ray photoelectron spectroscopy (XPS). Both labelling strategies resulted in good yields (>55%).

Conclusions

PEG stabilized and thiolated iodo-cobalt bis(dicarbollide) functionalized radiolabelled AuNRs were prepared. These could be appropriate boron-rich drug candidates with application in BNCT. Biodistribution studies in healthy and tumour-bearing animals are planned for the next future.

References

1. B. Nikoobakht, MA. El-Sayed, Chem Mater. 2003, 15, 1957.

Acknowledgement

This project was partially funded by Ministerio de Ciencia e Innovación (Grant number CTQ2009-08810).

Figure 1
Synthesis of functionalized COSAN derivatives; (i) THP, dimethylsulphate, H2SO4; (ii) KSAc; (iii) NaOMe, MeOH; (iv) CH2Cl2, I2; (v) NaOMe, MeOH. White dots: B or B-H; black dots: C-H; gray dots: Co3+

Figure 2
Radiolabelling either at the gold core or at the COSAN shell of AuNRs with 124I (stars).

6:46 PM
Array — Therapy monitoring and cell tracking of immune cells in cancer with Fluorine-19 MR Imaging (#251)

W. Reichardt1, 2, 3, D. von Elverfeldt1, E. Fischer1, C. Weidensteiner1

1 University of Freiburg, Medical Physics, Department of Radiology, Faculty of Medicine, Freiburg, Baden-Württemberg, Germany
2 German Consortium for Translational Cancer Research (DKTK), Heidelberg, Baden-Württemberg, Germany
3 German Cancer Research Center (DKFZ), Heidelberg, Baden-Württemberg, Germany

Introduction

The tracking of immune cells in vivo is getting increasingly important with the rise of  immune therapy1. Antibodies can activate a variety of immune cells. This leads to a massive infiltration of activated immune cells into the tumor and lymph nodes. Per-fluorinated carbons (PFC) which are taken up primarily by monocytes after i.v. injection can serve as a tracer for immune cell tracking. Therefore the signal from the exogenous tracer can be detected and localized with high sensitivity using 19F MRI if the number of cells loaded with PFC-tracer in the region of interest is sufficient³.

Methods

MR Imaging: A receive surface coil was built by the Medical Physics Group of the University Medical Center Freiburg and operated with a double-tuned 19F-1H birdcage resonator for transmission. Imaging (Fig.1) was performed on a 9.4 T Bruker animal scanner (Bruker, Ettlingen, Germany). 50 nl of the PFC was placed next to the tumor in a vial as a reference. The 19F-MRI signal intensities were normalized to the reference. Animal Model: A subcutaneous tumor model was generated in mice. After randomization five animals were treated with vehicle, 10 animals were treated with a combination of  anti-PD-L1  and anti-CD40 immune-stimulating antibodies.Three days after treatment all animals received the contrast agent PFC  (120µl  i.v.) . Five days after treatment animals were analyzed by MRI.

Results/Discussion

Statistics (Fig.2) showed a difference in the mean normalized signal intensity between the two groups indicating differences in immune cell infiltration after antibody treatment compared to control in s.c. tumor models in immune competent mice using PFCs as tracer. Additional analysis of tumor, liver and spleen showed a large difference in the spleen volume between the two groups while there was no significant difference in the normalized spleen intensity or the normalized liver intensity values. Tumor volume was smaller in the treated group. The results for the tumor volume indicate a tumor shrinking effect of the therapy. However, the mean normalized tumor signal intensity was lower in the treated group. This finding was not expected after treatment with immune-stimulating antibodies. However, as the spleen recorded a significant increase in volume while showing constant signal intensity it seems likely that a large part of activated immune cells is migrating to the spleen.

Conclusions

A 19F-MRI coil was built and successfully tested at a small bore animal scanner Bruker Biospec 9.4T. In vitro and in vivo test scans using phantoms and mice were performed to test and optimize the MR-protocol. The results showed that it is possible to detect dynamic changes in the migratory pattern and activation of immune cells in tumor models undergoing immune therapy. This might be useful to differentiate pseudoprogression due to the invasion off immune cells from real progression of a tumor.

References

  1. Ahrens ET, Bulte JW. Tracking immune cells in vivo using magnetic resonance imaging. Nat Rev Immunol. 2013 Oct;13(10):755-63. doi: 10.1038/nri3531. Epub 2013 Sep 10. Review. PubMed PMID: 24013185; PubMed Central PMCID: PMC3886235.
  2. Schwab, L, Goroncy, L, Palaniyandi, S., Gautam, S., Triantafyllopoulou, A., Mocsai, A, Reichardt, W., Karlsson, FJ, Radhakrishnan, SV, Hanke, K, Schmitt-Graeff, A, Freudenberg, M, von Loewenich, FD, Wolf, P, Leonhardt, F, Baxan, N, Pfeifer, D, Schmah, O, Schönle, A, Martin, SF, Mertelsmann, R, Duyster, J, Finke, J., Prinz, M., Henneke, P., Häcker, H., Hildebrandt, G.C, Häcker, G, Zeiser, R. Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance GvHD via tissue damage. Nature Medicine 20: 648-54, 2014
  3. Janjic JM, Srinivas M, Kadayakkara DK, Ahrens ET. Self-delivering nanoemulsions for dual fluorine-19 MRI and fluorescence detection. J Am Chem Soc. 2008; 130:2832–2841.

Figure 1:
Overlay of T2 RARE Image and the 19F image. Note the distribution of PFC positive cells in the tumor primarily in the rim. Next to the tumor the reference can be seen. 19F images were acquired with a RARE sequence: TR/TE/FA = 3000ms/76ms/90° matrix 64x64, FOV= 40 mmx40 mm, 12 axial slices with 3 mm thickness (30min.) 50 nl of the PFC was placed next to the tumor in a vial as a reference.

Figure 2:
a) Box plot of normalized tumor signal ROI values in both groups. b) Box plot of tumor volume values in both groups

 

6:54 PM
Array — Magnetic Particle Imaging Guided Heating In Vivo : Gradient Fields Localize Heating To Tumor while Sparing Non-specific Nanoparticle Accumulations (#192)

Z. W. Tay1, P. Chandrasekharan1, D. Hensley1, X. Y. Zhou1, B. Zheng1, R. Dhavalikar2, A. Chiu-Lam2, C. Rinaldi2, S. M. Conolly1

1 University of California, Berkeley, Bioengineering, Berkeley, California, United States of America
2 University of Florida, Gainesville, Florida, United States of America

Introduction

Magnetic Fluid Hyperthermia (MFH) is promising for cancer therapy but one challenge is focusing AC fields for selective heating of tumors with magnetic nanoparticles (MNP) while sparing healthy tissues with non-specific accumulations such as the clearance organs (liver) [1]. Magnetic Particle Imaging (MPI) is a new tracer imaging modality [2] whose signal generation is similar to how MFH generates heat [3-6]. MPI gradients localize signal & heating by locking MNP rotation. We show the first MPI image-guided localized heating of mice xenograft tumors while sparing MNPs cleared to the liver.

Methods

Superparamagnetic Iron Oxide Nanoparticles (SPIONs) from University of Florida was used. The in vitro study used a 3 x 3 grid of 0.1 ml vials separated edge-to-edge by 7 mm. The in vivo study used 7 – 9 week old athymic nude mice with MDA-MB-231-luc xenografts. 1.5 mg Fe of SPION was injected intratumorally and/or injected in the tail-vein and allowed to clear to the liver. MPI imaging used a 6.3 T/m field-free-line scanner at 20 kHz 20 mT. Based on the MPI image-guidance, targeted heating was performed on a co-registered 2.35 T/m field-free-line MPI setup optimized for 354 kHz 14 mT excitation [4]. In-vivo temperatures was measured by NeoptixTM fiber optic temperature probes. IVIS Lumina VivovisionTM  was used for bioluminescence and KubtecTM Cabinet X-ray for the anatomic references.

Results/Discussion

Fig 1 shows a MPI theranostics workflow where we achieved in vivo heating of tumor but spared the liver. Step 1) MPI image scan visualizing the SPION biodistribution in the tumor(s) (and clearance organs).  2) User selects a region to heat, and MPI gradients shift the field-free-line to target  3) Thermal dose planning based on MPI image 4) Localized heating. In vitro results (Fig 2a) show heating of the central vial with negligible temperature rise in the surrounding vials spaced 7 mm away, demonstrating highly localized heating to < 7 mm. With a 6.3 T/m gradient, this can be improved to 3 mm localization. In vivo results (Fig 2b) show good correlation of SAR deposition with MPI image intensity, enabling thermal dose planning from the MPI image alone. Fig 2c: To evaluate localization of therapy, we used dual tumor mice (both with SPIONs) but heating was targeted at only one tumor. Bioluminescence images show a dramatic decrease in activity only at the targeted tumor.

Conclusions

MPI enables (1) high contrast imaging of SPIONs  (2) thermal dose planning due to the quantitative nature of MPI images  (3) spatial localized heating of SPIONs through user-directed location of the field-free-line. In addition, real-time temperature monitoring could be achieved through MPI thermometry [7], enabling closed-loop feedback during treatment. Furthermore, localized heating / actuation could be leveraged with thermally or mechanically sensitive liposomes for user-directed localization of drug release in vivo. All these features make MPI a promising cancer theranostics platform. 

References

1. Thiesen B, Jordan A. Clinical applications of magnetic nanoparticles for hyperthermia. Int J Hyperthermia. 2008 Sep;24(6):467–474.

2. Gleich B, Weizenecker J. Tomographic imaging using the nonlinear response of magnetic particles. Nature. 2005 Jun 30;435(7046):1214–1217.

3. Dhavalikar R, Rinaldi C. Theoretical predictions for spatially-focused heating of magnetic nanoparticles guided by magnetic particle imaging field gradients. J Magn Magn Mater. 2016 Dec 1;419:267–273.

4. Hensley DW, Tay ZW, Dhavalikar R, Zheng B, Goodwill P, Rinaldi C, Conolly S. Combining magnetic particle imaging and magnetic fluid hyperthermia in a theranostic platform. Phys Med Biol. 2016 Dec 29;

5. Murase K, Aoki M, Banura N, Nishimoto K, Mimura A, Kuboyabu T, Yabata I. Usefulness of Magnetic Particle Imaging for Predicting the Therapeutic Effect of Magnetic Hyperthermia. Open Journal of Medical Imaging. Scientific Research Publishing; 2015;5(02):85.

6. Tasci TO, Vargel I, Arat A, Guzel E, Korkusuz P, Atalar E. Focused RF hyperthermia using magnetic fluids. Med Phys. 2009 May;36(5):1906–1912.

7. Perreard IM, Reeves DB, Zhang X, Kuehlert E, Forauer ER, Weaver JB. Temperature of the magnetic nanoparticle microenvironment: estimation from relaxation times. Phys Med Biol.

Acknowledgement

We would like to acknowledge NIH funding and the A*STAR NSS-PhD and Siebel Scholars fellowship (ZW Tay).

Experimental Data from a Theranostics Workflow for MPI Image-Guided, Localized Heating
First, an MPI image at low frequency (20 kHz) is taken of the mouse. Negligible heating occurs. Thermal dose planning based on MPI image intensity is performed. Next, the field-free-line gradient (FFL) is aligned to the target tumor. This prevents SPION rotation & heating everywhere but the target during the 354 kHz heat scan, localizing heat to the tumor while sparing the liver.

Spatial Localization of Tumor Therapy in a Dual Tumor Mouse
a.  3 x 3 phantom shows MPI gradients (2.3 T/m) can localize heating to < 7 mm.    b.  MPI image intensity correlates well with measured in vivo SAR, enabling thermal dose planning from the MPI image.   c. MPI localized heating of one out of two tumors. Tumor bioluminescence images show marked decrease in bioluminescence in the targeted tumor only, verifying spatial localization of therapy.

7:02 PM
Array — Preclinical development of copper-64 radiolabeled lipid nanoparticles for targeted delivery in atherosclerotic plaques (#168)

J. Vigne1, 2, 6, R. Aid2, 6, G. Even2, M. Escudé3, 4, N. Anizan2, 6, P. Oliva5, V. Mourier3, 4, A. Cordaro5, F. Hyafil1, 2, 6, C. Chauvierre2, F. Rouzet1, 2, 6, D. Letourneur2, D. Le Guludec1, 2, 6, C. Cabella5, I. Texier3, 4

1 Bichat University Hospital, Nuclear medicine department, Paris, France
2 INSERM U1148, Laboratory for vascular translational science, Paris, France
3 Univ. Grenoble Alpes, Grenoble, France
4 CEA LETI, Grenoble, France
5 Centro Ricerche Bracco, Bracco Imaging SpA, Turin, Italy
6 Fédération de Recherche en Imagerie Multimodalité, Université Paris Diderot, Paris, France

Introduction

Atherotrombosis is one of the world leading cause of death globally [1]. Recent results have shown accumulation of Lipidots™, 50 nm diameter nanoparticles composed of a lipid core stabilized by a biocompatible surfactant shell, in structures containing a high content of lipids [2]. Thus the Lipidots™ shell was modified to enable the chelation of positron emitter radiometals such as Copper-64 to obtain PET probes. The proof-of-concept of radiolabeled Lipidots™ to achieve targeted delivery purposes of atherosclerotic plaques in a preclinical model of atherosclerosis was demonstrated.

Methods

After synthesis, introducing additional amphiphilic DOTA chelates, nanoparticles hydrodynamic diameter and polydispersity index (PDI) were measured using dynamic light scattering. Radiolabeling consisted in adding 200 MBq of 64CuCl2 to a mix of nanoparticles in ammonium acetate 0.1 M pH 7 and heating at 60°C during 25 min. Radiochemical purity was assessed by thin layer chromatography (TLC). ApoE KO mice aged over 35 weeks were used as preclinical model of atherosclerosis and injected with 20 MBq of Lipidots™. Control groups consisted in injecting Lipidots™ to WT mice and in injecting the same mixture except without particles in ApoE KO mice. PET/MRI acquisitions were realized 24 h after injection then mice were sacrificed. Autoradiography and Oil Red O staining were performed on aortas.

Results/Discussion

Functionalized Lipidots™ hydrodynamic diameter was 50.0 ± 0.9 (mean ± SD) nm and PDI was 0.084 ± 0.01. Radiochemical purity assessed by TLC was over 99% and pH was between 6 and 7 on final product 1 hour post-radiolabeling and filtration. PET/MRI acquisitions revealed a predominant liver and spleen uptake of 64Cu-Lipidots™, blood pool signal was still detected 24 hours post-injection. Autoradiography performed on aortas dissected from ApoE KO mice injected with particles exhibited different focal uptake that colocalized perfectly with the lipid mapping realized with Oil Red O staining. This pattern was not observed in control groups. Whole aortas standardized signal intensities from autoradiography were 8.6 ± 0.39 (n=4) for ApoE mice KO Lipidots™ group, 4.0 ± 0.96 for ApoE mice without Lipidots™ group (n= 2; p < 0.05) and 1.8 ± 0.03 for WT Lipidots™ group (n=3; p < 0.001). 

Conclusions

Lipidots™ were efficiently radiolabeled by 64Cu to yield PET contrast agents with appropriate diameter and PDI, and seemed to significantly accumulate in atherosclerotic plaques in ApoE KO mice. Further studies will be performed using dye-loaded and 64Cu labelled nanoparticles to validate the suitability of these nanosystems in conditions where atherosclerotic plaques are involved. These nanoparticles could constitute an interesting tool for targeted delivery and imaging purposes in atherosclerosis.

References

1. WHO | Cardiovascular diseases (CVDs) [Internet]. WHO. [cited 2017]. Available from: http://www.who.int/mediacentre/factsheets/fs317/en/

2. J. Mérian, R. Boisgard, X. Decleves, B. Thezé, I. Texier, B. Tavitian, J. Nucl. Med. 2013, 54, 1996-2003

Acknowledgement

This work was supported by the EU (“NanoAthero” project FP7-NMP-2012-LARGE-6-309820). CEA-LETI/DTBS is part of the Arcane Labex program, funded by the French National Research Agency (ARCANE project n° ANR-12-LABX-003).

Authors thank the Accelerator for Research in Radiochemistry and Oncology at Nantes Atlantic  (ARRONAX, Nantes, France) for Copper-64 supply.

Scheme and transmission electron microscopy of the Lipidots™ nanoparticles.

Left : Scheme of Lipidots™, 50 nm diameter nanoparticles composed of a lipid core stabilized by a biocompatible surfactant shell. Lipidots™ were formulated as previously described [2], introducing additional amphiphilic DOTA chelates (0.6% of dried formulation) in the nanoparticle shell.

Right : Transmission electron microscopy of Lipidots™.

Autoradiography and Oil Red O staining of mice aortas.

A, B, C and D left, autoradiography after 20h exposure of aortas lied in "en face" position.

A : WT mouse aorta from the control group receiving 64Cu radiolabeled Lipidots.

B : ApoE KO mouse aorta from the control group receiving the same mixture except particles.

C and D left : ApoE KO mice receiving 64Cu radiolabeled Lipidots.

D right : Oil red O staining of the same aorta represented in D left.

 

7:10 PM
Array — In vivo imaging of polymersome uptake and distribution in cells and tumor bearing mice (#5)

S. J. Roobol1, 2, T. A. Hartjes3, R. M. de Kruijff6, J. D. M. Molkenboer-Kuenen7, S. Heskamp7, G. Torrelo-Villa6, R. Kanaar4, 1, A. B. Houtsmuller3, D. C. van Gent1, A. G. Denkova6, M. E. van Royen3, J. Essers1, 4, 5

1 Erasmus MC, Molecular Genetics, Rotterdam, Netherlands
2 Erasmus MC, Radiology & Nuclear Medicine, Rotterdam, Netherlands
3 Erasmus MC, Optical Imaging Centre, Rotterdam, Netherlands
4 Erasmus MC, Radiation Oncology, Rotterdam, Netherlands
5 Erasmus MC, Vascular Surgury, Rotterdam, Netherlands
6 Delft University of Technology, Radiation Science and Technology, Delft, Netherlands
7 Radboud University Medical Center, Radiology & Nuclear Medicine, Nijmegen, Netherlands

Introduction

Polymersomes, composed of amphiphilic block copolymers PB-b-PEO (polybutadiene - d - polyethylene oxide), have emerged as  promising robust customizable nano-carriers for high-LET radionuclides in radionuclide therapy. In this study, we analyzed on the uptake mechanism of polymersomes in cells in vitro using live cell microscopy and distribution in vivo using optical and SPECT/CT imaging of these novel nano-carriers.

Methods

Polymersomes were formed using a solvent displacement methodology. Characterization was done by Cryo-TEM and Dynamic Light Scattering. Polymersomes were  loaded with 111In or fluorescent PKH dyes for subsequent in vivo optical and SPECT imaging in MDA-MB-231 tumor bearing and control Balb/c nude mice. Confocal microscopy and fluorescence flow cytometry were used to quantify cellular uptake and analyze kinetics of polymersomes. We compared fibroblast, cancer and macrophage cell lines. Co-localization experiments were performed using PNT2C2 cells transfected and incubated with Rab4A, Rab7 or Lysotracker, respectively.

Results/Discussion

Polymersomes of 80nm were cleared from circulation within 1 hour. Interestingly, control mice showed longer circulating polymersomes compared to tumor bearing mice (139 v.s. 7 minute half-life, respectively). Biodistribution analysis showed high uptake in liver, spleen and bone-marrow compared to limited uptake in the tumor.  Live cell imaging demonstrated gradual intracellular uptake over time, cell cycle-dependent uptake kinetics and in addition we analyzed microtubule-mediated processing of polymersomes.  Co-localization showed polymersomes entering the endocytosis pathway through early endosomes (Rab4) and were transferred to late-endosomes (Rab7) and lysosomes (Lysotracker) after which they reside in a peri-nuclear localization. High-throughput analysis showed cell line specific uptake kinetics, evidenced in competition assays using macrophage cell lines.

Conclusions

Whole animal imaging showed very fast clearance of polymersomes by both the liver and spleen. With a combination of confocal, spinning disk and high-throughput microscopy we determined the timeframe, cell cycle-specificity and localization of fluorescent polymersome uptake in various cell types by endocytosis. To avoid healthy-organ damage using polymersomes this a specific accumulation might be countered by increasing the PEG-chain length. Future experiments using active targeting of the vesicles will give more insight in how polymersomes could be used in the clinic.

References

Wang G, de Kruijff RM, Rol A, Thijssen L, Mendes E, Morgenstern A, et al. Retention studies of recoiling daughter nuclides of 225Ac in polymer vesicles. Appl Radiat Isot. 2014 Feb;85:45-53.

 

 

In vitro analysis of polymersome uptake

(A,B) PNT2C2 cells stably expressing CAAX-GFP as membrane marker were incubated with polymersomes and followed over time. A, uptake over a time course of three hours. B, stills of dividing cells showing dramatic increase of uptake after division. (C) Competition assay using U2OS and J774 (white arrows) indicating heavy competition between cell lines. Polymersomes were labelled with PKH26 (red).

In vivo analysis of polymersome distribution and blood circulation.
(A) Balb/c nude mice bearing MDA-MB-231 tumors were injected with polymersomes (80nm, 111In-labaled, 15-20 MBq) and imaged at 4  and 24 hours post injection (h.p.i.) using SPECT/CT. (B) Both healthy control and tumor bearing mice were injected as before. At indicated time-points blood was sampled. Plot shows measured activity in blood samples. 

6:15 PM
emptyVal-1 — Imaging Cardiac Remodeling: Clinical Perspectives

F. Hyafil

6:35 PM
FS-02-2 — Imaging cardiac metabolism and remodeling with MRI (#583)

J. J. Prompers1

1 University Medical Center Utrecht, Radiology, Utrecht, Netherlands

Content

Alterations in myocardial energy metabolism have been implicated in the pathogenesis of heart failure. Studies have shown that the balance of substrate use (i.e. fatty acids, glucose, ketone bodies) is disturbed in heart failure.1-3 While our knowledge of myocardial substrate metabolism largely originates from perfused hearts, the ex vivo setup does not completely mimic the in vivo situation, where substrate availability can change depending on disease conditions. In this case, magnetic resonance spectroscopy (MRS) offers a powerful tool to study myocardial energy metabolism in vivo (Figure 1), which also allows longitudinal studies to map changes during disease progression and treatment. Furthermore, cardiac cine magnetic resonance imaging (MRI) can also be implemented to correlate the MRS findings with cardiac function. In this lecture, I will review methods and applications of cardiac MRS in probing myocardial energy metabolism in vivo in small animal models of heart failure.4 I will discuss 1H MRS to measure myocardial lipids and total creatine pool size, 31P MRS to measure cardiac energy homeostasis, and hyperpolarized 13C MRS to study substrate utilization in real time.

References

1. Glatz, J., et al. Cardiovasc. Drugs Ther. 2006; 20(6):471-476.

2. Abdurrachim, D., et al. Cardiovasc. Res. 2017; 113(10):1148-1160.

3. Aubert, G., et al. Circulation 2016; 133: 698–705.

4. Bakermans, A. J., et al. Prog. Nucl. Magn. Reson. Spectrosc. 2015; 88-89:1-47.

Acknowledgement

None

Figure 1
Overview of MR techniques to assess cardiac metabolism, structure and function.

6:55 PM
FS-02-3 — Imaging perfusion, metabolism and innervation with PET (#595)

A. Saraste1

1 Turku PET Centre, University of Turku, Turku, Finland

Content

Positron emission tomography (PET) myocardial perfusion imaging is being increasingly used for the detection of coronary artery disease. In the presence of significant left ventricle dysfunction, the assessment of myocardial ischemia and viability by PET plays a role in the identification of patients who may benefit from revascularization. In addition to these, PET imaging may play a role in the assessment of underlying pathophysiology and therapeutic options in heart failure. PET imaging with tracers of cardiac energy and substrate metabolism (such as 11C labeled acetate and analogues of glucose or fatty acids) have been used to show effects of therapies in heart failure. Studies have shown the ability of sympathetic innervation imaging to assess the risk of cardiac death, arrhythmia, and disease progression. New tracers have been tested for the assessment of angiogenesis and other mechanisms involved in myocardial repair after infarction. I will review clinical and experimental aspects of PET imaging in myocardial remodeling and heart failure.

References

Saraste A, Knuuti J. PET imaging in heart failure: the role of new tracers. Heart Fail Rev. 2017;22:501-511

7:15 PM
FS-02-4 — New Imaging Opportunities in CardiomyopathiesNew Imaging Opportunities in Cardiomyopathies (#586)

J. T. Thackeray1

1 Hannover Medical School, Department of Nuclear Medicine, Hannover, Lower Saxony, Germany

Content

  While advances in revascularization and medical therapy have lowered mortality rates after acute myocardial infarction, patients bear higher risk to develop heart failure. As such, advancing cardiovascular disease remains a significant burden on the health care system. Novel strategies to attenuate ventricular remodelling can alleviate the progression of terminal heart failure, but conventional diagnostic tests are often insufficient to identify patients who will most benefit from targeted intervention or to monitor therapeutic efficacy. This represents a significant challenge for the characterization of novel targeted drugs that may be costlier and more personalized than standard medical care. Molecular imaging offers a non-invasive means to evaluate physiologic parameters that may predict ventricular remodelling and progressive heart failure.

  For example, local tissue inflammation after myocardial infarction leads to worse patient outcome and is the target of several emerging treatment strategies. Characterization of inflammation has relied on 18F-FDG imaging, which has inherent limitations due to robust cardiomyocyte uptake and poor specificity of inflammatory cells. A wider range of tracers targeting specific markers of inflammatory leukocytes have gained traction in preclinical and clinical studies. Likewise, other molecular markers of neovasculature, fibroblasts, or extracellular matrix contributing to ventricular remodelling may be interrogated to predict subsequent functional outcome. Development of novel and specific therapies to support endogenous healing may be aided by targeted imaging agents, which provide not only a surrogate indicator of therapeutic efficacy, but also identify the appropriate targeting and timing of optimal treatment. Common targets for imaging and therapies may also introduce a new paradigm in clinical evaluation, where imaging endpoints may serve as ancillary indicators of therapeutic success or failure in clinical trials. This approach may also assist in the selection of appropriate patient populations for a specific intervention, working toward personalized precision medicine.

  A number of obstacles remain before wider clinical deployment of novel radiotracers and imaging techniques, including i) absolute, reliable, and reproducible quantification, ii) clear demonstration of added value for patient risk stratification, and iii) the ability to assess not only progression of disease, but also response to therapy. This lecture will address evolving approaches for molecular imaging in progressive heart failure and cardiomyopathy, with particular focus on inflammation, and provide perspective on the design and execution of molecular imaging research in cardiovascular disease.

8:30 AM
emptyVal-1 — Introductory talk by Vladimir Ponomarev - New York, USA

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

8:50 AM
PS-23-2 — Visualising regulatory T cell trafficking in pre-clinical humanised mouse transplantation models using SPECT/CT imaging (#106)

J. Jacob1, G. O. Fruhwirth3, R. I. Lechler1, L. A. Smyth1, 2, G. Lombardi1

1 King's College London, School of Immunology and Microbial Sciences, London, London, United Kingdom
2 University of East London, School of Health Sport and Bioscience, London, London, United Kingdom
3 King's College London, Rayne Institute, Imaging Sciences, London, London, United Kingdom

Introduction

Success of solid organ transplantation is hindered by attack from the recipient’s immune system which leads to rejection. Prolonged transplant survival can be achieved, in murine models, by favouring a regulatory environment via adoptive transfer of ex-vivo expanded regulatory T-cells (Tregs). Despite ongoing clinical trials using polyclonal Tregs, details of transferred Treg longevity and trafficking remain elusive. Here, we address whether longitudinal whole body nuclear imaging of radiolabelled human Tregs in vivo may help address the aforementioned points.

Methods

hNIS-GFP+ Tregs were generated and tested for phenotypic markers, suppressive capacity and ability to take up 99mTcO4-. hNIS-GFP+Tregs and autologous CD25- PBMCs were i/v injected into immunodeficient BRG (BALB/cRag2−/−gc−/−) or NSG (NOD/scid/IL-2Rg−/−) mice transplanted with human skin. Both BRG and NSG mice do not produce mature T or B cells, but NSG mice have an additional defect in innate immunity. Additionally, BRG mice were treated with or without anti-Gr1/Ly-6G antibody to deplete innate cells such as neutrophils. hNIS-GFP+ Tregs were tracked in vivo by SPECT/CT using 99mTcO4-  on days 0, 3, 8, 16, 25 and 40, and their location verified ex vivo by histology and flow cytometry. Ex vivo gamma-counting of transplant tissues confirmed in vivo imaging results.

Results/Discussion

Ex vivo expanded hNIS-GFP+ Tregs labelled with 99mTcO4 exhibited significant radioactive uptake in comparison to untransduced Tregs. Importantly, hNIS-GFP+ Tregs also retained their phenotype and suppressive ability following radioactive uptake (Fig.1). In vivo hNIS-GFP+ Tregs were observed as early as on day 3 post transfer in the skin graft and remained detectable until day 40 (Fig.2). Subsequent retrieval of hNIS-GFP+ Tregs from the graft confirmed Treg presence in the tissue, with a mean SUV of 0.61 at day 40 after injection (Fig.2C). Interestingly, we observed that the presence of mouse granulocytes impacted on Treg migration, as mice treated with anti-Gr1 antibody displayed delayed Treg presence in grafts, also seen with NSG mice (day 40).

Conclusions

This is the first study to show that SPECT/CT imaging using human hNIS-GFP reporter gene technology allows longitudinal Treg tracking in a transplant setting.

References

1. Regulatory T cells: tolerance induction in solid organ transplantation. Vaikunthanathan, T. et al. 2017. Clinical and experimental immunology. 189, 2, p197-210.

2. A whole-body dual-modality radionuclide optical strategy for preclinical imaging of metastasis and heterogeneous treatment response in different microenvironments. Fruhwirth, G. et al. 2014. Journal of Nuclear Medicine. 55, 4, p. 686-694

Acknowledgement

We acknowledge support from the Medical Research Council (MRC) for funding the PhD studentship, British Heart Foundation and Cancer Research UK.

Human Tregs can be transduced to express hNIS

hNIS-GFP Tregs can be visualised within the human skin graft of transplanted mice

9:00 AM
PS-23-3 — Multimodal Assessment of Orbital Immune Cell Infiltration and Tissue Remodeling During Development of Graves’ Disease by 1H/19F MRI (#130)

U. Flögel1, A. Schlüter2, C. Jacoby1, S. Temme1, P. J. Banga2, A. Eckstein2, J. Schrader1, U. Berchner-Pfannschmid2

1 Heinrich Heine University, Experimental Cardiovascular Imaging, Düsseldorf, Germany
2 University Hospital Essen, Molecular Ophthalmology, Essen, Germany

Introduction

Graves’ disease is an autoimmune condition of the thyroid gland, leading to overproduction of thyroid hormones. A frequent complication is Graves’ orbitopathy (GO), where inflammation of the orbit associated with increased adipogenesis and deposition of hyaluronan results in detrimental remodeling of the orbital soft tissue (1,2). Since current diagnostics are limited in encompassing the complex phenotype of GO, the present study aimed to evaluate key molecular and cellular features of GO by simultaneous monitoring of alterations in morphology, inflammatory patterns, and tissue remodeling.

Methods

To this end, we utilized a murine model of GO induced by immunization with a human thyrotropin receptor (TSHR) A-subunit plasmid (3,4). Altogether 52 mice were used: 27 GOs and 25 controls immunized with b-galactasidose plasmid (Ctrl). From these, 17 GO and 12 Ctrl mice were subjected to multimodal MRI at 9.4T, while 23 mice only underwent histology. Beyond anatomical 1H MRI, we employed T2 mapping for visualization of edema, chemical exchange saturation transfer (CEST) for detection of hyaluronan, and 19F MRI for tracking of in situ labeled immune cells after intravenous injection of perfluorcarbons (PFCs). The full experimental protocol took around 60-90 min and was well tolerated by all mice.

Results/Discussion

1H/19F MRI demonstrated substantial infiltration of PFC-loaded immune cells in peri- and retro-orbital regions of GO mice (Fig. 1), while healthy Ctrls showed only minor 19F signals. In parallel, T2 mapping indicated onset of edema in peri-orbital tissue and adjacent ocular glands (P=0.038/0.017, Fig.1), which were associated with enhanced orbital CEST signals in GO mice (P=0.031). Concomitanty, a moderate expansion of retrobulbar fat (P=0.029) was apparent, but no signs for extraocular myopathy were detectable. 19F MRI-based visualization of orbital inflammation exhibited the highest significance level to discriminate between GO and Ctrl mice (P=0.006, Fig. 2), and showed the best correlation with the clinical score (P=0.0007). Infiltration patterns of inflammatory cells were corroborated by histology and examination of excised tissue after fixation: 3D high resolution 1H/19F MRI (isotropic voxel size 40 µm) confirmed the prevalent PFC deposition in the peri-orbital tissue of GO mice.

Conclusions

In the present study, we demonstrate that multimodal 1H/19F MRI permits a comprehensive analysis of several hallmarks of GO pathology with simultaneous assessment of orbital immune cell infiltration, development of edema, alterations in the extracellular matrix, and quantification of fat and muscle dimensions. In particular, 19F MRI allowed a sensitive demarcation of inflammatory foci in the orbit, even when other markers indicated only weak or no signs of tissue alterations.

References

  1. Bahn RS. Graves’ ophthalmopathy. N Engl J Med. 2010;362(8):726–738.
  2. Shan SJC, Douglas RS. The pathophysiology of thyroid eye disease. J Neuroophthalmol. 2014;34(2):177–185.
  3. Moshkelgosha S, So P-W, Deasy N, Diaz-Cano S, Banga JP. Cutting edge: retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves’ orbitopathy induced by thyrotropin receptor plasmid in vivo electroporation. Endocrinology. 2013;154(9):3008–3015.
  4. Berchner-Pfannschmidt U, Moshkelgosha S, Diaz-Cano S, et al. Comparative assessment of female mouse model of Graves’ orbitopathy under different environments, accompanied by proinflammatory cytokine and T-cell responses to thyrotropin hormone receptor antigen. Endocrinology. 2016;157(4):1673–1682.

Acknowledgement

We gratefully thank Bodo Steckel (Heinrich Heine University), Mareike Horstmann (University of Duisburg-Essen, Germany) and Alexandra Brenzel (Imaging Center Essen IMCES, University Duisburg-Essen) for excellent technical assistance. The study was supported by Deutsche Forschungsgemeinschaft grants BE 3177/2-1 (UBP), FL 303/6-1 (UF), TE 1209/1-1 (ST), and by funding from the Stiftung Universitätsmedizin Essen (AE) and Interne Forschungsförderung Essen (AS).

Figure 1: 1H/19F MRI and T2 maps in GO and Ctrl mice
(A) 1H/19F MRI (left) reveals marked 19F signal intensity in peri-orbital regions of GO mice (top) while in corresponding T2 maps (right) only minor abnormalities compared to Ctrl mice (bottom) are visible. (B) Examples of merged 1H/19F MRIs with detectable 19F patterns in retro-orbital regions which was associated with an increase of T2 above 60 ms in the surrounding ocular gland (~57 ms in Ctrl)

Figure 2: Quantification of MRI data for GO and Ctrl mice
(A) 19F integral in vicinity of the orbit, (B) T2 of the Harderian gland, (C) CEST contrast in the orbit, and (D) fat volume surrounding the optic nerve; n = 17/12 for GO/Ctrl mice in A+D, 15/11 in C, and 11/9 in B; *P<0.05, **P<0.01; CI = confidence interval.

9:10 AM
PS-23-4 — Multicolor 19F-MRI for in vivo Imaging of immune cells activity (#400)

C. Chirizzi1, D. De Battista1, R. Furlan1, P. Metrangolo2, G. Comi1, F. Baldelli Bombelli2, L. Chaabane1

1 Ospedale San Raffaele, Institute of Experimental Neurology (INSPE), Milan, Italy, Italy
2 Politecnico di Milano, SupraBioNano Lab, Department of Chemistry, Materials, and Chemical Engineering “Giulio Natta”, Milan, Italy, Italy

Introduction

Inflammation is a dynamic process associated with several neurologic disorders and characterized by the involvement of different immune cells. MRI is a suitable imaging tool for in vivo investigation and demonstrated great potentials to image inflammation with the use of fluorine probes (1-4). In the present work, we extended 19F-MRI towards multicolor imaging using two different perfluorocarbons (PFCs) with two distinct resonance frequencies and a high fluorine payload. Furthermore, multicolor 19F-MRI was used to monitor in vivo the immune system activation in mice model of multiple sclerosis

Methods

Using two different PFCs, 19F-droplets were produced including fluorescent dyes for cytometric analysis (FCM). All in vivo experiments were performed at 7T in healthy and Experimental Autoimmune Encephalomyelitis (EAE) C57BL/6 mice. MRI at different fluorine frequencies of the PFCs and of hydrogen was acquired using a 3D-FSE sequence in all animals at different days post immunization (dpi). Before any signs of disease (4dpi), mice received a first dose of PFC and at disease onset (11dpi) the second PFC was administered. At disease peak (18dpi), animals received an additional dose of the first PFC. Mice were followed by MRI up to 22dpi. At the end of the study several organs were collected and processed for FCM to identify 19F-labeled cells.

Results/Discussion

Both 19F-droplets ( diameter = 180-200 nm) were clearly differentiated in vivo thanks to their single dominant resonance with 19 ppm of chemical shift allowing multicolor 19F-MRI without overlaps or artefacts. Fluorine signal was observed throughout the brain or localized in specific areas at the acute phase (Fig. 1a-b). Indeed, differences of 19F-signal were found in relation to the phases of EAE disease compared to healthy mice (Fig. 1c).

MRI observations were confirmed by FCM analysis of the brain where 10 to 20 % of cells were found positive to the second 19F-droplets in EAE mice while no cells were positive in healthy mice. These 19F-loaded cells were identified as monocytes, dendritic cells and microglia. In lymphoid organs, FCM analysis confirmed the presence of an inflammatory process with a high increase of monocytes compared to healthy mice (2 to 8 fold increase) and also a high 19F-uptake (2 to 5 fold increased versus healthy controls) with the second PFC at EAE onset

Conclusions

The present work demonstrates the potentiality of multicolor 19F-MRI with gain in sensitivity to track immune cells activation during different phase of disease progression in a model of multiple sclerosis. Interestingly, the proposed multicolor imaging method could be used for longitudinal in vivo investigations of cellular events which is of particular interest for the investigations of new therapies.

References

1. Temme S and al., 19F magnetic resonance imaging of endogenous macrophages in inflammation. WIREs Nanomed Nanobiotechnol; 2012.

2. Weise G et al., In vivo imaging of inflammation in the peripheral nervous system by 19F MRI. Experimental Neurology; 2011.

3. Caruthers SD et al., In vitro demonstration using 19F magnetic resonance to augment molecular imaging with paramagnetic perfluorocarbon nanoparticles at 1.5 Tesla. Invest Radiol; 2006.

4. Partlow KC et al. 19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons. FASEB J; 2007.

Acknowledgement

This study is supported by the Italian Multiple Sclerosis Foundation (FISM)

 

Figure 1. In vivo 19F-MRI.

Combined MR images acquired at different frequency (gray: 1H; green; PFC1; red: PFC2) in two EAE (a) and in a healthy mouse (b) at different phases of disease. (c). Comparison of signal from both fluorine tracers quantified in brain of healthy and EAE mice.

 

9:20 AM
PS-23-5 — Targeting activated synovial fibroblasts using photodynamic therapy in experimental arthritis (#274)

D. N. Dorst1, M. Rijpkema1, M. Buitinga2, M. Brom1, D. L. Bos1, A. Freimoser3, C. Klein3, B. Walgreen4, P. M. van der Kraan4, M. I. Koenders4, M. Gotthardt1

1 Radboud university medical center, Radiology and nuclear medicine, Nijmegen, Netherlands
2 KU Leuven, Leuven, Belgium
3 Roche Pharmaceutical Research and Early Development, Innovation center Zurich, Schlieren, Switzerland
4 Radboud umc, Experimental rheumatology, Nijmegen, Netherlands

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting synovial joints. In RA activated synovial fibroblasts (SF) are actively contributing to this inflammation. SF are characterized by the expression of Fibroblast Activation Protein (FAP). Here, we investigated the potential of photodynamic therapy (PDT) targeting FAP to selectively induce cell death in these cells. In targeted PDT, a light-sensitive molecule is delivered to a target cell and activated with light of a specific wavelength. This causes cell death through the production of reactive oxygen species (ROS).

Methods

Both ITC-DTPA and the photosensitizer IRDye700DX were conjugated to 28H1 (28H1-700DX). In vitro PDT assays were performed with 3T3 fibroblasts stably transfected with FAP. 3T3-FAP cells were incubated with 28H1-700DX or a control for 4 hours, and exposed to varying 690 nm light exposures. Subsequently, cell viability was measured using the CellTiter-Glo assay. For in vivo biodistribution, 5 days after onset of antigen-induced arthritis (AIA) C57Bl6 mice were injected with 28H1 labelled with 111-In, with or without 700DX. Additionally, micro-SPECT/CT and fluorescence imaging were performed. For PDT, arthritic mice were injected at day 5 of AIA with 28H1-700DX or PBS and exposed to 50 or 90 J/cm2 light 24 hours post injection. Joints were isolated at day 10 for histological analysis.

Results/Discussion

To assess PDT efficacy, we applied 13.7 J/cm2 light exposure to 3T3-FAP cells incubated with 6.67 pM 28H1-700DX, which significantly reduced cell viability (89.27%+/-2.48 compared to control (p<0.001)). No cell death was observed with the control 700DX-conjugate or with 3T3 fibroblasts not expressing FAP.

Conjugating the anti-FAP antibody to 700DX changed the in vivo biodistribution of the antibody, with a higher accumulation in the liver (27.06±0.95 %ID/g vs. 6.08±0.42 %ID/g with control (p<0.001)) and lower blood levels (5.32±0.36 %ID/g vs. 12.72±0.80 %ID/g with control (p<0.001)). Accumulation in the arthritic joints was not significantly different. The fluorescent signal still visible in the inflamed knee joint 24h post injection indicates that there is intact tracer accumulation, as previous experiments indicated that the fluorescent signal and the ability of 700DX to produce ROS are linearly correlated. Histological analysis of the PDT-treated mouse knee joints is ongoing.

Conclusions

We have demonstrated fibroblast-specific cell death using 700DX-conjugated 28H1 PDT, indicating FAP-based targeted PDT as a promising new tool in treating RA. Furthermore, we demonstrated that adding 700DX results in faster liver clearance of the antibody conjugate, but does not affect uptake in the inflamed knee joint. Visualization of fluorescent signal from the 28H1-700DX construct indicates that  the photosensitizer is intact and capable of producing ROS at the site of inflammation. Future research will further elucidate the applicability of our conjugate for PDT in animal models of RA.

Increased uptake of the 28H1-700DX tracer in an inflamed joint visualized by fluorescent signal.
Fluorescence measured at 700nm using the IVIS® Spectrum in vivo imaging system in paws of a mouse with antigen induced arthritis 24 hours post injection of the 28H1-700DX construct. Note the higher fluorescent signal in the inflamed knee joint (left) compared to the control knee joint (right).

9:30 AM
PS-23-6 — Molecular imaging of elastin and collagen deposition in renal fibrosis (#235)

M. Baues1, Q. Sun2, 3, B. Klinkhammer2, 3, J. Ehling1, 2, P. Boor2, 3, F. Kiessling1, T. Lammers1

1 Uniclinic RWTH Aachen, Institute for Experimental Molecular Imaging, Aachen, Germany
2 Uniclinic RWTH Aachen, Institute of Pathology, Aachen, Germany
3 Uniclinic RWTH Aachen, Department of Nephrology and Immunology, Aachen, Germany

Introduction

Millions of patients suffer from chronical kidney disease (CKD). The best predictor for CKD progression is fibrosis assessment. Biopsies have remained the gold standard, even if highly invasive and moderately informative. Fibrosis refers to deposition of extracellular matrix (ECM) components, such as elastin and collagen. Fibrogenesis is characterized by disease state-specific ECM compositions. Based on these notions, we employed the molecular imaging agents: ESMA [1] and CNA35 [2], which specifically target elastin and collagen, for the diagnosis and staging of renal fibrosis (Fig.1A).

Methods

The agents were evaluated using magnetic resonance imaging (MRI) and hybrid computed tomography - fluorescence molecular tomography (CT-FMT). This was done in different mouse models: unilateral ureteral obstruction (UUO), ischemia/reperfusion injury (I/R) and adenine-containing diet (Fig.1B [3]). Findings were verified by laser ablation inductively coupled mass spectrometry (LA-ICP-MS) and two-photon laser scanning microscopy (TPLSM).

Results/Discussion

Western blot and immunohistochemistry confirmed gradual deposition of elastin during disease progression (Fig.2A). For the elastin-specific contrast agent ESMA, normalized signal intensities in T1-weighted MRI as well as T1 relaxometry acquisition with echo time analysis, revealed significant differences in fibrotic vs. healthy kidneys 24 h after i.v. injection, which was clearly stronger than the difference observed for unspecific Gd-DTPA (Fig.2B). The metal quantification via LA-ICP-MS reflected the MRI signal intensities and visualized highest Gd concentrations in the fibrotic kidneys injected with ESMA (Fig. 2C). For the Cy7- and Alexa488-labeled collagen-specific agent CNA35 CT-FMT imaging revealed high accumulation in fibrotic (F) kidneys while only moderate amounts accumulate in the contralateral healthy (H) kidney (Fig.2D-E). TPLSM confirmed these findings, showing highly specific binding of CNA35 to perivascular collagen fibers at 4 and 24 h after i.v. injection (Fig.2F).

Conclusions

We established probes and protocols for molecular imaging of CKD. Our findings lay the basis for the establishment of elastin- and collagen-based imaging biomarkers that hold potential for non-invasive, quantitative and longitudinal analysis of renal fibrosis.

References

[1] Makowski M et al. Nat Med 2011, 17:383-388

[2] Sanders H et al. Chem Commun 2011, 47:1503-1505

[3] Ehling J et al. J Am Soc Nephrol 2016, 27:520-532

Acknowledgement

Supported by ERC (StG309495-NeoNaNo), DFG (SFB/TRR57) and START (124/14, 152/12).

Figure 1
Imaging biomarkers and mouse models for diagnosis and staging of kidney fibrosis.

Figure 2
Molecular imaging of elastin with ESMA and collagen with CNA35 in kidney fibrosis.

9:40 AM
PS-23-7 — Tracking the Delivery and Assessment of the efficacy of Fluorescent Glucocorticoid Hybrid Nanoparticles in Experimental Mouse Models of Inflammation (#413)

J. Napp1, 2, 3, A. M. Markus1, J. G. Heck4, C. Dullin2, C. Feldmann4, F. Alves1, 2, 3

1 MPI of Experimental Medicine, Translational Molecular Imaging Group, Göttingen, Germany
2 University Medical Centre Goettingen (UMG), Institute of Diagnostic and Interventional Radiology, Göttingen, Germany
3 University Medical Centre Goettingen (UMG), Clinic of Hematology and Medical Oncology, Göttingen, Germany
4 Karlsruhe Institute of Technology, Institute of Inorganic Chemistry, Karlsruhe, Germany

Introduction

Effective treatment of a disease obviously depends on the delivery of the therapeutic agents to the site of action. Therefore, the aim of the study was to evaluate the monitoring of the in vitro and in vivo distribution, delivery and uptake and the assessment of the efficacy of inorganic-organic hybrid nanoparticles (IOH-NPs) composed of an anti-inflammatory glucocorticoid, betamethasonphosphate (BMP) and a near-infrared fluorescent (NIRF) dye DY-647 (BMP-IOH-NPs)1.

Methods

BMP-IOH-NPs uptake by MH-S macrophages was analyzed with NIRF- and electron-microscopy. Lipopolysaccharide (LPS)-stimulated cells were treated for 48h with BMP-IOH-NPs (1x10-5-1x10-9 M), BMP or dexamethasone (DM) and drug efficacy was assessed by measurements of interleukin 6 (IL-6). Mice with Zymosan-A- induced paw inflammation were intraperitoneally treated with BMP-IOH-NPs (10 mg/kg) and mice with OVA-induced allergic airway inflammation (AAI)2 were treated intranasally with BMP-IOH-NPs, BMP or DM (2.5 mg/kg). Delivery was monitored by in vivo optical imaging (OI). Efficacy was assessed in vivo via paw volume measurement with µCT and ex vivo via paw weight quantification or in the AAI model by cell counts in bronchio-alveolar lavage (BAL) fluid histology and x-ray based lung-function³.

Results/Discussion

In vitro, an uptake of BMP-IOH-NPs by MH-S cells was observed during the first 10 min of incubation, with NPs load increasing over time. The anti-inflammatory effect of BMP-IOH-NPs on MH-S cells was dose dependent and comparable to that of DM and BMP (Fig. 1).

In vivo, the Zymosan-A injections induced inflammatory paw swelling (222 mm³ and 0.229 g in controls) was significantly reduced in mice treated with BMP-IOH-NPs (180 mm³ and 0.186 g) (Fig. 2A). OI showed accumulation of BMP-IOH-NPs within the lung of AAI mice 1h after instillation, detectable for at least 4h in vivo (Fig. 2B). BMP-IOH-NPs were preferentially taken up by peribronchial and alveolar M2 macrophages which were CD68+CD11c+ECF-L+MHCII- and podoplanin-proS-PClow. Treatment of AAI mice with BMP-IOH-NPs, but not with BMP, significantly reduced the number of eosinophils in BALs and immune cell infiltration in lungs, with an efficacy higher than the one of the golden standards, DM and BMP (Fig. 2C).

Conclusions

We show that glucocorticoids such as BMP applied in form of IOH-NPs allow efficient treatment of inflammatory disease by release of the active drug and simultaneous non-invasive monitoring of the delivery of the NPs by OI.

References

1Heck et al., J Am Chem Soc. 2015; 137:7329-36

2Markus et al., ACS Nano. 2015; 9:11642-57

3Dullin et al., J Synchrotron Radiat. 2015; 22:1106-11

Acknowledgement

Technical assistance: B. Heidrich, S. Garbode, B. Jeep, S. Wolfgramm

Fig. 1: In vitro uptake and efficacy of BMP-IOH NPs.

Fig. 2: Assessment of the in vivo efficacy and monitoring the distribution of BMP-IOH-NPs

9:50 AM
PS-23-8 — Evaluation of different treatments effectiveness preventing the development of pulmonary fibrosis with Micro-CT imaging. (#448)

J. Sadoine1, J. Avouac2, 3, M. Elhai2, 3, A. Cauvet2, S. Pezet2, Y. Allanore2, 3

1 Université Paris Descartes, EA2496 & Plateforme Imageries du vivant, Montrouge, France
2 Université Paris Descartes, INSERM U1016 CNRS UMR8104, Paris, France
3 Université Paris Descartes, Service de Rhumatologie A, Paris, France

Introduction

The X-ray micro-tomography (micro-CT) allows acquisition of structural images in order to obtain, with high resolutions, spatial representations of scanned (bio)-materials. Recent technical advances provide fast image acquisition allowing a long-term follow up of pathologies in small animal models. This technique is very sensitive and allows the evaluation of lung pathology and particularly lung fibrosis. Indeed, in the present study we describe the development of an image analysis method to explore the effect of three treatments against lung fibrosis associated with preclinical X-ray imaging.

Methods

We work with the Fra-2 mouse model, which is characterized by the spontaneous development of a progressive non-specific interstitial pneumonia. Tridimensional images were acquired with Micro-CT. Mice were anesthetized and respiratory frequency was recorded during the acquisition and only the inhalation was reconstructed. Fibrosing alveolitis was evaluated using micro-CT 2 d before mice were sacrified. Means of lung density of both groups were determined by evaluating all CT scans acquired from the apices to the bases of the lungs. Furthermore, the volume of functional lung parenchyma corresponding to functional respiratory capacity was drawn manually, excluding fibrotic area and vessels. The efficacy of 3 treatments (OX40L, IVA337 & MMP10) was evaluated on these parameters.

Results/Discussion

In a first study, the efficacy of a targeted therapy against OX40L was explored. Micro-CT revealed higher lung density consistent with fibrosing alveolitis in Fra-2 mice treated with control mAb than in C57/BL6 mice (P = 0.007); this lung density was decreased significantly in Fra2 mice treated with the anti-OX40L mAb (P = 0.004). Fra-2 mice had a functional residual capacity equal to 44.7% of lung volume versus 77.4% in control mice (P < 0.001). Fra-2 mice receiving anti-OX40L mAb had a functional residual capacity of 71.4% of lung volume. After, we tested the efficacy of IVA337 in the Fra-2 mouse model, Fra-2 mice treated with IVA337 100 mg/kg displayed a significant 21% decrease in lung density as compared with Fra-2 mice receiving the vehicle (p<0.05). Consistent with this finding, functional residual capacity increased significantly by 30% in mice treated with IVA337 100 mg/kg (p<0.05). Moreover, MMP10 inhibition failed to improve significantly fibrosing alveolitis in Fra-2 model.

Conclusions

Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. We demonstrate that treatment with 100 mg/kg IVA337 prevents also lung fibrosis in the Fra-2 mouse model. The present work shows how X-ray micro-CT imaging and image segmentation/quantification developped for the 3 differents study projects are suitable technique and method to quantify specific parameters (as tissue density or functionnal residual capacity) associated to lung pathologies as pulmonary fibrosis.

References

[1] Elhai M, Avouac J, Hoffmann-Vold AM, Ruzehaji N, Amiar O, Ruiz B, Brahiti H, Ponsoye M, Fréchet M, Burgevin A, Pezet S, Sadoine J, Guilbert T, Nicco C, Akiba H, Heissmeyer V, Subramaniam A, Resnick R, Molberg Ø, Kahan A, Chiocchia G, Allanore Y.OX40L blockade protects against inflammation-driven fibrosis.  Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):E3901-10.

[2] Avouac J, Konstantinova I, Guignabert C, Pezet S, Sadoine J, Guilbert T, Cauvet A, Tu L, Luccarini JM, Junien JL, Broqua P, Allanore Y. Pan-PPAR agonist IVA337 is effective in experimental lung fibrosis and pulmonary hypertension.  Ann Rheum Dis. 2017 Aug 11. pii: annrheumdis-2016-210821. doi: 10.1136/annrheumdis-2016-210821.

[3] Avouac J, Guignabert C, Hoffmann-Vold AM, Ruiz B, Dorfmuller P, Pezet S, Amar O, Tu L, Van Wassenhove J, Sadoine J, Launay D, Elhai M, Cauvet A, Subramaniam A, Resnick R, Hachulla E, Molberg Ø, Kahan A, Humbert M, Allanore Y. Stromelysin-2 (MMP-10), a novel mediator of vascular remodeling underlying pulmonary hypertension associated with systemic sclerosis.  Arthritis Rheumatol. 2017 Aug 13. doi: 10.1002/art.40229.

Acknowledgement

We thank the following individuals for excellent technical assistance: ..... and Prof. Catherine Chaussain & Dr. Lotfi Slimani (Dental School of the Paris Descartes University, EA 2496 & Life Imaging Facility of Paris Descartes University (Plateforme Imageries du Vivant - PIV)).

Inhibition of OX40L prevents the development of fibrosing alveolitis: CT-scan data
A, Fibrosing alveolitis was observed in Fra-2 mice receiving control IgG. B, Representative images of functional residual capacity (in blue) in different mice; bronchi are in white. C, Increasing lung density in Fra-2 mice treated with control IgG compared with Fra-2 mice treated with anti-OX40L mAb or Control mice. D, Residual lung volume.**P < 0.01; ***P < 0.001; two-sided Mann–Whitney test.

IVA-337 100 mg/kg prevents lung fibrosis in Fra-2 transgenic mice: Evaluation by CT-scan.

A, Representative pictures of micro-computed tomography. B, Decreased lung density at micro-computed tomography in Fra-2 transgenic mice treated with IVA337 100 mg/kg compare to vehicle-treated mice. C, Representative pictures of functional residual capacity in different mice in blue. D, Reduced residual lung volume. Values the mean ± SEM. Statistics: One way ANOVA test. * p<0.05

6:15 PM
emptyVal-1 — Introductory Talk by Greetje Vande Velde - Leuven, Belgium

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

6:35 PM
PS-11-2 — Elucidation of oropharyngeal and tracheal human papillomavirus infection by in vivo bioluminescence imaging (#276)

S. Schelhaas1, 3, M. Becker2, 3, S. Eligehausen1, M. Schelhaas2, 3

1 Westphalian Wilhelms-University Münster, European Institute for Molecular Imaging, Münster, Germany
2 Westphalian Wilhelms-University Münster, Institute of Cellular Virology, Münster, Germany
3 Westphalian Wilhelms-University Münster, Cluster of Excellence 'Cells in Motion', Münster, Germany

Introduction

Human papillomaviruses (HPV) have not only been recognized as the etiological cause of cervical cancer, but they are also associated with oropharyngeal cancer and recurrent juvenile papillomatosis of the upper respiratory tract. Incidents of oropharyngeal cancers caused by HPV are notably increasing, whereas the rare juvenile papillomatosis is complicated to treat and may result in airway obstruction and death due to recurrence. To date, no animal model exists to evaluate HPV infections in these tissues in vivo

Methods

We employed HPV type 16 (HPV16) pseudovirions, which are capable of infecting cells and tissues, but are abrogated in completion of the viral life cycle and cannot cause pathogenicity or carcinogenesis. Instead, the particles harbor pseudogenomes that allow reporter gene expression, such as the firefly luciferase (lucf). We infected isoflurane-narcotized BalbC mice with HPV-lucf virions via the nose or through a tracheal tubus. Bioluminescence imaging (BLI) was performed using an IVIS Spectrum. Tissue was collected to verify the presence of mCherry-expressing pseudovirions by immunohistochemistry.

Results/Discussion

HPV16-lucf infected cells expressed firefly luciferase, as has been verified by BLI in cell culture experiments. After nasal virus application, infection of the snout was detected by in vivo BLI, whereas tracheal infection was observed after tracheal virus application only. Interestingly, with both infection methods, bioluminescence signal in the lung was observed. The peak light emission occurred two days after infection. At this time point, mCherry expressing HPV pseudovirions were detected in the lung and in the lacrimal glands after nasal infection. 

Conclusions

We demonstrate that HPV pseudovirions are capable of infecting respiratory tissues of mice. In the future, this will allow in depth preclinical work on HPV infections in the oropharynx and in the trachea. HPV infection of the lung was surprising, as epidemiologically no pathogenic HPV infections of this organ have been reported. This indicates that HPV infections of the lung may be abortive in nature. Interestingly, as infectivity of HPV pseudovirions in the lung appears high, it may be worthwhile to explore HPV16 as vehicle for novel gene therapies in the lung.

Acknowledgement

This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC1003 – CiM), University of Münster, Germany and within the Infect-ERA initiative by funding from the Federal Ministry for Education and Research (BMBF, 031L0095A).

6:45 PM
PS-11-3 — In vivo imaging of B. pertussis infection and interactions in the airways of non-human primates in a model of whooping cough (#41)

T. Naninck1, C. Mayet1, V. Contreras1, S. Langlois1, L. Bossevot1, L. Coutte2, C. Locht2, N. Klimova1, 3, P. Sebo3, R. Le Grand1, C. Chapon1

1 CEA, IDMIT, Fontenay aux roses, France
2 Institut Pasteur de Lille, INSERM U1019, Lille, France
3 Institute of Microbiology, CASL, Prague, Czech Republic

Introduction

Whooping cough, due to Bordetella pertussis infection, is today a public health problem. Recent studies on non-human primates (NHP) indicate that acellular vaccines protect from symptoms but not from infection, confirming clinical data1,2. To develop more effective vaccines, a better understanding of the bacterial interactions with the host is needed. We thus developed fluorescence imaging techniques including in vivo Fibered Confocal Fluorescence Microscopy (FCFM)3 to explore the respiratory tract and assess interactions between B. pertussis and antigen presenting cells (APCs) in baboons.

Methods

FCFM in airways was first evaluated ex vivo on NHP explants. A solution of anti-HLA-DR mouse antibody labelled with AlexaFluor647 and a solution of acriflavine were dropped topically in the bronchus of a lung lobe and in a tracheal ring to label specifically the APCs and the other cellular structures, respectively. For in vivo studies, GFP-expressing B. pertussis was inoculated by intranasal and intra-tracheal routes in young baboons. Besides, monoclonal anti-HLA-DR antibody, labelled with AF647, was administered by topical application in the trachea to specifically target and label APCs. FCFM, coupled with bronchoscopy, was performed in the lower respiratory tract at day -2, 2, 7, 14 and 21 post-infection. Validation by immunohistofluorescence was then performed on tissues post-mortem. 

Results/Discussion

Animals infected with a GFP-expressing B. pertussis B1917 strain developed the classical clinical symptoms for whooping cough as previously described in baboons infected with wild-type strains4,2. We were also able to specifically label and detect cells of interest like APCs in the airways of NHP ex vivo using FCFM (Figure 1). Furthermore, in vivo FCFM coupled with bronchoscopy allowed us to detect bacterial and APC localizations and interactions in the lower respiratory tract of young baboons after B. pertussis-GFP infection in baboons (Figure 2 A-B). Ex vivo analyses also confirmed the interactions between B. pertussis and APCs in lungs (Figure 2 C) and allowed the detection of the bacteria 27 days post-infection in bronchi (Figure 2 D).

 

Conclusions

These findings confirm previous published in vitro data about strong interactions between Bordetella pertussis and APCs5. This approach using fluorescence imaging will then be a useful tool to describe the mechanisms of action of the bacteria during infection to develop more effective vaccines against pertussis. Moreover, this imaging protocol may also be implemented to study many diverse respiratory infectious diseases like tuberculosis or influenza for instance.

References

1.         Klein, N. P., Bartlett, J., Rowhani-Rahbar, A., Fireman, B. & Baxter, R. Waning Protection after Fifth Dose of Acellular Pertussis Vaccine in Children. N. Engl. J. Med. 367, 1012–1019 (2012).

2.         Warfel, J. M., Zimmerman, L. I. & Merkel, T. J. Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model. Proc. Natl. Acad. Sci. U. S. A. 111, 787–792 (2014).

3.         Thiberville, L. et al. In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy. Am. J. Respir. Crit. Care Med. 175, 22–31 (2007).

4.         Warfel, J. M., Beren, J., Kelly, V. K., Lee, G. & Merkel, T. J. Nonhuman Primate Model of Pertussis. Infect. Immun. 80, 1530–1536 (2012).

5.         Lamberti, Y., Gorgojo, J., Massillo, C. & Rodriguez, M. E. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival. Pathog. Dis. 69, 194–204 (2013).

Ex vivo imaging of airways by Fibered Confocal Fluorescence Microscopy

Fig. 1: Imaging of airways by Fibered Confocal Fluorescence Microscopy (FCFM). Ex vivo FCFM pictures on tracheal (A,B) and lung (C,D) explants. Prior to ex vivo imaging, tissues were stained with acriflavine (green) and with either anti-human HLA-DR AF647 (A,C) or isotypic control -IgG2a AF647 antibodies- (B,D) (red).

In vivo imaging with FCFM coupled with bronchoscopy in B. pertussis B1917-GFP infected baboons
Fig.2: In vivo FCFM (A) coupled with bronchoscopy (B) of the trachea of baboons 2 days post-infection with B.pertussis B1917-GFP.  (APCs in red; bacteria in green). Ex vivo confocal microscopy in lung tissue of (C) the interactions between APCs (CD163+, in red) and GFP-B.pertussis (green) after lung-bacteria co-culture and (D) the B.pertussis bacteria detected post-mortem with anti-LOS-A antibody.

6:55 PM
PS-11-4 — Multimodal imaging allows non-invasive assessment of the progression of lung and brain infection in mouse models of Cryptococcus neoformans infection (#231)

L. Vanherp1, A. Ristani1, J. Poelmans1, A. Hillen1, M. Brock2, K. Lagrou3, G. Janbon4, U. Himmelreich1, G. Vande Velde1

1 KU Leuven, Imaging and Pathology, Biomedical MRI, Leuven, Belgium
2 University of Nottingham, School of Life Sciences, Fungal Biology Group, Nottingham, United Kingdom
3 KU Leuven, Microbiology and Immunology, Clinical Bacteriology and Microbiology, Leuven, Belgium
4 Pasteur Institute, Mycology, RNA Biology of Fungal Pathogens, Paris, France

Introduction

Fungal infections caused by Cryptococcus species typically start in the lungs but can disseminate to the brain via the bloodstream. Preclinical studies assessing disease progression and dissemination are often limited to end-point analysis of the fungal burden or histology, which precludes longitudinal studies. By combining lung computed tomography (CT) and brain magnetic resonance imaging (MRI) with bioluminescence imaging (BLI), our aim was to gain insights in the progression of both lung and brain infection in different mouse models of C. neoformans infection.

Methods

As model of disseminated disease, Balb/C mice (n=3) were infected by intravenous injection of 50 000 cells of a newly engineered luciferase-expressing C. neoformans strain (NE1270). Mice were scanned with BLI and brain MRI (9.4T or 7T, Bruker Biospec) on day 3, 5 and 7. To study lung infection and a potential progression to brain infection, mice were infected with 500 or 50 000 bioluminescent C. neoformans cells via intranasal or oropharyngeal route (n=3-6 per group). These mice were scanned using BLI (1-2x/week), lung CT (1/week) and brain MRI (1/week). After sacrificing, ex vivo BLI was performed and the fungal burden in the organs was quantified (colony-forming unit counting). Additional animals were sacrificed at intermediate time points to correlate imaging readouts with fungal load.

Results/Discussion

In the model of disseminated disease, brain infection could be detected with BLI on day 3, while MRI only showed brain lesions on day 5. In addition, bioluminescent signals could be observed in other organs such as the spleen and kidneys. In the intranasal model, the progression of lung disease could be followed by BLI, while CT confirmed the presence of lung lesions (Fig. 1). Concurrently, we observed an increase in the BLI signal from the nose region. This complicated further assessment and quantification of brain involvement, as MRI could detect brain lesions in some animals. Mice infected via the oropharyngeal route did not present with this nose signal. Currently, we are investigating whether BLI can be used to further define the timeframe of lung to brain transition in this model. Quantification of the BLI signal and lesion volumes (CT/MRI) in the lungs (intranasal model) or the brain (intravenous model) showed a strong correlation with the fungal burden in the respective organs.

Conclusions

The combination of anatomical imaging techniques (CT/MRI) with BLI allowed monitoring of the progression of lung and brain infection. Imaging readouts were found to correlate strongly with the fungal burden in these organs, indicating that this approach is a non-invasive alternative that can lead to longitudinal insights in the evolution of the fungal load. Future studies will focus on the application of these techniques to further narrow down the timeframe of blood-brain barrier crossing in cryptococcosis models and to non-invasively assess therapeutic approaches.

Acknowledgement

This work was supported by funding provided by the European ERA-NET project CryptoVIEW.

Multimodal imaging of pulmonary Cryptococcus infection
BLI and CT allowed non-invasive assessment of the progression of lung disease in the intranasal model after infection with 50 000 or 500 bioluminescent Cryptococcus cells. A) BLI showed a progressing lung disease and signal from the nasal region. B) CT showed the presence and increase of hyperintense lesions in the infected lungs.

7:05 PM
PS-11-5 — Dynamic assessment of immune cell recruitment in pulmonary aspergillosis models using perfluorocarbon particles and 19F MRI (#142)

S. Saini1, J. Poelmans1, S. Liang1, 2, R. Verbeke3, B. Attili4, G. Vande Velde1, H. Korf5, I. Lentacker3, S. de Smedt3, K. Lagrou6, U. Himmelreich1

1 KU Leuven, Biomedical MRI unit, Leuven, Belgium
2 Philips Research China, Shanghai, China
3 University of Gent, General Biochemistry and Physical Pharmacy, Gent, Belgium
4 KU Leuven, Radiopharmaceutical Research laboratory, Leuven, Belgium
5 KU Leuven, Hepatology laboratory, Leuven, Belgium
6 KU Leuven, Clinical Bacteriology and Mycology, Department of Microbiology and Immunology, Leuven, Belgium

Introduction

Depending on the type of immunosuppression, progression of pulmonary fungal infection can lead to invasive pulmonary aspergillosis (IPA) in immunocompromised subjects, recruiting certain types of innate immune cells to lungs. It is crucial to understand particularly the interplay between the host’s immune system & pathogen. We aimed to understand the dynamics of disease progression & migration of inflammatory immune cells to the lungs in A. fumigatus infection mouse models using two clinically applied immunosuppressive (IS) drugs with the help of fluorine magnetic resonance imaging (19F MRI).

Methods

For immunosuppression in Balb/c mice, hydrocortisone acetate (9mg/mouse) was injected s.c. in the HCA group (n=10) on d-3/d -1. Alternatively, cyclophosphamide (200mg/mouse) was i.p. injected on d-4/d-1 (CY group, n=10) prior to infection. On d0, A. fumigatus (fluc+) infection was induced by intranasal administration of 1x106 (HCA group) or 5x105 spores (CY group), retrospectively [1]. Infected immunocompetent (I-IC group, n=4) received 1x106 spores. Non-infected IC mice (n=3) served as control. 19F MRI was performed with a purpose-built double-tuned coil 1H/19F coil commencing 1h post IV injection (d0/d1) of PFCE particles [2] containing fluorosurfactant Zonyl® FSP (ZPFCE) using Bruker 9.4T scanner. In vivo CT was performed on d1/d3. Ex vivo BLI was performed on d3 on the excised lungs.

Results/Discussion

The HCA group showed higher pulmonary 19F MRI signal intensity compared to the CY group, corresponding to higher in vivo ZPFCE labeled immune cell infiltration post fungal infection. We also observed transient 19F signal in the lungs of I-IC group, which was dissolved after d2 (fig 1). No detectable 19F MRI signal was observed in the lungs of N-IC group. 19F hot spots were also identified in the ‘lymph node’ region of HCA group and were not found in I-IC, CY or control groups. We noticed differences in ex vivo BLI in two different immunocompromised groups where a significantly larger increase in BLI signal intensity from lungs was observed in CY group when compared to the HCA group. No BLI signal was found in I-IC and N-IC mice groups indicating clearance of fungal burden from the lungs by the immune system. Quantitative CT imaging data confirmed lesion development in both HCA & CY groups but not in control group indicating differences in terms of lesion volume from whole lungs(fig 2).

Conclusions

We showed distinctive interactions between host & pathogen in IPA models by tracking pulmonary recruitment of immune cells (19F MRI), which correlated with fungal load of A. fumigatus (CT, BLI & CFUs). Our findings indicate that the HCA group developed excessive inflammation & less fungal infection in contrast to the CY group where infection predominates [3], based on peculiar immune environment created by different clinical IS drugs. This indicates the potential of 19F MRI for providing sensitive and quantitative data on immune cells infiltration in infectious diseases models in vivo.

References

[1] Poelmans J, Hillen A, Vanherp L, Govaerts K, Maertens J, Dresselaers T, Himmelreich U, Lagrou K, Vande Velde G. Longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging. Laboratory Investigation (2016) 96, 692–704.

[2] Dewitte H, Geers B, Liang S, Himmelreich U, Demeester J, De Smedt SC, Lentacker I. Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and 19F-MRI tracking of dendritic cells. Journal of Controlled Release 169 (2013) 141–149.

[3] Helioswilton SC, Tonani L, Ribeiro Barros Cardoso C and Regina Von Zeska Kress M. The Immune Interplay between the Host and the Pathogen in Aspergillus fumigatus Lung Infection. BioMed Research International (2013) 693023, 14.

Acknowledgement

The research leading to these results was supported by Marie Curie’s ITN BetaTrain project and was partly funded by FWO.

Fig. 1: In vivo longitudinal 19F MRI follow-up of immune cell in A. fumigatus model
19F MRI signal is shown as hot spots and was overlaid on anatomical MRI (1H) images from hydrocortisone acetate (HCA), cyclophosphamide (CY) and infected immunocompetent (I-IC) groups. The non-infected control group did not show detectable 19F MR signal in the lungs, (L=lungs, H= heart, R= 30mM reference).

Fig.2: In vivo quantification of A. fumigatus infected mice models using computed tomography & CFUs
Quantitative CT data showing the differences in lesion volume on day 1 and day 3 post infection in HCA (hydrocortisone acetate) model and CY (Cyclophosphamide) model. Colony forming units (CFUs) were counted from the homogenised lungs from all the experimental models  and cross validated with the in vivo 19F and CT data.

7:15 PM
PS-11-6 — Neutrophil dynamics in the pulmonary vasculature (#281)

J. Secklehner1, 2, J. B. G. Mackey1, 2, K. De Filippo2, J. Vuononvirta2, M. B. Headley3, M. F. Krummel3, N. Guerra4, L. M. Carlin1, 2

1 Cancer Research UK Beatson Institute, Glasgow, United Kingdom
2 Imperial College London, Inflammation, Repair and Development, London, United Kingdom
3 University of California, San Fransisco, Department of Pathology, San Fransisco, California, United States of America
4 Imperial College London, Life Sciences, London, United Kingdom

Introduction

Lung immune cells must be regulated to limit pathology while mounting a robust defence. Paradoxically, the immune system can both antagonise and benefit tumours. In cancer, neutrophils are associated with poor prognosis and linked to lung metastasis in breast cancer. During severe and/or chronic inflammation and cancer, blood neutrophils increase, where a subset are developmentally immature. Neutrophil maturation is essential to the development of effector mechanisms, however, methods for identifying murine immature neutrophils and analysing their activity in vivo are required.

Methods

Neutrophils are easily activated ex vivo; hence, we used lung intravital microscopy (IVM) to visualize interactions in vivo. We analysed localization, speed and duration of neutrophil: NK cell interactions in live mice in homeostasis or acute endotoxin-induced lung inflammation.  We used confocal laser-scanning microscopy of the live mouse lung in vivo and ex vivo, agarose inflated precision cut lung slices with multicolour labelling by fluorescent antibodies and dyes, transgenic reporter mice and adoptive transfer of cells in combination with multicolour flow cytometry to investigate the regulation and behaviour of lung leukocytes.

Results/Discussion

Immature and mature neutrophils have been identified degree of Ly6G. We confirmed their phenotypes based on nuclear morphology and recent mitotic activity within bone marrow. We developed methods that distinguish neutrophil maturity for their direct study in vivo by IVM of the lung and other experiments to test behaviour. Interactions with other immune cells can regulate neutrophils. Natural Killer (NK) cells are enriched in the lung where they may form a ‘resident’ intravascular population. We hypothesised NK cells regulate neutrophils in the lung vasculature. We found that lung NK:neutrophil interactions frequently occur for 5-10 minutes and remarkably, at times, material transfers from the neutrophils to NK cells. As predicted, endotoxin led to a rapid increase in neutrophil numbers in the lung, but, importantly, in the NK cell-depleted group this effect was substantially intensified. Cell-tracking of neutrophils revealed that NK cells also affect steady-state neutrophil motility.

Conclusions

We propose that NK:neutrophil interactions in the pulmonary vasculature mediate alterations to neutrophil behaviour and may signify a check-point for restricting neutrophilic infiltration during lung inflammation. This might enable us to develop new strategies to locally modify neutrophil behaviour in the lung without affecting systemic function. Additionally, we are now able to identify immature and mature neutrophils efficiently in the vasculature and we are using these methods to study their behaviour and putative roles in pathology.

Acknowledgement

Medical Research Council, Imperial College London, National Heart & Lung Institute Foundation and Cancer Research UK

7:25 PM
PS-11-7 — Red-light-switchable antibiotics: towards theranostics of bacterial infections (#37)

W. Szymanski1, 2, M. Wegener2, M. J. Hansen2, A. J. M. Driessen3, B. L. Feringa2

1 University Medical Centre Groningen, Department of Radiology, Groningen, Netherlands
2 University of Groningen, Centre for Systems Chemistry, Groningen, Netherlands
3 University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Groningen, Netherlands

Introduction

Medical treatments employ using bioactive compounds that evoke a pharmacological response by interacting with molecular targets in the human body. The selectivity of this interaction is crucial and the lack of it leads to the emergence of severe side-effects in the body and toxicity in the environment.[1]

Theranostics[2] aims at solving this issue using a combination of: advanced molecular diagnostics, which allows the doctors to precisely locate the disease; and locally-activatable treatment which can be locally activated, avoiding the emergence of side effects, toxicity and drug resistance.

Methods

Here, new photopharmacological[3] antibiotics will be introduced[4] that can be reversibly activated using deep-tissue-penetrating red light. The activating light could be delivered externally to an infection site discovered by imaging (yellow arrows, Fig. 1). Alternatively, optical imaging agents for infection can be used, and the light emitted from them can be used for unbiased local activation of the antibiotic (red arrow, Fig. 1).

Trimethoprim was chosen as the bioactive component. It is active against a broad spectrum of Gram-positive and Gram-negative bacteria and widely used in the clinic. It was modified with photoswitchable units (azobenzenes). Subsequent modification of the photoswitch moiety lead to structures that allowed us to control their activity with visible light.

Results/Discussion

A library of photoresponsive antibiotics was created and screened for the bactericidal activity against a model E. coli CS1562 strain. For the best compound, irradiation with red light at 652 nm effected photoisomerization to a photostationary state (PSS) of cis:trans = 87:13 (Fig. 2A).

In antibiotic activity assays, one half of a divided stock solution in DMSO was irradiated for 2.5 h with red light at 652 nm, before treating bacteria with the two separate samples in two-fold dilution series. To our delight, this experiment revealed a dramatic photoactivation effect: Whereas non-irradiated compound remained largely inactive with a MIC50 > 80 μM (Fig. 2B), red light-irradiated compound induced bacteriostasis down to 20 μM, with an observed MIC50 of 10 μM (Fig. 2B). It is worth noting at this point that photoisomerization with red light at close proximity also works effectively in aqueous medium.

Conclusions

We successfully developed diaminopyrimidines bearing azobenzene photoswitches, whose activity can be controlled by light. Remarkably, these compounds allowed for the full in situ photocontrol of antibacterial activity with green and violet light, making it possible to trigger both the activation and deactivation in the presence of bacteria. Apart from showcasing the activation of a biological agent otherwise inactive within the investigated concentration range, we were able to do so while also shifting the wavelength of activation from the UV range into the near-infrared therapeutic window.

References

[1]    Lancet (London, England) 2000, 356, 1255–9.
[2]    Bioconjug. Chem. 2011, 22, 1879–903.
[3]    a) J. Am. Chem. Soc. 2014, 136, 2178–91; b) Nat. Chem. 2013, 5, 924–8; c) Angew. Chem. Int. Ed. 2016, 55, 10978-10999.
[4]    J. Am. Chem. Soc. 2017, DOI: 10.1021/jacs.7b09281

Acknowledgement

M. W. gratefully acknowledges a postdoctoral fellowship of the German Research Foundation (WE 5922/1). We gratefully acknowledge generous support from NanoNed, The Netherlands Organization for Scientific Research (NWO-CW, Top grant to B. L. F. and NWO VIDI grant no. 723.014.001 for W. S.), the Royal Netherlands Academy of Arts and Sciences (KNAW), the Ministry of Education, Culture and Science (Gravitation programme 024.001.035) and the European Research Council (Advanced Investigator Grant no. 694345 to B. L. F.).

Figure 1
Activation principles for light-controlled antibiotics in theranostic approach.

Figure 2
Structure of the optimised design, together with photostationary states at different wavelengths (A) and difference in antibacterial activity (growth curves) for the irradiated and non-irradiated samples.

7:35 PM
PS-11-8 — In Vivo Tracking and Quantification of Inhaled Aerosol using Magnetic Particle Imaging towards Inhaled Drug Delivery Monitoring (#449)

Z. W. Tay1, P. Chandrasekharan1, X. Y. Zhou1, D. Hensley2, B. Zheng1, S. M. Conolly1

1 University of California, Berkeley, Bioengineering, Berkeley, California, United States of America
2 Magnetic Insight, Inc, Alameda, California, United States of America

Introduction

Pulmonary delivery of therapeutics is attractive but it is challenging to monitor and quantify the delivered aerosol / powder [1]. Currently, SPECT and PET are used but require inhalation of radioactive tracers [2]. Magnetic particle imaging (MPI) is an emerging medical imaging technique [3] that produces a sensitive tracer image [4] of superparamagnetic iron oxide nanoparticles (SPION) with zero ionizing radiation and robust imaging in the lung [5-7]. By mixing SPIONs into the aerosol, we can image & quantify aerosol mass deposited, delivery efficiency and evaluate lung clearance in vivo. 

Methods

Aerosol with droplet size smaller than 4.0 microns was generated by the Kent Scientific AeronebTM Nebulizer. Micromod PerimagTM SPIONs were added to the mix (final concentration of 5 mg / ml Fe) before aerosolization to provide an MPI-visible label of the aerosol. 40-week-old female Fischer 344 rats (180 - 200 g) were used. Delivery of the aerosol (net 0.06 mg Fe) was conducted by either forced ventilation (endotracheal intubation) or pulsed flow directed into the mouth cavity. Ventilation rate was varied and delivery efficiency evaluated. MPI imaging (respiratory gating) used a custom-built 3D 6.3 T/m field-free-line MPI scanner [8].  X-ray imaging was performed on a Kubtec Xpert 40. Timecourse, quantitative imaging of the lung and droppings was done to evaluate lung clearance. 

Results/Discussion

The lung phantom experiment in Fig 1b used aerosolized Doxorubicin HCl as a model drug and SPIONs mixed into the aerosol. The SPIONs have minimal effect on the droplet density / aerodynamic performance at the 5 mg/ml. MPI and Fluorescence Imaging shows that MPI image intensity is linearly quantitative of the Doxorubicin HCl deposited in the lung phantoms. Because MPI can image at depth without any tissue attenuation effects [9], MPI can track and quantitate aerosolized drug deposition. In vivo MPI scans in Fig 1c demonstrate that MPI can track and quantify the SPION biodistribution after delivery by aerosol. MPI images of rats with two forced ventilation rates shows that slower rates result in better delivery of aerosol throughout the lung while fast ventilation results in focal deposition. Timecourse imaging over 2 weeks (Fig 2) shows that MPI enables sensitive and quantitative measurement of SPION clearance from lung through the GI tract and into the droppings. 

Conclusions

MPI imaging is robust and quantitative in the lung and has zero ionizing radiation compared to current nuclear medicine methods for aerosol tracking. MPI monitoring of the mucociliary clearance is useful for long-term controlled release applications. SPIONs can also produce heat via RF excitation [10] to actuate drug release or perform hyperthermia therapy. With high sensitivity and high contrast images, MPI ventilation imaging could offer quantitative monitoring of the distribution and efficacy of drug aerosol delivery methods to inform and improve inhalable drug-delivery treatments.

References

  1. Ibrahim M, Verma R, Garcia-Contreras L. Inhalation drug delivery devices: technology update. Med Devices. 2015 Feb 12;8:131–139. 
  2. Dolovich M, Labiris R. Imaging drug delivery and drug responses in the lung. Proc Am Thorac Soc. 2004;1(4):329–337. 
  3. Gleich B, Weizenecker J. Tomographic imaging using the nonlinear response of magnetic particles. Nature. 2005 Jun 30;435(7046):1214–1217. 
  4. Graeser M, Knopp T, Szwargulski P, Friedrich T, von Gladiss A, Kaul M, Krishnan KM, Ittrich H, Adam G, Buzug TM. Towards Picogram Detection of Superparamagnetic Iron-Oxide Particles Using a Gradiometric Receive Coil. Sci Rep. 2017 Jul 31;7(1):6872. 
  5. Nishimoto K, Mimura A, Aoki M, Banura N, Murase K. Application of Magnetic Particle Imaging to Pulmonary Imaging Using Nebulized Magnetic Nanoparticles. Open Journal of Medical Imaging. Scientific Research Publishing; 2015;5(02):49.
  6. Zheng B, Yu E, Orendorff R, Lu K, Konkle JJ, Tay ZW, Hensley D, Zhou XY, Chandrasekharan P, Saritas EU, Goodwill PW, Hazle JD, Conolly SM. Seeing SPIOs Directly In Vivo with Magnetic Particle Imaging. Mol Imaging Biol. 2017 Jun;19(3):385–390
  7. Bulte JWM, Walczak P, Janowski M, Krishnan KM, Arami H, Halkola A, Gleich B, Rahmer J. Quantitative “Hot Spot” Imaging of Transplanted Stem Cells using Superparamagnetic Tracers and Magnetic Particle Imaging (MPI). Tomography. 2015 Dec;1(2):91–97.
  8. Yu EY, Chandrasekharan P, Berzon R, Tay ZW, Zhou XY, Khandhar AP, Ferguson RM, Kemp SJ, Zheng B, Goodwill PW, Wendland MF, Krishnan KM, Behr S, Carter J, Conolly SM. Magnetic Particle Imaging for Highly Sensitive, Quantitative, and Safe in Vivo Gut Bleed Detection in a Murine Model. ACS Nano. 2017 Nov 30
  9. Saritas EU, Goodwill PW, Croft LR, Konkle JJ, Lu K, Zheng B, Conolly SM. Magnetic particle imaging (MPI) for NMR and MRI researchers. J Magn Reson. 2013 Apr;229:116–126. 
  10. Hensley DW, Tay ZW, Dhavalikar R, Zheng B, Goodwill P, Rinaldi C, Conolly S. Combining magnetic particle imaging and magnetic fluid hyperthermia in a theranostic platform. Phys Med Biol [Internet]. 2016 Dec 29; 

Acknowledgement

We would like to acknowledge NIH funding and the A*STAR NSS-PhD and the Siebel Scholars fellowship (ZW Tay).

Magnetic Particle Imaging for Evaluation of Aerosol Drug Delivery Efficiency
a.  Photo of MPI scanner used.   b.  MPI image intensity is linear (R= 0.97) with the Doxorubicin HCl fluorescence (model drug) for DOX-SPION aerosol deposited on lung phantoms, indicating that the MPI image intensity can quantify the deposited drug.  c.   MPI can image and evaluate the aerosol delivery efficiency : inhaling the aerosol too fast results in focal deposition and poor C/P ratio. 

Magnetic Particle Imaging of the Timecourse Clearance of Deposited Aerosol From The Lung
a. Magnetic Particle Imaging (MPI) images show steady mucociliary clearance of deposited aerosol-SPIONs from the lung and into the GI tract to be excreted in droppings. MPI's high contrast and sensitivity enables tracking of the SPIONs at every stage of the clearance pathway, and is thus useful for long-term, controlled-release drugs. b. MPI's quantitative nature is useful for clearance kinetics. 

4:00 PM
emptyVal-1 — Introductory Talk by Bénédicte Jordan - Louvain, Belgium

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

4:20 PM
PS-07-2 — Multimodal mass spectrometry imaging for assessment of efficacy and mechanism of action of Oncology combination therapies (#351)

S. Ling1, 2, G. Hamm1, U. Polanska2, A. Dexter4, R. Steven4, S. Barry3, J. Bunch4, R. W. Goodwin1

1 AstraZeneca, DSM IMED, Cambridge, United Kingdom
2 AstraZeneca, DS IMED, Cambridge, United Kingdom
3 AstraZeneca, Oncology IMED, Cambridge, United Kingdom
4 National Physical Laboratory, Teddington, United Kingdom

Introduction

Tumour Microenvironment Heterogeneity impacts drug delivery, metabolism and resistance and drug interventions in turn regulate tumour metabolism. Analysis of impact of tumour metabolic heterogeneity in tissue has previously been challenging. As part of the CRUK Grand Challenge, multimodal Mass Spectrometry Imaging enables identification of endogenous metabolite biomarker changes altered in response to Oncology combination therapies to give new insights into drug mechanism of action, response and resistance.

Methods

Cross-site and cross-platform multimodal Mass Spectrometry Imaging was used to compare the monotherapy and combination efficacy of a combination therapy targeting the PI3K-AKT-mTOR pathway. As well as evaluation of drug delivery and potential regional accumulation of compound, targeted analysis of molecular biomarkers were used to assess predicted changes in glucose uptake, inflammatory processes and cholesterol biosynthesis and untargeted analysis including statistical identification of discriminative metabolites between samples and database search was used to identify de novo endogenous metabolite biomarkers.

Results/Discussion

Although it was not possible to define any differential tumour regions based on H&E, unsupervised clustering based segmentation enabled identification of distinct tissue regions based on similar changes in metabolite profile. These revealed ‘hotspots’ of potential resistance within tumour microenvironment, despite homogenous distribution delivery of the compounds. Different statistical methods such as distance-based, Manhattan or Euclidean K-Means clustering or a tSNE based deep learning approach all highlighted identical sub regions of the tissue and a greater number of metabolic differences were identified as driving tissue region differences than treatment differences.

Conclusions

We have shown that MSI metabolomics analysis of metabolic phenotypic response enables detection of greater heterogeneity in tumour response than visible by traditional pathology methods such as H&E. Combining this with biomarker information from Imaging Mass Cytometry (IMC) to identify the cell types and phenotypes responsible for the differential metabolic states and responses observed with combination therapies will drive our understanding of the impact of tumour heterogeneity on drug efficacy in vivo and in patients.

4:30 PM
PS-07-3 — Metabolic reprogramming in the heart and lung in a murine model of pulmonary arterial hypertension (#147)

J. L. Izquierdo-Garcia1, 2, T. Arias1, 2, Y. Rojas1, 2, J. Ruiz-Cabello3, 2

1 Centro Nacional Investigaciones Cardiovasculares (CNIC), madrid, Spain
2 CIBER Enfermedades Respiratorias (CIBERES), Madrid, Spain
3 Universidad Complutense de Madrid, Facultad de Farmacia, Madrid, Spain

Introduction

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary circulation that produces gradual narrowing of small pulmonary arteries, leading to progressive increase of pulmonary vascular resistance and, ultimately, right ventricular failure and death. Similar to cancer cells, pulmonary endothelial cells of patients with PAH show increased glycolysis and altered glucose oxidation1. The aim of this study is to study in situ the metabolic reprogramming associated with PAH development in the lung and the heart by metabolomic profiling and molecular imaging.

Methods

PAH was induced in mice (HPX+SU) by chronic hypoxia exposure plus treatment with SU5416. Control mice (NMX) were kept in a regular oxygenated room. In vivo 18F-FDG PET imaging was conducted prior to the induction of hypoxic conditions and after one, two, and three weeks of hypoxia (n=8) or normoxia (n=8). 25 animals per group were sacrificed at 3 weeks after treatment. Lung and heart tissue were analyzed by HR-MAS Magnetic Resonance Spectroscopy. Principal Component Analysis was performed to determine the differences between groups. PAH was characterized by echocardiography and histology analyses.

Results/Discussion

Metabolomic profiling of lung, right ventricle (RV) and left ventricle (LV) samples discriminated between groups (Fig. 1). In lung tissue, we identified significant alterations in glycolytic and glutaminolysis pathways, and the alteration of lipid metabolism. Furthermore, biomarkers of cell proliferation, such as glycine and choline metabolism were identified in lung tissues. Metabolic reprograming was also confirmed in heart samples. Lactate and alanine concentrations, endpoints of glycolytic oxidation, were found increased in PAH mice. Glutamine and taurine concentrations were identified as significant biomarkers of RV hypertrophy.

The altered or adapted energy metabolism was confirmed using in vivo PET imaging. Lung FDG uptake in HPX+SU mice significantly increased after the first week versus basal conditions and compared to NMX mice. FDG uptake was also significantly increased in HPX+SU ventricles versus basal conditions and NMX mice (Fig. 2) after first week of hypoxia exposure.

Conclusions

We demonstrated that the PAH mouse model induced by the combination of hypoxia and SU5416 treatment reproduces the metabolic abnormalities observed in the RV and pulmonary circulation in PAH patients. Specifically, we detected an upregulation of glycolysis and the presence of glutamine and fatty acid anaplerotic pathways in lung vasculature. We also detected some cell proliferation biomarkers that may represent new therapeutic targets for PAH. In addition, we monitored the specific RV metabolic alterations induced by pulmonary overpressure.

References

1-Xu W et al. PNAS 2007; 104: 1342-1347.

Acknowledgement

Funded by grants ITN-FP7-608027, FP7-PEOPLE-600396, FP7-PEOPLE-291820 and SEV-2015-0505.

Metabolomic profiling of lung and heart samples.
Principal components analysis (PCA) performed on 1H-MRS data of lung (A), right ventricle (RV) (B) and left ventricle (LV) (C) reveals a clear separation between normoxia (NMX) and hypoxia and SU 5416 treatment (HPX+SU) groups. Metabolites with the potential to distinguish the groups were quantified in the lung (D), the RV (E) and the LV (E). NMX: lung&RV=15; LV=10. HPX+SU: lung=22; RV=14; LV=10

PET/CT images of the left and right ventricles and lung parenchyma.
Representative images of NMX (A) and HPX+SU (B) mice at the end of the three-week protocol. 18F-FDG uptake in HPX+SU mice significantly increased after the first week versus the initial pre-hypoxic exposure conditions and compared to NMX in the lung (C) and in the right (D) and left (E) ventricles. The dotted line indicates basal conditions.

4:40 PM
PS-07-4 — Using 7T MRI and intravital microscopy for multimodal imaging of BAT activity in Type I and Type II diabetes mouse models (#466)

C. Jung1, M. Kaul1, H. Ittrich1, G. Adam1, J. Heeren2, M. Heine2

1 University Hospital Hamburg, Diagnostic and Interventional Radiology and Nuclearmedicine, Hamburg, Hamburg, Germany
2 University Hospital Hamburg, Biochemistry and Molecular Cell Biology, Hamburg, Hamburg, Germany

Introduction

The aim of the study was to determine the metabolic activity of brown adipose tissue (BAT) and its dependence on signalling pathway mediated by the anabolic hormone insulin and morover in type I and type II diabetes mouse models. Therfore we used superparamagnetic iron oxide nanoparticles (SPIO – for MRI) or quantum dots (QD – for intravital microscopy (IVM)) embedded into triglyceride-rich lipoproteins (TRL) to visualise lipid metabolism.

Methods

C57BL/6J wild-type were either treated with Alloxan which is selectively toxic to pancreatic beta cells (100µl iv; Alloxan monohydrate, Abcam) to induce type I diabetes or received a 35% Lard-based high fat diet to induce type II diabetes. To inhibit insulin secretion diazoxide was used. BAT activity was stimulated by treatment with the β3 receptor agonist CL316,243. All mice were starved for 4 hours before imaging. MRI at 7T ClinScan (Bruker) was performed before and 20 minutes after iv injection of TRL-SPIOs using a T2*w Multiecho-GRE sequence (TR/TEfirst 400/2ms, ETL 12). IVM analysis was performed for real time imaging of TRL-QD uptake into BAT. In order to quantify TRL clearance, the fate of radioactively labelled TRLs were analysed under the same experimental conditions.

Results/Discussion

While control mice showed a significant signal drop after CL treatment, no significant signal difference in BAT before and after the injection of TRL-SPIO was detectable neither for type I nor for type II diabetes mouse model. Inhibition of insulin signalling resulted in a significant lower uptake of TRL-SPIO into BAT. Analogy real time IVM analyses showed a clear reduction of TRL-QD in case of diabetes disease mouse models. MRI and IVM results were confirmed by quantitative metabolic studies using radioactive lipid tracers. In both setups diabetes type I and type II lead to a reduction of TRL uptake into BAT.

Conclusions

β3-receptor activation via CL with following acute insulin release lead to BAT activation, which can be visualised in vivo by MRI using TRL-SPIO. However, in case of diabetic disease, the uptake of TRL into BAT is diminished, indicating a loss of BAT activity in this case. Thus, MRI can visualize physiological lipid processing in the vascular endothelium of activated BAT.

 

Regulation of TRL uptake into activated BAT by the anabolic hormon insulin
Catabolic, cold-activated brown adipose tissue (BAT) burns triglycerides stored in lipid droplets for heat production. Consequently, endogenous lipid stores need to be replenished by anabolic processes. β3 receptor activation leads to FFA release from white adipose tissue. These FFAs provoke insulin secretion and insulin acts on BAT during triglyceride-rich lipoprotein (TRL) and FFA uptake.

MRI analysis in vivo of brown adipose tissue

In vivo MRI analysis showed a strong signal drop in BAT with a corresponding significant increase of delta R2* after TRL-SPIO iv injection under CL conditions. After Diazoxide treatment TRL-SPIO uptake into BAT and delta R2* was diminished.  

4:50 PM
PS-07-5 — The metabolic glucose profile of the spleen as a surrogate of peripheral immune cell activation after immune checkpoint blockade in melanoma bearing mice (#267)

B. F. Schörg1, J. Schwenck1, 2, P. Knopf1, S. Boecke3, W. Ehrlichmann1, G. Reischl1, D. Thorwarth3, M. Roecken4, C. la Fougère2, B. J. Pichler1, M. Kneilling1

1 Eberhard Karls University of Tübingen, Department of preclinical Imaging and Radiopharmacy / Werner Siemens Imaging Center, Tübingen, Baden-Württemberg, Germany
2 Eberhard Karls University of Tübingen, Department of Nuclear Medicine, Tübingen, Germany
3 Eberhard Karls University of Tübingen, Section for Biomedical Physics, Department of Radiation Oncology, Tübingen, Germany
4 Eberhard Karls University of Tübingen, Department of Dermatology, Tübingen, Germany

Introduction

The modulation of immune checkpoints (ICP, like CTLA-4 or PD-1) to re-activate exhausted T cells is a promising therapeutic strategy for cancer patients but as single treatment only effective in <40% of melanoma patients. 18F-FDG-PET imaging may provide an important contribution for treatment monitoring and for identification of non-responders. We aimed to analyze changes of the metabolic profile of the spleen in response to a new combination of local tumor irradiation (LIR) and PD-L1 + LAG-3 blockade by 18F-FDG PET in melanoma-bearing mice as a surrogate of peripheral immune cell activation.

Methods

We treated B16-F10-luc melanoma (expressing luciferase, luc)-bearing mice with a single LIR (10 Gy) of the tumors followed by weekly administrations of anti-PD-L1/LAG-3 mAbs (PDL1/LAG3; n=10). To examine the glucose metabolism of the tumors and especially of the spleens during the treatment, we performed 18F-FDG PET/MRI scans: 6 days post tumor inoculation (dpi; 1 day before LIR), 13 and 20 dpi. Spleen volume was measured by MRI. Tumor growth was determined by volumetric measurements and bioluminescence imaging (BLI); Mice were sacrificed 21 dpi, organs were collected for histopathology. We calculated the spleens 'total lesion glycolysis' (TLG, 18F-FDG SUV*spleen volume) to compare the therapy effects on its metabolism. Animals receiving LIR + isotype mAbs (ISO; n=5) were used as control.

Results/Discussion

We successfully established a new therapeutic approach combining LIR+PDL1/LAG3: 50% of the mice exhibited significantly reduced tumor growth (responders, 82±25 vs. 490±112 mm3; p<0.01) with stable BLI signal intensity compared to LIR+ISO treated mice; non-responders showed tumor progression comparable to control mice. Contrary, we observed an increased 18F-FDG-tumor uptake in all groups, suggesting a non-effective therapy in responding mice. The spleen volume of LIR+ISO treated mice decreased significantly to 84±5% at 20 dpi compared to baseline (p<0.05), while the spleens of the LIR+PDL1/LAG3 group were less affected. In contrast, we observed an increase in TLG exclusively in the spleens of LIR+PDL1/LAG3-treated mice 20 dpi compared to the baseline (responders: +40±6%; p< 0.001; non-responders: +70±23%). In line with our preclinical data, retrospective clinical studies assume an association of the therapeutic effects with changes of the 18F-FDG uptake in secondary lymphatic organs.

Conclusions

We showed that our new combination of LIR+PDL1/LAG3 mAbs is highly effective in 50% of mice with aggressive melanoma. Moreover, we have proven that the spleens’ glucose metabolism is generally affected by checkpoint inhibitor treatment (CIT) in both, responders and non-responders. Considering the increased 18F-FDG PET uptake in melanomas of responding mice, most probably mimicking progressive disease due to the highly activated infiltrating T cells with enhanced glucose metabolism, the metabolic 18F-FDG profile of the spleen may be a new approach to identify CIT responders.

5:00 PM
PS-07-6 — eNOS-/- mice fed with HFD develop progressive non-alcoholic fatty liver disease (NAFLD) which is partially reversible with antihypertensive and hypoglycemic therapy (#110)

B. Lavin1, M. E. Andia2, T. Eykyn1, A. Phinikaridou1, A. Xavier2, R. M. Botnar1, 2

1 King's College London, School of Biomedical Engineering Imaging Sciences, London, United Kingdom
2 Pontificia Universidad Católica de Chile, Radiology department, School of Medicine, Santiago, Chile

Introduction

Non-alcoholic fatty liver disease (NAFLD) is considered a key determinant for metabolic syndrome1. However, the cause and treatments are still controversial. Nitric oxide (NO) plays important roles in the pathophysiology of the vasculature and liver2 and its absence promotes systemic alterations3, 4. We investigated its role in intraperitoneal and liver fat accumulation (1) after administration of the inhibitor of endothelial NO synthesis (L-NAME)4 in wild type mice and (2) the effect of pharmacological treatments for type 2 diabetes (Metformin) and hypertension (Losartan) in eNOS-/- fed HFD.

Methods

Study design is summarized in Fig.1A. In vivo MRI: Whole body 3-point Dixon images were obtained using a 3T clinical MR scanner (Achieva, Philips Healthcare, Best, NL) and a 47mm single-loop surface coil. The acquisition parameters include TR/TE1/TE2/TE3=15/2.3/3.5/4.7ms; flip angle 10º; in plane resolution=0.17x0.17x0.5mm. Ex vivo NMR: Total liver fat/water ratio and extracted liver lipid metabolite profiles were studied using MR spectroscopy at 9.4T (Bruker). The acquisition parameters include TR=3.45s, Spectral BW/resolution=5597/0.64Hz, Phase cycles=8, NSA=64, Flip angle=90º. Animal treatments: All treatments were administered in water as follow: L-NAME (0.5mg/mL), Metformin (2mg/mL), Losartan (0.3mg/mL). Histology: Liver fat accumulation was analyzed using Trichrome stain.

Results/Discussion

eNOS-/- mice fed with HFD had increased body weight compared to chow. WT mice fed with HFD had a similar increase in body weight when treated with L-NAME (Fig 1B). Importantly, eNOS-/- mice treated with Metformin, Losartan or a combination of both did not display increased weight compared to those fed chow (Fig 1C). Representative Dixon images are shown in Fig 1D.

eNOS-/- mice fed with HFD had significantly more intraperitoneal fat and liver fat-fraction than WT fed with HFD and compared to normal chow groups. WT mice fed HFD and treated with L-NAME displayed similar fat accumulation to eNOS-/- mice (Fig. 2A-2B). Importantly, when treated with Losartan and Metformin, lower amounts of intraperitoneal fat and liver fat-fraction were observed in eNOS-/- mice fed with HFD, with a slightly synergistic effect when both treatments were co-administered (Fig 2C-2D). Our in vivo findings were corroborated by ex vivo NMR of liver water/fat ratio (Fig 2E-2F) and by histology (Fig. 2G).

Conclusions

Using in vivo MRI we demonstrated that liver fatty acid accumulation is accelerated in eNOS-/- mice fed with HFD, which could be replicated in WT mice fed with L-NAME. Intraperitoneal and liver fat accumulation could be partially normalized after treatment of hypertension (losartan) and type 2 diabetes (metformin). Our results show the relevance of noninvasive monitoring of liver fat fraction and body composition to evaluate the response and adherence to pharmacological treatments for NAFLD.

References

1Cohen JC, Science 2011; 2Iwakiri Y, Trends Pharmacol Sci 2015; 3Takahashi T, J Diabetes Res 2014; 4Sheldon RD, J Appl Physiol 2014; 5Suda O, Circulation 2002

Acknowledgement

(1) The British Heart Foundation (RG/12/1/29262). (2) CONICYT-PIA, Anillo ACT1416. PuenteUC P1712/2017.

Figure 1
(A) Study design. (B) Weight of the WT and eNOS-/- mice fed with normal chow and HFD. (C) Weight of the WT and eNOS-/-fed with HFD and treated with Losartan and/or Metformin. (D) Fat-only images from Dixon acquisition of all eNOS-/- and WT mice groups.

Figure 2
Intra-abdominal fat volume (A-C) and liver fat-fraction (B-D) obtained from 3-point Dixon sequence in WT and eNOS-/- mice fed with normal chow or HFD ± treatment. Liver water/fat ratio measured by NMR (E-F) in WT and eNOS-/- mice fed with normal chow or HFD ± treatment. (G) Trichrome staining of all eNOS-/- and WT mice groups.

5:10 PM
PS-07-7 — [18F]FDG-PET tracer uptake pattern is a major driving source for dynamic metabolic brain connectivity (#309)

M. Amend1, T. M. Ionescu1, B. Biswal2, H. F. Wehrl1, B. J. Pichler1

1 Eberhard Karls University Tuebingen, Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Tuebingen, Germany
2 New Jersey Institute of Technology, Department of Biomedical Engineering, Newark, United States of America

Introduction

PET derived metabolic brain connectivity has been described as a promising biomarker for certain neurological pathologies1. However, static PET without temporal information was acquired and network analysis was performed only at group level. It remains unclear whether fMRI derived resting state functional connectivity is associated with an underlying synchrony in brain metabolism. To allow a better comparison with time course derived resting state fMRI data, but also single subject metabolic network characterization, we used simultaneous PET/MRI to evaluate dynamic metabolic connectivity.

Methods

40 male Lewis rats (322±55g) were anesthetized (1.3% isoflurane) and physiology was monitored. EPI BOLD sequences (TE: 18ms, TR: 2500ms) were acquired with a 7T preclinical Bruker scanner using RT surface coils. Simultaneously to 60min fMRI scans, 30 rats received bolus [18F]FDG injections (856±27µCi) and dynamic PET images were recorded. 10 rats received 100min PET/fMRI scans. Here, [18F]FDG was constantly infused over the entire scan time (start activity: 4053±21µCi; infusion rate: 0.8µl/min). Based on group-mean matrices generated for both functional and metabolic connectivity, data  were evaluated for consistency, the impact of PET-tracer kinetics and inter-individual differences in PET-tracer activity. Finally, functional fMRI and metabolic PET connectivity readouts were compared.

Results/Discussion

fMRI connectivity was consistent over the entire scan (Fig. 1A). Calculated mean correlation strength (Fig. 1C) and edge numbers (Fig. 1D) provided stable values resulting in excellent within-scan consistency (Fig. 1E). PET-derived metabolic connectivity using bolus injection was also robust between-group (ICC=0.892). However, strongly decreased connectivity was detected in the latter periods of the scan (Fig. 1B). Decreases in correlation strengths (Fig. 1C) and edge numbers (Fig. 1D) were observed resulting in poor within-scan consistency (Fig. 1E). Metabolic connectivity derived from short time frames yielded stronger correlation strengths (Fig. 1C) and higher edge numbers (Fig. 1D) compared to longer framings. Applying constant tracer infusion, described decreases of within-scan consistency were removed resulting in excellent ICC of 0.812. Connectivity obtained via fMRI and PET showed fair overlap (DICE=0.47) with similar clustering patterns of positive and negative correlations.

Conclusions

We report dependencies of dynamic metabolic connectivity on time-varying tracer delivery and tracer uptake characteristics, since changes in tracerkinetics over time have significant impact on the connectivity readout. Due to stable kinetics, constant tracer infusion is an ideal approach to compare both temporal metabolic and functional connectivity. Resulting [18F]FDG-PET and resting state fMRI derived complementarities due to different underlying temporal resolutions and biochemical processes may lead to a better understanding of neurological disorders targeting energy demanding brain hubs.

References

1) Eckert T, Tang C and Eidelberg D. Assessment of the progression of Parkinson´s disease: a metabolic network approach. Lancet Neurol 2007; 6: 926-932

Figure 1: Within-scan consistency of results measured at group-level
BOLD-fMRI (A) and [18F]FDG-PET scans (B) were split in three 20 minute blocks and correlation matrices were computed. (C) Mean correlation strength of the weighted matrices for each 20-minute block. (D) Number of obtained edges for every 20 minute block. (E) Consistency quantification for BOLD-fMRI and [18F]FDG-PET scans reconstructed in different framings using ICC and Dice coefficient.

5:20 PM
PS-07-8 — Anatomical & Molecular Imaging To Undress The Naked Mole Rat (#303)

A. Kirby2, M. Pamenter2, A. Shuhendler1, 3

1 University of Ottawa, Chemistry & Biomolecular Sciences, Ottawa, Ontario, Canada
2 University of Ottawa, Biology, Ottawa, Ontario, Canada
3 University of Ottawa Heart Institute, Ottawa, Ontario, Canada

Introduction

The naked mole rat (Heterocephalus glaber) is amongst the most hypoxia-tolerant mammals identified, living in underground burrough systems that are chronically low on oxygen [1]. How these highly social animals persist and thrive in these conditions is still not fully understood. A recent report suggests that naked mole rat metabolism is rewired for fructose-driven glycolytic respiration in order to minimize the lethal effects of oxygen deprivation [2]. Herein, we present the first molecular imaging-based study of metabolism of the naked mole rat under normoxic and hypoxic conditions.

Methods

Naked mole rats were housed in colonies at the University of Ottawa prior to beginning the study. Pairs were removed from their colonies, approximately matched for age and weight, and were subjected to repeated imaging evaluation by MRI and [18F]-FDG PET. MRI was performed on 3T (MR Solutions Inc.) and 7T (GE/Aglient MR901) scanners for anatomical reference, acquiring T1- and T2-weighted images through Fast Spin Echo or three dimensional Gradient Echo sequences, respectively. Echocardiography was also performed using a VisualSonics 770 system. Following maintenance of animals in normoxia or acute hypoxia (7% O2, 60 min) and injection of 200 microCi [18F]-FDG PET, dynamic scans were acquired (45 min). Radiotracer uptake and distribution were analyzed using VivoQuant software.

Results/Discussion

In order to investigate the metabolism of the naked mole rat using moleculr imaging techniques, the imaging response of the animal under various conditions (e.g. fed vs. fasted and method of anesthesia) was investigated in terms of [18F]-FDG uptake. In order to properlyt interpret PET data, the anatomy of the naked mole rat was studied as no references beyond the head are published [3]. From the anatomical MR images, we noted interesting organ features, including a larger heart and smaller brain size relative to mice, and an extensive brown adipose tissue distribution in the neck. We also found a thicker myocardium with a very large papilary muscle in the left ventricle. Finally, we noted a significant difference in [18F]-FDG uptake between normoxic and acute hypoxic conditions, with a significant loss of brown adipose tissue uptake, and a significant increase in heart, brain, and liver uptake.

Conclusions

Using standard anatomical and molecular imaging techniques, unique anatomical and molecular information has been learned about the naked mole rat. The reasons for these features are currently being explored from the behavioural/physiological through to the molecular level with efforts to rationalize these difference in terms of the unique lifestyle of this subterranian mammal. Importantly, a rapid and significant change in glucose utilization patterns has been observed following acute hypoxia, suggesting reliance upon glucose as a fuel during phases of limited oxygen availability.

References

[1] Chung D, et al. (2016). Naked mole rats exhibit metabolic but not ventilatory plasticity following chronic sustained hypoxia. Proceedings of the Royal Society B. 283(1827):20162016

[2] Park TJ, et al. (2017) Fructose-driven glycolysis supports anoxia resistance in the naked mole rat. Science. 356(6335):307-11

[3] Seki F, et al. (2013) Multidimensional MRI-CT atlas of the naked mole-rat brain (Heterocephalus glaber). Frontiers in Neuroanatomy. 7:45

Acknowledgement

This work was supported by an NSERC Discovery Grant RGPIN 2015-05796 (A.J.S.), the Canada Research Chairs Program 950-230754 (A.J.S.), the Canadian Foundation for Innovation (A.J.S.), and the Canadian Institutes of Health Research PJT376892 (A.J.S.).

Glucose utilization before and after acute hypoxia in the naked mole rat
Naked mole rats were assessed in normoxia (20% O2, left) or after acute hypoxia (60 min at 7% O2, right) for glucose utilization by [18F]-FDG PET. Maximum intensity projections of the animals are shown with signal averaged between 25 and 45 min after injection of 200 microCi radiotracer. A significant change in uptake is noted for organs identified.

Whole-body anatomical maps of the naked mole rat at 7T
Whole body MR images were acquired on anesthetized naked mole rats at 7T. Both T1- (left) and T2-weighted (right) images were acquired to delineate tissue compositions. Both sagittal and coronal views are provided.

2:30 PM
emptyVal-1 — Introduction by Albert D. Windhorst

2:45 PM
ES-02-2 — Fluorinated gold nanoparticles as contrast agents in 19F-MRI and for protein corona evaluation (#604)

M. Carril1, 2

1 CIC biomaGUNE, San Sebastian, Spain
2 Ikerbasque, Basque Foundation for Science, Bilbao, Spain

Objectives

The aims of this talk are the following:

  • To learn about 19FMRI as a complementary technique to 1HMRI and potential applications such as the use of OFF/ON fluorinated probes.
  • To understand what nanoparticles can offer in the field of imaging probes for MRI.
  • To acquire chemical tools for the synthesis of fluorinated ligands and gold nanoparticles for magnetic resonance. Probe’s requirements and tips to tackle with hydrophobic and volatile fluorinated compounds.
  • To characterize nanoparticles. How we can use Nuclear Magnetic Resonance to characterize small nanoparticles.
  • To be aware of the importance of protein corona regarding the fate and behaviour of nanomaterials in vivo.
  • To learn how we could use fluorinated nanomaterials to study protein corona.

Content

In the last decades, nanotechnology has widely contributed to the field of preclinical contrast-enhanced imaging, and nowadays nanoparticles (NPs) as imaging probes are ubiquitous in MRI studies, among other imaging technologies. Fluorine 19 (19F) based MRI is a re-emerging field with promising features which complement proton-based traditional MRI. The most interesting advantage of 19F over 1H is the negligible endogenous 19F-MRI signal, for which any detectable signal can only come from an exogenous probe. However, in order to achieve a quality of image similar to that obtained with conventional MRI, a high load of fluorine atoms with the same resonance frequency is required. One of the main challenges in this field at the moment is the improvement of existing contrast agents in order to increase the SNR and circumvent the intrinsic hydrophobicity of fluorinated probes. Taking this into account, the use of NPs bearing a high number of identical fluorinated ligands could be an appealing strategy to increase the local concentration of chemically equivalent fluorine atoms. In this context, we have designed novel fluorine labels for nanoparticles [1] and general labelling tools. In addition, the lack of background fluorine signal in physiological environments allowed for the study of protein corona, that is, the spontaneous adsorption of proteins on the nanoparticles’ surface when they come into contact with blood. The measurement of the diffusion of fluorinated nanoparticles in complex media by 19F NMR permits the calculation of their hydrodynamic size which can give information regarding the presence of a protein corona [2]. For the first time a technique such a magnetic resonance that could be translated to in vivo has been used for the study of the protein corona, hence we are now getting closer to protein corona potential detection in vivo by 19F MRI and hence to overcome the limitations of in vitro measurements.

Relevant Publications

J. Ruiz-Cabello, B.P. Barnett, P.A. Bottomley, J.W.M. Bulte. Fluorine (19F) MRS and MRI in biomedicine. NMR in biomedicine, 2011, 24, 114.

I. Tirotta, V. Dichiarante, C. Pigliacelli, G. Cavallo, G. Terraneo, F. Baldelli Bombelli, P. Metrangolo, G. Resnati. 19F Magnetic Resonance Imaging (MRI): From design of materials to clinical applications. Chemical Reviews 2015, 115, 1106.

M. Carril. Activatable probes for diagnosis and biomarker detection by MRI. Journal of Materials Chemistry B, 2017, 8, 4332.

J. Estelrich, M.J. Sanchez-Martin, M.A. Busquets. Nanoparticles in magnetic resonance imaging: from simple to dual contrast agents. International Journal of Nanomedicine, 2015, 10, 1727.

P. Pengo, L. Pasquato. Gold nanoparticles protected by fluorinated ligands: Synthesis, properties and applications. Journal of Fluorine Chemistry, 2015, 177, 2.

C. Carrillo-Carrion, M. Carril, W.J. Parak. Techniques for the experimental investigation of the protein corona. Current Opinion in Biotechnology, 2017, 46, 106.

References

[1] O. Michelena, D. Padro, C. Carrillo-Carrión, P. del Pino, J. Blanco, B. Arnaiz, W. J. Parak, M. Carril. Novel fluorinated ligands for gold nanoparticle labelling with applications in 19F MRI. Chemical Communications, 2017, 53, 2447.

[2] M. Carril, D. Padro, P. del Pino, C. Carrillo-Carrión, M. Gallego, W.J. Parak. In situ detection of the protein corona in complex environments. Nature Communications, 2017, 8, 1542.

Acknowledgement

Funding support from MINECO (CTQ2015-68413-R) and Ikerbasque Basque Foundation for Science is acknowledged.

3:15 PM
ES-02-3 — Radiolabeling Nanoparticles for Nuclear Imaging (#600)

R. T. M. de Rosales1

1 King's College London, School of Biomedical Engineering & Imaging Sciences, London, United Kingdom

Objectives

  • To learn the basic principles of nuclear imaging (PET and SPECT), and its role in nanoparticle/nanomedicine development and potential clinical applications.
  • To gain knowledge of the different methods for nanoparticle radiolabeling (inorganic and organic materials)
  • To understand the limitations of PET and SPECT in the context of other clinical imaging techniques, and the potential new roles for multimodal imaging probes based on nanoparticle technologies.

Content

Imaging methods that report on nanoparticle/nanomedicine biodistribution and pharmacokinetics in vivo should allow us to identify, monitor and improve the efficacy of therapeutic/diagnostic probes based on nanoparticle technologies. To achieve this there is a need to develop simple and effective labelling methods for non-invasive imaging techniques and answer questions such as: How much nanopaticle/nanomedicine is/stays in the target? Do they migrate to potentially sensitive organs? How are they cleared?.

Nuclear imaging techniques such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET) are particularly well suited to answer these questions. In particular, PET is the only clinically available imaging technique that allows quantitative analysis of imaging signals with exquisite sensitivity (signal/background ratios) and adequate spatial/temporal resolution at the whole-body level. Taking examples from my own research group [1-5] and other researchers in the field, in this lecture I will describe examples on how different radiolabelling methods have been used to track nanoparticles and nanomedicines in preclinical and clinical studies, as well as their limitations and advantages.

Relevant Publications

  • Abou, D. S.; Pickett, J. E.; Thorek, D. L., Nuclear molecular imaging with nanoparticles: radiochemistry, applications and translation. Br J Radiol 2015, 88, 20150185.
  • Llop, J.; Gomez-Vallejo, V, Isotopes in Nanoparticles: Fundamentals and Applications. Pan Stanford 2016.
  • Smith, B. R.; Gambhir, S. S., Nanomaterials for In Vivo Imaging. Chem. Rev. 2017 , 117 (3), 901–986.
  • Burke, B. P.; Cawthorne, C.; Archibald, S. J., Multimodal nanoparticle imaging agents: design and applications. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 2017, 375, 20170261.
  • Lamb, J.; Holland, J. P., Advanced Methods for Radiolabeling Multimodality Nanomedicines for SPECT/MRI and PET/MRI. J Nucl Med 2018, 59, 382-389.

 

References

  1. Abdollah, M. R. A. et al., Fucoidan Prolongs the Circulation Time of Dextran-Coated Iron Oxide Nanoparticles. ACS Nano 2018, 12, 1156-1169.
  2. Edmonds, S. et al., Exploiting the Metal-Chelating Properties of the Drug Cargo for In Vivo Positron Emission Tomography Imaging of Liposomal Nanomedicines. ACS Nano 2016, 10, 10294-10307.
  3. Sandiford, L. et al., Bisphosphonate-anchored PEGylation and radiolabeling of superparamagnetic iron oxide: long-circulating nanoparticles for in vivo multimodal (T1 MRI-SPECT) imaging. ACS Nano 2013, 7, 500-512.
  4. T. M. de Rosales, R. et al., 99mTc-bisphosphonate-iron oxide nanoparticle conjugates for dual-modality biomedical imaging. Bioconjug Chem 2011, 22, 455-465.
  5. T. M. de Rosales, R. et al., Synthesis of 64Cu(II)-bis(dithiocarbamatebisphosphonate) and its conjugation with superparamagnetic iron oxide nanoparticles: in vivo evaluation as dual-modality PET-MRI agent. Angew Chem Int Ed Engl 2011, 50, 5509-5513.

Acknowledgement

Work in my group is funded by a Cancer Research UK Multidisciplinary Project Award (C48390/A21153), the KCL and UCL Comprehensive Cancer Imaging Centre funded by CRUK and EPSRC in association with the MRC and DoH (England), the Wellcome EPSRC Centre for Medical Engineering at King’s College London (WT 203148/Z/16/Z) and the King’s College London & Imperial College London EPSRC Centre for Doctoral Training in Medical Imaging (EP/L015226/1).

3:45 PM
ES-02-4 — Synthesis and application of RGD-targeted optical imaging agents (#588)

J. - L. Coll1

1 INSERM, Institute for Advanced Biosciences, La Tronche, France

Objectives

Tumor angiogenesis is very important to promote tumor initiation, progression and dissemination, and was initially listed as one of the six hallmarks of cancer. Due to the specific hostile tumor microenvironment (hypoxia, low pH and high interstitial fluid pressure), the structure and function of tumor vessels are different from those of normal vessels.

Integrins are heterodimeric cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. There are at least 18α and 8β subunits that can dimerize to form more than 24 combinations that yield functional cell surface receptors.

Integrins have several functions and are in particular important regulators of cells’ migration and invasion.

Integrin αvβ3 is overexpressed in endothelial cells during angiogenesis, while it is not detected in quiescent endothelial cells of established blood vessels. Because of this pattern of expression, and because it may be directly accessible from the blood stream for tumor targeting, Integrin αvβ3 became rapidly a very attractive target. Furthermore, the crystal structure of the extracellular segment of αvβ3 allowed a good characterization of its ligand-binding domain that reacts with the tri-peptide sequence RGD (Arg-Gly-Asp). A very large series of peptides and peptidomimetics containing this sequence have been developed and used for cancer diagnosis and therapy. The RGD peptide is probably the more widely investigated peptide ligand for the delivery of drugs, imaging agents and NPs to the tumor vasculature and to the tumor cells.

Content

Wee have been using a cyclic RGD motif to deliver different drugs, contrast agents and nanoparticles of different sizes and composition. In particular we are using it for non invasive, near-infrared, optical guided surgery (1, 2, 3,4). More recently we investigated how RGD-targeted nanoparticles intereract with their target cells. 

During this presentation, I will present relevant examples of RGD-based nanosystems used for drug delivery as well as for delivering contrast agents for imaging and finally for theranostics applications. I will in particular critically address the benefits and limits of using this peptide motif for integrin-targeting using different systems presented in the litterature.

Relevant Publications

Arosio, D., and Casagrande, C. (2016) Advancement in integrin facilitated drug delivery. Adv Drug Deliv Rev 97, 111-143

References

[1] I. Atallah, C. Milet, M. Henry, V. Josserand, E. Reyt, J.L. Coll, A. Hurbin, C.A. Righini, Near-infrared fluorescence imaging-guided surgery improves recurrence-free survival rate in novel orthotopic animal model of head and neck squamous cell carcinoma, Head Neck 38 Suppl 1 (2016) E246-55.

[2] J. Choi, E. Rustique, M. Henry, M. Guidetti, V. Josserand, L. Sancey, J. Boutet, J.L. Coll, Targeting tumors with cyclic RGD-conjugated lipid nanoparticles loaded with an IR780 NIR dye: In vitro and in vivo evaluation, Int J Pharm 532(2) (2017) 677-685.

[3] D. Duret, A. Grassin, M. Henry, T. Jacquet, F. Thoreau, S. Denis-Quanquin, J.L. Coll, D. Boturyn, A. Favier, M.T. Charreyre, "Polymultivalent" Polymer-Peptide Cluster Conjugates for an Enhanced Targeting of Cells Expressing alphavbeta3 Integrins, Bioconjug Chem 28(9) (2017) 2241-2245.

[4] A. Karageorgis, M. Claron, R. Juge, C. Aspord, F. Thoreau, C. Leloup, J. Kucharczak, J. Plumas, M. Henry, A. Hurbin, P. Verdie, J. Martinez, G. Subra, P. Dumy, D. Boturyn, A. Aouacheria, J.L. Coll, Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model, Mol Ther 25(2) (2017) 534-546.

[5] T. Jia, J. Choi, J. Ciccione, M. Henry, A. Mehdi, J. Martinez, B. Eymin, G. Subra, J.L. Coll, Heteromultivalent targeting of integrin alphavbeta3 and neuropilin 1 promotes cell survival via the activation of the IGF-1/insulin receptors, Biomaterials 155 (2018) 64-79.

Acknowledgement

This work was supported by the Institut National du Cancer (INCA-PLBio16-085), and the “Fondation ARC pour la Recherche sur le Cancer (PGA1-20160203791) ».

We thank our collaborators from the Institute of Biomolecules Max Mousseron (IBMM; Pr Gilles Subra), from the Department de Chimie Moléculaire (DCM, Dr Didier Boturyn) and from the OPTIMAL Grenoble small animal optical imaging facility (Dr Véronique Josserand).

Special thanks finally to the different researchers involved in the work of our team: Dr Tao JIA, Dr Jungyoon CHOI, Dr Lucie SANCEY, Dr Xavier LEGUEVEL, Dr Amandine HURBIN, Dr Véronique FRACHET, Dr Annie MOLLA, Pr Christian RIGHINI, Dr Ihab ATALLAH, Mr Maxime HENRY and Mr Thibault JACQUET

4:15 PM
emptyVal-2 — BREAK

5:00 PM
ES-02-6 — Nanomedicines: Hypes, hopes and future directions (#570)

F. Kiessling1

1 RWTH Aachen, ExMI, Aachen, Germany

Objectives

Nanoparticles are frequently suggested as “magic bullets” to detect, characterize and treat diseases. However, despite thousands of papers only few nanomedicines successfully passed the translational process and are in clinical routine use.

Therefore, the aim of this talk is to tackle the following learning objectives:

· to identify indications for diagnostic and therapeutic nanomedicines

· to discuss reasons for translational failures of nanomedicines

· to understand the pharmacokinetic demands on diagnostic and therapeutic nanomedicines

· to discuss rational strategies for the development of nanomedicines and their translation to the clinic

· to understand the value of combination therapies

Content

There are various medical applications for nanoparticles (NP), e.g. as components of skin cremes, vaccines, drug delivery systems, plasma expanders and imaging agents.

The precise definition of the requirements on a nanomedicine (NM) from the chemical, biological and medical perspective should be the first step in its development. Unfortunately, due to insufficient interaction between medical doctors, biologists, and chemists these requirements are often incompletely set. In particular, pharmacokinetic demands are regularly not sufficiently taken into account, which are very different for diagnostic, theranostic or therapeutic systems. NP larger than 5 nm tend to be removed by the reticulo-endothelial system (RES). Thus, tissues belonging to the RES but also macrophages in tumors and inflammatory lesions can be targeted with such NP. Adding stealth properties to NP increases their circulation time giving them more time to extravasate in tissues with high vessel permeability. This so-called EPR based accumulation is the basis for most tumor-targeted NM. However, the therapeutic benefit over small probes is often only moderate since EPR is variable among patients and even heterogeneous within the same tumor. Theranostic agents and companion diagnostics can help to preselect patients and to individualize therapy. In addition, in tumors larger NP tend to accumulate just outside the vasculature but do hardly penetrate the stroma and thus do not reach the cancer cells. As a consequence, refining the balance between accumulation and penetration may be therapeutically superior over just maximizing accumulation. Here, active targeting can substantially improve NM retention and cellular internalization but does not solve problems associated with insufficient delivery. Therefore, combination therapies, where small drugs or physical interventions are used to prime vessels and the tumor microenvironment, may help exploiting the full therapeutic potential of NM.

Relevant Publications

 

  1. Tsvetkova Y., Beztsinna N., Baues M., Klein D., Rix A., Golombek S., Al Rawashdeh W., Gremse F., Barz M., Koynov K., Banala S., Lederle W., Lammers T., Kiessling F. (2017) Balancing Passive and Active Targeting to Different Tumor Compartments Using Riboflavin-functionalized Polymeric Nanocarriers. Nano Lett, 17:4665‑4674
  2. Kunjachan S., Pola R., Gremse F., Theek B., Ehling J., Moeckel D., Hermanns-Sachweh B., Pechar M., Ulbrich K., Hennink W.E., Storm G., Lederle W., Kiessling F., Lammers T. (2014) Passive vs. active tumor targeting using RGD- and NGR-modified polymeric nanomedicines. Nano Lett, 14:972-81
  3. Ojha T., Pathak V., Shi Y., Moonen C., Hennink W., Storm G., Kiessling F., Lammers T. (2014) Pharmacological and Physical Vessel Modulation Strategies to Improve EPR-mediated Drug Targeting to Tumors. Adv Drug Deliv Rev, 119:44-60
  4. Kunjachan S., Ehling J., Storm G., Kiessling F., Lammers T. (2015) Non-invasive Imaging of Nanomedicines and Nanotheranostics: Principles, Progress and Prospects. Chem Rev, 115:10907-10937
  5. Kiessling F., Mertens M.E., Grimm J., Lammers T. (2014) Nanoparticles for imaging: top or flop? Radiology, 273:10-28
  6. Lammers T., Yokota-Rizzo L., Storm G., Kiessling F. (2012) Personalized nanomedicine. Clin Cancer Res, 18:4889-94

 

5:30 PM
emptyVal-3 — Panel Discussion

Why is there limited application of particle based imaging probes in humans, whereas the success in a pre-clinical is obvious?

What makes a good particle based imaging probe to a success?

Challenges in evaluation of particle imaging probes.

(...)

4:00 PM
emptyVal-1 — Introductory Lecture by Marleen Keyaerts - Brussels, Belgium

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

4:20 PM
PS-08-2 — Discriminating Radiation Necrosis from Recurrent Tumor with [18F]PARPi and Amino Acid PET in Mouse Models (#516)

P. L. Donabedian1, S. Kossatz1, B. Carney1, 2, J. A. Engelbach3, 4, S. A. Jannetti2, 1, W. Weber1, J. Garbow3, 4, T. Reiner1

1 Memorial Sloan-Kettering Cancer Center, Radiology, New York, New York, United States of America
2 Graduate Center of the City University of New York, Ph.D Program in Chemistry, New York, New York, United States of America
3 Washington University, Radiology, St. Louis, Missouri, United States of America
4 Washington University, Alvin J. Siteman Cancer Center, St. Louis, Missouri, United States of America

Introduction

Radiation necrosis can appear similar to recurrent tumor on standard imaging. Current algorithms for this differential diagnosis require one or more follow-up imaging studies, long dynamic acquisitions, or complex image postprocessing. The best PET imaging protocol available for this setting is amino acid PET, which has not seen widespread adoption in the clinical environment. Using mouse models of both glioblastoma and radiation necrosis, we tested the potential of poly(ADP-ribose) polymerase (PARP)-targeted PET imaging with [18F]PARPi to better discriminate radiation necrosis from tumor.

Methods

The mouse model of radiation necrosis was generated by 60-Gy stereotactic irradiation of the left cerebral hemisphere. The glioblastoma mouse model was generated by intracranial injection of U251 MG cells.  PARP1 expression of both models was determined using immunohistochemistry. We chose [18F]Fluoroethyltyrosine ([18F]FET) as an amino acid PET tracer.  Both tracers were synthesized using cyclotron-produced fluorine-18, and DCE-MR and PET/CT imaging carried out on paired cohorts of mice. Analysis of PET data was carried out to quantify differences in tracer uptake in lesioned and contralateral brain regions between [18F]FET-PET and [18F]PARPi-PET in both glioblastoma and radiation necrosis mice.

 

Results/Discussion

In mice with experimental radiation necrosis, lesion uptake on [18F]PARPi-PET was barely higher than contralateral (lesion: 0.23±0.29 %ID/ccmean; contralateral: 0.15±0.20 %ID/ccmean; p = 0.73), while [18F]FET-PET clearly delineated the contrast-enhancing region on MR with a region of uptake averaging 1.7 times background (lesion: 9.7±3.2 %ID/ccmean; contralateral: 5.9±1.7 %ID/ccmean; p = 0.14). In mice bearing focal intracranial U251 xenografts, Visual delineation of the tumor from background was much easier on PARPi-PET than FET-PET, and both lesion-to-contralateral activity ratios (max/max, p = 0.034) and tumor-to-background ratios (max/mean, p = 0.009) were higher on PARPi-PET than FET-PET.

Conclusions

We present preclinical data showing that experimental murine radiation necrosis is not significantly [18F]PARPi-avid, and that [18F]PARPi-PET outperforms [18F]FET-PET in distinguishing radiation necrosis from focal intracranial xenografts. Efficient discrimination between recurrent tumor and radiation necrosis represents an added value for [18F]PARPi-PET used for postsurgical treatment planning and patient selection for treatment with pharmacological or radiotherapeutic PARP inhibitors.

References

1. Walker, A. J. et al. Postradiation imaging changes in the CNS: how can we differentiate between treatment effect and disease progression? Futur. Oncol. 10, 1277–1297 (2014).

2. Carney, B. et al. Non-invasive PET Imaging of PARP1 Expression in Glioblastoma Models. Mol. Imaging Biol. 18, 386–392 (2016).

3. Jiang, X. et al. A gamma-knife-enabled mouse model of cerebral single-hemisphere delayed radiation necrosis. PLoS One 10, 1–13 (2015).

Acknowledgement

This work was supported by the National Institutes of Health grants R01CA204441, R21CA191679 and P30CA008748. We also thank the MSKCC Center for Molecular Imaging and Nanotechnology, Small Animal Imaging Core Facility, Radiochemistry & Molecular Imaging Probes Core Facility and Molecular Cytology Core Facility.

DCE-MR and PARPi- and FET-PET/CT of radiation necrosis and tumor.
Left: Axial slices of DCE-MR (left columns) and fused PET/CT images (right columns) of animals with radiation necrosis (A) or U251 tumors (C) injected with [18F]PARPi (top rows) or [18F]FET (bottom rows). Right: Lesioned-to-contralateral (L/CL) whole-hemisphere %ID/ccmax ratios in radiation necrosis (B), U251 tumor (D), and treatment naive mice [18F]FET-PET and [18F]PARPi-PET images.

4:30 PM
PS-08-3 — Omics landscape of FDG-PET based heterogeneity in solid tumors. (#514)

M. A. Jarboui1, J. A. Disselhorst1, M. A. Krueger1, C. Trautwein1, P. Katiyar1, B. J. Pichler1

1 Werner Siemens Imaging Center, Eberhard Karls University Tuebingen, Tuebingen, Baden-Württemberg, Germany

Introduction

The heterogeneity observed within solid tumors and the myriad of genetic alterations shape tumors evolution and their molecular environment. Despite the development of quantitative analytical methods in molecular system biology, several studies struggle to capture the biological signature of tumor heterogeneity. Accordingly, we developed an image-guided milling machine (IGMM) that allows for accurate delineation of tumor tissues based on tracers uptake with subsequent multiplex omics analysis.

Methods

FDG-PET imaging was performed on an MMTV-PyMT breast cancer mouse model with highly heterogeneous tumors. Imaging data were used to delineate 131 regions of interest (ROIs) with differential FDG uptake in 24 mice. ROIs were isolated with our designed IGMM. Using a multiplex extraction method, metabolites and total proteins were isolated. Metabolites were analyzed using either, the Biocrates targeted metabolomic platform where metabolites were detected by LC-MS analysis (ABSCIEX QTRAP 6500) or by untargeted 1H NMR spectroscopy (600 MHz Bruker Avance III). Equal amounts of total protein from each ROI were acquired using the high-resolution LC-MS LTQ Orbitrap Fusion (Thermo Scientific). Acquired MS spectra were further analyzed using Maxquant label-free quantification algorithm (LFQ).

Results/Discussion

Unbiased PCA analysis of high versus low FDG uptake regions showed a clear separation based on protein and metabolites abundance. We detected alterations of specific tumor driver proteins, transcription factors, and interferon-induced proteins. The expression of the transcription factor Sp1, a hallmark of oncogenesis and zinc finger proteins, the largest transcription factor family, correlate with increased FDG uptake. Targeted metabolomic analysis showed an increase in glycogenic amino-acids and polyamines. While spermine, spermidine, kynurenine and putrescine levels positively correlate with FDG uptake, histamine and serotonin were negatively correlated. Free carnitine and short chains acylcarnitines were abundant in high uptake regions, unlike the long chains. Untargeted NMR data are currently under investigation, partial analysis of 10 samples subset of 10 metabolites show a strong positive correlation of taurine,  alanine, creatine/creatinephosphate and lactate with FDG uptake.

Conclusions

Our analysis shows a specific proteomic and metabolomic profile defined by the FDG-PET uptake of tumor regions within oncogenic tissue. Regions of high FDG uptake were characterized by high transcriptional activation, increased beta-oxidation, active amino-acid metabolism and polyamines accumulation. As we are finalizing data analysis and integration, our investigation provides a detailed molecular landscape of solid tumor heterogeneity and is the first attempt to use imaging-guided isolation of tumor regions in tandem with proteomics/metabolomics investigation.

4:40 PM
PS-08-4 — Effects of spacer region on the development of an αCD20 CAR construct (#519)

W. Al Rawashdeh1, J. Brauner1, S. Rüberg1, N. Mockel-Tinbrink1, T. Toepfer1, C. Barth1, D. Lock1, D. Schneider2, G. Rauser1, M. Jurk1, A. Kaiser1, O. Hardt1

1 Miltenyi Biotec, Bergisch Gladbach, Germany
2 Lentigen Corporation, Gaithersburg, United States of America

Introduction

Chimeric antigen receptor (CAR) modified T cells have emerged as the hottest topic in cellular immunotherapy as of late. T cells transduced with second generation CARs have demonstrated remarkable complete remission rates in patients with refractory B cell malignancies and the potency and persistence of CAR T cells have been shown to depend, in part, on the activating and co-stimulatory domains of the CAR construct. However, the choice of the spacer is also critical. Here, two αCD20 CAR T cell constructs, differing from the spacer region, were investigated as preclinical candidates.

Methods

Autologous T cells were transduced using lentiviral vectors to express one of two αCD20 CARs with different spacer domains using a 12-day TCT process on the CliniMACS Prodigy®, which performs all manufacturing steps in a single automated and closed system1. Cytotoxicity and cytokine release tests were assessed in vitro1. In vivo efficacy and persistence studies were performed using NSG mice and i.v. injection of hCD20+ lymphoma CDX (Raji), while sensitivity and specificity studies were performed using s.c. co-injection of wildtype and CD20-transduced melanoma CDX (Mel-526) in different percentages along with the CAR T cells. Therapy dose was normalized to 1x106 CD20 CAR+ cells in all in vivo studies. In vivo imaging was performed using IVIS Lumina III and XenoLight RediJect D-Luc Ultra.

Results/Discussion

TCT runs yielded ~6x109 CD3+ cells (viability ≥ 97%) and transduction efficiencies ˃10% (release specification) were always achieved. In vitro, cytokine release and cytotoxicity upon co-culture with CD20+ cells were comparable for both constructs (Fig. 1). However, CART-1 failed to produce any therapeutic effect in 2 independent in vivo studies (n=5, n=5) revealing a tumor burden similar to Mock-treated (n=5, n=7) and untreated controls (n=6, n=7) (Fig. 2). In contrast, CART-2 , efficiently eradicated hCD20+ cells (n=6, n=7). CART-2 cells persisted in vivo and were detected in blood, spleen and bone marrow many weeks post therapy, suggesting a durable clinical response. CART-2 did not cause any weight loss and no pathological alterations were found in 8 major organs by ex vivo histopathological analysis, indicating lack of toxicity. CART-2 displayed high specificity and sensitivity, where 2% hCD20+ cells were sufficient to delay tumor growth while hCD20- cells were not killed (n=30).

Conclusions

The impact of the different spacers on efficacy was not accurately reflected by in vitro studies and was only observed in vivo. This outcome motivates the efforts to find improved in vitro predictive assays. 

References

1. Lock. D, Mockel-Tenbrink. N, Drechsel. K, Barth. C, Mauer. D, Schaser. T, Kolbe. C, Al Rawashdeh. W, Brauner. J, Hardt. O, Pflug. N, Holtick. U, Borchmann. P, Assenmacher. M, Kaiser. A. “Automated manufacturing of potent CD20 directed CAR T cells for clinical use”. Hum. Gen. Ther. (2017) Oct;28(10):914-925

Fig. 1: Comparison of in vitro cytotoxicity of T cells transduced with the different αCD20 CAR const
CART-1 and CART-2 T cells demonstrated strong in vitro killing of CD20+ Jeko-1 cells, ~100% killing at 5:1 E:T ratio, in comparable to Mock-GFP T cells. In vitro killing of CART-1 and CART-2 was very similar.  

Fig.2: In vivo efficacy second study.
Total flux (p/s) shows that CART-2 achieved a highly significant therapeutic effect (p<0.01). Mice treated with CART-2 cells were the only group to achieve complete remission where the BLI signal was comparable to background (< 1x106 p/s) starting day 15, while CART-1 and Mock-GFP T cells had no therapeutic effect as the increase in tumor burden was similar to the untreated group (Tumor only).

4:50 PM
PS-08-5 — Characterizing, imaging and targeting triple negative breast cancer tumors and metastases (#542)

F. De Lorenzi1, L. Rizzo1, S. Von Stillfried2, F. Gremse1, C. Rijcken3, C. - C. Glüer4, F. Kiessling1, T. Lammers1

1 Institute for Experimental Molecular Imaging, Aachen, Germany
2 RWTH Aachen University, Pathology Institute, Aachen, Germany
3 Cristal Therapeutics, Maastricht, Netherlands
4 Kiel University, Department of Radiology and Neuroradiology, Kiel, Germany

Introduction

The lack of clinically established targeted therapies for triple negative breast cancer (TNBC) implies the need to systematically characterize primary tumors and secondary lesions in order to identify new therapeutic targets1. We here developed an optically imageable pre-clinical TNBC model which spontaneously metastasizes to distant organs, allowing non-invasive and longitudinal monitoring of cancer progression. Multiple imaging modalities were employed to characterize human and murine TNBC lesions as well as to address the biodistribution and targeting efficacy of nanomedicines.

Methods

4T1/iRFP breast cancer cells (680 nm) were orthotopically implanted in female nude mice. Solid tumor growth and metastases were monitored non-invasively through hybrid computer tomography and fluorescence molecular tomography (CT-FMT). Upon CT-based identification of metastases, mice were injected with fluorescently labeled micelles (750 nm) and monitored for nanoparticle (NP) biodistribution. Before sacrifice, mice were injected with lectin and excised organs were scanned by fluorescence reflectance imaging (FRI) to assess the colocalization of metastases (680 nm) and nanocarriers (750 nm). Human and murine primary and secondary lesions were stained for the characterization of vascular and stromal parameters, which taken together determine the targeting efficiency of NP on EPR-basis.

Results/Discussion

In vivo (passive) target site accumulation of micelles was seen over time in primary tumors (yellow arrows) and metastases (red arrows) (Fig 2A-B) over healthy organs (Fig 2C). The micelles signal (750 nm) overlaps with that of malignant cells (680 nm), at metastases which were macroscopically recognized at bright field images (Fig 2C). Histological characterization of the vasculature and stromal components (Fig 2D-I) gave evidence that metastases are more extensively vascularized than solid tumors, and comprise a denser ECM matrix (collagen fibers crosslinking through LOX). These factors might determine the targetability of malignant lesions. Additionally, further investigation of integrin targets on both murine and human primary tumors and metastases is currently ongoing through histopathology as well as proteomics and mass spectrometry-“based” methods.

 

Conclusions

This study evidences that metastases at different organs can be efficiently targeted with micellar nanomedicines, with a preferential accumulation at primary tumors in comparison to secondary lesions. Such systematic characterizations of microenvironmental factors influencing EPR and target site accumulation are of high relevance for the clinical translatability of nanomedicines and shall bring valuable insights into the discovery of new biomarkers for TNBC.

References

1"Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease", G. Bianchini et al., Nature Reviews Clinical Oncology volume13, pages674–690(2016)

Multimodal and non-invasive metastasis imaging

5:00 PM
PS-08-6 — Intratumoral activity distribution of 177Lu-PSMA-617 in a mouse model of prostate cancer (#543)

A. Örbom1, S. - E. Strand2, O. Vilhelmsson Timmermand1

1 Lund University, Oncology and Pathology, Lund, Sweden
2 Lund University, Medical Radiation Physics, Lund, Sweden

Introduction

Despite the promising results of PSMA-targeted peptide radionuclide therapy of disseminated prostate cancer, and its rapid translation into the clinic, very little is published on the small-scale activity, and consequently absorbed dose, distribution. A handful of studies have examined patient biopsies [1-2], but the limited literature using preclinical models seem to omit this aspect [3]. This study aims to study the intratumoral activity distribution of a therapeutic PSMA-targeted peptide, and compare it to histology, antigen expression and some diagnostic PET-tracers.

Methods

BALB/c nude male mice were given subcutaneous tumor xenografts of human PSMA-expressing LNCaP cells. PSMA-617 (ABX, Radeberg, Germany) was labeled with 177Lu and the mice (n=8, n=7 with tumors) were given 20 MBq by i.v. injection. The animals were sacrificed at 20 min (n=3, n=2 with tumors), 70 min (n=2), 90 min (n=1), and 120 min (n=2) after which blood, tumor, kidneys and salivary glands were collected, weighed and their activity measured by gamma counter. One kidney and part of the tumor was frozen on dry ice and cryosectioned at 10 µm thickness. Autoradiography was performed using a double-sided silicon strip detector (Biomolex Imager 700, Biomolex, Oslo, Norway) with a 50 µm intrinsic spatial resolution [4] after which the same tissue sections were stained with hematoxylin and eosin.

Results/Discussion

The percent injected activity per gram was low at all time-points in salivary glands (<1.1% IA/g), which do not express PSMA in this animal model, and blood (<1.7% IA/g). Median kidney uptake was 72.4% IA/g at 20 min p.i., 108.2% IA/g at 70 min p.i., and 24.8% IA/g at 120 min p.i. For the tumor, median uptake was 16.7% IA/g at 20 min p.i., 10.1% IA/g at 70 min p.i. and 21.0% IA/g at 120 min p.i. Activity distribution in the kidney was almost entirely in the cortex, where it exhibited a spotty uptake pattern at all time-points, possibly indicating uptake in the glomeruli (Figure 1). The tumor activity distribution appears to become more homogeneous over time, however there are still areas with viable tumor cells with comparably low uptake at 120 min p.i. (Figure 2).

Conclusions

The small-scale activity distribution influences the absorbed dose delivered to both tumor and risk organs and is relevant to the choice of fractionation schedule and radioprotective measures. This study has begun to investigare the small-scale activity distribution of 177Lu-PSMA-617 in a mouse model of prostate cancer and currently ongoing work will increase the sample sizes as well as include comparisons with uptake of some clinical tracers using multi-radionuclide imaging and with antigen distribution etc using immunohistochemistry.

References

1. Pyka, Thomas, et al. "68Ga-PSMA-HBED-CC PET for differential diagnosis of suggestive lung lesions in patients with prostate cancer." Journal of Nuclear Medicine 57.3 (2016): 367-371.

2. Schottelius, Margret, et al. "[111 In] PSMA-I&T: expanding the spectrum of PSMA-I&T applications towards SPECT and radioguided surgery." EJNMMI research 5.1 (2015): 68.

3.Benešová, Martina, et al. "Preclinical evaluation of a tailor-made DOTA-conjugated PSMA inhibitor with optimized linker moiety for imaging and endoradiotherapy of prostate cancer." Journal of Nuclear Medicine 56.6 (2015): 914-920.

4. Örbom, Anders, et al. "Characterization of a double‐sided silicon strip detector autoradiography system." Medical physics 42.2 (2015): 575-584.

Figure 1
Activity distribution within kidneys of mice injected with 177Lu-PSMA-617.

Figure 2
Bottom: Activity distribution within tumors of mice injected with 177Lu-PSMA-617. Top: The same tissue sections stained with hematoxylin and eosin.

5:10 PM
PS-08-7 — Generation of Trimodal Imaging Stem Cells for Quantification and Optimization of Stem Cell Cancer Therapy (#526)

M. Zaw-Thin1, R. Bofinger2, J. Connell1, P. S. Patrick1, D. Stuckey1, H. C. Hailes2, A. B. Tabor2, M. F. Lythgoe1, T. L. Kalber1

1 University College London, Centre for Advanced Biomedical Imaging, London, United Kingdom
2 University College London, Department of Chemistry, London, United Kingdom

Introduction

Stem cells have been used as selective anticancer agents1. In vivo imaging of stem cell distribution informs on methods needed to enhance cell migration to tumours. However, imaging cells distribution throughout the body is challenging and no single imaging modality can provide a complete answer. The aim of this study was to develop stem cells labelled with novel bimodal nanoparticles (SPECT/MRI) in combination with luciferase (Bioluminescence - BLI) to assess transplanted cell distribution from different injection routes and the ability of cells to home to tumours.

Methods

111In-SPION nanoparticle: DOTA was functionalised with an amine to form peptide bonds with carboxyls on superparamagnetic iron oxide nanoparticles (SPION)2. SPION were radiolabeled with 111In (HEPES-pH 5.5) with magnetic purification.

Cell labelling: luciferase positive human adipose derived stem cells (ADSC) were incubated overnight with 111In-SPION at 37 °C (Figure 1a). 1.5x105 ADSCs were injected intravenously (i.v) or intracardially (i.c) into NOD/SCID mice and imaged with SPECT/CT (Nanoscan-Mediso), MRI (ICON-Bruker) and BLI (IVIS-PerkinElmer) at day 0, 1, 3 and 7 after injection.

Tumour migration: 1.5x105 ADSCs were stimulated with IL-6 and SDF-1α (50 ng/ml)3 and injected i.v or i.c into NOD/SCID mice bearing a murine 4T1 orthotopic breast tumour and imaged with BLI as above.

Results/Discussion

SPECT/CT images from cell distribution study suggested that 1h after i.v injection, ADSCs were in lungs (50%) and liver (10%). This correlated with BLI and MRI (Figure 1b, c), which showed a reduction in T2 in the liver. 1h after i.c injection, SPECT/CT images showed ADSC distribution was predominantly in liver (42%), lungs (6%), kidney (2%) and brain (1%). This also correlated with BLI and MRI (Figure 1e, f), which showed focal hypointensities in the brain and kidney. SPECT and MRI co-registered images of liver and brain suggested the cells were dual labelled (Figure 1d, g).

BLI images from the tumour migration study suggested that there were ADSCs presence within the tumour as early as 1h after i.c injection, (Figure 2b). However, ADSCs were only detected within the tumour at day 3 after i.v injection (Figure 2a).

Conclusions

These results demonstrate the advantages of combining a genetic BLI reporter with a novel SPECT/MRI nanoparticle to quantitatively assess whole body distribution patterns of ADSC after different injection routes. The results from the tumour migration study suggest that the i.c injection route provides much more efficient ADSC delivery to tumour tissue. Future experiments will utilise this trimodal imaging strategy to quantitatively assess ADSC homing after cytokine stimulation and via different injection routes.

References

1) Sage et al.Thorax 2014;69:638. 2) Mitchell et al. Biomaterials 2013;34:1179. 3) Shi et al. Haematologica 2007;92:897.

Figure 1
(a) Diagram of ADSC labelled 111In-SPION & luciferase, (b, e) SPECT/CT images of ADSC distribution 1h after intravenous (i.v) (lung & liver) and intracardiac (i.c) injection (lungs, liver & kidneys), (c, f) BLI images of viable cells after i.v (lung) and i.c injection (brain, liver & kidneys), (d, g) Co-registered SPECT/MRI images of dual labelled cells after i.v (liver) and i.c injection (brain).

Figure 2

BLI images of 4T1 breast tumour bearing NOD/SCID mice showing the presence of ADSCs in the tumour (a) day 3 after intravenous injection and (b) 1h after intracardiac injection.

 

5:20 PM
PS-08-8 — In vivo monitoring of intracellular pO2 in response to CAR T cell immunotherapy against glioma (#540)

F. Chapelin1, W. Zhu2, H. Okada3, E. T. Ahrens2

1 University of California San Diego, Bioengineering, La Jolla, California, United States of America
2 University of California San Diego, Radiology, La Jolla, California, United States of America
3 University of California San Francisco, Neurological Surgery, San Francisco, California, United States of America

Introduction

Hypoxia is associated with tumor recurrence, and malignant progression1. Monitoring tumor pO2 levels can provide a preclinical biomarker for the effectiveness of emerging immunotherapies2. Perfluoro-crown-ether (PCE) nanoemulsion (NE) dissolves O2, causing a linear increase in the 19F spin-lattice relaxation rate (R1) with increasing pO2. PCE NE can intracellularly label tumor cells ex vivo pre-implantation. Using 19F MRI/MRS, we tested the hypothesis that an increase in pO2 is commensurate with CD8+ T cell apoptotic processes in a mouse model of glioma treated with human CAR T cells.

Methods

U87 glioma cells expressing EGFRvIII and luciferase were labeled ex vivo with PCE NE3 in media. Human T cells were transduced with a CAR vector4 to express a surface antibody against EGFRvIII. Female SCID mice (N=15) received unilateral subcutaneous injections of 5×106 PCE-labeled glioma cells. All mice were subjected to MRI and BLI four days post-tumor implantation, then received intravenous cell therapy (day 0). Groups 1-3 (N=5 per group) received 20×106 CAR T cells, 20×106 untransduced T cells, and no T cells, respectively. MRI and BLI scans were acquired on days 1, 3, 7, and 10. The 19F R1 was measured over entire tumor using PRESS to yield pO2 values, calculated with a calibration curve5. Tumors and spleens were harvested and fixed for histological correlation.

Results/Discussion

Prior to implantation, U87-EGFRvIII-Luc cells were labeled ex vivo with PCE NE to level ~7×1012 F-atoms/cell measured via 19F NMR. Following sub-cutaneous injection, labeled glioma cells appear as an MRI 19F hotspot with SNR~10 (Fig. 1A). Longitudinal in vivo measurements show a transient spike in tumor pO2 approximately three days after CAR T cell infusion (R1=0.99±0.12 s-1, pO2=134±25 mmHg) compared to untransduced T cells (pO2=61±20 mmHg) and control (pO2=40±9 mmHg, p = 0.026, Fig. 1B). These data suggest specific CAR T cell homing to the tumor tissue, presumably initiating a target killing cascade, and altering intracellular pO2. Longitudinal bioluminescence measurements show significant tumor regression 7 days post CAR treatment compared to  both control groups (p=0.012, Fig. 1C).  Histopathological staining confirmed the presence of CAR T cells in greater amounts than untransduced T cells in the tumors at day 3 post-infusion (data not shown6), consistent with the MRS results.

Conclusions

In this study, we show that 19F NE enables temporal measurements of tumor cell oxygen tension in response to CAR T cell therapy. Peak pO2 three days post-infusion suggests significant CAR T cell infiltration and tumor cell killing. Overall, these data support the view that 19F pO2 MRI and MRS can serve as a biomarker for cell-mediated apoptosis and provide insight into the modes of action of engineered T cell immunotherapy against cancer.

References

1 Tatum, J. L. et al. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol 82, 699-757, (2006).

2 Matsuo, M. et al. Magnetic resonance imaging of the tumor microenvironment in radiotherapy: perfusion, hypoxia, and metabolism. Semin Radiat Oncol 24, 210-217, (2014).

3 Kadayakkara, D.K.K., et al. In vivo observation of intracellular oximetry in perfluorocarbon-labeled glioma cells and chemotherapeutic response in the CNS using fluorine-19 MRI. Magn Reson Med 64(5): 1252–1259, (2010).

4 Ohno, M. et al. Expression of miR-17-92 enhances anti-tumor activity of T-cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts. J Immunother Cancer 1, 21, (2013).

5 Zhong, J. et al. In vivo intracellular oxygen dynamics in murine brain glioma and immunotherapeutic response of cytotoxic T cells observed by fluorine-19 magnetic resonance imaging. PLoS One 8, e59479, (2013).

6 Chapelin, F. et al. Fluorine-19 nuclear magnetic resonance of chimeric antigen receptor T cell biodistribution in murine cancel model. Scientific Reports, 18;7(1):17748 (2017).

Figure 1: In vivo longitudinal pO2 and luminescence changes in U87 tumors.
(A) In vivo 1H/19F MRI image showing labeled U87 cancer cells in mouse flank. (B) pO2 measurements following delivery of CAR T cells or controls. A significant increase in tumor pO2 in CAR T cell-treated animals is observed at day 3 (*, p = 0.026). (C) BLI shows twice lower radiance in CAR-treated animals compared to controls at day 7 (*, p = 0.01), representing significant tumor growth reduction.

1:30 PM
emptyVal-1 — Introductory Talk by Jolanda de Vries - Nijmegen, The Netherlands

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

1:50 PM
PS-15-2 — Positron emission tomography imaging of OX40+ activated T cells to monitor and predict response to an in situ tumor vaccine. (#69)

I. S. Alam1, 5, A. T. Mayer1, 5, I. Sagiv-Barfi2, K. Wang3, O. Vermesh1, 5, D. K. Czerwinski2, E. M. Johnson1, 4, 5, M. L. James1, 4, 5, R. Levy2, S. S. Gambhir1, 5

1 Stanford University, Radiology, Stanford, California, United States of America
2 Stanford University, Oncology, Stanford, California, United States of America
3 Harbin Medical University, Imaging Center, Harbin, China
4 Stanford, of Neurology and Neurological Sciences, Stanford, California, United States of America
5 Stanford University, Molecular Imaging Program, Stanford, California, United States of America

Introduction

Monitoring and predicting outcomes to cancer immunotherapy is challenging due to the highly varying and complex spatiotemporal dynamics of immune response in the tumor microenvironment. T cell activation is considered critical to treatment success across many classes of cancer immunotherapy and is likely more prognostic of treatment outcome than the presence of tumor infiltrating T cells alone. We report a novel PET tracer (64Cu-DOTA-mAbOX40) that enables non-invasive and longitudinal imaging of OX40, a cell surface marker expressed on antigen-specific activated T cells [1].

Methods

Anti-OX40 monoclonal antibody was DOTA-conjugated, radiolabeled with 64Cu and evaluated in vitro to assess its specificity for activated vs. resting murine T cells (supplementary Fig. A). Female BALB/c mice bearing dual A20 lymphoma tumors on the shoulders were administered low dose CpG oligonucleotide (50ug in PBS, n=7-10) or PBS alone directly in the left tumor only (vehicle control, n=7-10). PET/CT imaging of mice with 64Cu-DOTA-mAbOX40 (3.0-4.1MBq, i.v.) was performed at an early (day 2) and late (day 9) time point post-treatment initiation ( supplementary Fig. B). Flow cytometry analyses of tumor draining lymph nodes(TDLN), tumors, spleen from vehicle and CpG-treated mice was also performed at day 2 and 9 to determine OX40 expression alongside other T cell markers (CD3/4/8,44 and 25).

Results/Discussion

Early time point imaging post CpG administration revealed increased radiotracer uptake in the CpG-treated tumor [CpG 10.3±0.7; Veh 7.5±0.3 %ID/g; p<0.05] and associated TDLN [CpG 12.92 ± 1.15; Veh 10.67 ± 0.94%ID/g; p<0.01] (Fig. 1A- B). An increase in the frequency of OX40+CD3+ expression in these tissues vs untreated sites (p<0.05) and vehicle cohorts was further confirmed by FACS (Fig. 1C-D) suggesting in situ CpG vaccination triggered a local induction of cellular immune response. ViSNE, a visualization technique for high-dimensional cytometry data, showed that OX40+ cells were highly restricted to clusters associated CD4 helper T cells. Changes in OX40+ CD3 frequency preceded the increase in the overall proportion of CD3+ T cells within CpG-treated tumors. Importantly, early OX40-PET signal (mean %ID/g) in the local tumor environment was predictive of response at late time points [r2=0.746] with higher accuracy than anatomical  measurements (Supplementary Fig. C-D).

Conclusions

To our knowledge, this is the first report of an OX40 PET tracer that enables specific imaging of OX40+ effector T cell driven immune responses. In vivo, OX40-PET coupled with immunological and statistical techniques revealed new insights into response following in situ tumor vaccination with CpG, an adjuvant immunotherapy currently in clinical trials [2]. OX40-ImmunoPET provides a readily translatable approach for monitoring activated T cells with high sensitivity and specificity for clinical cancer immunotherapy strategies. 

References

[1] Weinberg AD, Morris NP, Kovacsovics-Bankowski M, Urba WJ, Curti BD. Science gone translational: the OX40 agonist story. Immunological reviews. 2011;244(1):218-231.

[2] Brody JD, Ai WZ, Czerwinski DK, et al. In situ vaccination with a TLR9 agonist induces systemic lymphoma regression: a phase I/II study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2010;28(28):4324-4332.

Acknowledgement

The authors would like to acknowledge the Stanford Center for Innovation in In-Vivo Imaging (SCI3). This work was supported in part by funding from the Ben & Catherine Ivy Foundation (SSG), the Canary Foundation (SSG), NCI R01 1 CA201719-02 (SSG), and the Leukemia and Lymphoma Society (RL).

Figure 1: OX40-ImmunoPET and characterization of OX40 expression in an in situ cancer vaccine model

A) PET/CT (VRT) images at day 2/day 9 post therapy (injected tumors-white arrows; distal tumor-blue arrows) B. 64Cu-DOTA-mAbOX40 uptake [mean +/- SEM, n=3-7). Two-way ANOVA with Bonferroni post-test for multiple comparisons.  C-D. OX40+ subset is restricted to CD4+ T cells and increases locally with CpG. One-way ANOVA with Bonferroni post-test. ****p < 0.0001 ***p < 0.001; ** p < 0.01; *p < 0.05. 

Supplementary Figure

A) PMA/Ionomycin or Dynabead activated T cells, showed increased OX40 expression vs. resting cells(p<0.001). Higher 64Cu-DOTA-mAbOX40 uptake in activated vs resting cells is significantly reduced in blocked & OX40-/- T cells. B) In vivo study design. C) Linear regression model: early PET signal in the local tumor is more predictive of response at late time point vs. D) Day 2 anatomic measurements.

2:00 PM
PS-15-3 — MicroSPECT/CT imaging to monitor subsequent changes in tumor PD-L1 expression after radiotherapy (#195)

P. J. Wierstra1, J. D. M. Molkenboer-Kuenen1, G. Sandker1, J. Bussink2, M. Gotthardt1, E. H. J. G. Aarntzen1, S. Heskamp1

1 Radboud University Medical Centre, Radiology and Nuclear Medicine, Radboud Institute of Molecular Life Sciences, Nijmegen, Netherlands
2 Radboud University Medical Centre, Radiation Oncology, Nijmegen, Netherlands

Introduction

Immune checkpoint inhibition (ICI) therapies have proven to be effective anti-cancer treatments. However, not all patients respond to these drugs and are exposed to unnecessary side-effects while alternative therapy is delayed. Increasing tumor PD-L1 expression with radiotherapy could be a strategy to optimize ICI treatment response. However, the expression of PD-L1 is a dynamic process, which can change during disease progression and treatment[1, 2]. Here, we investigated the effect of radiotherapy on tumor PD-L1 expression using microSPECT/CT imaging in mice with syngeneic tumors.

Methods

BALB/c mice were injected with tumor cell lines with differential expression of PD-L1; colorectal cancer CT26 cells (n=12) and C57/Bl6 mice were inoculated with melanoma B16-F1 (n=12) or Lewis lung carcinoma LLC1 cells (n=12) on the right hind legs. In half of the mice, tumors were irradiated with a single dose of 10 Gy. The next day, mice were injected with 23.8 ± 1.7 MBq 111In-anti-murine PD-L1 (111In-mPD-L1, 30 µg). After 24 h, microSPECT/CT imaging and ex vivo biodistribution studies were performed; together with immunohistochemical analysis of tumor PD-L1.

Results/Discussion

Uptake of 111In-mPD-L1 was significantly increased in CT26 tumors after irradiation (26.3 ± 2.0 vs. 17.1 ± 3.1%ID/g, p = 0.003). A smaller, but significant effect was observed for LLC1 (15.7 ± 1.8 versus 12.3 ± 1.7 %ID/g, p = 0.033). For B16-F1 tumors, the difference in tracer uptake between irradiated vs. non-irradiated tumors was not significant (16.7 ± 3.5 vs. 14.9 ± 6.8 %ID/g). Uptake in draining lymph nodes of the tumor was increased in LLC1 and B16-F1 tumor bearing mice (LLC1: 11.6 ±1.7 versus 9.0 ± 0.8 %ID/g, p = 0.036, B16-F1: 13.1 ± 1.7 versus 7.6 ± 1.2 %ID/g, p = 0.002). No significant differences in splenic uptake were observed. Immunohistochemical staining showed a striking upregulation of tumor PD-L1 in CT26 tumors, and moderate increase in LLC1 tumors, which was related to increased PD-L1 expression.

Conclusions

In this study we demonstrated that radiation induced an upregulation of PD-L1 expression in CT26 and LLC1 xenografts. Also, we demonstrated that this dynamic expression of PD-L1 can be monitored and quantified non-invasively using 111In-mPD-L1 mAb imaging. Studies on the effects radiotherapy on PD-1L expression are ongoing and will allow rational design of novel combination therapies in which radiotherapy complements ICI. Visualization of PD-L1 has the potential to determine when a window of ICI treatment opportunity occurs, during which patients are likely to respond to better to ICI therapy.

References

  1. Taube, J.M., Unleashing the immune system: PD-1 and PD-Ls in the pre-treatment tumor microenvironment and correlation with response to PD-1/PD-L1 blockade. Oncoimmunology, 2014. 3(11): p. e963413.
  2. Vilain, R.E., et al., Dynamic changes in PD-L1 expression and immune infiltrates early during treatment predict response to PD-1 blockade in melanoma. Clin Cancer Res, 2017.

Comparison in 3 cell lines of radiotherapy induced changes in tumor PD-L1 expression
MicroSPECT/CT images (top panel) of mice with irradiated(10 Gy) and non-irradiated tumors 1 day post injection of 30 µg 111In-anti-mPD-L1. Middle panel shows the immunohistochemical analysis of PD-L1 expression of these tumors and the bottom panel shows the quantification of the uptake of 111In-anti-mPD-L1 in tumors, lymph nodes, and spleen.

2:10 PM
PS-15-4 — Immune imaging of human PD-L1 levels in cancer using single domain antibodies (#290)

K. Broos1, Q. Lecocq1, J. Bridoux2, G. Raes3, 4, C. Xavier2, M. Keyaerts2, 5, N. Devoogdt2, K. Breckpot1

1 Vrije Universiteit Brussel, Laboratory for molecular and cellular therapy, Jette, Belgium
2 Vrije Universiteit Brussel, 2In Vivo Cellular and Molecular Imaging, Jette, Belgium
3 Vrije Universiteit Brussel, 3Cellular and Molecular Immunology, Brussels, Belgium
4 Vlaams Instituut voor biotechnologie, 4Myeloid Cell Immunology Lab, Ghent, Belgium
5 UZ Brussel, 5Nuclear Medicine Department, Jette, Belgium

Figure 1

(A) SPECT/CT images to determine the uptake of 99mTc-labeled Nb K2 in athymic nude mice bearing PD-L1 negative ( PD-L1-; left; n=4)  or PD-L1 positive (PD-L1+ ; right; n=6) 624 mel-cells (n = 6).

(B) Gamma counting to determine the %IA/g uptake of sdAb K2 in a PD-L1 negative (n=4) or PD-L1 positive tumor (n=6).

2:20 PM
PS-15-5 — Molecular imaging of anti-EGFR UniCAR immunotherapy (#242)

R. Bergmann1, S. Albert2, A. Feldmann1, N. Berndt3

1 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Saxony, Germany
2 University Cancer Center (UCC) ‘Carl Gustav Carus’ TU Dresden, Tumor Immunology, Dresden, Saxony, Germany
3 Nationales Centrum für Tumorerkrankungen, DKFZ, Heidelberg, Baden-Württemberg, Germany

Introduction

Adoptive T-cell therapy with chimeric antigen receptor (CAR) engineered T cells (CAR T cells) has shown clinical efficacy in hematologic malignancies with more modest responses when targeting solid tumors. However, CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells cross react with healthy tissues. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner that will be reported.

Methods

The UniCAR-binding domain is based on the mAb anti-La 5B9 that recognizes a continuous sequence of ten amino acids (E5B9 tag) of the nuclear protein La/SS-B. The TM consists of an anti EGFR domain that was conjugated with the E5B9 tag. The in vivo killing of A431-Luc+ cells was measured by luciferase assay. Conjugation of the TM with p-SCN-Bn-NODAGA resulted in a protein with 3 attached NODAGA moieties and was 64Cu-labeled according standard methodologies reaching specific activities larger than 80 GBq/µmol TM. The 64Cu-TM were i.v. and s.c. injected without or together with A431 and/or UniCAR T-cells in NMRI nu/nu mice; and the in vivo kinetics was measured by small animal PET.

Results/Discussion

The 64Cu-TMs were fast distributed in the body after i.v. injection. Main acceptors of the 64Cu-TMs are the kidneys; after 4 h 20 ± 3%ID of the TMs were trapped in the kidney cortex, while 10 ± 9%ID were eliminated into the urine, and 8 ± 3%ID were accumulated in the liver. The 64Cu-TMs accumulated in the tumors with a peak time of 1.7 h with an activity concentration of 2.5 ± 0.8 (SUV) that was changed to 0.33 ± 0.03 after 28 h. The delivery half-lifes of the 64Cu-TMs from the subcutaneous injection sites were 1.7 h (64Cu-TM), 7.0 h (64Cu-TM & UniCAR T-cells), 15.5 h (64Cu-TM & UniCAR T cells & A431 cells), and 19.4 h (64Cu-TM & A431 cells),  the TMs delivered into the circulation were immediately trapped by the kidney; as result the activity in the kidneys increased linearly over the time. The luminescence imaging of the tumors showed the efficient killing by the UniCAR system.

Conclusions

The 64Cu-TMs have a well-defined clearance rate from the circulation, the subcutaneous injection site and from the tumors. The combination of different imaging technologies and the knowledge about the 64Cu-TM kinetics in vivo, accounting for all involved potential interactions with the respective UniCAR components, should allow defining optimal conditions for the application of UniCARs in immunotherapy, like the time of activation of the UniCAR T cells, and consequently interrupting an ongoing therapy if necessary in case of severe side effects occur.

Kinetics of [64Cu]Cu-α-EGFR TM (pro) (NODAGA)1.2
Kinetics of [64Cu]Cu-α-EGFR TM (pro) (NODAGA)1.2 in selected organs and tissus and orthogonal images of the TM accumulation in the NMR Foxn1 nu/nu mouse bearing a A431 tumor after 90 min.

Retargeting Therapy of EGFR-positive tumor cells
Luminescence images of the subcutaneous injection sites of the control and treatment groups in the UnCAR anti-EGFR therapy.

2:30 PM
PS-15-6 — In vivo tracking of CAR-T by [18F]BF4-PET/CT in human breast cancer xenografts reveals differences in CAR-T tumour retention. (#112)

E. Kurtys1, L. Lim1, F. Man1, A. Volpe1, G. O. Fruhwirth1, 2

1 King's College London, 1. Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom
2 King's College London & UCL, Comprehensive Cancer Imaging Centre King's College London & UCL, London, United Kingdom

Introduction

Genetically modified T cells are emerging anti-cancer immunotherapeutics. Challenges for T cell immunotherapy of solid tumours include on-target off-site toxicities against healthy tissues, in vivo relocalization reducing on-site efficacy, and cytokine storm. Non-invasive cell tracking can characterise in vivo cell therapy distribution and relocalization kinetics as well as identify off-site targets.

Our goal was to quantify tumour retention and potential relocalization of an anti-ErbB family chimeric antigen receptor T cell (CAR-T) in mouse models of triple-negative human breast cancer (TNBC.)

Methods

The T1E28z CAR-T1 was made in vivo traceable by PET using the human sodium iodide symporter (NIS) as a reporter, which was additionally fused to red fluorescent protein (RFP)2 to aid preclinical studies. NIS-RFP:CAR-T were generated by lentiviral transduction, selectively expanded via co-expressed IL-4:IL-2/15 receptor chimera3, and fully characterized in vitro for reporter (expression, localization, radiotracer uptake) and CAR function (IFNγ release, tumour cell killing). MDA-MB-436 and MDA-MB-231 breast tumours were established orthotopically in young-adult female immunodeficient NSG mice. NIS-RFP:CAR-T was intratumorally injected. Tumour retention was quantified by PET/CT over weeks. Subsequent to animal culling, tissues were collected for ex-vivo analyses (γ-counting, histology).

Results/Discussion

In vivo traceable NIS-RFP:CAR-T were found to be fully functional (reporter and CAR) without negative impacts of radiolabelling (Fig.1). NIS-RFP:CAR-T were successfully detected at various concentration levels using [18F]BF4- PET/CT imaging. Only live NIS-RFP:CAR-T were detectable due to NIS function being coupled to an intact Na+/K+-gradient. Intratumourally administered NIS-RFP:CAR-T were alive in vivo for at least 15 days. In MDA-MB-436 tumours (Fig.2), [18F]BF4- uptake significantly increased 7d post administration (13.0±1.9, N=5 vs. 9.0±3.1, N=9, P=0.0194) suggesting on-site expansion of traceable NIS-RFP:CAR-T cells. After 15d, [18F]BF4- uptake was comparable to day 1 levels (8.86±2.8, N=9, P=0.9493). Interestingly, the [18F]BF4- uptake was significantly lower after 15d in highly metastatic MDA-MB-231 cells (20.3±9.6% of day 1; P=0.0069; N=4), suggesting relocalization or on-site cell therapy death. Ex vivo γ-counting and histology confirmed in vivo imaging results.

Conclusions

NIS reporter gene imaging by [18F]BF4- -PET is a sensitive tool for monitoring anti-cancer T cell therapy in breast cancer. PET imaging informed on T cell tumour presence, which is a prerequisite for their therapeutic effect. In vivo imaging allowed quantification of NIS-RFP:CAR‑T retention in primary tumours and identified tumour model-specific differences. Further research is required to elucidate the molecular reasons for the observed differences of anti-ErbB CAR-T retention in TNBC models.

References

1Davies et al., Mol Med, 2012; 18:565-76.

2Diocou et al, Sci Rep 2017; 7(1):946

3Wilkie S. et al. J Biol Chem 2010; 285(33):25538-44.

Acknowledgement

We would like to thank Dr Maher for the T1E28z CAR, and Cancer Research UK, EPSRC, Worldwide Cancer Research and King’s Health Partners for financial support.

Fig 1 In vitro expansion and validation of NIS-RFP:CAR-T cells.

Fig.2 PET imaging and ex-vivo biodistribution of [18F]BF4- in the MDAMB436 xenograft tumour model.

2:40 PM
PS-15-7 — Metabolic profiling of secondary lymphatic organs by 18F-FDG-Positron Emission Tomography (PET)/CT in checkpoint inhibitor immunotherapy (CIT) patients with metastatic melanomas as a novel tool to predict the immune response (#401)

J. Schwenck1, 2, B. F. Schörg1, F. Fiz2, K. Wistuba-Hamprecht3, A. Forschner3, T. Eigentler3, B. Weide3, C. Garbe3, M. Röcken3, C. Pfannenberg4, B. J. Pichler1, C. la Fougère2, M. Kneilling1, 3

1 Eberhard Karls University, Werner Siemens Imaging Center, Tübingen, Baden-Württemberg, Germany
2 Eberhard Karls University, Department of Nuclear Medicine and Clinical Molecular Imaging, Tübingen, Baden-Württemberg, Germany
3 Eberhard Karls University, Department of Dermatology, Tübingen, Baden-Württemberg, Germany
4 Eberhard Karls University, Department of Diagnostic and Interventional Radiology, Tübingen, Baden-Württemberg, Germany

Introduction

CIT prolongs the overall survival in the majority of patients with metastatic melanoma but still a large percentage of patients does not benefit for unknown reasons. Although the knowledge about the exact mechanisms of CIT is limited, there is evidence that a systemic immune response is needed to enable an effective CIT-induced anti-tumor response. Aim of our retrospective study was to identify CIT responders by analysis of CIT-specific alterations of the glucose metabolism in secondary lymphoid organs such as the spleen of metastatic melanoma patients by 18F-FDG-PET.

Methods

In experimental tumor models we were able to differentiate between effective and non-effective immunotherapies by analyzing the 18F-FDG-uptake in the spleen. Thus, we  investigated retrospectivly 18F-FDG-PET/CT scans of 38 patients with metastatic melanoma pre- and post-therapy with CTLA-4 or PD-1 Ab (21 responder: 5x nivolumab; 7x pembrolizumab; 9x ipilimumab; 17 non-responder: 2x nivolumab; 11x pembrolizumab; 4x ipilimumab). Regions of interest (ROI) in the spleen were defined in the CT images and copied to the coregistered PET for semiquantitative analysis. Total lesion glycolysis (TLG) was calculated by multiplication of the spleen volume and the SUVmean.

Results/Discussion

We observed no significant differences between responders and non-responders in the baseline 18F-FDG-PET/CT-scans before CIT) neither by analyzing the spleen volume (221±18 cm3 vs. 209 ±22 cm3) nor the spleen 18F-FDG-uptake (SUVmean: 1,74±0,06vs.1,72±0,05; TLG: 384±37 vs. 359±36).

After onset of CIT the follow up 18F-FDG-PET/CT-scans provided a comparable increase in spleen volume in responders (+8±6%) and non-responders (+7±5%), but 15 out of 21 responders showed an a higher 18F-FDG uptake in spleen when compared to the baseline 18F-FDG-PET/CT-scans. The mean standard uptake values in the spleen of responders increased by +10±9% SUVmean, while hardly any change were observed in non-responders (SUVmean -1,3±2,6%). Furthermore, the total lesion glycolysis (TLG) in the CIT-responders increased stronger (+25±22%) than in non-responders (+6±6%).

Conclusions

The results of our retrospective study imply an association of the CIT-induced anti-tumor therapy effect with metabolic changes in secondary lymphatic organs, which was detectable by non-invasive 18F-FDG-PET/CT. Whether this may represent a novel powerful tool to monitor CIT-induced systemic immune responses, has to be uncovered by preclinical research focusing on the exact mode of action of the CIT-induced systemic immune response and prospective clinical studies to evaluate the prognostic value.

2:50 PM
PS-15-8 — Visualizing hypoxia-mediated immunosuppression in bone metastasis (#458)

B. Weigelin1, 2, M. Giampetraglia1, W. Tian1, E. Dondossola1, D. Hutmacher3, X. Lu4, R. de Pinho4, C. Logothetis1, P. Friedl2, 1

1 MD Anderson Cancer Center, Genitourinary Medical Oncology and Koch Center, Houston, United States of America
2 Radboud University Medical Center, Cell Biology, Nijmegen, Netherlands
3 Queensland University of Technology, Brisbane, Australia
4 MD Anderson Cancer Center, Cancer Biology, Houston, United States of America

Introduction

For many cancer types, immunotherapy has the potential to reach long-lasting remission in patient subsets. However, bone metastases typically resist immunotargeting, implicating the bone stroma as contributor to cancer resistance. Despite high vascular density, local oxygen tension in the bone marrow is overall low and heterogeneous (0.5 – 5 % in mice). Using advanced microscopy, we here tested whether hypoxia, via activation of hypoxia-inducible factor-1 alpha (Hif-1a) contributes to local cancer resistance towards T cell-mediated effector function in bone.

Methods

B16F10 melanoma and PPSM prostate cancer cells expressing the ovalbumin antigen were confronted with activated OT-1 CTL in vitro and in vivo. By combining a collagen-based organotypic assay and an automated image segmentation workflow, we developed a 3D medium-throughput screening assay to identify immunosuppressive conditions derived from the bone microenvironment. Results were validated in bone metastatic lesions, relating CTL position and function to vascularization, hypoxia and Hif-1a expression using bone clearing (Bone CLARITY) and 3D reconstruction of the mouse tibia by multiphoton microscopy. To monitor CTL killing efficacy in live tissue, cancer cells were implanted in a subcutaneous tissue-engineered bone ossicle which allows for intravital imaging at single-cell resolution.

Results/Discussion

Using the 3D cytotoxicity assay, we found that prostate cancer cells develop resistance towards CTL-mediated killing at oxygen levels of 1.5 % while lower oxygen levels (0.5 %) were required to induce resistance in melanoma cells. Resistant cancer cell subsets showed nuclear accumulation of Hif-1a and re-sensitization was achieved by blocking Hif-1a activation using glyceryl trinitrate. Resistance was not associated with upregulation of PD-L1 and CTL remained negative for the immune checkpoint molecules PD1 and VISTA. Monitored through an imaging window or by 3D reconstruction after tissue clearing, PPSM/OVA cells were poorly targeted by CD8 CTL. CTL infiltration was dependent on the size of the lesion and the antigen-specificity of the CTL. Large, established lesions typically excluded CD8 T cells while small, early microlesions were efficiently infiltrated. Both, large and small lesions were resistant despite systemic application of OVA-specific adoptively-transferred OT1 CTL.

Conclusions

By combining in vitro screening, ex vivo whole-organ 3D reconstruction using tissue clearing, and in vivo dynamic intravital imaging, this workflow will serve to identify the cellular mechanisms that compromise immune-effector function and provide rationales for therapeutic combinations to enhance immunotherapy in bone.

6:15 PM
emptyVal-1 — Introductory Talk by Go van Dam - Groningen, The Netherlands

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

6:35 PM
PS-09-2 — Standardization of intra-operative fluorescence imaging systems using a multi-parameter solid phantom (#402)

D. Gorpas1, 2, M. Koch1, 2, M. Anastasopoulou1, 2, U. Klemm1, V. Ntziachristos1, 2

1 Helmholtz Zentrum München, Institute of Biological and Medical Imaging (IBMI), Neuherberg, Bavaria, Germany
2 Technical University of Munich, Chair of Biological Imaging, Munich, Bavaria, Germany

Introduction

Fluorescence guided surgery is emerging into a significant tool for oncology. Limitations in human vision and tactile feedback require post-surgical pathology assessment [1]. Fluorescence imaging has shown promising results in clinical studies. Nevertheless, it has not yet been integrated into the standard clinical practice. This, in part, is due to the lack of standardization methods that enable repeatability of studies and comparison of systems [2]. In this work, we propose a novel composite phantom that allows characterization of intra-operative fluorescence imaging systems.

Methods

We used transparent polyurethane for the phantom matrix, while organic quantum dots, TiO2 nanoparticles, and nigrosin in the phantom matrix and Hemin in the wells simulated fluorescence, scattering and absorption respectively. The phantom wells assess sensitivity, fluorescence and optical resolution, light cross-talk, and enable flat-fielding (Fig. 1a-b). To test the phantom, we employed a hybrid imaging system (Camera I), consisting of an electron multiplying CCD (EMCCD) for fluorescence detection and a CCD camera for color imaging [3]. A second system (Camera II), without the color channel, was also employed to demonstrate the phantom application for benchmarking (Fig. 1c-d). Following phantom imaging we segmented the wells [4] and quantified the corresponding SNR and contrast.

Results/Discussion

The results demonstrate the phantom application to characterize an imaging system, as well as to benchmark systems of markedly different specifications. Specifically for Camera I, besides the quantification of the assessment metrics, imaging of the phantom further enables registration between fluorescence and color data, and augmentation of color images with fluorescence information. Images are also corrected for possible vignetting due to illumination inhomogeneities (Fig. 1e-f). The comparison of the two systems showed that Camera I can achieve ~30% higher SNR and ~45% higher contrast than Camera II for all wells assessing sensitivity (Fig. 2). Nevertheless, Camera II can perform equivalently to Camera I when its working distance is reduced by ~40% and 2× binning is applied (Fig. 2). To identify which of the acquisition settings of Camera II result into equivalent to Camera I performance, we adopted a least squares method between all metrics quantified through the phantom.

Conclusions

Lack of fluorescence imaging standardization is one of the major factors delaying clinical translation of this exciting technology [1, 5]. Most current approaches quantify one performance parameter [5]. The study herein introduced a multi-parametric phantom for the characterization of fluorescence imaging systems. This procedure can also guide the configuration of different systems for comparable performance. The latter is essential for multicenter clinical trials. We envision that composite phantoms will become important assets for clinical translation of fluorescence molecular imaging.

References

[1] M. Koch, and V. Ntziachristos, 2016, "Advancing Surgical Vision with Fluorescence Imaging", Annu Rev Med, 67(1): 153-164.
[2] A. V. Dsouza, H. Lin, E. R. Henderson, K. S. Samkoe, and B. W. Pogue, 2016, "Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging", J Biomed Opt, 21(8): 080901.
[3] J. Glatz, J. Varga, P. B. Garcia-Allende, M. Koch, F. R. Greten, and V. Ntziachristos, 2013, "Concurrent video-rate color and near-infrared fluorescence laparoscopy", J. Biomed. Opt., 18(10): 101302.
[4] H. Bay, A. Ess, T. Tuytelaars, and L. Van Gool, 2008, "Speeded-Up Robust Features (SURF)", Comput. Vis. Image Und., 110(3): 346-359.
[5] B. Zhu, J. C. Rasmussen, and E. M. Sevick-Muraca, 2014, "A matter of collection and detection for intraoperative and noninvasive near-infrared fluorescence molecular imaging: To see or not to see?", Med. Phys., 41(2): 022105.

 

 

Acknowledgement

The authors would like to thank Dr. Pilar Beatriz Garcia-Allende for her contribution during the phantom preparation. The research leading to these results has received funding by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich-824 (SFB-824), subproject A1 and from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 644373 (PRISAR).

Fig. 1. Application of composite standardization phantom for fluorescence camera assessment.
(a) Phantom. (b) Phantom wells. Sensitivity vs red-optical properties and blue-depth; purple-resolution; pink-cross-talk; green-illumination; cyan-matrix. (c) Camera I. (d) Camera II. D: diffuser; F: filter; DM: dichroic mirror; RL: relay lens. (e) Intensity of scattering wells (top left); light profile (top right); raw image (bottom left); corrected image (bottom right). (f) Augmented image.

Fig. 2 Quantitative comparisons between Camera I and Camera II.
(a) SNR, and (b) contrast from the nine wells with different optical properties. (c) SNR, and (d) contrast from the nine wells with different depths from the phantom’s top surface. In all panels, x -axis labeling corresponds to the labeling of phantom elements shown in Fig. 1(b).

6:45 PM
PS-09-3 — [18F]fluoroethyltyrosine-induced Cerenkov luminescence improves image-guided surgical resection of glioma (#93)

D. Lewis1, 2, R. Mair1, A. Wright1, K. Allinson4, S. Lyons1, T. Booth1, J. Jones1, R. Bielik1, D. Soloviev1, 2, K. M. Brindle1, 3

1 University of Cambridge, Cancer Research UK - Cambridge Institute, Cambridge, United Kingdom
2 Cancer Research UK – Beatson Institute, Glasgow, United Kingdom
3 University of Cambridge, Department of Biochemistry, Cambridge, United Kingdom
4 Cambridge University Hospitals NHS Foundation Trust, Department of Pathology, Cambridge, United Kingdom

Introduction

The extent of surgical resection is significantly correlated with outcome in glioma 1, however current intraoperative navigational tools such as fluorescence imaging with 5‑aminolevulinic acid (5-ALA) are effective only in a subset of patients 2,3.  We show here that a new optical intraoperative technique, Cerenkov luminescence imaging (CLI) following intravenous injection of O‑(2-[18F]fluoroethyl)-L-tyrosine (FET), can be used to accurately delineate glioma margins, performing better than the current standard of fluorescence imaging with 5-aminolevulinic acid (5-ALA).

Methods

Rats implanted orthotopically with U87, F98 and C6 glioblastoma cells were injected with FET and 5-aminolevulinic acid (5-ALA).  Positive and negative tumor regions on histopathology were compared with CL and fluorescence images.  The capability of FET CLI and 5-ALA fluorescence imaging to detect tumor was assessed using receptor operator characteristic curves and optimal thresholds (CLIOptROC and 5-ALAOptROC) that separated tumor from healthy brain tissue were determined.  These thresholds were used to guide prospective tumor resections, where the presence of tumor cells in the resected material and in the remaining brain were assessed by Ki-67 staining.

Results/Discussion

FET CLI signal was correlated (y = 1.06x – 0.01; p < 0.0001; Fig 1a) with signal in preoperative PET/MR images (Fig 1b,c) and with expression of the amino acid transporter SLC7A5 (LAT1). FET CLI (AUC = 97%) discriminated between glioblastoma and normal brain in human and rat orthografts more accurately than 5-ALA fluorescence (AUC = 91%), with a sensitivity >92% and specificity >91% (Fig 1d-i), and resulted in a more complete tumor resection (Fig 2). 

Conclusions

FET CLI can be used to accurately delineate glioblastoma tumor margins intraoperatively, performing better than the current standard of fluorescence imaging following 5-ALA administration, and is therefore a promising technique for clinical translation.

References

1. Hervey-Jumper, S.L. & Berger, M.S. Maximizing safe resection of low- and high-grade glioma. Journal of neuro-oncology 130, 269-282 (2016).

2. Lau, D., et al. A prospective Phase II clinical trial of 5-aminolevulinic acid to assess the correlation of intraoperative fluorescence intensity and degree of histologic cellularity during resection of high-grade gliomas. Journal of Neurosurgery 124, 1300-1309 (2016).

3. Jaber, M., et al. The Value of 5-Aminolevulinic Acid in Low-grade Gliomas and High-grade Gliomas Lacking Glioblastoma Imaging Features: An Analysis Based on Fluorescence, Magnetic Resonance Imaging, 18F-Fluoroethyl Tyrosine Positron Emission Tomography, and Tumor Molecular Factors. Neurosurgery 78, 401-411 (2016).

Figure 1

CLI with [18F]FET more accurately delineates GBM from brain tissue than 5-ALA imaging. (a) FET CLI in tumor slices correlates with PET signal. (b) CE T1-w MRI of a U87 GBM and (c) FET PET/ T2-w MRI. H&E of a U87 GBM (d), with FET CLI (e), 5-ALA (g) and segmentation. (h) ROC curves for FET CLI (blue) and 5-ALA (red) (n≥5). (i) AUC of the ROC, sensitivity and specificity with FET CLI and 5-ALA (n≥5)

Figure 2
Image-guided surgical resection is improved using CLI of [18F]-FET (a) compared to fluorescence imaging with 5-ALA (b).  Images acquired before and after two surgical resections of the tumor. (c) Ki-67 staining of the resected tumor specimens (#) and (##), showing positive staining for tumor cells and negative Ki-67 in the remaining brain (###). 

6:55 PM
PS-09-4 — PET/CT and near-infrared fluorescence imaging of colorectal cancer using a single injection of a dual-labeled cRGD-based tracer (#547)

B. Sibinga Mulder1, H. Handgraaf1, D. Vugts2, C. Sewing2, M. Stammes3, L. de Geus-Oei4, A. Windhorst2, M. Bordo5, J. Mieog1, C. van de Velde1, J. Frangioni5, A. Vahrmeijer1

1 LUMC, Surgery, Leiden, Netherlands
2 VUMC, Nuclear Medicine, Amsterdam, Netherlands
3 LUMC, Radiology, Leiden, Netherlands
4 LUMC, Nuclear Medicine, Leiden, Netherlands
5 Curadel, Boston, United States of America

Introduction

Hybrid tracers, allowing both positron emission tomography (PET) and near infrared fluorescence (NIRF) imaging, can aid in accurate preoperative surgical planning and intraoperative real-time NIRF detection of the tumor. cRGD peptide targets integrins associated with angiogenesis (e.g. αvβ3) and has been used successfully for both PET and NIRF imaging. This study evaluates the hybrid tracer ZW800F-cRGD-[89Zr]Zr-DFO for PET and NIR fluorescence imaging in human colorectal xenografts.

Methods

An in vitro binding assay was performed and subsequently 10 nmol ZW800F-cRGD-Zr-DFO was injected in mice (n=7) bearing orthotopic colorectal tumors (HT29-luc2). NIRF imaging for detection of the tumor specific signals was performed at 4 and 24 h, the biodistribution was determined. For assessment of the stability of the signal, NIRF imaging was performed up to 168 h in mice bearing subcutaneous tumors. Subsequently, ZW800F-cRGD-Zr-DFO was synthesized and labeled with 89Zr. Finally, 10 nmol ZW800F-cRGD-[89Zr]Zr-DFO (3 MBq) was injected to mice (n=8) bearing orthotopic colorectal tumors (HT29-luc2). PET/CT was performed at 1, 4 and 24 h post injection. Biodistribution was performed at 4 and 24 h post injection.

Results/Discussion

The in vitro binding assay demonstrated an almost linear increase in fluorescence intensity with increasing concentrations. Sufficient fluorescent signals were measured in the tumors of the mice injected with ZW800F-cRGD-Zr-DFO (emission peak ~800nm). The fluorescence signal of ZW800F-cRGD-[89Zr]Zr-DFO remained stable after labelling with zirconium-89 and PET/CT at 4 h allowed clear visualization of the colorectal tumors. Biodistribution at 4 h showed the highest uptake of the tracer in kidneys and sufficient uptake in the tumor. At 24h the uptake in the tumor was still sufficient.

 

Conclusions

This study shows the feasibility of PET and fluorescence imaging of tumors with a single injection of ZW800F-cRGD-[89Zr]Zr-DFO, allowing preoperative surgical planning followed by intra-operative imaging.

Tumor specific signals
Concordant bioluminescent, fluorescence and PET/CT tumor specific signals 4 h after injection with ZW800F-cRGD-[89Zr]Zr-DFO.

7:05 PM
PS-09-5 — Development and characterization of a multi-modality anti-PSMA targeting agent for imaging, surgical guidance, and targeted photodynamic therapy of PSMA-expressing tumors (#75)

S. Lütje1, 2, S. Heskamp1, G. M. Franssen1, C. Frielink1, A. Kip1, G. Fracasso3, K. Herrmann2, M. Gotthardt1, M. Rijpkema1

1 Radboudumc, Radiology and Nuclear Medicine, Nijmegen, Netherlands
2 University Hospital Essen, Nuclear Medicine, Essen, Germany
3 University of Verona, Pathology and Diagnostics, Verona, Italy

Introduction

Prostate cancer (PCa) recurrences after surgery frequently occur. To improve management of PCa, the multi-modality anti-PSMA targeting agent 111In-DTPA-D2B-IRDye700DX was developed and characterized in vivo. This agent can be used for both pre- and intra-operative tumor localization, image-guided surgery, and eradication of (residual) tumor tissue by PSMA-targeted photodynamic therapy (tPDT), which is a highly selective cancer treatment based on targeting molecules conjugated to photosensitizers that can induce cell destruction upon exposure to near-infrared (NIR) light.

Methods

The anti-PSMA monoclonal antibody D2B was conjugated with IRDye700DX and DTPA and subsequently radiolabeled with 111In. To determine the optimal time point for tPDT, BALB/c nude mice with PSMA+ s.c. LS174T-PSMA xenografts received 30 µg of the conjugate intravenously (8 MBq/mouse) followed by µSPECT/CT, near-infrared fluorescence imaging, and biodistribution at 24, 48, 72, and 168 h p.i.. Tumor lesions were resected under image-guidance using intraoperative NIR fluorescence imaging. Tumor growth of LS174T-PSMA xenografts and overall survival of mice treated with 80 µg of the conjugate followed by 1-3 times of NIR light irradiation (100 or 150 J/cm2) at 24 h p.i. was compared to control mice that received either NIR light or PBS alone.

Results/Discussion

Highest specific tumor uptake was observed at conjugate doses of 80 µg/mouse. Biodistribution revealed no significant difference in tumor uptake in mice at 24, 48, 72, and 168 h p.i.. PSMA+ tumors were clearly visualized with both µSPECT/CT and NIR fluorescence imaging. Median survival of mice treated with 80 µg of DTPA-D2B-IRDye700DX and 3x150 J/cm2, 1x150 J/cm2, 3x100 J/cm2 of NIR light at 24 h p.i. was significantly improved compared to both control groups (70, 61, and 36 days vs. 16 days for both controls, respectively, p=0.0009). Treatment with 3x150J/cm2 resulted in significantly prolonged survival compared to treatment with 3x100J/cm2 (p=0.0224). No significant difference was observed between mice that receive 3x150J/cm2 or 1x150J/cm2 (p=0.6714).

Conclusions

Proof-of-principle was provided that 111In-DTPA-D2B-IRDye700DX can be used for pre- and intra-operative detection of PSMA+ tumors with radionuclide and fluorescence imaging, surgical guidance, and PSMA-targeted PDT. The optimal conjugate dose for PSMA-tPDT in this setting is 80 µg/mouse, the optimal time point for NIR light irradiation is 24 h p.i.. In vivo, PSMA-tPDT resulted in significant prolongation of median survival.

Imaging and targeted photodynamic therapy of PSMA-expressing tumors
1) Near-infrared fluorescence image (left) and microSPECT/CT image of a mouse with a s.c. PSMA+ LS174T-PSMA tumor performed at 24h after injection of 111In-DTPA-D2B-IRDye700DX (80μg/mouse, 8MBq). 2) Kaplan-Meier survival curve of mice treated with 80μg of DTPA-D2B-IRDye700DX and different NIR light irradiation intensities.

7:15 PM
PS-09-6 — Image-Guided Pathology for Evaluation of Resection Margins in Locally Advanced Rectal Cancer using the Near-Infrared Fluorescent Tracer Bevacizumab-800CW (#496)

S. J. de Jongh1, J. J. J. Tjalma1, M. Koller2, M. D. Linssen3, A. Jorritsma-Smit3, A. Karrenbeld4, K. Havenga2, P. H. J. Hemmer2, E. G. E. de Vries5, G. A. P. Hospers5, B. van Etten2, V. Ntziachristos6, G. M. van Dam2, W. B. Nagengast1

1 University Medical Center Groningen, Gastroenterology and Hepatology, Groningen, Netherlands
2 University Medical Center Groningen, Surgery, Groningen, Netherlands
3 University Medical Center Groningen, Clinical Pharmacy and Pharmacology, Groningen, Netherlands
4 University Medical Center Groningen, Pathology, Groningen, Netherlands
5 University Medical Center Groningen, Medical Oncology, Groningen, Netherlands
6 Technical University of Munich, Helmholtz Center, Institute for Biological and Medical Imaging, Neuherberg, Germany

Introduction

Negative circumferential resection margins (CRM) are the cornerstone for curative treatment of patients with locally advanced rectal cancer (LARC). Unfortunately, perioperative techniques for evaluation of resection margins are lacking, whereas standard histopathological examination is time-consuming. In this study, we evaluated the feasibility of optical molecular imaging as a tool for evaluation of resection margins at the surgical theater, i.e. Image-Guided Pathology (IGP), to improve clinical decision making.

Methods

Fluorescence imaging data of fresh surgical specimens and subsequent bread-loaf slices from patients with LARC (NCT01972373) were analyzed as a side study. All patients were administered intravenously with 4.5 mg of the fluorescent tracer bevacizumab-800CW 2-3 days prior to surgery. Seven patients met the inclusion criteria for correlation of fluorescence intensities in fresh surgical specimens with histology, to evaluate resection margins. For analysis of bevacizumab-800CW localization in bread-loaf slices, sufficient data was available from 17 patients. A receiver operating characteristics (ROC) curve was plotted to determine the mean fluorescence intensity (MFI) cut-off value for tumor detection.

Results/Discussion

Using IGP, in one patient a histologically confirmed tumor-positive CRM was predicted correctly at the surgical theater (Figure 1). Tumor-negative CRMs were predicted correctly in four patients using IGP. One tumor-positive CRM could not be detected; however, this positive margin was based on the presence of only an isolated microscopic tumor deposit in the CRM. One close CRM (1.4 mm) was identified as tumor-positive. Optical imaging enabled a clear differentiation between tumor and surrounding tissue in the bread-loaf slices (n=42) of all 17 patients ex vivo. In our limited sample size, an optimal MFI cut-off value of 5085 was determined based on the ROC curve, with a sensitivity and specificity of 98.2% and 76.8% respectively.

Conclusions

We demonstrate for the first time the potential of IGP for identification of positive resection margins directly after surgery in patients with LARC. Clearly, this might change current peri-operative decision making with regard to additional targeted resections or intraoperative brachytherapy. Based on the initial results from this study, a standardized methodology was developed to confirm these findings in a subsequent larger IGP study.

7:25 PM
PS-09-7 — Tumor-Specific Uptake of the Near-Infrared Fluorescent Anti-EGFR Antibody Panitumumab-IRDye800 in Patients with Head and Neck Squamous Cell Carcinoma: Safety and Feasibility Results from our Phase I Study (#310)

N. S. van den Berg1, N. Teraphongphom1, R. W. Gao1, S. Hong1, B. Martin2, V. Divi1, M. J. Kaplan1, R. Ertsey1, N. J. Oberhelman4, G. Lu1, C. S. Kong2, A. D. Colevas3, E. L. Rosenthal1

1 Stanford University, Otolaryngology - Head and Neck Surgery, Stanford, California, United States of America
2 Stanford University, Pathology, Stanford, California, United States of America
3 Stanford University, Medicine, Stanford, California, United States of America
4 Stanford University, Surgery, Stanford, California, United States of America

Introduction

Surgery remains the cornerstone of cancer treatment. Yet, radical tumor resection cannot always be achieved, and often healthy tissue has to be resected as well, leading to high morbidity. Real-time intraoperative fluorescence imaging may provide a solution. In the current study panitumumab, an anti-epidermal growth factor receptor (EGFR) antibody, was coupled to the near-infrared (NIR) dye IRDye800. Besides safety evaluation, we evaluated panitumumab-IRdye800 for intraoperative real-time fluorescence-based navigation to primary and metastatic head-and-neck squamous cell carcinoma (HNSCC).

Methods

We conducted an open-label, dose escalation clinical trial of panitumumab-IRdye800 in 21 HNSCC patients. Cohort 1 (n=3) received a microdose (0.06mg/kg). Cohort 2A (n=5), 2B (n=7) and 2C (n=6) received 0.5mg/kg, 1.0mg/kg or 50mg (flat dose) of panitumumab-IRDye800, respectively. Ccohort 2A and 2B patients also received 100mg unlabeled panitumumab. Patients were followed for 30 days after infusion, and adverse events were recorded. Surgery will be performed 2-5 days post-panitumumab-IRDye800 infusion. Prior ot standard of care surgery, real-time intraoperative NIR fluorescence imaging was used to evaluate tumor and lymph node (LN) visibility. Specimen fluorescence intensities were correlated with histology and immunohistochemistry for amongst others EGFR.

Results/Discussion

No dose-limiting toxicities occurred. One grade 1 adverse event was observed. Intraoperative imaging showed clear demarcation between cancerous and normal tissue, and ex vivo imaging findings correlated well with intraoperative findings. Cohort 1 was not analyzed, as it was primarily to establish safety. The average tumor-to-background ratio (TBR) was 5.40±0.68 for cohort 2A, 5.44±0.70 for cohort 2B and 6.53±1.25 for cohort 2C. Correlation of fluorescence intensities with tumor location resulted in a sensitivity and specificity of 92.03% and 78.07% for cohort 2A, and 91.86% and 91.17% for cohort 2B and 97.44% and 87.97% for cohort 2C. Positive predictive value were 68.32%, 85.51% and 87.85%, and negative predictive value found were 95.02%, 95.63%, and 97.96% for cohort 2A, 2B and 2C, respectively. EGFR expression positively correlated with fluorescence intensity.

Conclusions

Results from this first-in-human trial using panitumumab-IRDye800 suggest that it is a safe, and highly specific and sensitive optical imaging agent to aid in real-time intraoperative detection and surgical resection of both primary and metastatic HNSCC.

Schematic overview of the clinical trial
Eligible patients with HNSCC will receive a systemic infusion with panitumumab-IRdye800. 2-5 days later, the patient will undergo surgery whereby intraoperative NIR fluorescence imaging is performed to look at tumor and lymph node fluorescence. Following imaging, standard-of-care surgery will be performed. Fluorescence imaging findings will be correlated to histology and immunohistochemistry. 

7:35 PM
PS-09-8 — An instantaneous staining approach for tumor delineation in freshly excised biospecimens (#323)

S. Kossatz1, A. Strome1, W. Weber1, 3, S. Patel2, T. Reiner1, 3

1 Memorial Sloan Kettering Cancer Center, Radiology, New York, New York, United States of America
2 Memorial Sloan Kettering Cancer Center, Surgery, New York, New York, United States of America
3 Weill Cornell Medical College, Radiology, New York, New York, United States of America

Introduction

Molecularly specific delineation of tumors in the diagnostic and intraoperative setting remains an unmet clinical need with the potential to identify malignant growths faster and with higher accuracy than standard practice. Here, we present PARPi-FL, a fluorescent inhibitor of the DNA repair enzyme PARP1, as ultrafast topical contrast agent for whole tissues and sections to differentiate tumor vs. non-tumor tissues. We tested its ability to instantaneously stain tumor cells in xenografts of oral squamous cell carcinoma (OSCC), esophageal adenocarcinoma (EAC), and clinical biospecimens of OSCC.

Methods

PARPi-FL is a cell permeable, fluorescently labelled PARP inhibitor (excitation/emission max.: 503 nm/515 nm). We optimized the topical staining protocol on cryosections and fresh tissue samples of FaDu (OSCC) and OE19 (EAC) xenografts towards nuclear staining with low cytoplasmic background using confocal microscopy. Different staining times (1-10 min), concentrations (50-1000 nM) and washing protocols were tested. The topical staining outcome was validated against systemic PARPi-FL injection. We translated the optimized staining to human biospecimens of OSCC and evaluated the ability to outline tumor margins via microscopic and macroscopic imaging. We confirmed presence of tumor foci in tissues by H&E histopathology and PARP1 expression of cells by immunohistochemistry (IHC).

Results/Discussion

Optimized topical PARPi-FL staining in xenografts resulted in staining of tumor cell nuclei, but not the cytoplasm or stromal cells, allowing clear identification of tumor areas in cryosections and fresh tissues. Total staining time was 5 min for cryosections and 15 min for fresh tissue using 100 nM PARPi-FL. Higher concentrations increased cytoplasmic uptake. Topical staining quality and intratumoral distribution were comparable to intravenous PARPi-FL injection in FaDu and OE19 xenografts. The staining protocol was effectively translated to human biospecimens of OSCC without loss of quality (n=5 completed, ongoing). We identified tumor foci in both fresh tissue samples and cryosections, based on PARPi-FL accumulation in tumor cell nuclei, while uptake in normal epithelium and mucosa was low (Figure 1). According to the histopathological evaluation, 100% of SCC regions in the studied cohort were visualized by PARPi-FL staining and showed a positive IHC staining of PARP1.

Conclusions

We show that PARPi-FL topical staining is suitable for instantaneous tumor cell identification in live patient biospecimens. Widespread expression of PARP1 in tumors was confirmed in our patient cohort. This technology can be used in a diagnostic setting, as well as on excised tissue during surgery to identify positive margins. Importantly, the quick turnaround time for results allows for an immediate feedback to the physician, reducing diagnostic delays and repeated patient visits.

Figure 1.
Rapid PARPi-FL staining of fresh tissue or cryosections in a patient sample of OSCC allowed for identification of tumor foci due to accumulation in tumor cell nuclei, but not surrounding normal cells. The biopsy was split in three parts to carry out staining of fresh tissue, cryosections and histopathology. Images were acquired on a confocal microscope. Green: PARPi-FL, blue: Hoechst DNA stain.

2:30 PM
SpotSymp 02-1 — Where do we stand in the clinic? (#618)

A. Vahrmeijer1

1 Leiden University Medical Center, Surgery, Leiden, Netherlands

Content

Due to its relatively high tissue penetration, near-infrared (NIR; 700-900 nm) fluorescent light has the potential to visualize structures that need to be resected (e.g. tumors, lymph nodes) and structures that need to be spared (e.g. nerves, ureters, bile ducts). NIR fluorescence imaging using non-targeted fluorescent probes has been extensively studied in the last decade. Although proven feasible, tumor-specific imaging can be dramatically enhanced using tumor-specific fluorescent contrast agents. Clinical translation of these agents is challenging, and hurdles have to be overcome. In this overview we recapitulate the key regulations for first-in-human studies with fluorescent agents, provide insight in different strategies for swift clinical translation and discuss how clinical introduction of these fluorescent agents was achieved. We will review the key results from these studies and from recent clinical studies using tumor targeted contrast agents (antibody or peptide based), discuss the advantages and limitations of the technology, and suggest various imaging system and contrast agent parameters that could be optimized in future trials. Moreover, a clear roadmap for clinical translation of targeted probes will be presented.

3:00 PM
SpotSymp 02-2 — Real-time Quantitative Fluorescence-Guided Surgery (#610)

S. Gioux1

1 University of Strasbourg, ICube Laboratory, Strasbourg, France

Content

There is a pressing clinical need to provide image guidance during surgery. Currently, assessment of tissue that needs to be resected or avoided is performed subjectively leading to a large number of failures, patient morbidity and increased healthcare cost. Because near-infrared (NIR) light propagates deeply within living tissues and interacts with molecular constituents, it offers unparalleled capabilities for objectively identifying healthy and diseased tissue intraoperatively. These capabilities are well illustrated through the ongoing clinical translation of fluorescence imaging during oncologic surgery. In this work, we will review our efforts to provide real-time image-guidance during surgery using NIR diffuse optical imaging. We will present our latest methods and results in wide-field quantitative intraoperative imaging, with a particlar focus onto real-time quantitative fluorescence imaging.

References

1. Valdes PA, Angelo JP, Choi HS, Gioux S. qF-SSOP: real-time optical property corrected fluorescence imaging. Biomed Opt Exp 2017; 8(8): 3597-605.

2. van de Giessen M, Angelo JP, Vargas C, Gioux S. Real-time, profile-corrected single snapshot imaging of optical properties. Biomed Opt Exp 2015; 6(10): 4051-62.

3. Gioux S, Choi HS, Frangioni JV. Image-guided surgery using invisible near-infrared light: fundamentals of clinical translation. Mol Imaging 2010; 9: 237-255.

Acknowledgement

Funding for this research was provided by European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under grant agreement No 715737 (QuantSURG); National Institute of Health (NIH) (K01-DK-093603, F31-DK-105839, R01-EB-011523); National Science Foundation (NSF) (DGE-1247312); France Life Imaging; University of Strasbourg IdEx and ICube Laboratory.

3:30 PM
SpotSymp 02-3 — Designing a Clinical Trial with Impact (#587)

M. Koller1

1 UMCG, Surgery, Groningen, Netherlands

Content

The impact of a clinical trial is not only related to the Impact Factor of a journal in which the paper is published. Many other factors are important that determines the impact of a clinical trial. In this lecture I would like to give an overview on these different factors, and how these factors can be translated to the design of a clinical trial. Furthermore, based on our 10 years experience in clinical trials in the field of optical imaging I will elaborate on the progress made in the field and what is needed to keep making progress in the future.

4:00 PM
SpotSymp 02-4 — Clinically Relevant Imaging Strategies in Endoscopy. (#578)

S. Rogalla1, 2

1 Stanford University, Radiology, Stanford, California, United States of America
2 Stanford University, Medicine, Stanford, California, United States of America

Content

Malignant neoplastic lesions account for around 600,000 deaths in the United States and is the second leading cause of death in Western Countries after cardiovascular disease. A majority of cancer types present at readily accessible surfaces offer unique detection opportunities. Effective clinical management of cancer is facilitated by early detection, when full surgical resection is possible, prior to invasion into adjacent tissue or significant intravasation into blood vessels leading to metastasis or by optimized surgical resection aiming for a R0, defined as zero tumor rest, resection without harming functional tissue. Good prognosis with long-term disease-free survival is therefore more likely following early detection when progression is limited.

At present, detection of several types of cancer largely relies on routine inspection with the naked eye (e.g. skin and oropharynx) or simple white light tools (e.g. cervix and colon). Emerging optical tools based on differential refraction, absorption, reflection, scattering or fluorescence of carcinomas relative to normal tissues enable label-free visualization of neoplasia. However, the differences in intrinsic optical properties of normal and malignant tissues can be subtle, and relying on these may lead to high miss rates. Enhanced optical contrast offered by molecularly targeted agents can be used to improve early detection, and given that optical imaging and sensing tools can be readily combined, integrated systems that image over a range of scales, or detect multiple parameters, can be developed to aid in early detection or optimized image-guided resection improving the signal to noise ratio. Moreover, diagnosis is, at present, made by histologic examination of tissue biopsies following identification of suspicious lesions. Miniature and handheld microscopic imaging tools have recently been developed, and integration of these tools with wide-field optical surveillance devices offers both rapid detection and confirmatory histologic examination at the point-of-care offering guidance for biopsy and/or resection. A wide-variety of targeted probe strategies have been described with demonstrated benefit in preclinical models and in a limited number of human studies.

Here I present an overview of integrated multimodality optical imaging strategies and sensing tools that use combinations of intrinsic and extrinsic optical contrast for early detection or margin delineation for carcinomas at epithelial surfaces and brain tumors. I will discuss these new technologies, such as fluorescence and Raman-based optical imaging and show that they have utility in detecting the most common cancer types to improve the overall-survival of patients.

Acknowledgement

Will Foundation, Kenneth Rainin Foundation, NIH

4:30 PM
emptyVal-1 — BREAK

5:00 PM
SpotSymp 02-6 — Optoacoustic clinical imaging: current and future perspectives (#580)

C. Zakian1

1 Helmholtz Zentrum Munich, IBMI, Munich, Germany

Content

Multispectral optoacoustic imaging has emerged as a promising non-invasive clinical and pre-clinical modality suitable for vascular and microvascular structure mapping along with hemoglobin metabolism as well as lipid and water distribution in tissue.  This technique relies on intrinsic tissue chromophore light absorption followed by the emission and detection of ultrasound waves.

The current state-of-the-art optoacoustic devices, such as MSOT (multispectral optoacoustic tomography) and RSOM (raster-scanning optoacoustic mesoscopy) are based on extensive and continuous engineering progress resulting in improved laser sources, ultrasound detectors and scanning mechanisms. These advances enabled and propelled the translation of this technique into the clinics.

In this review, we present exciting new insights into the clinical applications of MSOT and RSOM in dermatology, gastroenterology, oncology, endocrinology and cardiovascular diseases .  We discuss unique clinical capabilities of these optoacoustic modalities in psoriasis assessment, skin and breast cancer detection, assessment of periphery vascular disease, muscular metabolism and thyroid imaging.

We will further highlight current challenges and future directions, with special emphasis on GI endoscopic applications, currently tackled by the EC research project ESOTRAC, an international multi-disciplinary consortiums aiming at improving early detection of esophageal cancer.

Acknowledgement

ESOTRAC has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 732720.

5:30 PM
SpotSymp 02-7 — Update on Targeted PhotoDynamic Therapy (#599)

M. Gotthardt1

1 Radboud university medical centre, Department of Radiology and Nuclear Medicine, Nijmegen, Gelderland, Netherlands

Content

Tracer molecules can in most cases be used for radionuclide as well as fluorescence imaging, but they also have potential for use in photodynamic therapy. Here, the development and translation of a tracer targeting the GLP-1 receptor is described starting from selection of an optimal ligand to clinical translation for radionuclide imaging of tumors, diabetes and congential hyperinsulinism. The achievement of the EU FP7 BetaCure project will be presented in respect to development of this probe for image guided surgery and photodynamic therapy. Finally, a perspective towards future clinical implementation of such multi-purpose ligands within the context of precision medicine treatment of tumours and other diseases is presented.

6:00 PM
SpotSymp 02-8 — Intra-Operative Nuclear Imaging: Current Applications, Limitations and Future Strategies (#593)

I. Alam1

1 Stanford University, Radiology Dept, Radiology, Stanford, California, United States of America

Content

Nuclear imaging modalities, single photon emission computed tomography (SPECT) and positron emission tomography (PET), are valued for their sensitive imaging of molecular processes and targets and their limitless depth of detection. They are frequently used pre-operatively for the diagnosis, localization and staging of solid tumors informing both surgical and therapeutic management.

Due to geometric constraints of SPECT and PET scanners, the real-time localization of radioactive tracers within the intraoperative setting has only been possible because of portable hand-held probes for radio-guided surgery (RGS). Typically RGS involves the use of a hand-held gamma radio-detection probe to detect SPECT isotopes such as 99mTc, injected directly into or next to suspected lesions prior to surgery and has become standard-of-care in a defined set of surgical procedures e.g. SLN biopsy in breast cancer and malignant melanoma. Despite their utility, the lack of image documentation in the use of these probes, has in recent years fueled the development of small gamma cameras for intra-operative use, which have improved surgical accuracy as demonstrated in various clinical studies. In contrast, RGS using PET tracers, is not yet well established though two classes of hand-held PET probes have been developed and evaluated to date; a dedicated PET gamma probe, designed specifically to detect the high-energy 511 keV photons and a PET positron detection probe. Often used in conjunction with 18Fluorodeoxyglucose (18F-FDG), these studies have at times yielded limited results, partly due to the limitations of the radiotracer itself. The use of more specific PET tracers as well as ongoing hardware and software development for these RGS technologies are expected to enhance spatial resolution and improve their accuracy and utility.

The current use of RGS probes and the evolution of nuclear imaging systems compatible with the intra-operative setting will be discussed as well as their advantages and limitations. In this regard, intraoperative guidance with nuclear imaging could benefit greatly from multimodal approaches, made possible due to recent advances in technology. Recent examples of such hybrid intra-operative approaches will be discussed and their exciting potential to enhance the utility of nuclear approaches in the future.

6:30 PM
emptyVal-2 — BREAK

7:00 PM
SpotSymp 02-10 — Bimodal probes for guided interventions (#607)

M. Schottelius1

1 TU München, Chair for Pharmaceutical Radiochemistry, Garching, Bavaria, Germany

Objectives

Over the last decades, radioguided surgery has evolved as a reliable tool for the sensitive intraoperative detection and identification of tissues of interest, based on the pre- or intraoperative specific accumulation of a dedicated radiotracer in these tissues. Based on the excellent tissue penetration of gamma radiation, valuable information about the anatomical location of the diseased tissue is obtained, greatly facilitating surgical procedures and improving their outcome.

However, the inherent disadvantage of using intraoperative radioguidance alone is its limited spatial resolution. The use of hybrid tracers combining gamma emission and fluorescence in one (targeted) molecule merges the best of both modalities - by additionally providing the high spatial and temporal resolution of fluorescence imaging (far-red and near-infrared), allowing for highly sensitive real-time visualization directly correlated to the surgical field. The unquestionable utility of such a hybrid approach has initially been established by the introduction of ICG-[99mTc]nanocolloid (ICG: indocyanine green) for sentinel lymph node biopsy in different cancer types, demonstrating substantially improved sensitivity for the optical SLN detection in the hybrid setting. This finding has triggered the recent development of a broad variety of targeted hybrid nuclear/NIR-probes for different cancer types, ranging from full-size antibodies to small molecules (peptides, inhibitors).

Especially for the latter, the choice of the radiolabeling strategy and the fluorescent dye have tremendous impact on the overall performance of the hybrid tracer, including its targeting efficency, its physicochemical characteristics, in vivo stability, its tendency towards plasma protein binding and tracer pharmacokinetics.

Content

This contribution aims at providing a comprehensive overview over the different aspects of hybrid tracer development, with a certain focus on the selection criteria for the most suitable fluorescent dye for a given targeting vector and application, in combination with a given radiolabeling methodology. Our own recent data on hybrid PSMA-ligands will be presented to illustrate the complex interplay between ligand structure and tracer performance, and will be complemented by findings from other groups, highlighting selected aspects of this vibrant field of research and its potential for clinical translation.

7:30 PM
SpotSymp 02-11 — Defining the Cutting Edge: The use of molecular imaging for image guided surgery and tumor treatment. (#575)

J. P. Basilion1

1 Case Western Reserve University, Department of Radiology, Cleveland, United States of America

Content

A challenge for surgical removal of cancer is to maximize the removal of the cancerous tissue while minimizing removal of normal tissues.  This is critical for a number of prevalent cancers.  Several investigators have shown the utility of systemically delivered optical imaging probes to image tumors and guide surgical removal in small animal models of cancer and recently first-in-man studies have demonstrated feasibility in Europe and the USA. However, to date there are no FDA approved cancer-selective optical imaging probes that can be used to guide surgery.

The future direction of this field is to develop and translate into clinical use effective optical imaging probes for real-time assessment of surgical margins during tumor resection.  Here we demonstrate methods for imaging tumors margins during surgery that have a shorter time to clinical translation. Specifically, we will show that optical imaging probes topically applied ex vivo to resected tumor and surrounding normal tissue can rapidly differentiate between tissues. In contrast to systemic delivery of optical imaging probes which label tumors uniformly over a long period of time (i.e. hours), topical probe application results in rapid and robust probe activation that is detectable as early as 5 minutes following application.

We have also moved to implement a theranostic approach for image guided surgery. Using Prostate specific membrane antigen (PSMA), a well-established biomarker for prostate cancer, we have developed a PSMA-targeted photodynamic therapy (PDT) conjugate, PSMA-1-Pc413, which has demonstrated the ability to effectively inhibit prostate tumor growth after irradiation with 672nm laser light. Using this molecule for image guided surgery (IGS) we have exploited the PDT component to improve IGS. Our data demonstrate that mice undergoing imaged-guided surgery had less residual tumor in the surgical cavity than mice that had surgery under white light only and that IGS followed by PDT significantly improved survival. Kaplan-Meier survival curves showed that image guided surgery can improve the survival of mice (57%) compared to white light surgery followed by PDT (33%) and white light surgery without PDT (40%). PDT after image-guided surgery further improved the survival rate to 75%.

4:00 PM
emptyVal-1 — Introductory Talk by Bettina Weigelin Houston, USA

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

4:20 PM
PS-20-2 — Intravital Multiphoton Endomicroscopy – Technical Feasibility and Cancer Applications (#65)

S. Weischer1, G. - J. Bakker1, J. Heidelin3, V. Andresen3, B. Messerschmidt4, P. Friedl1, 2

1 RIMLS, Radboud Institute of Molecular Life Science, Department of Cell Biology, Nijmegen, Netherlands
2 UT MD Anderson Cancer Center, Department of Genitourinary Medical Oncology, Houston, Texas, United States of America
3 Lavision Biotec GmbH, Bielefeld, Germany
4 Grintech GmbH, Jena, Germany

Introduction

Besides the peritumor tissue, the tumor core represents a biologically active compartment with frequent occurrence of malnutrition, hypoxia, internal bleedings and necrosis as well as a resource of invasive tumor cells [1 - 3]. However, the biology of the tumor remains largely unresolved due to the inability of commonly used microscopy approaches to visualize deep events beyond 300 µm in established tumor lesions.

Methods

To overcome the current limitations of near-infrared multiphoton microscopy, we developed a rotational multiphoton endomicroscope consisting of a side-view GRIN optics. The GRIN lens (0.28NA, FOV 150x150µm) is embedded in a smooth polyimide tubing with 420µm diameter. The endomicroscope was inserted into live tumors (“optical biopsy”) and enabled intravital imaging of deep tumor regions. The endoscope allowed imaging with cellular resolution (1.2µm lateral, 25µm axial) up to 3mm of depth, including multicolor fluorescence and second harmonic generation of fibrillar collagen. Here, we apply the endomicroscope to determine tissue tolerability and intra-tumor dynamics of B16F10 melanoma tumors in the deep mouse dermis.

Results/Discussion

By rotation combined with z-step scanning, 360˚panoramas from 100 µm-thick tissue from inside the tumor were obtained. Insertion and rotation inside the tumor showed largely friction-free gliding towards the tissue. Extensive technical analysis showed that (1) tumor development is unaltered by the microscopy procedure, (2) blood vessels in vicinity of the injection site remain perfused and non-leaky beyond control values, (3) circulating tumor cell numbers after endomicroscopy were not increased and (4) nearly border-free wound healing within 2 days post microscopy. Kinetic timelapse to assess intratumor cell dynamics revealed that the majority of tumor cells in the tumor core were stationary, however occasional individual tumor cells (3-10%) migrated with speeds of up to 1µm/min with variable persistence. In addition, significant tumor cell subsets were identified in association with the tumor vasculature, including intravascular resident and circulating cells.

Conclusions

Intratissue rotational endomicroscopy provides direct and poorly traumatic access to previously unresolved tissue types beyond 1 mm of penetration depth with (sub)cellular resolution, including vascular perfusion and cell migration in the tumor core. Beyond imaging intratumor biology and treatment responses, multiphoton endomicroscopy will be amenable to a broad range of applications including body cavities, parenchymatous organs positioned in deep body regions (pancreas, liver) and deep neuroimaging.

References

[1] Friedl, P. and K. Wolf (2003). Nat Rev Cancer 3(5): 362-374.

[2] Hanahan D. and R.A. Weinberg (2011). Cell 144: 646-74

[3] Deryugina, E. I. and W. B. Kiosses (2017). Cell reports 19(3): 601-616.

Acknowledgement

We would like to thank Anke Kraeplin (Grintech, Jena, Germany), Esther Wagena, Manon Vullings and Carmen Lucia Pontes (RIMLS, Nijmegen, NL) for technical support. The work was funded by an ERC Consolidator grant.

4:30 PM
PS-20-3 — Deep imaging of single-cell function with two-photon microscopy in vivo (#254)

A. Birkner1, C. H. Tischbirek1, H. Jia1, Z. Varga1, V. Bonfardin1, M. Tohmi1, B. Sakmann1, A. Konnerth1

1 Institute of Neuroscience, Technical University Munich, Munich, Bavaria, Germany

Introduction

Two-photon (2P) microscopy is one of the key methods for investigating neural function on a single-cell and subcellular level in vivo. However, 2P-microscopy is limited in imaging depth and a special challenge is imaging dense cell populations, in which typically the maximum imaging depth is about 300 µm below the brain surface. In the mouse cortex, this corresponds to the upper three cell layers, out of six. To increase the maximum imaging depth and access all six cortical layers, we combined red-shifted 2P-excitation wavelengths with approaches for a reduction of out-of-focus fluorescence.

Methods

We used two-photon calcium imaging to record activity from neural cells in the mouse cortex in vivo. To image dense cell populations with single cell resolution, a synthetic calcium indicator was pressure-injected into the brain tissue (1). Moreover, individual cells were filled with a calcium dye with single-cell electroporation, to be able to record activity from subcellular structures (2). To relate fluorescence transients to action-potential output, two-photon imaging was combined with cell-attach recordings. The morphology of single cells was reconstructed, either with 2P-stacks from the in vivo experiments, or by electroporating biocytin into single-cells, immunohistochemical postprocessing of the tissue and imaging with a confocal microscope.

Results/Discussion

We used red-shifted two-photon (2P) excitation wavelengths, in combination with an approach to reduce background fluorescence. The red-shift was made possible by using a new calcium indicator, named Cal-590. We characterized its 2P-excitation spectrum and its calcium-sensing properties in the mouse brain in vivo (3). Moreover, to minimize background fluorescence, an approach for the targeted-delivery of the dye to specific cortical layers was developed. Thereby, just the cells of interest in the cortical layer of choice were labeled (4). With this combination of the red-shift with the depth-restricted staining we were able to image dense cell populations down to the deepest cortical layer, layer 6, at 900-1000 µm below the brain surface. We used our method to identify functionally unique cells within a dense cell population, which were subsequently stained with a second, spectrally different, calcium dye. Subcellular structures of these target cells can then be functionally analyzed.

Conclusions

In conclusion, we developed a simple and flexible approach for deep two-photon imaging, offering new tools for the functional analysis of neural circuits on a single-cell level and for addressing new scientific questions.

References

(1) Stosiek et al., PNAS. 2003
(2) Kitamura et al., Nat. Methods, 2008
(3) Tischbirek, Birkner et al., PNAS, 2015
(4) Birkner, Tischbirek et al., Cell Calcium, 2016

Deep and dual-color two-photon calcium imaging
Depth-restricted dye loading of a red-shifted calcium indicator (Cal-590) is used to image deep cortical layers of the mouse brain in vivo. Functionally unique cells are identified within this cell population and targeted for single-cell electroporation with a second, spectrally different, calcium indicator.

4:40 PM
PS-20-4 — Light Sheet Fluorescence Microscopy of Expanded Dentate Gyrus Tissue Samples allows Super Resolution Connectomics (#144)

M. K. Schwarz1, I. Pavlova1, J. E. Rodriguez2, J. Buergers2, U. Kubitscheck2

1 University Clinic of Bonn, Epileptology/Functional Neuroconnectomics, Bonn, Germany
2 Rheinische Friedrich-Wilhelms-University Bonn, Institute of Physical and Theoretical Chemistry, Bonn, Germany

Introduction

Critical details of neuronal connectivity occur on length scales of ~100 nm. Structures small like this can optically only be resolved using super resolution light microscopy. This is not feasible for the reconstruction of extended neuronal networks, because all available super resolution approaches are restricted to thin samples of ~20 µm and neuronal networks often extend over several mm. A novel tissue expansion procedure combined with light sheet fluorescence microscopy can bypass these constraints and allow

super resolution connectomic analysis of large 3D tissue samples [1, 2, 3].

Methods

Expansion microscopy. Fluorescently labeled, sodium acrylate embedded and enzymatically treated tissue samples (proteinase K) are physically expanded by repeated addition of destilled water before imaging. Prior to tissue expansion, the virally transduced (recombinant adeno-associated virus, rAAV) EGFP expressing neurons are labeled with conventional antibodies (anti-chicken EGFP, Alexa 488 anti-chicken), immersed in sodium acrylate and covalently attached to the polymer matrix using MA-NHS. [1]. High resolution LSFM of expanded tissue samples. Experiments are performed using an experimental LSFM microscope setup [3] that was slightly modified by installing a long distance objective (25x) with a numerical aperture of 1.1 to achieve super resolution of expanded samples.

Results/Discussion

We here combined tissue expansion with light sheet fluorescence microscopy to allow fast and gentle imaging of very large brain tissue samples [2, 4]. Mouse dentate gyrus tissue samples containing EGFP labelled granule neurons were expanded and imaged utilizing a purpose build light sheet fluorescent microscope [3]. Tissue expansion yielded a virtual lateral and axial optical resolution of 75 and 250 nm respectively, with depths up to effectively 2 mm, thus allowing to optically resolve fine structural details of single granule cells, as well as extended granule cell networks (Fig. 1, A, B). Exploiting state-of-the art genetic labeling approaches to selectively label specific subsets of neurons (GCs) we could generate a structural map of sub-regions of the mouse hippocampus in super resolution (Fig .1, A, B). Our initial analysis revealed an interesting conserved pattern of dendritic constrictions that is generally present on distal parts of dentate gyrus granule cell dendrites.

Conclusions

This combination of tissue expansion and light sheet fluorescence microscopy represents an ideal tool to obtain super-resolved 3D images of extended neuronal networks at imaging rates exceeding those of classical point-scanning instruments by a factor of ~20. Here, we were able to identify structural motives -dendritic constrictions- that might separate single dendrites into distinct functional compartments contributing to their computing capacity. These "dendritic constrictions" might have a function in scaling the somatic signal integration of differential synaptic entorhinal inputs.

References

[1] Chen, F., Tillberg, P.W., and Boyden, E.S. (2015). Optical imaging. Expansion microscopy. Science 347, 543–548.

[2] Desphande t., Li T., Herde MK., Vatter H., Schwarz MK., Henneberger C., Steinhaeuser C., Bedner P. (2017) „Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy. Glia 65(11): 1809-1820.

[3] Baumgart, E., and U. Kubitscheck. 2012. Scanned light sheet microscopy with confocal slit detection. Opt. Expr. 20: 21805–21814

[4] J. Doerr, M. K. Schwarz, D. Wiedermann, A. Leinhaas, A. Jakobs, F. Schloen, I. Schwarz, M. Diedenhofen, N. C. Braun, P. Koch, D. A. Peterson, U. Kubitscheck, M. Hoehn, and O. Brüstle (2017), “ Whole-brain 3D mapping of human neural transplant innervation,“ Nat. Comm. 8, 19 January 2017.

Acknowledgement

We thank Christian Henneberger and Michel Herde for helping to successfully setup tissue expansion and Tony Kelly and Heinz Beck for helpful discussions. This work is financially supported by grants from the German Research Foundation DFG (SCHW 1578/2-1; SCHW 1578/3-2)

Fig. 1: Light sheet fluorescent imaging of EGFP labeled granule cells in the mouse dentate gyrus.
Mouse dentate gyrus tissue samples containing  fluorescently labelled granule neurons after tissue expansion imaged with a light sheet fluorescent microscope. The virtual lateral and axial optical resolution of 75 and 250 nm respectively, allows to optically resolve extended granule cell networks (A) as well as fine structural details of single granule cells and their neurites (B).

4:50 PM
PS-20-5 — Multiscale optical imaging of 10 nm polymer and 100 nm liposome accumulation in the brain upon temporarily opening up the blood-brain barrier using ultrasound and microbubbles (#55)

J. - N. May1, S. K. Golombek1, D. Rommel2, R. Pola3, A. J. C. Kühne2, F. Gremse1, F. Kiessling1, T. Lammers1, 4

1 Uniklinik RWTH Aachen, Institute for Experimental Molecular Imaging, Aachen, North Rhine-Westphalia, Germany
2 RWTH Aachen, DWI – Leibniz Institute for Interactive Materials, Aachen, North Rhine-Westphalia, Germany
3 Academy of Sciences, Institute of Macromolecular Chemistry, Prague, Czech Republic
4 Utrecht University, Department of Pharmaceutics, Utrecht, Netherlands

Introduction

The blood-brain barrier (BBB) is a major obstacle for drug delivery into the brain. The combination of ultrasound (US) and microbubbles (MB) can induce a spatially and temporarily controlled opening of the BBB, allowing for the extravasation of drugs and delivery systems out of the blood vessels into the brain. In this study, we used US and MB to permeate the BBB, and employed multimodal and multiscale optical imaging, together with fluorophore-labeled polymers and liposomes to assess the impact of nanocarrier size on the efficiency of sonoporation-mediated drug delivery to the brain.

Methods

Treated as well as control CD-1 nude mice received an i.v. injection of polymeric PBCA-based MB and pHPMA polymers (10 nm) or liposomes (100 nm), both labeled with Alexa488 and Cy7. Upon US treatment, the accumulation of the two nanocarriers was longitudinally monitored using computed tomography-fluorescence molecular tomography (CT-FMT). Prior to sacrifice, rhodamine-labeled lectine was injected to stain blood vessels. Ex vivo analysis included fluorescence microscopy (FM), confocal microscopy (CM) and stimulated emission depletion nanoscopy (STED). Possible side effects and the overall extent of BBB opening were also investigated, using H&E and immunofluorescence (directed against extravasated endogenous IgG) stainings. 

Results/Discussion

The success of BBB opening upon applying US and MB was confirmed via the increased number of blood vessels showing IgG extravasation in sonoporated vs. control animals. Based on H&E stainings, the treatment did not induce obvious brain damage. While the extravasation and penetration of polymers upon sonoporation was clearly visible using all optical imaging modalities, the extravasation of liposomes could only be detected using more accurate/sensitive techniques such as confocal microscopy and STED nanoscopy (Figure 2 A-D). Using software tools to quantify the extent of extravasation and penetration depth, we confirmed that 10 nm polymers penetrated more efficiently and deeper into the brain than 100 nm liposomes.

Conclusions

US and MB can be employed to efficiently open up the BBB, enabling the delivery of drugs and drug delivery systems to the brain. Multimodal and multiscale optical imaging show that relatively small (10 nm polymers) carrier materials accumulate more efficiently and penetrate deeper into the brain than relatively large (100 nm liposomes) drug delivery systems.

Acknowledgement

This work is supported by the ERC (starting grant 309495: NeoNaNo) and the DFG (La2937/1-2 and SFB1066).

Figure 1: Study design and applied imaging modalities.

Figure 2: Accumulation and extravasation of liposomes and polymers upon BBB opening.

5:00 PM
PS-20-6 — Mass spectrometry imaging after intratracheally instillation of nanoparticles in lung tissue (#344)

A. - C. Niehoff1, D. Dietrich2, A. Vennemann3, M. Wiemann3, M. Sperling2, U. Karst2

1 Shimadzu Europa GmbH, Duisburg, North Rhine-Westphalia, Germany
2 University of Münster, Institute of Inorganic and Analytical Chemistry, Münster, North Rhine-Westphalia, Germany
3 IBE R&D gGmbH, Münster, North Rhine-Westphalia, Germany

Introduction

The uptake of cerium oxide nanoparticles (CeO2-NP) e.g. from diesel exhaust via inhalation may pose a health hazard for humans. As CeO2-NP elicit lung inflammation and fibrosis, knowledge about both, their distribution in the lung and their biological effects is needed. Here we combined the imaging techniques laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) to study the distribution of CeO2-NP and changes of phospholipid distribution in the lung.

Methods

Different doses of cerium oxide nanoparticles were intratracheally instilled into rat lungs. The lungs were removed after different incubation times and cryo-sections were prepared. Laser ablation experiments were performed line-by-line with a 213 nm Nd:YAG laser using a spot diameter of 50 μm and subsequent merging reveals the distribution of cerium in the tissue. For MALDI-MS experiments dihydroxybenzoic acid and 9-aminoacridine were applied via sublimation and recrystallization to detect phospholipids in positive and negative ion mode.

Results/Discussion

Cerium distribution pattern as well as the distribution of naturally occurring elements such as phosphorus were depicted by means of LA-ICP-MS. Cerium distribution changed over time from a largely even to a more concentrated distribution pattern. Alterations of the phosphorus distribution were spatially linked to the cerium distribution, especially at high doses and after longer incubation times. MALDI-MS experiments suggest that zones with increased phosphorus concentration were co-localized with elevated levels of phospholipids. Phospholipids were identified via exact mass and fragmentation experiments.

Conclusions

Results show that combining MALDI-MS and elemental imaging techniques can provide new insights into local changes caused by nanoparticles in tissues.

5:10 PM
PS-20-7 — In situ multi-elemental imaging of human specimens with laser spectrometry : a future gold standard for medical diagnosis ? (#278)

B. Busser1, 2, M. Leprince3, S. Moncayo3, F. Pelascini4, J. Charles1, J. L. Coll2, V. Bonneterre1, V. Motto-Ros3, L. Sancey2

1 Grenoble Alpes University Hospital, Grenoble, France
2 Grenoble Institute for Advanced Biosciences, Grenoble, France
3 Institut Lumière matière, Villeurbanne, France
4 Critt Matériaux alsace, Schiltigheim, France

Introduction

The physiological and pathological roles of both endogenous and exogenous chemical elements are of major interest for the medical community. Elemental imaging of biological tissues is currently a technological challenge, generally requiring complex instruments with restricted accesses. We recently developed an all-optical method, fully compatible with standard microscopy systems, for multi-elemental imaging of biological tissues. In this work, we used LIBS to image the elemental composition of various human samples of medical interest including skin, lymph node, lung and kidney tissues.

Methods

We upgraded our Laser Induced Breakdown Spectroscopy (LIBS) instrument to work faster and to image the elements contained in paraffin-embedded samples, which are the most frequent form of clinical specimens (surgical resections or biopsies). Our LIBS system allows the in situ imaging and quantification of the elements of the periodic table within biological/human tissues, with ppm-scale sensitivity and a pixel size of up to 10x10 µm². A laser is focused on the sample surface, which generates a plasma. Elemental images (maps) are obtained by scanning the surface of the sample. Different spectrometers are used to collect the signal of various metallic elements such as Fe, Ca, Na, P, Mg, Zn, Al, Mn, Co, Si, Cr, Ti and Cu contained in the tissues of interest.

Results/Discussion

The proof-of-concept was obtained by studying the bio-distribution several metal-based (Gd, Au, Ag, Pt) nanoparticles in vivo in 2D and in 3D and in mice (Fig.1). The next step was to analyze human samples. Consequently, we worked on human skin biopsies and we showed that several endogenous elements were differentially expressed between normal and cancer tissues1 (also see Fig. 1). Very recently, we described that LIBS elemental imaging was able to reveal the presence of exogenous metals within cutaneous and lymph nodes tissues of human origin2. We imaged the presence of high levels of aluminium within a post-vaccinal cutaneous granuloma (Fig.2) 2. We also found several metal particles within lung tissues and thoracic lymph nodes from patients with sarcoidosis, or with other respiratory dieases induced by exposure to dust. We are currently performing complementary experiments on pathological specimens of medical interest in normal or cancer tissues such as brain, lung, and skin.

Conclusions

We describe recent results obtained with LIBS for the multi-elemental imaging of biological tissues for medical applications. This laser spectrometry technique is highly versatile because almost any element can be imaged with high sensitivity. Besides, this technique is complementary to optical microscopy used by medical pathologists for routine diagnostic activity. Multi-elemental imaging with LIBS can help pathologists establish or confirm diagnoses for a wide range of medical applications, particularly when the nature of external agents present in tissues needs to be investigated.

References

1. Moncayo et al, Spectrochimica Acta Part B: Atomic Spectroscopy, 2017, doi.org/10.1016/j.sab.2017.04.013

2. Busser et al, Modern Pathology, 2017, doi:10.1038/modpathol.2017.152

Acknowledgement

This work was supported by the ITMO Cancer et ITMO Technologies pour la santé de l'alliance nationale pour les sciences de la vie et de la santé (AVIESAN), the Institut National du Cancer (INCa) and INSERM within the project LAST (#PC201513). This work was also supported by the french national grant ANR-17-CE18-0028 “MEDI-LIBS”.

Different applications of LIBS multi-elemental imaging
LIBS multi-elemental imaging can help to monitor the kinetics of metal-based nanoparticles in 2D and in 3D. This may be of interest for pre-clinical investigations in animal tissues. More recently, we developped the use of LIBS imaging for human paraffin-embedded tissues. The aim is to help thd pathologists in their routine diagnostic work.

Aluminium (Al) accumulation in local skin reactions to Al-adsorbed immunotherapies

Histopathological morphology of a cutaneous granuloma (A) and a skin pseudolymphoma (B) with corresponding elemental images obtained after LIBS (right panels). LIBS analysis revealed high levels of Aluminium (in green) in the immunohistiocytic areas. Sodium (Na, in red), enabled the visualization of the global tissue architecture. Scale bar: 1 mm. (adapted from reference 2)

1:30 PM
emptyVal-1 — Introductory Talk by Cornelius Faber - Münster, Germany

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

1:50 PM
PS-03-2 — In vitro and in vivo validation of a potential PET tracer targeting α-synuclein (#528)

L. Kuebler1, K. Herfert1, S. Buss1, A. Maurer1, R. Stumm1, F. Schmidt4, A. Leonov2, S. Ryazanov2, A. Giese3, C. Grießinger2, B. J. Pichler1

1 Eberhard Karls Univeristy of Tuebingen, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tuebingen, Baden-Württemberg, Germany
2 Max Planck Institute for Biophysical Chemistry, Goettingen, Germany
3 Ludwig-Maximilians-University, Center for Neuropathology and Prion Research, Munich, Germany
4 MODAG GmbH, Munich, Germany

Introduction

Imaging α-synuclein (αSYN) pathology to distinguish synucleinopathies from other neurodegenerative disorders is still challenging and relevant PET tracers for an early detection and differential diagnosis are missing. A potential compound, anle138b, was shown to interfere with the pathological aggregation of αSYN [1]. We developed novel lead structures and tested them towards binding affinity and selectivity in a filter binding assay (FBA). One promising candidate was then labeled with C-11 for PET imaging. The brain delivery, clearance rate and metabolites were determined in healthy mice.

Methods

In vitro saturation FBAs were performed using recombinant human αSYN, Aß1-42 and tau46 fibrils and a mix of αSYN fibrils, monomers and oligomers. Incubation with increasing concentrations (0.02nM–48nM) of the tritiated compound provided total binding, and non-specific binding was obtained with an excess of the cold compound. KD-values were calculated from non-linear regression. 60 min PET scans were performed after iv injections of 16±3MBq of the C-11-labeled compound in mice. For the metabolite and biodistribution study, two mice were injected with 41±13MBq of the tracer and sacrificed 5 and 15 min after injection. Blood was collected and the mice were perfused with PBS. The right brain hemisphere was homogenized for HPLC. The left hemisphere and other organs were measured in a γ-counter.

Results/Discussion

Our compound showed high affinity for pure αSYN fibrils (KD < 2 nM) and for αSYN monomeric and oligomeric structures (KD < 1 nM). Binding experiments using Aß1-42 and tau46 fibrils revealed a 100- and 17fold lower affinity. Radiolabeling resulted in radiochemical yields of 35 % and specific activities were 25.8 ± 10.0 GBq/µmol. The dynamic PET data revealed a good blood brain barrier penetration, a fast clearance from the brain and a peak SUV value of 1.5-1.8 in the mouse brain. We further observed two metabolites, one was only present in the plasma and another one was present in the plasma and brain.

Conclusions

Our work provides very encouraging in vitro and in vivo data of a potential new PET tracer to image αSYN depositions in the brain. Future experiments aim to further validate the tracer in human and animal brain tissues with confirmed αSYN pathology and optimize radiosynthesis protocols to increase the specific activity.

References

[1] Wagner J, Ryazanov S, Leonov A, Levin J, Shi S, Schmidt F, et al. Anle138b: A novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson’s disease. Acta Neuropathol. 2013;125:795–813.

2:00 PM
PS-03-3 — Increased functional connectivity in the default mode-like network is related to behavioural outcome in a neurodevelopmental model with relevance for schizophrenia (#390)

S. Missault1, 2, C. Anckaerts2, S. Ahmadoun1, I. Blockx2, K. Bielen3, D. Shah2, S. Kumar-Singh3, A. Van der Linden2, S. Dedeurwaerdere1, M. Verhoye2

1 Experimental Laboratory of Translational Neurosciences, University of Antwerp, Translational Neurosciences, Wilrijk, Belgium
2 Bio-Imaging Lab, University of Antwerp, Biomedical Sciences, Wilrijk, Belgium
3 Laboratory of Cell Biology and Histology, University of Antwerp, Veterinary Sciences, Wilrijk, Belgium

Introduction

Maternal immune activation (MIA) is an important risk factor for schizophrenia. Functional dysconnectivity and NMDA receptor hypofunction have been postulated to be key features in the pathophysiology of schizophrenia. The aim was to investigate functional connectivity (FC) changes in default mode-like network, and NMDAR function in adult rat offspring prenatally exposed to an immune challenge, and to determine whether they relate to behavioural outcome. Finally, we explored whether MIA offspring exhibit a different pathophysiology depending on the maternal response to the immune stimulus.

Methods

Pregnant rats were injected with Poly I:C or saline. 6h post-injection, maternal serum was collected for cytokine analysis and 24h post-injection maternal weight response was recorded. Based on the maternal weight response, offspring were divided into 3 groups: controls (n=11), and offspring of dams that gained and lost weight post-MIA (Poly I:C WG, n=12; Poly I:C WL, n=16). Male adult offspring were subjected to resting-state functional MRI, diffusion tensor imaging (DTI), pharmacological fMRI with 0.2mg/kg NMDAR antagonist MK-801 (7T Bruker PharmaScan), and behavioural testing.

Results/Discussion

Pregnant dams that lost weight post-MIA displayed a more pronounced increase of the chemokine RANTES vs. controls than rats that gained weight post-MIA. Region of interest- and seed-based analysis of resting-state fMRI data revealed that Poly I:C WL offspring exhibited increased FC in the default mode-like network (Fig.1). Comparison of the BOLD response before and after (20-30 min post) MK-801 administration showed that Poly I:C WG offspring had a much smaller response to the NMDAR antagonist vs. controls (Fig.2). Voxel-based analysis of DTI data revealed no differences in MIA offspring vs. controls. Behavioural deficits were subtle with the most pronounced deficit being an increased anxiety. Hypersynchronicity in the default mode-like network was associated with behavioural outcome in MIA offspring.

Conclusions

MIA offspring displayed a differential pathophysiology dependent on the maternal response to the immune challenge. FC in the default mode-like network was related to the behavioural outcome in MIA offspring.

Acknowledgement

This study was supported by the Research Foundation Flanders (FWO) (G.0586.12) and Molecular Imaging of Brain Payhology (BRAINPATH) (612360).

Fig.1. Increased functional connectivity (FC) in the default mode-like network (DMN) of Poly I:C WL
A. ROI-based analysis revealed increased FC within the DMN in adult MIA offspring vs. controls, which was most pronounced in Poly I:C WL offspring. B. Average group statistical seed-based FC maps with posterior parietal cortex as seed region (one-sample t-test, FWE corrected, p<0.05, minimal cluster size k=10).

Fig.2. Pharmacological MRI with NMDA receptor antagonist MK-801.
Average group statistical difference maps of BOLD signal pre – post MK-801 administration (uncorrected, p<0.01, minimal cluster size k=10). MIA offspring showed a significantly smaller response to the NMDAR antagonist compared to controls, which was most pronounced in Poly I:C WG offspring.

2:10 PM
PS-03-4 — Longitudinal in vivo MRI assessment of functional connectivity and structural integrity in the TgF344-AD rat model of Alzheimer’s Disease (#395)

C. Anckaerts1, I. Blockx1, P. Summer2, J. Michael2, C. Kreutzer2, H. Boutin3, S. Couillard-Despres2, M. Verhoye1, A. Van der Linden1

1 Bio-Imaging Lab, University of Antwerp, Wilrijk, Belgium
2 Institute of Experimental Neuroregeneration; Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria
3 Wolfson Molecular Imaging Centre, University of Manchester, Manchester, United Kingdom

Introduction

Recently, the TgF344-AD rat model of AD, bearing mutant human amyloid precursor protein (APP) and Presenilin 1 (PS1) genes was first described by Cohen et al1. According to this publication, this model manifests the full spectrum of AD pathology similar to human AD, i.e. progressive cerebral amyloidosis, tauopathy, neuronal loss and age-dependent cognitive decline. Here, we further examined AD-related pathology in this rat model by means of resting state functional MRI (rsfMRI) and diffusion tensor imaging (DTI) to evaluate functional connectivity (FC) and structural integrity, respectively.

Methods

Female TgF344-AD rats (NWT=12, NTG=11) were scanned on a 7T MR system in a longitudinal manner (6, 10, 12, 16 and 18 months). Rats were anaesthetized using a mixture of medetomidine (0.05mg/kg s.c. bolus; 0.01mg/kg/h s.c. infusion) and a low dose of isoflurane (0.4%). Each imaging session consisted of a rsfMRI scan (2D GE-EPI; TR 2s; TE 29ms; 20 axial slices; 300 repetitions; (0.234 x 0.234 x 0.8)mm³), a DTI scan (2D DW-SE-EPI; TR 7.5s; TE 26ms; δ 4ms; Δ 12ms; b-factor 800 s/mm²; 60 diffusion directions; 20 slices; (0.234 x 0.234 x 0.8)mm³) and a T2-weighted 3D anatomical scan (RARE, TR 3.185s; TE 44ms; RARE factor 8; (0.11 x 0.25 x 0.20) mm³). Samples for ex vivo analyses were acquired at 10, 16 and 18-20 months.

Results/Discussion

Analysis of the rsfMRI data revealed significantly reduced FC strength in TG animals (Fig. 1), affecting regions characteristically involved in AD such as the hippocampus and cingulate cortex. These differences in FC between TG and WT were found to be the greatest at 10 months. A voxel-based analysis of the DTI data indicated progressive changes of the structural integrity of the brain (Fig. 2). As such, increased mean diffusivity (MD) was found in the sensory cortex of TG animals, whereas decreased values were present in the ventricular area. Furthermore, TG rats had reduced fractional anisotropy (FA) in the sensory cortex and several white matter structures, whereas increased FA was present in the corpus callosum of TG rats. These DTI changes were exacerbated in advanced stages of the disease (16-18 months). Initial histological examination of the hippocampus indicated progressive gliosis and increasing amyloid plaque load between 10 and 20 months. Further analyses are still ongoing.

Conclusions

Already at early stages, TgF344-AD rats displayed a strong attenuation of the brain’s FC preceding major structural alterations of the brain. Further ex vivo analyses will help in explaining the observed in vivo MRI results.

References

1. Cohen et al., J Neurosci, 2013

Acknowledgement

This research was supported by the European Union’s Seventh Framework Programme (grant agreement number 278850; INMiND) and by the Fund for Scientific Research Flanders (FWO) (grant agreements G067515N and G057615N).

Figure 1. Functional connectivity changes in the TgF344-AD rat.
A-E. Average zFC matrices for WT (top) and TG animals (bottom) at each time point. Each square indicates the zFC between each pair of ROIs, with the colour scale indicating the strength of the connectivity.
F. Outcome of the statistical analysis showing significant genotype effects (bottom) and ageing effects (top). The colour scale indicates log-transformed p-values.

Figure 2. Structural alterations in the TgF344-AD rat.
Genotype differences for MD (A) and FA (B), obtained from a voxel-based statistical analysis. Results are shown on a study-specific 3D anatomical template, FWE p<0.05, minimum cluster size of 10 voxels. The colour scale represents t-values with red/yellow indicating higher values in TG animals, whereas blue colours represent lower values in TG animals compared to WT.

2:20 PM
PS-03-5 — Cannabinoid-induced changes in intrinsic connectivity of the adult rat brain are mediated by actomyosin contractility (#491)

C. Morisset1, 2, 3, J. Ferrier6, 5, 4, C. Demene1, 2, 3, A. Ricobaraza4, 5, T. Deffieux1, 2, 3, M. Tanter1, 2, 3, Z. Lenkei6, 5, 4

1 INSERM U979, Wave Physics for Medicine Lab, Paris, France
2 ESPCI Paris, Institut Langevin, Paris, France
3 CNRS, UMR 7587, Paris, France
4 CNRS, UMR 8249, Paris, France
5 ESPCI Paris, Brain Plasticity Unit, Paris, France
6 Center of Psychiatry and Neurosciences, INSERM U984, Paris, France

Introduction

The effects of exogenous cannabinoids on the central nervous system are mediated mainly by CB1R. They interfere with the endocannabinoid system and modify brain connectivity. We have previously described a novel molecular pathway dependent on CB1R signaling and actomyosin contractility. Here we asked whether this pathway may also induce changes in synaptic function and lead to modifications of resting-state functional connectivity in vivo by blocking neuronal actomyosin contractility using Neurelaxin-A in the living rat brain during cannabinoid treatment (CP 55, 940) of adult rats.

Methods

Experiments were performed on 12 male Dawley rats, anesthetized with an IP injection of 75 mg.kg-1 ketamine and 1 mg.kg-1 xylazine. Ultrasound imaging was performed in a coronal plane at Bregma–3.6mm. Ultrasound sequences are emitted by an array of 128 elements transducer connected to a modified ultrafast ultrasound scanner. Ultrasensitive Doppler images were acquired every second from plane waves compounding (5 angles, PRF=2500). After 10 minutes recording of baseline functional connectivity (FC), animals were then injected 5µL of either vehicle (n=4) or Neurelaxin-A 5mM (n=4) in the right cerebral ventricle. 15 minutes after the injection, a 10 minutes’ recording were performed then the rats were IP injected with 0.7 mg/kg of CP 55,940. Another 10 minutes’ recording was performed.

Results/Discussion

In rats receiving the vehicle, the cannabinoid CP 55,940 induced several alterations of the resting state functional connectivity compared to baseline. Overall, the dorsal hippocampus, a brain region particularly rich in CB1R seemed to be the main target of CP 55,940 regarding bilateral connectivity. A seed-based analysis of interhemispheric correlation was performed at each group (Neurelaxin-A or Vehicle-treated rats) for statistical comparison.. While the connectivity patterns remained similar in both conditions at baseline and after pretreatment, the functional connectivity strength between the right and left hippocampi was strikingly reduced after CP 55,940 injection, suggesting that cannabis decreases the interhemispheric connectivity of the hippocampus. Most importantly, actomyosin blockade using Neurelaxin-A pre-treatment was able to prevent this major alteration.

Conclusions

Neurelaxin-A lets us inhibit brain actomyosin contractility in vivo for the first time. This gives preliminary results arguing for a cannabinoid-induced alteration of FC that could be rescued by blocking actomyosin contractility. These results provide new insights into the effects of cannabinoids on brain function and highlight actomyosin contractility as a major effector of cannabis effects. As a strong correlation exists between psychotic pathologies and alteration of FC, it opens new perspectives in the understanding of both cognitive function and the pathogenesis of psychiatric disease.

Neurelaxin prevents cannabis-induced bilateral decorrelation

2:30 PM
PS-03-6 — BOLD fluctuations under 0.01 Hz detected in resting-state functional MRI (#315)

P. Pais1, Y. Jian1, J. Stelzer1, B. Edlow2, X. Yu1

1 Max Planck Institute - Cybernetics, High Field MRI, Tuebingen, Germany
2 Massachusetts General Hospital, Neurosciences intensive care unit, Boston, Massachusetts, United States of America

Introduction

Spectral analysis in resting-state fMRI (rs-fMRI) studies has mainly focused on the 0.01 to 0.1 Hz frequency range1-6. Frequencies under 0.01 Hz are typically regarded as artifacts from scanner instabilities or physiological noise7, and are routinely excluded from the rs-fMRI analysis. Here, we show robust ultra-slow rs-fMRI signal fluctuations of high regularity in the brain of rats under anesthesia, which seem to be independent from breathing-derived motion8 or cardiovascular oscillations9,10 and possibly indicate a peculiar brain state in the animals.

Methods

12 to 15 minutes of rs-fMRI data were acquired from anesthetized rats (under isoflurane, a-chloralose, medetomidine or urethane) using a 3D-EPI sequence with the following parameters: TE, 12.5 ms; TR, 1s; matrix size, 48x48x32; resolution, 400x400x600 µm. All images were acquired with a 12 cm diameter 14.1 T/26 cm magnet interfaced to an Avance III console. Trans-receiver surface coils were used to acquire the whole brain fMRI. Animals were mechanically ventilated, and a low dose of the paralytic agent pancuronium was used to prevent motion artifacts. Frequency decomposition analysis and power estimation of the 0.005-0.012 Hz frequency band were performed to map the ulstra slow oscillations (USO) in the rat brain.

Results/Discussion

Here we show that frequencies below the classical 0.01 Hz limit can be detected, with high amplitude and rhythmic pattern, in the Blood-Oxygen Level Dependent (BOLD) fMRI signal of animals receiving anesthesia (Fig.1). When present, these slow waves occured predominantly in the hypothalamic area, as confirmed with bandpass power analysis (Fig.2). The features of the reported USO (brain region predominance and apparent absence of correlation with motion or physiological artifacts), suggest that these oscillations might have a neural origin instead of being derived from MR hardware noise. Importantly, infraslow oscillations in a similar range were detected by EEG in brain-injured patients, which appeared related to modulations in the cortical excitability and have been hypothesized to emerge from a deep brain source11.

Conclusions

Our observations suggest that frequency components in the ultra-slow range may contribute to brain function. Future work will aim to clarify the source of these oscillations with neuronal and astrocytic calcium imaging and the utilization of cholinergic modulators to study reversibility of these oscillations in the rat brain.

References

1. Obrig, H. et al 2000; 2. Mitra, P. P. et al 1997; 3. Biswal, B. et al 1995; 4. Golestani, A. M. et al 2017; 5. Raichle, M. E. et al 2001; 6. Gohel, S. R. & Biswal, B. B. 2015; 7. Smith, A. M. et al 1999; 8. Van de Moortele et al 2002; 9. Cohen, M. A. & Taylor, J. A. 2002; 10. Kato, M. et al 1992; 11. van Putten, M. J. A. M. et al 2015.

Acknowledgement

Graduate Training Center of Neuroscience Tuebingen.

Figure 1. Slow oscillations detected in the rat.
USO identified under different anesthesia conditions.

Figure 2. Power of the 0.005 to 0.012 Hz band (USO) in the rat brain.
Power of the band-pass filtered functional map averaged from 6 rats that presented slow oscillation time courses. Abbreviations: M, mammillary nuclei, LH: lateral hypothalamus.

2:40 PM
PS-03-7 — An in-depth view of the mouse cerebral vascular architecture: a multiscale multimodal imaging approach. (#396)

R. Hinz1, J. R. Detrez2, L. Peeters1, C. Berghmans3, M. Verhoye1, A. Van der Linden1, W. H. De Vos2, G. A. Keliris1

1 Bio-Imaging Lab - University of Antwerp, Biomedical Sciences, Wilrijk, Belgium
2 Laboratory of Cell Biology and Histology - University of Antwerp, Veterinary Sciences, Wilrijk, Belgium
3 Molecular Imaging Center Antwerp - University of Antwerp, Faculty of Medicine, Wilrijk, Belgium

Introduction

Vascular abnormalities are hallmark comorbidities of a variety of neuropathologies. Hence, in-depth knowledge about the cerebral vascular architecture and its alterations in disease are quintessential. Here, we used a multi-scale approach combining, on the same animal, anatomical MRI and time of flight magnetic resonance angiography (TOF-MRA) with high-resolution whole brain microscopic imaging data in order to create a high resolution vascular atlas that could be co-registered to the Allen Brain space providing a link to additional functional, anatomical and genetic information.

Methods

Male C57BL/6 (N=6) mice of 12 weeks old were scanned on a 9.4T Biospec. MRI data acquisition included a 3D-T2 anatomical scan followed by three orthogonal orientated 2D-TOF-MRA to assess the macro vasculature. All scans were co-registered to the Allen Brain atlas space using ANTs. Micro vasculature was acquired with 3D microscopic imaging of a cleared mouse brain (iDISCO) with a double labeling of the vasculature using isolectin to stain the vascular wall combined with an albumin staining of the vessel lumen. Cleared samples were imaged on a light sheet microscope with a 1.8x effective magnification and a step-size of 5µm. Each subject’s microscopic imaging data was co-registered to its respective MRI anatomical dataset and subsequently co-registered to the Allen Brain atlas using ANTs.

Results/Discussion

The developed atlas provides a multi-scale representation of the vasculature of the mouse brain (Fig.1). More specifically, it contains the macro-vasculature acquired with TOF-MRA providing: a) a template of mainly the larger vessels and sinuses (Fig. 1A) and b) a vascular probability map that reflects spatial reliability across subjects with the large vessels presenting high spatial reliability in contrast to further more variable branches (Fig. 2). The atlas also contains a novel double-labeling of the micro-vasculature. Preliminary results show that isolectin staining was superior in labeling capillaries while albumin staining more successfully labeled the bigger vessels and could be used for alignment to the TOF-MRA. By combining both stainings an improved detection of the mouse cerebral vascular architecture can be achieved. Furthermore, the atlas also contains 3D-T2 weighted anatomical information which can be used as an access point for other MRI experiments.

Conclusions

This multi-scale approach provides not only the full vascular tree with unprecedented detail but also a means for co-registration and multivariate analysis of multimodal imaging information via the Allen Brain framework. Future work can further extend this atlas with quantitative computational tools able to identify vascular changes and abnormalities as often observed in neurodegenerative disorders.

Acknowledgement

This research was supported by Molecular Imaging of Brain Pathophysiology (BRAINPATH) under grant agreement number 612360 within the Marie Curie Actions-Industry-Academia Partnerships and Pathways (IAPP) program, by the Fund of Scientific Research Flanders (FWO G048917N) and Flagship ERA-NET (FLAG-ERA) FUSIMICE (grant agreement G.0D7651N).

Fig. 1. Multimodal imaging of brain vasculature.

A. TOF-MRA B. MIP image of the whole brain cerebral vasculature recorded in cleared mouse brain C. Zoom-in of the hippocampal region of the brain shown in B. Color bar: Depth coding of the vessels.

Fig. 2. Vascular probability map.
The spatial reliability of the vasculature. Lighter vessels are more spatially reliable. White vessels are present in all subjects.

2:50 PM
PS-03-8 — Multispectral-optoacoustic-tomography imaging of acute cerebral hypoxia and upregulation of matrix-metalloproteinase activity in a mouse model of cerebral ischemia and reperfusion (#11)

R. Ni1, M. Vaas1, W. Ren1, J. Klohs1

1 ETH Zurich & University of Zurich, Institute for Biomedical Engineering, Zurich, Switzerland

Introduction

Hemodynamic alternations and the subsequent inflammatory responses such as upregulations of matrix metalloproteinases (MMPs) play important roles in the pathophysiology of cerebral ischemia. In this study we aimed to detect in vivo the changes in cerebral tissue oxygenation and MMP activity during and after transient middle cerebral artery (MCA) occlusion (tMCAO) in mice using multispectral optoacoustic tomography (MSOT) co-registered with magnetic resonance imaging (MRI) to derive information on the ischemic lesion.

Methods

C57B6L/J mice underwent tMCAO or sham surgery (n = 39) were imaged by MSOT for cerebral hemodynamic changes during 1 h tMCAO or at 48 h after reperfusion. Brain MMP activities were detected by using MSOT with a MMP-activatable probe at 48 h after reperfusion [1]. Diffusion weighted imaging and T2-weighted MRI were performed at 7 T. The MSOT deoxy-, oxyhemoglobin and MMP images were co-registered with structural MR for lesion delineation and anatomical references. Ex vivo near-infrared imaging and triphenyltetrazolium chloride staining were performed with brain slices for visulization of MMP signal and ischemic lesions.

Results/Discussion

Reduced ipsi/contralateral ratio of tissue oxygen saturation was observed during acute tMCAO compared to sham-operated mice (52.5 ± 23.1 %, vs 98.6 ± 18.3 %, p = 0.0003), which recovered to normal at 48 h after reperfusion (99.9 ± 9.4 %, n = 9). Elevated ipsi-/contralateral MMP signal was detected at 48 h after reperfusion in the ipsi-lesion brain regions of tMCAO (4738.7± 2867.8 MSOT a.u., n = 5) compared to sham-operated mice (1138.4 ± 709.7 MSOT a.u., n = 4, p = 0.0479). The ex vivo near-infrared fluorescence imaging results demonstrated increased MMP signals in the core of the ischemic lesion as defined by triphenyltetrazolium chloride staining.

Conclusions

In conclusion, MSOT constitutes a useful tool for in vivo visualization of hemodynamic alternations and MMP activity. We demonstrated acute cerebral hypoxia and subsequent increase in MMP activity in the mouse brain after focal cerebral ischemia with reperfusion.

References

[1] J. Klohs et al., "In vivo near-infrared fluorescence imaging of matrix metalloproteinase activity after cerebral ischemia," J Cereb Blood Flow Metab 29(7), 1284-1292 (2009).

Acknowledgement

This work was funded by the University of Zurich and the ETH Zurich Foundation through a Seed Grant of "University Medicine Zurich/Hochschulmedizin Zürich" and by funding from the Olga Mayenfisch Stiftung.

Figure 1

Figure 1 In vivo assessment of brain oxygenation and matrix metalloproteinases with MSOT in tMCAO mouse at 48 h after reperfusion (a) Hb, HbO2 images, unmixed signal from HbO2 (red) and Hb (blue), scale 0-2×101 MSOT a.u,; (b) tMCAO mouse injected with MMP-activatable probe 48 h after reperfusion with unmixed signal from MMP (green) overlaid on T2-weighted MR image, scale 0-9×104 MSOT a.u,

6:00 PM
SG-04-1 — Allen brain atlas registration of mouse MR and histology images - what can we learn from it for basic stroke research, which challenges remain? (#616)

A. Hess1

1 University of Erlangen-Nuremberg, Institute of Pharmacology and Toxicology, Erlangen, Germany

Content

Brain atlases are of utmost importance for any brain imaging approach as soon as a certain structure, a ROI, has to be structurally identified. Until recently anatomical brain atlases have been derived from two dimensional histological sections, which were labelled by experts e.g. the very well-known and highly appreciated atlases for mouse and rat by Paxinos et al.. These histological atlases provide microscopic, i.e. cellular resolution, which was and still is of importance to sufficiently delineate the different substructures in the brain. At this cellular level histochemistry provided specific marker like Nissl staining or ACh-esterase staining serving as basic information for brain parcellation. This approach goes back to the times of Broadmans’ parcellation of the human brain. However, all histological atlases suffer, even due to technical advances, to a different degree from cutting and preparation/staining artefacts and most importantly, the full three dimensional context is lost. Several approaches have been worked out to cope with these constrains. Methodologically inherent in 3D imaging techniques like MRI, SPECT, PET or CT is the 3D context and the reduced, if not even artefact-free, data quality, but unfortunately with a lower spatial resolution. Technical improvements particular in the MRI field lead to much higher resolutions (groups of Johnson (Duke) and Henkelmann (Toronto) in the order of ten’s of microns. Based on these resolutions and improved imaging contrasts MRI based fully 3D brain atlases became available. Of note, after 3D imaging the brain in the skull most of the classical histological atlas generation approaches can be applied in addition. Therefore, multimodal brain atlases were generated combining the best from the two worlds like the Waxholm datasets as only one example. At this point, another methodological progress is striking: non-affine registration or warping approaches for building probabilistic atlases. Probabilistic atlases due to averaging across many individual subjects (after appropriate registration) provide much higher SNR in the datasets but even more important allow for a new branch of anatomical analyses, namely voxel-wise group statistics, which were introduced under the term “voxel based morphometry”. This has led to fundamentally new insights into neurobiological research and continues to impact particularly the field of brain pathologies, transgenic mouse research as well as neurodegeneration but also studies of brain plasticity. In addition and somewhat parallel was the usage of brain atlases in the context of functional imaging studies were activation biomarkers like 2DG in autoradiography, 2FDG in PET or fMRI BOLD activation markers were intended to be analyzed in a brain structure specific manner and compared between groups of subjects. To obtain a “match” between functional data, in general at a (much) lower resolution, and the brain atlases used different registration approaches are applied. Here the obtained quality of the “match” is of great impact for the final results. Automated functional analysis pipelines, which are used in a “black-box” manner, starting from the raw data as input and provide statistical differences at the output should be much more scrutinized or better falsified than is currently the case. Further mention should be made of the fact that more and more high resolution atlases are being made available for single brain structures / areas like the cerebral cortex (Amunts/Zilles group) or the rat hippocampus (Bjaalie group). Of note, from more and more species (digital) brain atlases are provided from insects (drosophila, honey bee), fishes, Gerbil to monkeys using state of the art imaging technologies (see e.g. Bakkers https://scalablebrainatlas.incf.org/ for an overview at lease for mammalian species). It is noteworthy that almost all these atlases are freely accessible to the scientific community (cf. http://www.nitrc.org). Finally and very recently additional data entities led to an enormous boost in dimensionality of digital brain atlases to name only a few topics: connectivity by diffusion tensor imaging (MRI DTI) or trace injections as Allen Brain Atlas, OMICS approaches like ViBrism and again the ABA or development (emap and ABA). Another new endeavor is to perform new multimodal parcellation schemes e.g. incorporate functional data like resting state fMRI data to obtain parcellations of brain structures at a much higher level fusing anatomical/histological and functional information. As a conclusion, it should be noted that the data on digital brain atlases, especially in the mouse through the flagship project Allen brain atlas, already allows in-silico studies on the anatomy and function of the brain. This is very effective, is cost-saving, reduces the number of laboratory animals and allows completely new research questions (cf. Ganglberger et al., NI 2017).

6:20 PM
SG-04-2 — Allen brain atlas registration of mouse MR and histology images - what can we learn from it for basic stroke research, which challenges remain? (#603)

P. Boehm-Sturm1

1 Charité – Universitätsmedizin Berlin, Department of Experimental Neurology and Center for Stroke Research Berlin, Berlin, Germany

Content

Registration to a standard brain atlas is the prerequisite for modern analysis of neuroimaging data, e.g. connectomics or voxel-based group statistics. Tools and pipelines for human neuroimaging data are quite prevalent but common agreements and workflows in the small animal imaging community are lacking. To date, the Allen brain atlas is the most comprehensive database of mouse brain anatomy and connectivity. We have developed MATLAB toolboxes1 for registration of mouse MR and histology images to this atlas based on the freely available SPMMouse2 and elastix3 packages. Examples will be given of how atlas registration can help to generate hypotheses in basic research of functional recovery after stroke in the mouse based on techniques that correlate data from behavioral testing with imaging data, e.g. voxel-based lesion symptom mapping4. To stimulate a discussion, some personal insight will be given into technical pitfalls of image registration, advantages and drawbacks of the Allen database, and challenges when using atlas registration-based techniques in models of disease.

References

1. Koch S et al. Atlas registration for edema-corrected MRI lesion volume in mouse stroke models. J Cereb Blood Flow Metab. 2017;0271678X1772663. Epub ahead of print.

2. Sawiak SJ et al. SPMMouse: A New Toolbox for SPM in the Animal Brain. Proc Int’l Soc Mag Res Med. 2009. p. 1086.

3. Klein S et al. elastix: a toolbox for intensity-based medical image registration. IEEE Trans Med Imaging. 2010;29(1):196–205.

4. Bates E et al. Voxel-based lesion-symptom mapping. Nat Neurosci. 2003;6(5):448–450.

Acknowledgement

This work was supported by the Federal Ministry of Education and Research (BMBF) (grant number 01EO0801, Center for Stroke Research Berlin) and Deutsche Forschungsgemeinschaft (DFG) (Excellence Cluster NeuroCure).

6:40 PM
SG-04-3 — Advancements in vascular imaging of the mouse brain. (#609)

R. Hinz1, J. R. Detrez2, L. Peeters1, M. Verhoye1, A. Van der Linden1, W. H. De Vos2, G. A. Keliris1

1 University of Antwerp, Bio-imaging Lab, Wilrijk, Belgium
2 University of Antwerp, Laboratory of Cell Biology and Histology, Wilrijk, Belgium

Content

The cerebral vasculature has a key supporting role by supplying the brain with oxygen, nutrients and removing brain metabolites. Any aberrations in this vascular system can have severe consequences on normal brain functioning. As seen in stroke, occlusion of vessels can lead to ischemia and eventually neuronal death. Furthermore, structural vascular abnormalities have been observed in many neuropathological diseases such as Alzheimer’s disease, Huntington’s disease, multiple sclerosis and brain tumors[1]. Therefore, it is essential to have knowledge of the whole brain’s vascular architecture.

Recent advances in preclinical imaging methods allow detection of whole brain vasculature on different imaging scales. Ex vivo histological imaging techniques allow the visualization of whole brain vasculature at resolutions < 3 µm. As the high resolution datasets are generally large, the processing of the data is time consuming. The datasets, however, allow precise investigation of the topology of the micro-vasculature and provide information on regional capillary densities[2]–[4]. In contrast to histological imaging, non-invasive in vivo imaging techniques have the advantage to assess the development of macro-vasculature and venous sinuses within the same subject over time. This is essential in studies investigating the effect of progressing neuropathology on cerebral vasculature [5].

Vascular imaging data usually have a high specificity allowing for extraction of the vasculature after noise reduction and filtering. Further processing can be undertaken to extract information on vascular branching and vascular density. Additionally, vascular imaging data can be co-registered to the Allen Brain Atlas, which gives access to and allows comparison with anatomical, genetic and connectivity information.

References

[1]        C. C. V Chen, Y. C. Chen, H. Y. Hsiao, C. Chang, and Y. Chern, “Neurovascular abnormalities in brain disorders: Highlights with angiogenesis and magnetic resonance imaging studies,” J. Biomed. Sci., vol. 20, no. 1, p. 1, 2013.

[2]        A. Paolo et al., “Whole-Brain Vasculature Reconstruction At the Single Capillary Level,” pp. 1–25, 2017.

[3]        B. Xiong et al., “Precise Cerebral Vascular Atlas in Stereotaxic Coordinates of Whole Mouse Brain,” Front. Neuroanat., vol. 11, no. December, pp. 1–17, 2017.

[4]        S. Xue et al., “Indian-ink perfusion based method for reconstructing continuous vascular networks in whole mouse brain,” PLoS One, vol. 9, no. 1, pp. 1–7, 2014.

[5]        N. Beckmann et al., “Age-dependent cerebrovascular abnormalities and blood flow disturbances in APP23 mice modeling Alzheimer’s disease.,” J. Neurosci., vol. 23, no. 24, pp. 8453–8459, 2003.

Acknowledgement

This talk was made possible by the Bio-imaging lab and Laboratory of Cell Biology and Histology - University of Antwerp and was supported by Molecular Imaging of Brain Pathophysiology (BRAINPATH) under grant agreement number 612360 within the Marie Curie Actions-Industry-Academia Partnerships and Pathways (IAPP) program, by the Fund of Scientific Research Flanders (FWO G048917N), Flagship ERA-NET (FLAG-ERA) FUSIMICE (grant agreement G.0D7651N) and by the European Union’s Seventh Framework Programme (FP7/2007–2013) under grant agreement number 278850 (INMiND).

1:30 PM
emptyVal-1 — Introductory Lecture by Hervé Boutin - Manchester, UK

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

1:50 PM
PS-14-2 — PET imaging of glioma associated-inflammation using the TSPO-negative human glioblastoma cell line LN18 (#145)

H. Pigeon1, C. Truillet1, E. Jaumain1, B. Jego1, F. Caillé1, A. Winkeler1

1 Imagerie Moléculaire In Vivo, Inserm, CEA, Univ. Paris Sud, CNRS, Université Paris Saclay, CEA/DRF/JOLIOT/SHFJ, Orsay, France

Introduction

Glioma associated-inflammation has been correlated to invasiveness, angiogenesis and poor prognosis. Studying glioma associated-inflammation, in particular microglia cells may help to better understand its role in tumour progression and treatment response. Imaging neuroinflammation is feasible using PET imaging of the 18 kDa translocator protein (TSPO), a well-known marker for neuroinflammation. As glioma cells may also express TSPO, exclusive visualization of microglia cells is challenging. Using the TSPOnegative glioma cell line LN18 we investigated glioma associated-inflammation only.

Methods

Direct inoculation of LN18 human glioma cells in the striatum of nude mice does barely yield tumour growth. We therefore injected LN18 glioma cells subcutaneously into flanks of nude mice. After tumour growth and serial passages in mice, a cell suspension of LN18 tumours was prepared and 2x105 cells were stereotactically implanted into the right striatum of nude mice (n=6). Tumour growth was monitored using 18F-DPA-714 PET. A volume-of-interest (VOI) analysis was performed on the tumour injected brain hemisphere and in the contralateral side and standardized uptake values (SUV) were calculated. PET findings were validated using autoradiography and immunofluorescence (IF) for human nestin (tumoral cells), CD11b (microglia) and murine TSPO (stromal host cells).

Results/Discussion

At 7 weeks post-implantation PET images show a ring-like 18F-DPA-714 signal in the glioma injected hemisphere, without 18F-DPA-714 uptake in the centre of the tumour. SUVs in the glioma injected hemisphere are significantly higher than in the contralateral site (p<0.0005) (Figure). Ex-vivo and in-vitro autoradiography confirm in vivo PET signal, with increased 18F-DPA-714 in the tumour rim, and lack of central 18F-DPA-714 signal. Interestingly, immunofluorescence (IF) staining confirmed increased murine TSPO expression mostly at tumour boundaries, which co-localizes with microglial cells suggesting that TSPO imaging of the LN18 human glioma model allows to visualize glioma associated-inflammation only (Figure).

Conclusions

We found a significantly higher tumour-to-contralateral 18F-DPA-714 uptake at 7 weeks post glioma cell implantation. The signal shows a ring-like structure that seems to reflect glioma associated-inflammation surrounding the tumour, as indicated by the presence of TSPOpositive microglia cells, which form dense boundaries around the tumour. These results suggest that the LN18 human glioma model allows to visualize exclusively glioma associated-inflammation in vivo using 18F-DPA-714 PET, which may help us to visualize changes regarding glioma associated-inflammation upon treatment.

Acknowledgement

This work was performed on a platform of France Life Imaging network partly funded by the grant “ANR-11-INBS-0006”. This research was partly funded by the EU 7th Framework Programme (FP7/2007-2013) under grant agreement n° 278850 (INMiND).

Figure LN18 tumour
In vivo imaging of the TSPOnegative human glioma LN18 using 18F-DPA-714 PET (A). Quantification of SUVs indicates a significantly higher uptake of 18F-DPA-714 in the tumour-injected brain hemisphere compared to the contralateral side (B). Immunofluorescence staining shows dense infiltration of TSPOpositive microglia into the LN18 tumour that form a thick boundary around the tumour (C).

2:00 PM
PS-14-3 — Non-invasive assessment of the onset and progression of amyotrophic lateral sclerosis in a hTDP-43A315T mouse model: a PET-MR study (#28)

A. Weerasekera1, 2, M. Crabbé1, 6, W. Gsell1, 2, D. Sima3, C. M. Deroose6, S. Van Huffel3, P. Van Damme4, 5, K. Van Laere1, 6, U. Himmelreich1, 2

1 KU Leuven, MoSAIC, Molecular Small Animal Imaging Centre, Imaging & Pathology, Leuven, Belgium
2 KU Leuven, Biomedical MR Unit, Imaging & Pathology, Leuven, Belgium
3 KU Leuven, ESAT - STADIUS, Stadius Centre for Dynamical Systems, Signal Processing and Data Analytics, Leuven, Belgium
4 University Hospitals Leuven, Department of Neurology, Leuven, Belgium
5 KU Leuven, Laboratory for Neurobiology, Department of Neurosciences, Leuven, Belgium
6 KU Leuven and University Hospitals Leuven, Division of Nuclear Medicine, Leuven, Belgium

Introduction

TDP-43 proteinopathy is a common pathological feature in patients with amyotrophic lateral sclerosis (ALS). 1 However, the molecular mechanism of how mutated TDP-43 triggers ALS remains poorly understood. In this regard, non-invasive imaging of TDP-43 transgenic mouse models can help to elucidate the pathophysiology2. Therefore, we investigated, for the first time, the effect of a hTDP-43A315T mutation on the aging mouse brain by using longitudinal in vivo PET-MR imaging, combining [18F]FDG-PET, magnetic resonance spectroscopy (MRS), diffusion tensor imaging (DTI) and perfusion MR imaging.

Methods

hTDP-43A315T (n=29) and control (n=10) mice underwent PET-MR imaging (7T Bruker Biospec - Albira Si PET insert) to study cerebral glucose metabolism at 4 and 8 months of age. A static 30 min [18F]FDG PET scan (11,4 ± 1.5 MBq, IV, fasted) was acquired simultaneously with a 3D T2w spin echo MRI using a RARE sequence. FDG/MR scans were coregistered to the Mirrione atlas for VOI and voxel-wise analysis. In parallel, mice were scanned at 2, 5, and 9-months of age using the following sequences: A) MRS: PRESS, TE/TR=20ms/1.8s, VOI=1.5x2.5x2 mm3, jMRUI-based quantification; B) DTI: EPI sequence, 12 dir, 6 b-values; C) Perfusion MR: single-slice FAIR RARE, 14 inversion times (9.4T Bruker). ROIs were placed in the motor cortex and hindbrain. Body weight and motor performance were monitored weekly.

Results/Discussion

[18F]FDG uptake was significantly decreased by 15.9±5,5% in the unilateral motor and somatosensory cortex of TDP-43 mice over time, as compared to controls (pFWE = 9,29.10-6; Fig. 1). On the other hand, glucose metabolism was increased by 8±4,5% in a cluster covering the bilateral substantia nigra, amygdala and reticular nucleus (pFWE=8,26.10-5; Fig. 1). Cortical Glx levels (glutamine+glutamate) were consistently elevated in TDP-43 mice (peak level at 5 months: +36%; p<0,001), while a transient Glx increase was noted in the hindbrain at 2 months of age (+32%; p<0,001), as compared to controls (Fig. 2). In addition, cortical creatine levels gradually increased over time and were significantly higher at 9 months of age (+19,7%; p=0,008), as shown by MRS. Both blood flow and quantitative DTI scalars remained stable in transgenic mice and controls. Weight also did not significantly differ between the TDP-43 and control group.

Conclusions

This study revealed that hTDP-43A315T overexpression affects cerebral energy metabolism, as reported by altered [18F]FDG uptake and creatine tissue levels. In addition, Glx neurotransmitter levels were significantly elevated in transgenic ALS mice, indicative of a disturbance in glutamatergic neuronal signaling. Moreover, these findings are largely in accordance with clinical reports, emphasizing the translational value of this transgenic model3,4. Correlations between imaging results, survival, and immunohistochemical analysis are currently in progress to further validate the present study.

References

1. Neumann, M. et al. Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. Science (80-. ). 314, 130–133 (2006).

2. Herdewyn, S. et al. Prevention of intestinal obstruction reveals progressive neurodegeneration in mutant TDP-43 (A315T) mice. Mol. Neurodegener. 9, 24 (2014).

3. Willekens, S. M. A., Van Weehaeghe, D., Van Damme, P. & Van Laere, K. Positron emission tomography in amyotrophic lateral sclerosis: Towards targeting of molecular pathological hallmarks. Eur. J. Nucl. Med. Mol. Imaging 44, 533–547 (2017).

4. Han, J. & Ma, L. Study of the features of proton MR spectroscopy ( 1 H-MRS) on amyotrophic lateral sclerosis. J. Magn. Reson. Imaging 31, 305–308 (2010).

Acknowledgement

* Both authors contributed equally to this abstract.

Figure 2

Figure 1

2:10 PM
PS-14-4 — Neuroimaging of subacute brain inflammation and microstructural changes predicts long-term functional outcome after experimental traumatic brain injury (#408)

S. Missault1, 2, C. Anckaerts2, I. Blockx2, S. Deleye3, D. Van Dam4, 5, N. Barriche1, G. De Pauw1, S. Aertgeerts1, F. Valkenburg4, P. P. De Deyn4, 5, 6, J. Verhaeghe3, L. Wyffels7, A. Van der Linden2, S. Staelens3, M. Verhoye2, S. Dedeurwaerdere1

1 Experimental Laboratory of Translational Neurosciences, University of Antwerp, Translational Neurosciences, Wilrijk, Belgium
2 Bio-Imaging Lab, University of Antwerp, Biomedical Sciences, Wilrijk, Belgium
3 MICA, University of Antwerp, Molecular Imaging Center Antwerp, Wilrijk, Belgium
4 Laboratory of Neurochemistry and Behaviour,, Institute Born-Bunge, University of Antwerp, Biomedical Sciences, Wilrijk, Belgium
5 Department of Neurology and Alzheimer Research Center, University of Groningen and University Medical Center Groningen (UMCG), Groningen, Netherlands
6 Department of Neurology and Memory Clinic, Hospital Network Antwerp (ZNA) Middelheim and Hoge Beuken; Biobank, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium
7 Department of Nuclear Medicine, University Hospital Antwerp, Edegem, Belgium

Introduction

There is currently a lack of prognostic biomarkers to predict the different sequelae following traumatic brain injury (TBI). The present study investigated the hypothesis that subacute neuroinflammation and microstructural changes predict chronic TBI deficits.

Methods

Young adult male Sprague-Dawley rats were subjected to Controlled Cortical Impact (CCI) injury, sham surgery or skin incision (naïve). CCI-injured (n=18) and sham rats (n=6) underwent positron emission tomography (PET) imaging with the translocator protein (TSPO) radioligand [18F]PBR111 (8.6±0.4MBq, Siemens Inveon PET/CT scanner) and diffusion tensor imaging (DTI) (7T Bruker PharmaScan) in the subacute phase (PET at 7&21 days post-injury, DTI at 4&18 days post-injury). CCI-injured, sham-operated and naïve rats (n=8) were subjected to behavioural testing in the chronic phase (5.5-10 months post-injury): open field and sucrose preference tests, two one-week video-EEG monitoring periods, each one followed by a pentylenetetrazole (PTZ) seizure susceptibility test, and a Morris water maze test.

Results/Discussion

Neuroimaging revealed brain inflammation (Fig.1), decreased fractional anisotropy and increases in mean, axial and radial diffusivity (Fig.2) in perilesional cortex & hippocampus of CCI rats vs. shams. Behavioural analysis revealed disinhibition, anhedonia, increased seizure susceptibility and impaired visuospatial learning in CCI rats. Subacute TSPO & changes in DTI metrics were significantly correlated with several chronic deficits. Importantly, not only absolute values at 4/7 or 18/21 days correlated with behavioural outcome, but also the relative change over time correlated well with chronic TBI sequelae. ROC curve analysis revealed that PET & DTI parameters have good sensitivity & specificity to distinguish between TBI animals with & without particular deficits. Stepwise regression analysis showed that, depending on the investigated deficit, PET or DTI alone, or the combination of these imaging modalities could very well predict the variability in the functional outcome data.

Conclusions

Taken together, both TSPO PET and DTI seem promising prognostic biomarkers to predict different chronic TBI sequelae.

Acknowledgement

This study was supported by ERA-NET NEURON/Research Foundation Flanders GA00913N and Molecular Imaging of Brain Pathophysiology (BRAINPATH) (612360).

Fig.1. PET imaging with [18F]PBR111 in CCI-injured rats.
A. Representative PET images of sham-operated and CCI-injured rats. Yellow: lesion, dark blue: perilesional cortex, light blue: contralateral cortex, purple: ipsilesional hippocampus, pink: contralesional hippocampus. B. Increased uptake of [18F]PBR111 in perilesional cortex and ipsilesional hippocampus of CCI-injured rats vs. shams, which is most pronounced at 7 days post-injury.

Fig.2. Diffusion tensor imaging in CCI-injured rats.
A. Representative fractional anisotropy (FA) and mean diffusivity (MD) maps of sham and CCI rats. Green: lesion, yellow: periles ctx, red: contralat ctx, ochre: ipsiles hippocampus, blue: contrales hippocampus. B-C. ROI-based analysis reveals decreased FA at 4 days post-CCI in perilesional cortex and hippocampus, as well as increased MD, which is most pronounced at 18 days post-injury.

2:20 PM
PS-14-5 — Intra-arterial MRI-guided infusion of thrombin to induce ischemic stroke in pigs (#175)

D. Golubczyk1, I. Malysz-Cymborska1, L. Kalkowski1, M. Zawadzki2, P. Holak3, J. Glodek3, K. Milewska1, M. Bogacki4, M. Janowski5, 6, 7, Z. Adamiak3, W. Maksymowicz1, P. Walczak1, 6, 7

1 University of Warmia and Mazury, Department of Neurology and Neurosurgery, Olsztyn, Poland
2 Central Clinical Hospital of Ministry of the Interior and Administration in Warsaw, Warsaw, Poland
3 University of Warmia and Mazury in Olsztyn, Department of Surgery and Roentgenology with the Clinic, Olsztyn, Poland
4 Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Department of Gamete and Embryo Biology, Olsztyn, Poland
5 Mossakowski Medical Research Centre Polish Academy of Sciences, Department of Neurosurgery, Warsaw, Poland
6 The Johns Hopkins University School of Medicine, Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, Maryland, United States of America
7 The Johns Hopkins University School of Medicine, Cellular Imaging Section and Vascular Biology Program, Baltimore, Maryland, United States of America

Introduction

Ischemic stroke represents the third leading cause of death and the leading cause of long-term disability in adults1. Relying solely on rodent models proved inadequate, as practically all clinical trials failed. There is urgent need, supported by STAIR2 and STEPS3, to develop large animal models. We decide to use catheter-based endovascular technique under X-ray navigation, and MRI for guiding cerebral vascular occlusion in pig. Real-time MRI allowed visualization and guided administration of intraarterial thrombus-inducing solution as well as detecting blockage of brain vasculature.

Methods

Animal procedures were approved by local ethics committee and were performed according to ARRIVE guidelines. Percutaneous puncture was made to insert introducer (5F,Prelude MeritMedical) into femoral artery followed by guiding catheter using C-arm guidance.  Microcatheter was placed in ascending pharyngeal artery (APA) proximally to the rete mirabile. Next phase of the experiment was made under real time guidance of 3T MRI scanner (Magnetom Trio, Siemens). To induce ischemia we explored utility of thrombin intra-arterial administration at two boluses. MRI protocol included dynamic GE-EPI for assessment of trans-catheter cerebral perfusion, GE-EPI for monitoring thrombin-mediated blood clotting as well as SWI, diffusion, T2w and T1w with contrast. Images were analyzed using AMIRA6.4.

Results/Discussion

Feraheme-enhanced perfusion MR scans showed brain territory supplied by the catheter infusion at three time points – baseline, thrombin administration, 30 min after stroke induction (Fig. 2A;B,C). Brain perfusion was dynamically quantified (Fig 2A’;2B’,2C’), what showed significant changes before thrombin administration (8,754.4 mm3) and after thrombus formation ( 6,484.59 mm3). T2w scan 17 hours post stroke induction confirmed ischemia (Fig.1H). BBB status didn’t change within initial 17 hours, followed by disruption observed in the cortex 20 hours later (Fig.1N).

Conclusions

Our study has demonstrated feasibility of an endovascular stroke model in pig. We were able to observe in real-time manner formation of thrombus resulting in blockage of cerebral perfusion and eventually stroke lesion. Our model produced relatively large infarct volume covering majority of MCA territory. Overall, we have developed novel model of ischemic stroke in pigs with high clinical relevance.

References

  1. Writing Group, M. et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation 133, e38-360, doi:10.1161/CIR.0000000000000350 (2016).
  2. Fisher, M. et al. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke 40, 2244-2250, doi:10.1161/STROKEAHA.108.541128 (2009).
  3. Savitz, S. I. et al. Stem Cell Therapy as an Emerging Paradigm for Stroke (STEPS) II. Stroke 42, 825-829, doi:10.1161/STROKEAHA.110.601914 (2011).

Acknowledgement

Funding: NCBIR EXPLOREME grant (STRATEGMED1/235773/19/NCBR/2016)

Figure 1.
SWI (A-E), T2w (F-J) and T1+Gd images (K-O) at different time points

Figure 2. Assessment and visualization of cerebral vascular occlusion
Segmentation and 3D reconstruction of perfusion at baseline (A) and during thrombin administration (B) with quantification in A’ and B’. (C) Feraheme perfusion volume after thrombin, 4D quantified in C’. (D) SWI 1.5 hours after thrombin showing persisting hypointensities in the MCA supply territory. Histogram for SWI contralateral (green) and ipsilateral (red) hemisphere (D’)

2:30 PM
PS-14-6 — Survival of stem cell graft in cortical stroke model in the mouse: a bioluminescence and magnetic resonance imaging study (#364)

A. Minassian1, S. Vogel1, M. Dobrivojević Radmilović1, D. Wiedermann1, M. Hoehn1, 2

1 Max Planck Institute for Metabolism Research, In-vivo-NMR Laboratory, Cologne, Germany
2 Leiden University Medical Center, Department of Radiology, Leiden, Netherlands

Introduction

Stroke is a leading cause of death and a major cause of severe disability. If treatment is not received within 3 hours after stroke onset, chances for recovery diminish. Hence, it has become essential to design a therapy that restores the neural circuitry lost during lesion progression. However, past studies were unable to document complete graft survival or even reported vitality loss. Here we report the first long term characterization of a cortical stroke model and monitor in vivo the vitality fate of neural stem cells engrafted adjacent to the ischemic territory in the mouse cortex.

Methods

34 nude mice (8 weeks old, 30 g) were divided in three groups: A) mice with cortical stroke only (n=11), B) animals with cortical stroke and neural stem cell engraftment (n=14), and C) for control, healthy mice with stem cell engraftment (n=9).  For group A and B, cauterization was used for permanent distal MCA occlusion. For group B and C, 1.5 x 105 human neural stem cells (H9 lineage), transduced for constitutive Luc2 expression for bioluminescence imaging (BLI), were implanted into the cortex medial to the lesion and in equivalent location in the healthy mice. Monitoring of lesion volume and location by T2-weighted (T2W) MRI at 9.4 T, and of graft vitality by repetitive BLI was continued for 3 months.

Results/Discussion

The ischemic lesion is highly reproducible in size and location, as determined on T2W-MRI incidence maps. Acutely, at 48 hours after stroke onset, the permanent distal MCA occlusion resulted in purely cortical ischemia (Fig. 1A). Co-registration of T2W anatomical scans to the mouse brain atlas identified the lesion localized in the right sensorimotor cortex but not involving striatal areas. After six weeks, vasogenic edema was resolved but a severe thinning of the affected cortex was prominent. In this process, hippocampal tissue expanded almost to the brain surface, replacing the space left by the shrinking cortex (Fig. 1B, arrow). The graft location in the cortex after 3 months is presented by HuNu, staining for human cell nuclei while the injection canal is visualized on acute MRI (Fig. 2A,B). BLI confirms stable graft vitality over the whole 3 months period, equal in healthy brain and in ischemic periphery (Fig. 2C).

Conclusions

For therapeutic success the graft must be placed at the ischemic target area, and graft vitality is required for the desired regenerative effect. Earlier histological studies only demonstrated cells at the end of observation period but could not quantify survival of the original graft over time. Here we report for the first time that quantitative long term graft survival is achieved, compatible with a clinically relevant stroke model with cortical involvement. This now allows to investigate the therapeutic influence of stem cells on functional and structural network recovery after stroke.

Acknowledgement

We thank Michael Diedenhofen for help with image analysis and Melanie Nelles for immunohistochemistry support. This work was financially supported by grants from the EU-FP7 programs TargetBraIn (HEALTH-F2-2012-279017) and BrainPath (PIAPP-GA-2013-612360).

Coronal T2-weighted MR multislice images at 48 h (A) and at six weeks (B) after stroke induction
At the acute time point (48h) the affected ischemic territory is visualized by T2-hyperintensity, caused mainly by vasogenic edema. In the chronic phase (6 weeks), edema had resolved by cortex was severely thinned and hippocampus was replaced filling the space generated by cortical loss.

Local and functional characterization of cortical stem cell graft
HuNu staining (A) marked the graft in the cortex. Localization of graft placement was noted by the needle injection canal on MRI (B). BLI measurements confirmed graft vitality with no loss during 3 months. Vitality was equally stable for engraftment into healthy cortex and adjacent to ischemic territory (C), supporting compatibility of graft viability with stroke over several months.

2:40 PM
PS-14-7 — In vivo observation of pro-inflammatory and protective phases of immune response during stroke: a combined bioluminescence and magnetic resonance imaging study (#230)

F. M. Collmann1, R. Pijnenburg1, S. Hamzei Taj1, A. Minassian1, M. Hoehn1, 2

1 Max Planck Institute for Metabolism Research, In-vivo-NMR Laboratory, Cologne, North Rhine-Westphalia, Germany
2 Leiden University Medical Center, Radiology, Leiden, Netherlands

Introduction

A growing focus during investigations into brain diseases and brain lesions is directed towards the role of inflammatory processes. Macrophages and microglia, major players during stroke, can develop pro-inflammatory, aggravating (M1-like) or anti-inflammatory, protective (M2-like) phenotypes.

Here, we decipher the time profiles of both activated phenotypes during stroke with bioluminescence imaging (BLI) after injecting lentiviral (LV) particles expressing Luc2 and eGFP reporters under Ym1 or iNOS control, representing M2-like and M1-like phenotypes, respectively.

Methods

We intrastriatally injected 2 µl of concentrated LV-Ym1-Luc2-T2A-eGFP (n=11) or LV-iNOS-Luc2-T2A-eGFP (n=5), 14 or 21 days (d) before stroke induction by middle cerebral artery occlusion (MCAO). BLI signals were recorded at several time points before and after MCAO using a Perkin Elmer IVIS Spectrum CT. Lesion size was monitored by magnetic resonance imaging (MRI) using a Bruker 11.7T scanner. BLI time profile was analysed for time points of maximal signal (“time to peak”) and for signal integration over time (“total immune response”) after stroke.

Results/Discussion

BLI signals were weak for both phenotypes before stroke (Fig. 1, shown for iNOS), but increased several fold after stroke induction (Fig. 2A, B). For Ym1 (M2-like phenotype), signal increase reached maximum on 11 d post stroke with an average 6-fold intensity. For iNOS (M1-like phenotype), maximal BLI signal appeared at variable time after stroke induction. Here, a strong inverse correlation of time to peak (R2 = 0.59; Fig. 2C) and a direct correlation of total immune response (R2 = 0.48; Fig. 2D) with lesion volume was observed. In contrast, the same immune response variables for the protective M2-like phenotype Ym1 showed no correlation with lesion volume (R2 = 0.28 and 0.19, respectively).

The individual immune response was followed over time discriminating between pro-inflammatory, aggravating and protective phenotypes of the immune cells (macrophages and microglia). Both phenotypes presented distinct behavior patterns.

Conclusions

The pro-inflammatory immune response aims at the damaged tissue. Thus, a correlation with lesion volume makes sense: larger lesions stimulate a faster response, which in turn may even induce growing lesion volumes. The anti-inflammatory, protective phenotype aims at the still viable ischemic periphery. In consequence, a relationship with the damaged tissue area is not expected. However, our in vivo strategy appears instrumental for monitoring future protective immune-therapies where induced shifts towards the M2 phenotype have been reported to improve neuronal survival and outcome (1).

References

  1. Hamzei Taj et al. Journal of Neuroimmune Pharmacology 2016; 11: 733-748

Acknowledgement

We thank Dr. Markus Aswendt for his helpful discussions during the early phase of this project, and Cordula Schäfer, Andreas Beyrau, Michael Diedenhofen, Ulla Uhlenküken and Fabian Distler for their professional technical support. This work was financially supported by grants from the EU-FP7 program TargetBraIn (HEALTH-F2-2012-279017) and BrainPath (PIAPP-GA-2013-612360).

Fig. 2 BLI quantification of pro- and anti-inflammatory phenotypes after stroke.

In both, Ym1 and iNOS groups, BLI signal increased after stroke (A, B). In the Ym1 group signal increased with significant changes from including 6 dps to 11 dps compared to time points before stroke (A). Error bars are presented as SEM. In the iNOS group, time to peak inversely correlated with T2 lesion volume (C), and total immune response revealed moderate correlation with T2 lesion volume (D).

Fig. 1 BLI signal increased in LV-iNOS-Luc2-T2A-eGFP injected mice with cortico-striatal lesions.

Before MCAO induction, BLI signal was low (top), but Luc2 signal increased strongly after stroke (bottom). T2 map measured at 7 dps of the same mouse, depicted in the photographs, is shown on the right. Abbreviations: dps, days post stroke; MCAO, middle cerebral artery occlusion.

2:50 PM
PS-14-8 — In-vivo Imaging of metabolism modulation in a model of neuroinflammation using CEST MRI methods (#524)

D. De Battista1, G. Comi1, L. Chaabane1

1 San Raffaele Hospital, Neuroscience/INSPE, Milano, Italy

Introduction

CEST is a versatile imaging tool for detecting exchangeable protons in hydroxyl (-OH), amine (-NH ), and amide (-C(O)NH-) groups in distinct molecules through transfer of signal loss between these protons and water. In particular, CEST imaging demonstrated to be sensitive to metabolites as glucose1 and glutamate2 but also to protein concentration (APT3) and pH (AACID4). In the present study, we explored the multiparametric CEST imaging in a model of multiple sclerosis (MS) that develops severe motor disability.

Methods

All MRI experiments were performed on a 7T-scanner. CEST imaging was first optimized on samples of bovine serum albumin or of glucose at different concentrations and pH. CEST Z-spectra were collected using a sequence with a selective saturation pulse (1.5 sec) followed by a Fast spin echo acquisition. A total of 48 saturation offsets were acquired between -7 to 7 ppm. Different power of saturation were also tested. Both magnetization transfer (MT) asymmetry and AACID were calculated after B0 corrections. For AACID, the difference of CEST effects of amine protons (2.75 ppm) and amide protons (3.50 ppm) was normalized by MT effects at 6 ppm. In vivo CEST-MRI was performed on the brain of dark agouti rats immunized with an emulsion of rat MOG (EAE, n=4) and on healthy rats (n=6).

Results/Discussion

In vitro tests confirms the sensitivity of CEST to the concentration of either amine, amide or glucose and the strong dependence of AACID to pH and independence on proteins concentration. In vivo, compared to healthy controls, MT asymmetry (MTasym) was significantly higher in the cerebellum of EAE rats (Fig. A-B). In particular, MTasym differences were significant for saturation offsets between 0.8 and 2.2 ppm, which is related to glucose (Fig. B). Indeed, the area under the curve (AUC) in that interval was almost doubled. Interestingly, this increase of MTasym was found in both visible T2-weighted lesions and normal appearing CNS (Fig. A) while the AUC correlated with the cumulative score of motor disability (Fig. C). And, no significant change of AACID was found in EAE compared to healthy rats.

Conclusions

Using CEST methods, we highlighted a significant increase of glucose level in the cerebellum of EAE rats with or without visible lesions. Interestingly, energy metabolism increase was also observed in the CSF from MS patients and was suggested to contribute to mitochondrial dysfunction and neuroaxonal degeneration. CEST might be a sensitive tool for the investigation of MS pathology. Complementary studies are ongoing to further confirm our observations.

References

1. van Zijl PCM, et al. (2007) Proc Natl Acad Sci USA. 104(11): 4359–4364.

2. Cai K, et al. (2012) Nat Med. 18(2):302–306.

3. Zhou J, et al. (2003) Nat Med 9(8):1085–90

4. McVicar N, et al (2014) J Cereb Blood Flow Metab 34:690–698.

5. Mathur D,et al. (2014) Front Neurol. 2014; 5: 250

In vivo CEST-MRI in a model of EAE
(A) T2 weighted images from EAE rats with different level of disease severity (CS, cumulative score) and lesions in the cerebellum (arrows). (B) MT asymmetry measured in the cerebellum of both EAE and healthy controls. The grey area represents the frequency shift interval for glucose in which the area under the curve (AUC) was calculated. (C) AUC correlation with disease disability (CumScore).

4:00 PM
PS-06-1 — Introductory Lecture by Xin Yu - Tübingen, Germany

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

4:20 PM
PS-06-2 — Virtual histology of animal and human brains with Alzheimer’s disease (#149)

F. Chauveau1, H. Boutin2, D. Meyronet3, H. Rositi4, C. Olivier5, H. Elleaume6, E. Brun6, F. Peyrin5, M. Wiart7

1 Univ. Lyon, Lyon Neuroscience Research Center; CNRS UMR5292; INSERM U1028; Univ. Lyon 1, Lyon, France
2 Univ. Manchester, Faculty of Biology Medicine and Health, Wolfson Molecular Imaging Center, Manchester, United Kingdom
3 Cardiobiotec CRB-HCL, Neuropathology department, Hospices Civils de Lyon, Lyon, France
4 Univ. Clermont Auvergne, Institut Pascal; CNRS UMR 6602; SIGMA-Clermont, Clermont-Ferrand, France
5 Univ. Lyon, CREATIS; CNRS UMR 5220; INSERM U1206; INSA-Lyon, Univ. Lyon 1, Lyon, France
6 Univ. Grenoble Alpes, EA-7442; ESRF, Grenoble, France
7 Univ. Lyon, CarMeN; INSERM U1060; INRA U1397; INSA-Lyon, Univ. Lyon 1, Lyon, France

Introduction

Synchrotron Radiation X-ray Phase Computed Tomography (SR-PCT) of brain tissue can reveal different image contrast depending on sample preparation. Indeed, we have shown that ethanol dehydration of brain samples reveals myelin as a hyper-intense signal while maintaining optimal detection of packed proteinaceous structures such as amyloid plaques [1]. The present work builds on this versatile “virtual histology” tool to characterize amyloid deposits in brain samples from Alzheimer’s disease (AD) animal models and patients.

Methods

Mono, bi-, and triple transgenic lines of mouse (PDAPP, APP-PS1, 3xTg [2]) were studied, along with one line of transgenic rats [3]. Human samples of frontal cortex were obtained from 6 sporadic AD cases and 1 familial AD case (Cardiobiotec, CRB-HCL, Lyon, France). All samples were scanned after dehydration in ethanol on beamlines ID19 and ID17 at ESRF. In line propagation imaging was performed at 26keV, voxel size of 6.5μm and 3m free space propagation on ID19 (34 keV, 6.5µm and 11m on ID17). Images were reconstructed using Paganin’s method [4].

Results/Discussion

All transgenic animal brains (Fig. 1) and human AD (Fig. 2) cases exhibited amyloid signals in the form of bright spots. Myelin-based hyper-intensities enabled a detailed visualisation of white-matter tracts and subsequent structural connectivity. Additionally, vessels appeared hypo-intense in perfused brains from animals (Fig.1) and hyper-intense in human autopsic brains (Fig. 2), probably because of the presence of blood-related iron in non-perfused tissue. Concurrent immunofluorescence analyses of these observations are in progress.

Conclusions

Whole-brain mouse acquisitions were performed faster (3-10 min) than previously described phase contrast approaches [5]. Human amyloid plaques were evidenced for the first time by SR-PCT. Dehydration-added myelin contrast may be used to highlight unexplored associations between amyloid deposition and myelin alterations.

References

[1] Rositi et al. Abstract #233, presented in European Molecular Imaging Meeting, Mar 18-20, Tübingen, Germany; 2015.

[2] Mucke et al. J Neurosci. 2000 ; Blanchard et al. Exp Neurol 2003 ; Oddo et al. Neuron. 2003.

[3] Cohen RM, et al. J Neurosci. 2013.

[4] Paganin et al. J Microsc. 2002.

[5] Noda-Saita  et al. Neuroscience 2006 ; Connor et al. NeuroImage 2009 ; Pinzer et al. NeuroImage 2012 ; Astolfo et al. J Synchrotron Radiat. 2016.

Acknowledgement

We are grateful to our collaborators (Nicolas Rama, Corinne Perrin, Karen Silva) for their help in gathering animal and human brain samples.

Figure 1.
Relevance of SR-PCT for characterization of vascular and myelin architecture in control mouse versus transgenic PDAPP mouse (right). Amyloid plaques are visible as bright spots on right panel.

Figure 2.
SR-PCT detects parenchymal (blue) and vessel-associated (red) amyloid plaques on human AD brain.

4:30 PM
PS-06-3 — Interrogation of neurobehavioral dynamics in freely swimming zebrafish larvae with an open-source tracking microscope (#354)

P. Symvoulidis1, 2, 3, A. Lauri1, 2, 3, A. Stefanoiu4, M. Cappetta1, 2, 3, S. Schneider3, H. Jia5, A. Stelzl1, 2, M. Koch1, 7, C. C. Perez1, 2, A. Myklatun1, 2, 3, S. Renninger6, A. Chmyrov1, 7, T. Lasser4, W. Wurst2, V. Ntziachristos1, 7, G. G. Westmeyer1, 2, 3

1 Helmholtz Zentrum Munich, Institute of Biological and Medical Imaging, Munich, Germany
2 Helmholtz Zentrum Munich, Institute of Developmental Genetics, Munich, Germany
3 Technical Universtiy of Munich, Department of Nuclear Medicine, Munich, Germany
4 Technical Universtiy of Munich, Computer Aided Medical Procedures, Munich, Germany
5 Technical Universtiy of Munich, Institute of Neuroscience, Munich, Germany
6 Champalimaud Centre for the Unknown, Lisbon, Portugal
7 Technical Universtiy of Munich, Chair for Biological Imaging, Munich, Germany

Introduction

Neuroimaging of distributed neuronal activity in freely moving animals is a long-standing goal in neuroscience. We demonstrate a tracking microscope for simultaneous imaging of neuronal activity and behavior in the freely swimming zebrafish larva, an important vertebrate model organism. We show how the microscope can be used in conjunction with genetically encoded calcium indicators for screening of neuroactive drugs with respect to their combined neuronal and behavioral effects as well as for mapping spontaneous and stimulus-induced neuronal activation patterns during naturalistic behavior.

Methods

We built the fluorescent tracking microscope to work without moving stages, objectives, or illumination to avoid confounding effects on the behavior of the reporter fish. Using a single objective, two images are acquired, one capturing the behavior over the entire arena (~12x12x2mm) in which the larvae can swim freely, the other one providing a fluorescent image with variable magnification up to 15x. The latter FOV is always centered and focused on the brain of the fish by dynamic adjustments of a 3D-scanning system consisting of a 2D-galvanometric mirror and an electrically tunable lens. The microscope is built from off-the-shelf components in a modular fashion that allows for easy addition of, e.g., excitation light sources for imaging or tracked interrogation.

Results/Discussion

We provide simultaneous calcium and behavioral imaging in olfactory and optical stimulation paradigms and in response to pharmacological intervention. When we triggered avoidance behavior in freely swimming larvae by infusing the aversive odorant cadaverine on one side of a bipartitioned swimming arena, we could observe calcium transients in the olfactory system of a fluorescent calcium indicator fish (Figure 1a,b) after each of the visits of the fish to the site of cadaverine infusion [1].

In response to changes in illumination intensity, we could observe exponentially decaying calcium transients in the pineal complex of freely swimming larvae which exhibited shorter swimming distances in the bright condition (Figure 2a).

Moreover, we observed transient increases of the swimming velocity in larvae after infusion of the neurostimulant 4-AP into the swimming arena that transitioned to a phase of inactivity while the calcium activity in several brain regions continued to rise (Figure 2b).

Conclusions

The open-source tracking microscope (NeuBtracker.org) allows for concurrent neuro- and behavioral imaging in unrestrained zebrafish larvae to reveal brain activity patterns during naturalistic behavior. The system enables long-term observation over developmental stages or high-throughput neurobehavioral screening of genetic or pharmacological interventions in a multi-well format. With the growing arsenal of fluorescent reporters and compatible optogenetic tools, spatiotemporal and multiplexed interrogation of signaling processes, also within other organ systems are attractive future prospects.

References

[1] P. Symvoulidis et al., “NeuBtracker—imaging neurobehavioral dynamics in freely behaving fish,” Nat. Methods, 2017.

Acknowledgement

We thank K. Kawakami, A. Muto, R. Portugues, L. Knogler, K. Asakawa, J. Ngai, M. Ahrens, H. Baier, K. Slanchev, and L. Godinho for generously providing zebrafish lines. We are grateful for support from the European Research Council under grant agreements ERC-StG: 311552 (G.G.W., A.L., P.S.) and the Helmholtz Alliance ICEMED (G.G.W.).

Figure 1: Neurobehavioral imaging of aversive behavior with the tracking microscope NeuBtracker
(a) Schematic of the tracking microscope  NeuBtracker showing the swimming arena and the two channels for behavioral (1x) and fluorescent neuronal imaging (up to 15x). (b) Swimming trajectory of zebrafish in response to an aversive odor (Cad) presented on one side of a partitioned arena. Fluorescent signal changes in the olfactory epithelium in response to visits to the site of Cad injection.

Figure 2: Neurobehavioral imaging of dark-light cycles and pharmacological stimulation
(a) Fluorescence signal changes obtained with NeuBtracker from the pineal complex of freely swimming larvae during repeated dark-light cycles. Corresponding behavioral readout (bottom). (b) Example fluorescent images before and after pharmalcological stimulation with the neurostimulant 4-AP. Signal time courses for different brain regions (middle) and simultaneously measured behavior (bottom). 
 

4:40 PM
PS-06-4 — Simultaneous fMRI and Fast-Scan Cyclic Voltammetry (#94)

L. Walton1, M. Verber2, R. M. Wightman2, Y. - Y. I. Shih1

1 Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Neurology, Chapel Hill, North Carolina, United States of America
2 University of North Carolina at Chapel Hill, Chemistry, Chapel Hill, North Carolina, United States of America

Introduction

BOLD fMRI measures cerebral blood oxygenation changes in response to stimuli, but the roles of vasoactive neurotransmitters in fMRI signal generation are poorly understood.1-3 Fast-scan cyclic voltammetry (FSCV) quantifies electroactive neurotransmitter and oxygen concentrations with high spatiotemporal resolution.4,5  Technical limitations have stymied simultaneous FSCV/fMRI use beyond proof-of-concept.6,7 This study addresses these limitations and shows simultaneous FSCV/fMRI both as an in vitro concentration calibration system and as an in vivo evoked tissue oxygen change detector.

Methods

In vitro: A dopamine-sensing voltage waveform was applied to carbon-fiber microelectrodes used in a flow-injection setup modified for MR use. Phosphate-buffered saline (pH 7.4) flowed to the electrode and dopamine HCl boluses were delivered via 6-port injection valve.

In vivo: A tungsten stimulating electrode was implanted into a rat brain near the ventral tegmental area (4s, 60Hz stim). A carbon-fiber microelectrode was implanted in the nucleus accumbens (NAc) and an Ag/AgCl electrode was implanted in the contralateral cortex. Oxygen-sensing FSCV and BOLD fMRI were acquired simultaneously (TR/TE=1000/15ms, matrix=80x80, FOV=2.56cm2, 5 slices). Contrast-to-noise ratios were calculated as [(Oxygen Peak Amplitude-Average Baseline)/Standard Deviation]*100.

Results/Discussion

To assess whether simultaneous FSCV/fMRI data could be collected, we synchronized FSCV data collection to MR TTL outputs. MR interference was eliminated after a 50ms wait time was introduced to interleave the encoding gradient and FSCV waveforms. To test whether physiologically relevant concentrations of neurotransmitters could be detected inside an MR bore, bolus injections of dopamine were introduced. Concentrations ≥50nM could be detected with optimized filtering. Calibration constants were >10nA/µM, consistent with the sensitivity of in vitro electrodes in a shielded environment.

We electrically evoked oxygen changes in the NAc, which scaled with stimulation intensity; however, a temporal offset was observed that may be attributed to the mismatched fMRI and FSCV detection volumes. FSCV had 5x higher temporal resolution than BOLD and superior contrast-to-noise ratios. BOLD and FSCV oxygen metrics significantly correlated, lending credence to our experimental setup.

Conclusions

We optimized a simultaneous FSCV/fMRI experimental setup for use in vitro and in vivo. FSCV is faster, more sensitive, and can detect electroactive neurotransmitters, but BOLD provides unbiased, brain-wide measurements. This multimodality holds great promise for BOLD noise removal and improving our current understanding of fMRI data with neurochemical context at multiple temporal and spatial scales.   

References

  1. Hillman EM. Coupling mechanism and significance of the BOLD signal: a status report. Annu Rev Neurosci 37, 161-181 (2014).

  2. Raichle ME, Hartman BK, Eichling JO & Sharpe LG. Central noradrenergic regulation of cerebral blood flow and vascular permeability. Proc Natl Acad Sci U S A 72, 3726-3730 (1975). 

  3. Darby JM, Yonas H, Marks EC, Durham S, Snyder RW & Nemoto EM. Acute cerebral blood flow response to dopamine-induced hypertension after subarachnoid hemorrhage. J Neurosurg 80, 857-864 (1994). 

  4. Rodeberg N, Sandberg S, Johnson J, Phillips PEM, Wightman RM. Hitchhiker’s Guide to Voltammetry: Acute and Chronic Electrodes for in Vivo Fast-Scan Cyclic Voltammetry. ACS Chem Neurosci 8(2), 221-234 (2017). 

  5. Zimmerman J, Wightman RM. Simultaneous electrochemical measurements of oxygen and dopamine in vivo. Anal Chem 63(1), 24-28 (1991). 

  6. Kimble CJ, Johnson DM, Winter BA, Whitlock SV, Kressin KR, Horne AE, Robinson JC, Bledsoe JM, Tye SJ, Chang SY, Agnesi F, Griessenauer CJ, Covey D, Shon YM, Bennet KE, Garris PA, Lee KH. Wireless instantaneous neurotransmitter concentration sensing system (WINCS) for intraoperative neurochemical monitoring. In Engineering in Medicine and Biology Society, 2009. EMBC 2009. Annual International Conference of the IEEE. 4856-4859 (2009). 

  7. Lowry JP, Griffin K, McHugh SB, Lowe AS, Tricklebank M, Sibson NR. Real-time electrochemical monitoring of brain tissue oxygen: a surrogate for functional magnetic resonance imaging in rodents. Neuroimage 52(2), 549-555 (2010). 

Acknowledgement

We thank the Shih lab members for their helpful discussions and critiques. We acknowledge the help of Philip Summers for 3D modeling the electrode holder prototype. Our team is supported by NIMH R01MH111429, R41MH113252, R21 MH106939, NINDS R01NS091236, NIAAA U01AA020023, R01AA025582, NICHD U54HD079124, American Heart Association 15SDG23260025, and Brain & Behavior Research Foundation. 

Figure 1. Optimizing simultaneous in vitro FSCV and fMRI.
(A) Carbon-fiber microelectrode tip (30x magnification). (B) 6-port valve flow-injection cartoon. Buffer (white) or dopamine (purple) from the sample loop flows to the microelectrode depending on valve position. (C) Schematic for interleaved dopamine or oxygen FSCV waveforms and fMRI (inset=shaded box). (D) Calibration plots with different noise level environments. Slope = dopamine sensitivity.

Figure 2. Simultaneous in vivo oxygen FSCV and BOLD fMRI.
(A) Sagittal view of FSCV implants and the applied oxygen waveform. (B) Timecourses of simultaneous evoked BOLD and FSCV oxygen changes. Red bar denotes stim duration. (C) FSCV was significantly more sensitive to evoked changes than BOLD (t-test, F=25.3, P<0.0001). (D) fMRI correlation coefficients derived from downsampled FSCV oxygen timecourses. (E) BOLD and time-corrected FSCV highly correlate.

4:50 PM
PS-06-5 — Optogenetic fMRI with ultrafast excitation by ChETA (#266)

F. Albers1, L. Wachsmuth1, S. Hamzei-Taj1, H. Lambers1, C. Faber1

1 University Münster, Clinical Radiology, Münster, Germany

Introduction

The combination of optogenetics and fMRI (ofMRI) enables manipulation of a genetically defined cell population and simultaneous brain-wide readout of local and network responses. The opsins ChR2 and C1V1, which are commonly used in ofMRI studies, may be stimulated with frequencies up to 100 Hz and 50 Hz, respectively. Consequently, stimulations with higher frequencies, i.e. mimicking electric deep brain stimulation, result in ill-defined activation patterns.  Here, we examined BOLD activation upon optogenetic stimulation of the ultrafast opsin ChETA [1] with frequencies up to 200 Hz.

Methods

Three weeks prior to MRI, rAAVs with constructs encoding for ChETA and C1V1 under CamKII-promoter were injected into the forelimb area of the cortex (S1Fl) of 4 female Fischer rats, each. GE-EPIs were acquired (TR = 1 s, TE = 18 ms, 350*325 μm² spatial resolution, slice thickness 1.2 mm, 9 contiguous slices) with different stimulations (5 s stim., 25 s rest, 7-200 Hz). Mean light intensities were kept below 22 mW/mm² to avoid heating artifacts [2]. To compare frequencies the duty cycle was kept at ~ 0.1. Data were preprocessed using SPM8 and then, using Matlab, time courses were extracted from activated regions and averaged across stimulation trials. Activated regions were determined by a t-test performed in ImageJ. BOLD amplitudes were normalized in each animal and changes compared.

Results/Discussion

In three of four animals BOLD responses upon optogenetic stimulation of ChETA could be detected (Fig. 1a). A wide range of stimulation frequencies (7 Hz, 9 Hz, 12 Hz, 20 Hz, 80 Hz, 100 Hz, 120 Hz, 150 Hz, 200 Hz with constant duty cycle of 0.1) lead to BOLD activation. BOLD amplitudes did not differ depending on stimulation frequency, showing that ChETA operates even at high frequencies of 200 Hz and that short pulse lengths of 0.5 ms are sufficient for activation (Fig. 1b). The temporal evolution of the BOLD signal did not depend on optogenetic stimulation frequency (Fig. 2a). Figure 2b shows BOLD time courses for C1V1 and ChETA responses, each averaged across four 10-min measurements. C1V1 responses showed a slower decay to baseline compared to ChETA responses (mean ± standard deviation: 16 ± 2 s for C1V1 and 14 ± 2 s for ChETA). Onset differences were not found to be significant, which may be due to the temporal resolution of the EPI sequence of 1 s.

Conclusions

Optogenetic stimulation with up to 200 Hz of the opsin ChETA causes BOLD responses in ofMRI experiments, without causing unwanted heating artefacts when mean intensities are taken into account. The channel kinetics of ultrafast opsins may lead to different timing properties of the BOLD response, when compared to slower opsins. These differences, however, were not resolved here unambiguously, and may be due to different expression rates or different activation patterns of the illuminated population.

References

[1] Gunaydin LA, Yizhar O, Berndt A et al: Ultrafast optogenetic control. Nat Neuroscience, 2010;13(3):387-92.

[2] Schmid F, Wachsmuth L, Albers F et al.: True and apparent optogenetic BOLD. Magnetic Resonance in Medicine, 2017;77(1):126-136.

Figure 1

(a) Exemplary BOLD map at 80 Hz optogenetic stimulation with 1.25 ms pulse length in rat S1FL. Colorbar gives t-value. (b) BOLD amplitudes, normalized in each animal, versus stimulation frequency, grouped for frequency ranges. Duty cycle was kept constant at 0.1, n gives number of measurements.

Figure 2

(a) Exemplary BOLD time courses upon optogenetic stimulation of ChETA with frequencies from 9 – 200 Hz.

(b) Averaged time courses (n=4, each) for ChETA and C1V1 activation with 9 Hz 10 ms-pulses stimulation. Mean ± SEM is shown. Bar indicates stimulation interval in (a) and (b).

5:00 PM
PS-06-6 — Dynamic Imaging of Manganese-Labeled Hydrogel for Interventional MRI-Guided Intrathecal Delivery of Stem Cells (#428)

I. Malysz-Cymborska1, D. Golubczyk1, L. Kalkowski1, S. Vieira2, 3, 4, M. Janowski5, 6, 7, J. Głodek8, P. Holak8, K. Milewska1, W. Maksymowicz1, T. Svendsen9, R. L. Reis2, 3, 4, J. M. Oliveira2, 3, 4, P. Walczak1, 5, 6

1 University of Warmia and Mazury, School of Medicine, Neurology and Neurosurgery, Olsztyn, Poland
2 University of Minho, 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, Guimaraes, Portugal
3 PT Government Associated Laboratory, ICVS/3B’s, Braga, Portugal
4 University of Minho, The Discoveries Centre for Regenerative and Precision Medicine, Guimaraes, Portugal
5 Johns Hopkins University, Russell H. Morgan Dept. of Radiology and Radiological Science, Baltimore MD, United States of America
6 Johns Hopkins University, Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, Baltimore MD, United States of America
7 Mossakowski Medical Research Center, Polish Academy of Sciences, NeuroRepair Department, Warsaw, Poland
8 University of Warmia and Mazury, Faculty of Veterinary Medicine, Department of Surgery and Roentgenology with the Clinic, Olsztyn, Poland
9 FMC BioPolymer, Sandvika, Norway

Introduction

Stem cell transplantation has been shown efficacious in animal models of neurological diseases; however, in disseminated pathology of ALS, broad delivery of cells remains to be a challenge. Infusion into cerebrospinal fluid spaces is promising however, to protect cells from sedimentation, we propose embedding cells in injectable alginate biomaterial [1]. Hydrogel biodistribution within injection into CSF is uncertain, therefore monitoring in real-time is highly desirable [2]. Here we report on application of Mn for interventional MRI, of intrathecal injection of alginate-embedded MSCs in pig.

Methods

For initial optimization of Mn2+ concentration in alginate hydrogels, serial dilutions of MnCl2 in H2O were scanned in the clinical MRI using T1 weighted sequence. For toxicity assessment porcine MSCs were either cultured as monolayer or embedded in LVM alginate and both formulations were supplemented with increasing concentrations of MnCl2. The composite solution was then cross-linked with cross-linker (CaM, NovaMatrix). The MTS assay was used as a readout.

For in vivo study, four Large White domestic pigs were used. Catheter was introduced via lumbar puncture under x-ray guidance into thoracic section of the spinal cord. Immediately before infusion, CaM was mixed with LVM/MnCl2 (MnCl2 0.1mM) hydrogel. Dynamic T1 images were acquired to monitor hydrogel biodistribution in real-time.

Results/Discussion

Mn2+ MRI of phantoms revealed gradual increase of the signal (hyperintensity) with maximum of the signal detected at 0.1mM MnCl2 (Fig1A). Toxicity assay with pMSC showed detrimental effect only for a monolayer cell culture with concentration-dependent response to MnCl2. Notably, this negative effect was not observed for same concentration of MnCl2 for cells embedded in LVM (Fig.1B).

In vivo study with intrathecal injection hydrogel/cell composite in swine demonstrated that gelation dynamics allowed for infusion of the biomaterial up to two minutes after mixing with the cross-linker. Dynamic imaging during hydrogel infusion revealed hyperintensity in the area of the catheter tip which further expanded rostrally and caudally over the infusion duration (6 min). The rostral-caudal distribution of the hydrogel ranged between 10-15 cm in tested pigs.

Conclusions

Mn2+ supplementation of alginate hydrogels is safe for transplanted cells and enables MRI visualization of biomaterial placement in real-time.

References

  1. Lee KY and David J. Mooney. Alginate: properties and biomedical applications. Prog Polym Sci. 2012 Jan; 37(1): 106–126.
  2. PanD, Schmieder AH, Wickline SA, and Lanza GM. Manganese-based MRI contrast agents: past, present and future. Tetrahedron. 2011 Nov 4; 67(44): 8431–8444.

Acknowledgement

Funding: NanoTech4ALS grant (12/EuroNanoMed/2016)

Figure 1

In vitro evaluation of MnCl2-alginates: A. MRI signal intensity analysis of serial MnCl2 dilutions in H2O,  B. pMSC proliferation in medium (green) or LVM hydrogel (blue) containing increasing MnClconcentrations.

Figure 2

Real-time MRI monitored intrathecal transplantation of LVM/MnCl2-alginates in pig : A. MRI dynamic T1 scans  during  LVM/MnCl2 transplantation,  B. Distance covered by the transplanted LVM/MnCl2 hydrogel, C. MRI T1 isotropic scans before and post injection of the alginate with MnCl2.

5:10 PM
PS-06-7 — Quantitative Cerebral Blood Volume and Flow Measurements by Magnetic Particle Imaging (#477)

R. Orendorff1, P. Keselman1, S. M. Conolly1, 2

1 University of California, Berkeley, Bioengineering, Berkeley, California, United States of America
2 University of California, Berkeley, Electrical Engineering and Computer Science, Berkeley, California, United States of America

Introduction

To diagnose cerebral vascular disorders, doctors use CT methods to image blood pools in the cerebral vasculature. These techniques work for large deficits like hemorrhagic stroke, but are ill-equipped to track blood changes over time due to tissue signal near the vasculature. CT methods also incur high radiation doses and use a contrast agent that is toxic for patients with chronic kidney disease [1]. Magnetic Particle Imaging (MPI) is a promising new modality that can image the cerebral vascular clearly as iron nanoparticles flow through the brain. Here we present perfusion imaging using MPI.

Methods

MPI [2] was performed on a Fisher-344 rat; a brief description follows. The rat was anesthetized using 3% isoflurane and a catheter was inserted into a tail vein. The rat was then placed in a MPI scanner with access to the catheter from outside the scanner. 0.2 mL of 5.7 mg Fe/mL Resovist iron nanoparticle solution was injected into tail vein over a period of six seconds.

Imaging was performed using a custom Berkeley field free point (FFP) scanner. This MPI scanner has a gradient strength of 7 T/m by 3.5 T/m by 3.5 T/m in the x, y, and z directions, respectively. A single transverse plane through the rat's brain was captured at a rate of one frame every 1.28 seconds for one minute. The field of view was 2.6 cm by 2.6 cm.

Results/Discussion

The time series images were processed to produce a map of the relative cerebral blood flow (rCBF) in the image. This calculation was done by finding the maximum rate of change of the iron signal through each pixel in the image, which was plotted to produce an rCBF map. The relative cerebral blood volume (rCBV) map was calculated by the integral of the concentration curve as the bolus passed through the brain.

The tracer changes over time were also visualized to determine the difference between the background signal and cerebral signal change over time. This analysis demonstrates that the particles were confined to the brain space, and indicate when the bolus passed through the cerebral vasculature. The large residual signal after the bolus has passed is due to recirculation of the particles in the blood, in the same manner as other tracer modalities. [4]

Conclusions

Here we present the world's first MPI perfusion imaging. We have calculated, for the first time, physiologically relevant parameters (rCBF, rCBV) from an MPI time course. This information is vital for the diagnosis and treatment of stroke and other diseases involving the cerebral vasculature. We have shown that MPI shows great promise to provide a safe, effective, and fast way in which to acquire this information.

References

[1] Krishna PR, et al. Indian Journal of Nephrology. 2009

[2] P.W. Goodwill et al. IEEE Trans. Med. Imaging. 2010

[3] T. Knopp et al. Magnetic Particle Imaging. 2012

[4] L. Axel, Radiology, 1980.

Acknowledgement

We are grateful for funding support from the Keck Foundation Grant 009323, NIH 1R01EB019458, NIH 1R24MH106053, and a UC Discovery Grant. Ryan Orendorff would like to thank the NSF GRFP for funding support. 

MPI Imaging Apparatus and raw time series iron concentration data through the cerebral vasculature
(a) Image of the Berkeley field free point (FFP) MPI scanner, named Gertrude. This scanner has a gradient field of 7 T/m by 3.5 T/m by 3.5 T/m respectively. (b) The time course plot shows a marked peak increase in signal as the particles first pass through the brain, followed by a slow decay as the particles recirculate in the bloodstream and are filtered out by the liver.

Relative Cerebral Blood Volume and Flow measurements by fast MPI
(a) A measure of the relative Cerebral Blood Flow (rCBF) for each pixel in the image, clearly showing high perfusion rates inside the brain. (b) A measure of the relative Cerebral Blood Volume (rCBV) for each pixel in the image, which is a measure of the amount of iron/blood owing through the brain as the rst bolus passes.

5:20 PM
PS-06-8 — Comparison of PET and MRI estimation of cerebral perfusion using multi parametric PET-MR in a non-human primate model of stroke (#54)

J. Debatisse1, 2, N. Makris3, N. Costes4, M. Verset5, O. Wateau5, K. Portier1, M. Aggour1, J. - B. Langlois4, C. Tourveille4, D. Le Bars4, T. Troalen2, H. Contamin5, T. - H. Cho3, 6, E. Canet-Soulas1

1 Univ Lyon, CarMeN Laboratory, INSERM, INRA, INSA Lyon, Université Claude Bernard Lyon 1,, Lyon, France
2 Siemens Healthcare SAS, Saint-Denis, France
3 CREATIS, CNRS UMR 5220, INSERM U1206, Université Lyon 1, INSA Lyon, Université Jean Monnet Saint-Etienne, Lyon, France
4 Cermep - Imagerie du vivant, Lyon, France
5 Cynbiose SAS, Marcy-L'Etoile, France
6 Department of Neurology, Hospices Civils de Lyon, Lyon, France

Introduction

Restauration of blood flow to an ischemic organ is essential to prevent irreversible tissue injury. To this end, reliable estimation of cerebral blood flow (CBF) is crucial for a precise diagnosis of acute ischemia. Recent introduction of PET-MRI hybrid technology allows the simultaneous acquisition of PET and MRI data. PET using [15O]H2O remains the reference method to quantitatively assess CBF but it can also be assessed using DSC-MRI (dynamic susceptibility contrast MRI) with an injection of a paramagnetic contrast agent.

Methods

Longitudinal cerebral perfusion assessment using a Biograph mMR PET-MRI scanner (Siemens Healthcare, Erlangen, Germany) was performed in a minimally invasive endovascular non-human primate (NHP – Macaca fascicularis) model of stroke (transient occlusion of the middle cerebral artery). Imaging data were acquired before ischemia (“baseline” imaging session, n=2 NHP) and after recanalization of the ischemic area (“post-reperfusion” imaging session, n=1 NHP). Kinetic modeling of the [15O]H2O PET was performed to compute CBF parametric maps, extracting an image derived input function from the carotid. DSC-MRI data were processed using the software Olea Sphere® (Olea Medical, La Ciotat, France) and 4 post-processing algorithms were compared: sSVD, cSVD, oSVD and the Bayesian method.

Results/Discussion

An automatic (clustering of arterial voxels) or manual selection of the arterial input function (AIF) was used to derive the MRI-CBF maps. Brain regions of interests (ROIs) consisting in 6-mm circles were manually placed in the affected and unaffected hemispheres (72 ROIs for NHP#1 and 83 ROIs for NHP#2). The MRI- and PET-CBF values obtained with the four algorithms were compared by linear correlation, and by Bland-Altman plots to characterize similarities and differences between methods. NHP#1 lesions observed on the diffusion-weighted imaging at the post-reperfusion imaging session, and corresponding PET- and MRI-CBF maps are represented on Figure 1. PET-CBF against Bayesian-based MRI-CBF values either with automatic or manual AIF show the best correlation (Figure 2A-2D). The slopes of the correlations show either an over- or underestimation of the MRI-CBF against the reference method PET-CBF. These observations are confirmed by the Bland-Altman plots shown in Figure 2E-2H.

Conclusions

The use of manual AIF demonstrated the lowest bias, inferior to 10%, whereas the automatic AIF produced higher bias in both baseline and post-reperfusion. Correlation with the automatic AIF and Bayesian method versus PET-CBF is close to the one obtained with manual AIF (R2=0.50 versus R2=0.47) but when looking at absolute values, manual AIF gives the closest estimation of MRI-CBF compared to PET-CBF. A larger sample is needed to confirm the value of the Bayesian algorithm for longitudinal studies of CBF and CBV.

Acknowledgement

The authors would like to thank Siemens Healthcare for providing the prototype sequence used in this work.

Figure 1
Illustrative maps of Diffusion Weighted Imaging (DWI), PET-CBF map, and MRI-CBF map (obtained with manual AIF and Bayesian method) in NHP#1 at the post reperfusion imaging session.

Figure 2

A-B: NHP#1 PET- and MRI-CBF correlations with auto AIF (A) or manual AIF (B) + Bayesian.

C-D: NHP#2 PET- and MRI-CBF correlations with auto AIF (C) or manual AIF (D) + Bayesian.

E-F: Bland-Altman plots at baseline in NHP#1 + #2 (n=155 ROIs) with auto AIF (E) or manual AIF (F) + Bayesian

G-H: Bland-Altman plots at post reperfusion in NHP#1 (n=72 ROIs) with auto AIF (G) or manual AIF (H) + Bayesian

8:30 AM
emptyVal-1 — Introductory Talk by Amnon Bar-Shir - Rehovot, Israel

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

8:50 AM
PS-22-2 — Unambiguous detection of atherosclerosis by pretargeted molecular imaging (#78)

I. Fernández-Barahona1, J. Pellico1, 2, J. Ruiz-Cabello2, 3, 4, F. Herranz1, 2

1 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
2 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES)., Madrid, Spain
3 CIC biomaGUNE. Ikerbasque, Basque Foundation for Science, Donostia-San Sebastián, Spain
4 Departamento Química Física II. Facultad de Farmacia. Universidad Complutense de Madrid, Madrid, Spain

Introduction

Pretargeted imaging is based on the use of bioorthogonal tracers that selectively accumulate upon reaction with a pre-modified biomolecule in vivo. To date, this promising approach has been especially used for cancer diagnosis however, to our knowledge, it has not been applied to atherosclerosis. Here, we synthesized a bioorthogonal nano-radiotracer for in vivo pretargeted molecular imaging in a mouse model of atherosclerosis. We based this approach in our new platform for molecular imaging, 68Ga core-doped Fe2O3 nanoparticles, which provide PET and T1-MRI signals.

Methods

In the first place, TCO moiety was covalently attached to E-06, a naturally occurring mouse monoclonal IgM antibody targeting oxidized LDL, oxidized HDL and proteins covalently modified by oxidized phospholipids.

Secondly, we synthesized a new 68Ga-based nano-radiotracer (NRT). Citrate-coated extremely small iron oxide nanoparticles core-doped with 68Ga. NRT synthesis was carried out very rapidly in a microwave oven, taking only 15 minutes to obtain the pure, ready-to-use sample. We next incorporated the tetrazine (TZ) moiety to the NRT surface through amide formation between benzylamino-tetrazine and the carboxylic acid groups in citrate.

Results/Discussion

After full NRT characterization, in vivo imaging experiments were carried out in ApoE-/- and C57BL/6 mice. 24 hours after Ab injection, 68Ga-NRT-TZ was intravenously administered; some ApoE-/- mice were inyected with 68Ga-NRT as a bioorthogonal reaction control. PET/CT imaging of ApoE-/- mice 1 hour post 68Ga-NRT-TZ injection revealed specific localization in several consecutive planes of the aortic arch. In contrast, post 68Ga-NRT-TZ injection images from control mice showed no noticeable signal in the aortic arch nor in the whole aorta. PET/CT signal was also absent from ApoE-/- mice injected with unmodified 68Ga-NRT.

The convenient relaxometric values of 68Ga-NRT-TZ motivated us to check the ability of MRI to detect NRT accumulation in the aorta. Ex vivo T1-weighted MRI of aortas from ApoE-/- mice injected with 68Ga-NRT or 68Ga-NRT-TZ were acquired. Clear hyperintense areas were evident in lesions, showing higher intensity areas for the ApoE-/- mouse injected with 68Ga-NRT-TZ.

Conclusions

The production of a new kind of bioorthogonal nano-radiotracer through the synergistic combination of nanotechnology and radiochemistry proves the feasibility of in vivo pretargeted imaging. Our results demonstrate the ability of this approach to unambiguously detect atherosclerosis.

 

References

1.           Pellico, J. et al. One-Step Fast Synthesis of Nanoparticles for MRI: Coating Chemistry as the Key Variable Determining Positive or Negative Contrast. Langmuir 33, 10239–10247 (2017). doi:10.1021/acs.langmuir.7b01759

2.           Pellico, J. et al. In vivo imaging of lung inflammation with neutrophil-specific 68Ga nano-radiotracer. Sci. Rep. 7, 13242 (2017). doi:10.1038/s41598-017-12829-y

3.           Pellico, J. et al. Fast synthesis and bioconjugation of 68 Ga core-doped extremely small iron oxide nanoparticles for PET/MR imaging. Contrast Media Mol. Imaging (2016). doi:10.1002/cmmi.1681

4.          Pellico, J. et al. Hybrid pretargeted molecular imaging of atherosclerosis with bioorthogonal nano-radiotracers. Bioconjugate Chemistry. (under review)

Acknowledgement

This study was supported by a grant from the Spanish Ministry for Economy and Competitiveness (MEyC) (grant number: SAF2016-79593-P) and from Carlos III Health Research Institute (grant number: DTS16/00059). We thank Simon Bartlett for editorial assistance and manuscript preparation. The CNIC is supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (MEIC award SEV-2015-0505).

Figure 1

Figure 2

9:00 AM
PS-22-3 — Specific quantification of 57Fe‑nanoparticles by combined MRI and mass spectrometric imaging (#146)

A. Beuker1, M. Masthoff1, R. Buchholz2, L. Wachsmuth1, F. Albers1, W. Heindel1, U. Karst2, M. Wildgruber1, C. Faber1

1 Translational Research Imaging Center, Institute for Clinical Radiology, University Hospital of Muenster, Muenster, North Rhine-Westphalia, Germany
2 Institute for Inorganic and Analytical Chemistry, University of Muenster, Muenster, North Rhine-Westphalia, Germany

Introduction

Iron oxide nanoparticles (ION) are common contrast agents for (pre-)clinical MRI. However, MRI signal is always influenced by iron from endogenous sources. Hence, an exact correlation of administered ION with the MRI signal by T2 quantification is not possible in vivo. Here, we combine non-radioactive 57Fe-based ION MRI with laser-ablation-mass-spectrometry (LA-ICP-MS) for specific differentiation between endogenous iron (56Fe) and applied ION. We assess distribution of administered ION, apply 57Fe-ION for cell tracking and aim to correlate local ION concentration with T2 values.

Methods

Healthy C57BL/6 mice were i.v. injected with novel 57Fe-ION (NanoPET, Berlin; dose: 2,5 ml/kg; 16,7mg Fe/ml). After 2h, 1d, 3d, 7d and 30d p.i. MRI with T2/T2*-mapping of liver, spleen, kidneys and brain was performed on a 9,4T small-animal MRI. Mice were sacrificed and organs of interest were extracted for LA-ICP-MS to quantify the amount of 57Fe and the 56Fe/57Fe isotope ratio. Distribution of 57Fe-ION was validated by histology via mac-3 and Prussian blue staining, too.

57Fe-ION were also evaluated for cell tracking in a mouse model of local inflammation. C57BL/6 mice were injected s.c. with 100µl of a polyacrylamide-gel (pellet) to induce sterile inflammation. 57Fe-ION were injected i.v. after 4h, followed by MRI (1d, 3d, 7d p.i.) and LA-ICP-MS, now including the subcutaneous pellet.

Results/Discussion

Specific detection and quantification of 57Fe-ION by ex vivo LA-ICP-MS in liver, spleen and kidneys (see Fig.1 a-c) enabled for a particle distribution study. Comparing the detected 57Fe-signal with histological staining indicated that most ION were first internalised by tissue macrophages in liver and spleen. Over time, 57Fe-signal increased in the red pulp of the spleen but decreased in macrophages (see Fig.1 c-d).

Distribution of 57Fe-ION by LA-ICP-MS was correlated to organ specific T2 times by in vivo MRI (see Fig. 2). Observed signal decrease in liver, spleen, kidney and brain just p.i. was mainly due to intravascular accumulation of ION. While T2 of the brain was recovering quickly and no long-term changes could be detected, T2 of the liver and spleen remained low indicating storage of applied ION. T2 of kidneys slowly recovered, due to clearance of ION.  

57Fe-ION were also detectable in the subcutaneous pellets, indicating migration of macrophages with internalised ION.  

Conclusions

Using novel 57Fe-ION enables for specific detection and quantification of applied ION. Hereby, studying distribution and long-term fate of ION becomes feasible. From increasing 57Fe-signal in splenic red pulp, we assume 57Fe-iron is deposited in endogenous iron stores over time.

Additionally 57Fe-ION can specifically validate and quantify ION based cell tracking, shown by detecting internalised 57Fe-ION in inflammatory tissue.  

Finally, our future aim is to correlate organ specific T2 times with the 57Fe-ION-amount and thus establish a specific validation of MR-iron quantification.

Fig. 1: Distribution maps for 57Fe in Laser-ablation-mass-spectrometry (LA-ICP-MS)
LA-ICP-MS of 10µm cryoslices was performed of explanted organs after in vivo intravenous application of 57Fe-ION. Exemplary semiquantitative distribution maps for 57Fe are shown for a) liver and b) kidney. Quantitative distribution maps for 57Fe in spleen are shown for c) 3d and d) 30d post injection.  Laser spot size was 25µm.

Fig. 2: Time course of T2-relaxation times in different tissues

T2-relaxation times for liver, spleen, kidneys and brain at different time points after 57Fe-ION i.v. injection (2h, 1d, 3d, 7d, 30d). Naïve animals did not receive any injection. Data is presented as mean ± standard deviation (at least n=3 for each time point). 

9:10 AM
PS-22-4 — Fishing for Aldehydes: Expanding the Probe Tackle Box with Conditional CEST-MRI Lures (#261)

M. Suchy1, T. Dang1, Y. Truong1, C. Lazurko1, W. Oakden3, W. Lam3, G. Facey1, G. Stanisz3, 4, A. Shuhendler1, 2

1 University of Ottawa, Chemistry & Biomolecular Sciences, Ottawa, Ontario, Canada
2 University of Ottawa Heart Institute, Ottawa, Ontario, Canada
3 Sunnybrook Research Institute, Physical Sciences, Toronto, Ontario, Canada
4 University of Toronto, Medical Biophyscis, Toronto, Ontario, Canada

Introduction

Aldehydes are regularly produced in cells through tightly regulated processes necessary for life. Low homeostatic concentrations are required for immune responses, genetic regulation, and signal transduction mechanisms [1]. Cell stress can throw aldehyde levels into dysregulation, resulting in the initiation and progression of a variety of disease and injury states [2]. While some aldehydes have been investigated for assessing injury [3] or cancer therapy response [4], for example, the use of reactive carbonyls as imaging biomarkers is infrequent and mostly limited to ex vivo detection.

Methods

Building upon previous work establishing rapid, catalyst-free trapping of aldehydes using N-amino anthranilic acids [5], we have developed activatable molecular MRI contrast agents providing imaging signal through chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) [6].  These hydrazine-containing agents, collectively termed Hydrazo-CEST, are CEST-inactive until they trap reactive carbonyls to form hydrazones (Fig. 1a). Upon hydrazone formation, proton exchange from the ring-proximal nitrogen falls into the CEST regime and produces high CEST-MRI contrast (%MTRasym>20%).

Results/Discussion

Using a variety of control probes, we have identified that both the hydrazone proton and the ortho-carboxylic acid are necessary for CEST-MRI. The CEST-MRI signal was sensitive to the electronics of both hydrazine and carbonyl components: the more electron-withdrawing the combined substituents, the lower the contrast (Fig. 1b), which was correlated with alterations in proton exchange rates, deviating from that required for contrast production. Aldehyde trapping proceeded rapidly under physiological conditions (kobs~0.1 min-1) and to near completion (>90%) within 15 min (Fig. 1c). The ortho­-carboxylic acid substantially increased the degree of reaction completion and the stability of the hydrazone product relative to control compounds lacking the ortho-carboxylic acid or containing an ortho-methyl ester. This effect derived from a key intramolecular hydrogen bond. Finally, we have verified that our N-amino anthranilic acid probes were biocompatible even at high doses (Fig. 1d).

Conclusions

The development of Hydrazo-CEST, comprised of a set of CEST-MRI probes conditionally activated when bound to bioactive carbonyls, and the rational investigation of the chemical determinants of CEST signal production, places the mapping of aldehydes in vivo by MRI within reach. These probes could provide novel diagnostic and prognostic strategies for diseases and injuries that are initiated and evolved through the biogenesis of aldehydes, including heart disease, neurodegeneration, and traumatic brain injury.

References

  1. Niki E, Free Radical Biology and Medicine (2009) 47:469-84.
  2. Ellis EM, Pharmacology & Therapeutics (2007) 115:13-24.
  3. Cebak JE et al., Journal of Neurotrauma (2016) 33:1-16; Halstrom A et al., Journal of Clinical Neuroscience (2017) 35:104-8.
  4. Gomes Junior AL, et al., Oxidative Medicine and Cellular Longevity (2015) 212964; Kumaraguruparan R et al., Clinical Biochemistry (2005) 38:154-8.
  5. Kool ET et al., JACS (2013) 135:17663-6; Kool ET et al., Organic Letters (2014)16:1454-7.
  6. Bar-Shir A et al., ACS Chemical Biology (2015) 10:1160

Acknowledgement

This work was supported by an NSERC Discovery Grant RGPIN 2015-05796 (A.J.S.), the Canada Research Chairs Program 950-230754 (A.J.S.), the Canadian Foundation for Innovation (A.J.S.), and the Canadian Institutes of Health Research PJT376892 (A.J.S.).

Summary of Performance of Hydrazo-CEST Probes

9:20 AM
PS-22-5 — Using single fluorinated agent for multiplexed imaging with 19F-CEST MRI (#337)

R. Shusterman-Krush1, L. Avram1, B. C. Gibb2, A. Bar-Shir1

1 Weizmann Institute of science, Rehovot, Israel
2 Tulane University, Chemistry, New Orleans, Louisiana, United States of America

Introduction

The complexity of biology attracts scientists from a wide range of fields. Although optical imaging sensors are widely used to study such complexity in a multicolor imaging fashion, their light signal source calls for alternatives. In MRI, diaCEST[1] and paraCEST[2] based probes have been used for multiplexed imaging by exploiting the Dw of a labile proton. Here, we show that combining the CEST approach with 19F-MRI allows obtaining “multicolor” imaging of multiple targets using a 19F-agent. Based on the distinct Dws obtained in 19F-MR, a novel platform for multiplexed MR imaging is presented.

Methods

All NMR and MRI experiments were performed on 9.4T scanners (Bruker, Germany). 19F-CEST NMR data were acquired as previously described[3]. 1H-MRI was acquired using a FLASH sequence. For 19F-MRI, RARE sequence was used TR/TE=6000/3.64 ms; 10 mm slice thickness; FOV=3.2×3.2 cm2; matrix size=64×64. For the 19F-CEST MRI the frequency of the pre-saturation pulse (B1=3.6 μT/ 3000 ms) was swept from Δω=+5ppm to Δω=-5ppm in 100Hz steps.

Results/Discussion

In the present study, two supramolecular systems composed of molecular hosts (either cucurbit[n]uril, CB[7];  or octa acid, OA, Fig. 1a) and 19F-guest (fluoroxene, Fig. 1a) were used. Initially, the 19F-CEST characteristics of CB[7]:fluoroxene and OA:fluoroxene systems in PBS were studied (Fig. 1b). A large CEST effect was obtained (45-50% signal change) for both studied samples. While for the CB[7]:fluoroxene system the effect was at Dw of +1.6 ppm (Fig. 1b, purple), the effect for OA:fluoroxene system was at Dw of -2.0 ppm (Fig. 1b, green). This observation encouraged us to use this platform for multiplexed 19F-MRI. A phantom of 4 different host:guest pairs (1:100) was prepared as shown in Fig. 2. 1H-MRI and 19F-MRI did not reveal any difference between the samples. However, by performing the 19F-CEST MRI experiment, a multicolor imaging characteristic was obtained. A pseudo-multicolor imaging map demonstrates the capability of using the proposed platform for multiplexed MR imaging.

Conclusions

We have demonstrated the feasibility of applying the 19F-CEST methodology on a variety of host:guest systems to amplify 19F-MR signals of low concentration targets (up to 500 nM of imaged target). By targeting the molecular hosts (CB[7] or OA) to multiple low-concentration targets the proposed platform has the potentiality to monitor such targets, simultaneously, using single 19F-probe.

References

[1]          G. Liu, M. Moake, Y.-e. Har-el, C. M. Long, K. W. Y. Chan, A. Cardona, M. Jamil, P. Walczak, A. A. Gilad, G. Sgouros, P. C. M. van Zijl, J. W. M. Bulte, M. T. McMahon, Magnetic Resonance in Medicine 2012.

[2]          G. Ferrauto, D. D. Castelli, E. Terreno, S. Aime, Magnetic Resonance in Medicine 2013.

[3]          L. Avram, M. A. Iron, A. Bar-Shir, Chemical Science 2016.

chemical structure and 19F-CEST NMR analysis

Figure 1: (a) Chemical structure of cucurbit[7]uril (CB[7]), octa acid (OA), and Fluoroxene, followed by a schematic description of the multicolored host-guest probes system. (b) 19F-CEST-NMR characterization of the CB[7]:fluoroxene (1:100 ratio) and OA:fluoroxene (1:100 ratio) systems.

19F-CEST MRI

Figure 2: (a) 1H-MRI and 19F-MRI of a phantom containing CB[6]:fluoroxene (blue), CB[7]:fluoroxene (magenta), β-cyclodextrin:fluoroxene (orange) and OA:fluoroxene (green) host-guest systems. (b) 19F-CEST map overlaid on 1H-MRI exhibiting significant effect for the CB[7]:fluoroxene (Dw=+1.6 ppm) and OA:fluoroxene (Dw=-2.0 ppm) systems. (c) The MTRasym plots obtained from the map shown in b.

9:30 AM
PS-22-6 — Extracellular glutamate and intracellular calcium recording with fiber optic and simultaneous fMRI (#386)

Y. Jiang1, X. Chen1, 2, P. Pais1, 3, X. Yu1

1 ​​Max Planck Institute for Biological Cybernetics, Tübingen, Germany
2 University of Tuebingen, Tübingen, Germany
3 Graduate Training Centre of Neuroscience, Tübingen, Germany

Introduction

Here, we expressed genetically encoded fluorescent reporter iGluSnFRfor extracellular glutamate (Glu) sensing and genetically encoded calcium indicator GCaMP6f for calcium sensing in both neurons and astrocytes, and applied two channel fiber optic recording system in combination with blood oxygenation level-dependent signal (BOLD) fMRI. This platform offers us a more direct interpretation of neuronal transient with fMRI, thus, would expand our understanding of the signal propagation through the neuron-glia-vessel network couple to BOLD fMRI signals.

Methods

All images were acquired with a 14.1 T/26cm horizontal bore magnet (Magnex), interfaced to an AVANCE III console (Bruker) and equipped with a 12 cm gradient set, capable of providing 100 G/cm with a rise time of 150 us (Resonance Research). A transreceiver surface coil was used to acquire fMRI images. fMRI scans with block design were performed using 3D Echo planar imaging sequence: TR, 1.5 s, TE,11.5 ms, 1.92X1.92X1.92 cm3, FOV, 48X48X48 matrix, 400X400X400 um3 spatial resolution. The reporter iGluSnFR and GCaMP6f were expressed by AAV5 virus in the two hemisphere forepaw somatosensory cortex (FP-S1) with Syn or GFAP promoter. Fiber optic (200 mm) was inserted into the area which expressed the cortex for fluorescent signal recording.

Results/Discussion

Neuronal calcium and Glu signals with simultaneous fMRI from the FP-S1 of two hemispheres were acquired, respectively. Evoked neuronal calcium and Glu spikes were shown to follow each electrical pulses (Fig 1A), while the Glu spikes have earlier onset time and faster time to peak response in comparison with neuronal calcium. Also, amplitude of the evoked Glu spike increased proportionally to the amplitude of BOLD signals as the function of the stimulation intensity (Fig. 1B). The simultaneous fMRI BOLD maps and the time course of BOLD signal were shown (Fig. 1C).

Similar to previous study2, the astrocytic calcium signal is an integrated unitary spike, which has slower onset than the Glu spikes(Fig. 2A). Interestingly, we also observed the baseline drop of the Glu signal during the stimulation, which shows earlier onset with extended longer tail than the astrocytic signal. Also noteworthy is that the BOLD signals detected from both hemispheres are similar to each other(Fig.2B).

 

Conclusions

Concurrent glutamate and calcium recording was established with the BOLD fMRI brain mapping in anesthetized rats. This platform would lead to a better understanding of neurovascular coupling through the neuro-glial-vascular network in the animal brain. Future study will further clarify the neurovascular coupling events in the neuro-glial-vascular network and specify the source for the Glu baseline drop of during stimulation.

References

1. Marvin, J. S., et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat methods 10, 162-170, doi:10.1038/nmeth.2333 (2013).

2. Wang, M., He, Y., &Yu, X. 2017. A novel role of intrinsic astrocytic calcium spikes to mediate brain states through central/dorsal thalamic nuclei. ISMRM 2017.

Acknowledgement

Tuebingen Universtity and Graduate Training Center of Neuroscience Tuebingen.

Fig 1. Characterizations Glu and neuronal calcium responses and BOLD signal in rat.
 (A) Temporal features of sensory response of the evoked Glu and calcium signal by forepaw stimuli (3mA, 2Hz,4s). (B) The evoked Glu spike increased proportionally to the amplitude of BOLD signals as the function of the stimulation intensity. (C) The fMRI images and representative time course of iGluSnFR (left hemisphere) and GCaMP (right hemisphere) expressed in rat somatosensory cortex.

Fig 2. Concurrent glutamate and astrocytic calcium recording with fMRI.

(A) The glutamate and astrocyte calcium transient (upper panel) induced by multiply stimuli (2 Hz,4s) and the overall signal time course (lower panel). (B) The whole brain fMRI map and time course of iGluSnFR and astrocytic GCaMP expressed in rat somatosensory cortex by evoked forepaw electrical stimulations.

9:40 AM
PS-22-7 — Sensing intracellular calcium ions using a manganese-based MRI contrast agent (#422)

A. Barandov1, B. B. Bartelle1, A. Jasanoff1

1 Massachusetts Institute of Technology, Department of Biological Engineering, Cambridge, Massachusetts, United States of America

Introduction

Calcium ions are essential to signal transduction in virtually all cells. Although optical probes for intracellular calcium imaging have been in use for decades, the development of probes for noninvasive monitoring of intracellular calcium signaling in deep tissue and intact organisms remains an important challenge [1]. To address this problem, we designed and synthesized a new class of contrast agents (ManICS1-AM) that can be internalized and trapped in cells, and that enables intracellular calcium levels to be monitored by magnetic resonance imaging (MRI).

Methods

MRI data were acquired on a 7 T Bruker Biospec system. HEK293 cells (Freestyle 293-F, Thermo Fischer Scientific) were cultured and prepared for relaxometry, cell uptake and calcium response assessments. For stimulation experiments, cells were incubated with 10 µM ManICS1-AM for 2 h to allow for effective labeling and AM ester cleavage. Pharmacological stimulation was conducted by adding 5-10 µM of stimulants thapsigargin, charbocol, calcimycin, or arachidonic acid (Sigma-Aldrich).

Results/Discussion

To enable readouts of intracellular Ca2+ fluctuations by MRI, we developed a cell permeable probe, ManICS1-AM, derived from our previously reported intracellular contrast agents [2] and calcium specific chelator [3]. ManICS1-AM is readily internalized and retains in cytosol upon enzymatic cleavage of acetomethoxy (AM) esters (Fig. 1). The calcium sensor, ManICS1, exhibits relaxivity of 3.8-5.1 mM-1s-1 in vitro, a 34% change dependent on Ca2+ concentrations over its full titration range from 0 to 1 mM (Fig. 2) and an EC50 of 5 µM once loaded into cells. At concentrations of 100 µM the agent is able to transduce 1µM intracellular calcium signals into 2% changes in T1-weighted MRI, which is easily detectable by fMRI. Importantly, our sensor shows signal changes of 7% as ManICS1-loaded cells were stimulated chemically (Fig. 2E-F). The sensor enables to map contrast agent-mediated calcium responses using rapid MRI pulse sequences with optimal spatiotemporal resolutions.

Conclusions

Here we are reporting the first example of intracellular calcium sensitive contrast agent, which is retained in cells through well-understood mechanisms and shows significant T1-weighted MRI signal changes to intracellular calcium fluctuations induced by chemical stimulants. Relying on our cell data, our calcium sensor ManICS1-AM is a powerful tool for monitoring calcium signaling events in brain which is the subject of our on going research.

References

[1] Gingerberger C. et al. Neuron, 2012, 73, 862.

[2] Barandov A. et al. J. Am. Chem. Soc. 2016, 138, 5483.

[3] Tsien R. et al. J. Cell Biol. 1982, 94, 325.

Acknowledgement

The authors thank funding came from the MIT Simons Center and NIH grants R21-MH102470 and U01-NS090451 to A.J.

Figure 1. Design of cell permeable sensors for calcium-dependent molecular fMRI.
The sensors consists of a cell permeable paramagnetic platform (black complex, examples used in ManICS shown at top right), a BAPTA-based calcium chelator (dark blue), and a linker connecting them (green).

Figure2. ManICS1 reports calcium-dependent MRI signal changes in cells.
(A) T1-weighted signal changes as a function of [Ca2+] in buffer. (B) Relaxivity values corresponding to the titration series in A. (C) Washout time course of sensors for preincubated HEK293 cells. (D) Distribution of manganese in cells incubated with sensors. (E) Ca2+ responses in cells labeled with ManICS1-AM (left) or Fura-2-AM (right). (F) Ca2+ response of cells incubated with ManICS1-AM.     

9:50 AM
PS-22-8 — Synthesis of fluorinated curcumin-based molecules for detecting amyloid plaques by 19F-MRI (#426)

R. Stefania1, L. Tei2, F. Garello1, U. Fasoglio1, M. Tripepi1, G. Forloni3, A. Deluigi3, C. Balducci3, E. Terreno1

1 University of Torino, Department of Molecular Biotechnology for Health Sciences, Torino, Italy, Italy
2 Università del Piemonte Orientale, Department of Sciences and Technologic Innovation, Alessandria, Italy, Italy
3 IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, , Department of Neuroscience, Milano, Italy, Italy

Introduction

The detection of Aβ plaques is one strategy for Alzheimer’s Disease (AD) diagnosis. Amyloid imaging can be successfully pursued by 19F-MRI using fluorinated curcumin-based compounds.1 The developed probes contained a limited number of 19F atoms/molecule, and, furthermore, they were directly conjugated to the aromatic portion of the molecule, thus potentially reducing the detection sensitivity due to the broadening of the 19F signal. This work aims at synthesizing a series of novel F-containing curcumins with a high number of 19F nuclei, suitably spaced from the aromatic part of the molecule.  

Methods

The preparation of F-containing curcumins (Fig. 1) started from the synthesis of mono and bi-carboxylic acid derivatives of curcumin followed by an amide coupling with two different perfluorinated amines, one of them incorporated a carboxylic functional group to improve solubility. All the compounds obtained were then purified by HPLC, dissolved at 10 mg/mL in DMSO or in a mixture of normal saline and Cremophor®, and characterized by 1H and 19F NMR. Following T1 and T2 measurement at 7 T, 19F-MR images of the synthesized compounds were acquired to prove their potential application in vivo. Finally, to verify the ability to bind Aβ plaques, different slices of APP-PS1 mouse brain tissues were incubated with the four compounds and then imaged by confocal microscopy.

Results/Discussion

With the exceptions of bi-F18 (characterized in DMSO), the compounds synthesized were soluble in normal saline added with Cremophor. T1 and T2 values obtained for the water soluble compounds lied in the proper range for ensuring a good MRI detection (Table 1), and a phantom containing the three compounds at 10 mg/mL was imaged in 10 min with a good signal-to-noise ratio, proportional to the number of fluorine atoms per molecule. The deposition of the compounds on brain slices explanted from APP-PS1 mice (transgenic AD model) showed a reduced affinity of the derivatives with respect to the parent curcumin. However, a qualitative assessment of the fluorescent signal allowed to rank the affinity of the compounds towards the plaques in the order: mono-F9 > mono-carboxy-F9 > bi-F18 > bi-carboxy-F18. Most likely, the higher affinity showed by the mono-derivatives is due to the preservation of one phenolic group in the aromatic portion of the structure involved in the binding to the plaque.

Conclusions

The compounds synthesized in this work displayed a good potential for in vivo applications either in terms of water solubility or 19F-MRI signal detection. As far as the affinity towards plaques is concerned, the preliminary results highlighted the importance to maintain at least one phenolic group of curcumin. On this basis, compound mono-F8 appears to be the more promising to be tested in vivo on preclinical AD models for amyloid imaging by 19F-MRI.

References

  1. Tooyama, I et al. Ageing Research Reviews 30 (2016): 85-94.

 

Figure 1
Chemical structure of synthesized 19F curcumin-based molecules

Table 1
T1 and T2 relaxation values of the four newly-synthesized compounds

1:30 PM
emptyVal-1 — Introductory Talk by Kai Licha - Berlin, Germany

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

1:50 PM
PS-01-2 — Lanthanide(III)-Based Molecules, Macromolecules and Nanomaterials: New Generation of the Near-Infrared Emitting Probes for Optical Imaging (#34)

I. Martinic1, S. V. Eliseeva1, T. Nguyen3, D. Gosset1, S. Routier2, F. Suzenet2, V. L. Pecoraro3, S. Petoud1

1 CNRS, Center for Molecular Biophysics, Orleans, France
2 University of Orleans, Institute of Organic and Analytical Chemistry, Orleans, France
3 University of Michigan, Department of Chemistry, Ann Arbor, Michigan, United States of America

Introduction

Fluorescence optical imaging is a highly sensitive technique. In particular, imaging in the near-infrared (NIR) region attracts significant attention due to reduced autofluorescence and light scattering. New generation of optical imaging agents, lanthanide(III) (Ln3+)-based probes (LnPs), possess unique optical properties e.g. sharp emission bands not affected by the microenvironment and high resistance toward photobleaching. Low absorbance of free Ln3+ requires the use of appropriate chromophores for sensitization of their luminescence.1 Herein, we present several families of LnPs.

Methods

Yb3+[Zn(II)MCpyzHA] was synthesized by reaction of pyrazinehydroxamic acid with Zn2+ and Yb3+ triflates. The L1Nd3+ complex was obtained by interaction between the ligand L1 and Nd3+ nitrate; AQ1,nOH-Yb@PS/PEG (n=4,8) nanoparticles by encapsulation of 1,4- or 1,8- dihydroxyanthraquinones and Yb3+ triflates in polystyrene beads and their functionalization with polyethylene glycol. (Ln8-)G3P-(aza-BODIPY)n (n=16,32) were synthesized by coupling of aza-BODIPY with generation-3 polyamidoamine (G3P) dendrimer and interaction with Yb3+ or Nd3+ nitrates. Absorption/excitation/emission spectra, luminescence lifetimes, quantum yields and photostabilities were measured. Uptake of LnPs by HeLa cells was analyzed by confocal and epifluorescence microscopies and by flow cytometry in the visible/NIR.

Results/Discussion

Novel LnPs have been designed and successfully used for visible/NIR in vitro imaging and their advantages over the current fluorescent probes have been demonstrated. In particular, i) non-permeable and photostable polymetallic metallacrown (MC) complexes, Yb3+[Zn(II)MCpyzHA], demonstrated their applicability for labelling of cell necrosis2; ii) monometallic L1Nd3+, the first LnP with excitation and emission wavelengths within the ideal diagnostic window, demonstrated a deeper penetration through tissues of different origin and a more sensitive detection; iii) polymetallic AQ1,nOH-Yb@PS/PEG nanoparticles represent a major breakthrough in the design of LnPs with improved properties by simplifying screening of efficient chromophores; and iv) polymetallic G3P dendrimers, (Ln8-)G3P-(aza-BODIPY)n, demonstrated possibility for tuning of photophysical properties, photostability and cellular uptake, by controlling the number of chromophores at their periphery and by presence and nature of Ln3+.

Conclusions

Presented monometallic and polymetallic Ln3+-based molecules, macromolecules and nanomaterials possess a superior and complementary optical properties over the currently available commercial fluorescent probes. Their unique potential makes them attractive candidates for advanced applications in optical imaging and therefore represents significant breakthroughs toward the creation of a new generation of the NIR optical imaging probes for in vitro and in vivo applications with possibilities for replacement of current probes.

References

1. I. Martinić, S.V. Eliseeva, S. Petoud, Journal of Luminescence, 189 (2017) 19-43.

2. I. Martinić, S.V. Eliseeva, T.N. Nguyen, V.L. Pecoraro, S. Petoud, JACS (2017), 139, 8388-8391.

Acknowledgement

This research has received funding from the European Community's Marie Curie Seventh Framework Programme (FP7/2007-2013: n° 316906 (ITN Luminet) and n° 611488 (IRSES Metallacrowns)), La Ligue Contre le Cancer, La Region Centre and ANR Lumiphage.

Figure 1. Crystal structure and/or schematic representation of LnPs.

Crystal structure of Yb3+[Zn(II)MCpyzHA] (A). Schematic representations of: L1Nd3+ (B), AQ1,4OH-Yb@PS/PEG nanoparticle (C) and Yb8-G3P-(aza-BODIPY)16 dendrimer (D).

 

Figure 2. Results of epifluorescence and confocal microscopies of HeLa cells incubated with LnPs.

Epifluorescence microscopy of: necrotic cells incubated with Yb3+[Zn(II)MCpyzHA] (λex: 447nm/60nm, λem> 805nm, τexp: 10s) (A) and viable cells incubated with L1Nd3+ (λex: 655nm/40nm; λem> 805nm, τexp: 20 s) (B). Confocal microscopy of viable cells incubated with: AQ1,4OH-Yb@PS/PEG (λex: 488nm; λem: 580–750nm) (C) and Yb8-G3P-(aza-BODIPY)16 (λex: 633nm; λem: 650-800nm) (D).

2:00 PM
PS-01-3 — Ex Vivo Tracking of Endogenous CO with Ruthenium(II) Complexes (#285)

J. A. Robson1, A. Toscani1, C. Torre2, C. Marín-Hernández2, M. Terencio2, M. Alcaraz2, R. Martínez-Máñez2, F. Sancenón2, J. Wilton-Ely1

1 Imperial College London, London, United Kingdom
2 Universitat Politècnica de València, Departamento de Química, Valencia, Spain

Introduction

In the body, endogenous CO acts as a gaseous signalling molecule serving many functions, including anti-inflammatory, antiapoptotic and anticoagulative roles.1 The potential of CO as a therapeutic agent has received significant attention, particularly since CO-releasing molecules (CORMs) are capable of liberating controlled amounts of CO in biological systems.2 One of the major obstacles limiting progress in understanding the biological role of CO and its therapeutic application is the lack of real-time methods to selectively track CO in biological systems.

Methods

Our novel molecular probes provide one- or two-photon fluorogenic detection of CO in biological environments using the IDA paradigm pioneered by Anslyn.3 In vitro studies use confocal and multi-photon microscopy to visualise the release of CO by carbon monoxide releasing molecules CORM-3 and hemin in live cells. Ex vivo studies on mice with a subcutaneous air pouch use Lipopolysaccharides (LPS) to induce an inflammatory reaction characterized by plasmatic exudation and migration of leukocytes to the cavity. LPS also induces HO-1 protein expression in leukocytes migrating to the air pouch exudates, causing them to exhibit higher levels of CO than would be expected in the absence of LPS. Exudates in the pouch are collected and visualized using two-photon microscopy.

Results/Discussion

Equipped with fluorophores suited for imaging in biological environments, new water-soluble ruthenium vinyl probes have been designed based on the system we have used for detection of CO in air.4 In the presence of CO in vitro, these probes exhibit a rapid and selective fluorogenic response, the probes are selective for CO even in high reactive oxygen species (ROS) environments. In addition, they avoid the drawbacks of current palladium-based systems such as slow response times, pH limitations and the toxicity of unligated Pd salts. Coupled with their pH stability and low toxicity, these attributes allow fluorescence imaging of range cellular environments and permit the visualisation of very low levels of CO in cells. For the first time, these two-photon fluorescent probe detected endogenous carbon monoxide ex vivo using mice with a subcutaneous air pouch.5

Conclusions

The ruthenium probes are nontoxic to cells and can be used in low doses for the in vitro two-photon fluorescence imaging of CO in cells in the presence of CORM-3 or hemin. Even more significantly, the probes can be used in ex vivo two-photon fluorescence, detecting CO in cells collected from the exudates of an air pouch inflammation mouse model. The combination of selectivity, sensitivity and inexpensive, straight-forward synthesis make the system described here a very attractive and efficient probe for the facile fluorogenic detection of this gas in realistic biological environments.

References

  1. Szabo, C. Nat. Rev. Drug. Discov. 2016, 15, 185−203.
  2. Motterlini, R.; Otterbein, L. E. Nat. Rev. Drug. Discov. 2010, 9, 728.
  3. Nguyen, B. T.; Anslyn, E. V. Coord. Chem. Rev. 2006, 250, 3118− 3127.
  4. Moragues, M. E.; Toscani, A.; Sancenón, F.; Martínez-Máñez, R.; White, A. J. P.; Wilton-Ely, J. J. Am. Chem. Soc. 2014, 136, 11930.
  5. Torre, C.; Toscani, A.; Marín-Hernádez, C.; Robson, J.A.; Alcaraz, M.;Sancenón, F.; Martínez-Máñez, R.; White, A. J. P.; Wilton-Ely, J. J. Am. Chem. Soc. Accepted. doi: 10.1021/jac7611158

 

Acknowledgement

I thank the EPSRC for PhD studentship

Mechanism

Overview

2:10 PM
PS-01-4 — Near-infrared fluorescent probe for detecting NF-kB activation in a mouse model of type 1 diabetes (#311)

T. Taghian1, V. G. Metelev1, 2, S. Zhang1, A. A. Bogdanov1

1 University of Massachusetts Medical School, Radiology, Worcester, Massachusetts, United States of America
2 M.V.Lomonosov Moscow State University, Chemistry, Moscow, Russian Federation

Introduction

Detecting inflammation markers in the endocrine pancreas during the silent phase of type 1 diabetes (T1D) will aid in diagnosis and enable early therapeutic interventions [1]. Transcription factor NF-kB plays a pivotal role in regulating beta cell function and is involved in development of T1D. We designed and synthesized near-infrared fluorescent oligodeoxyribonucleotide (NIR-ODN) probes for imaging NF-kB-DNA interactions [2], Fig.1. The goal of our research was to perform NIR imaging of pancreatic cells and whole pancreatic islets using NIR-ODN probe by using models of experimental diabetes.

Methods

Multiple low dose streptozotocin (MLD-STZ) T1D model was developed by using SKH1 mice which received 50 mg/kg/d STZ for 5 d. Pancreata were harvested on day 8 and snap frozen for sectioning. Islets and exocrine tissue fragments were separated using collagenase P with subsequent density centrifugation. Frozen non-fixed tissue sections were incubated with 40-250 nM Cy5.5-labeled NIR-ODN probe in the presence of Mg2+, DTT and tRNA for 4 h followed by staining for insulin. Additionally, tissue sections were examined after treating with anti-NF-kB p65phospho-(S536). Fluorescence images were acquired using 14-bit 20 MHz CCD and quantified using ImageJ. Electrophoretic shift (EMSA) results were obtained by using cell lysates and NIR-ODN probe, visualized and quantified using a NIR imaging system.

Results/Discussion

Following MLD-STZ treatment of mice, insulin-positive fraction of the STZ-treated pancreas on tissue sections was significantly decreased in observed area compared to control. However, blood glucose levels remained in the normal range throughout the study. Treated mice exhibited significantly higher level of NF-kB expression in the nuclei and cytoplasm of the islet cells vs. control as assessed by fluorescence images using anti-NF-kB p65phospho-(S536) and EMSA. In agreement with these results, Cy5.5 labeled NIR-ODN exhibited highly significant quantitative differences in binding to nuclei and cytoplasmic sites of STZ treated islet cells vs. control cells as demonstrated in frozen sections (Fig.2). Therefore, we demonstrated that the sensitivity of NIR-ODN duplex probe specific for mature NF-kB heterodimer was sufficient for detecting NF-kB expression and activation in MLD-STZ treated normoglycemic non-fixed pancreas in a mouse model of T1D, prior to the onset of diabetes.

Conclusions

Results of biochemical (EMSA) and fluorescence microscopy studies showed that NIR-ODN imaging probe was specific for NF-kB both in cell cytoplasm and nuclei of pancreatic cells. The binding to nuclei and cytoplasmic fraction of pancreatic islet cells correlated with the increased NF-kB activity/expression after MLD-STZ treatment damage to the pancreas. The tested NIR probe is not species-specific and can be used in studies of various animal models of diabetes as well as other inflammatory disease.

References

1. Mathis, D. et al. Nature, 2001; 414(6865):792-8. 2. Zhang, S. et al. Proc Natl Acad Sci, 2008; 105(11):4156-61.

Acknowledgement

NIH grants R01 DK095728, R01 EB000858 (AAB).

Figure 1
Molecular model of NIR-ODN probe. The probe is a 21-mer duplex labeled with Cy5.5 at internucleoside amino linker and stabilized with phosphorothioate bonds at the 5'-and 3'- termini, Cy5.5 atoms are rendered as spheres. 

Figure 2
NIR-ODN NF-kB binding to nuclei and cytoplasmic fractions of NFkB. A- MLD-STZ treated mouse islet cells in non-fixed tissue treated with NIR-ODN probe. B- control islet section treated with NIR-ODN. red - Cy5.5-NIR-ODN, green - anti-insulin, blue- DAPI. C- results of fluorescence intensity measurements performed using sections of MLD-STZ treated and control nuclei and cytoplasm (n=100-150/group).

2:20 PM
PS-01-5 — In vivo Cerenkov imaging of cellular energetics in breast cancer cells with different metastatic potential (#313)

A. D. Arroyo1, B. Moon1, A. V. Popov1, E. J. Delikatny1

1 University of Pennsylvania, Radiology, Philadelphia, Pennsylvania, United States of America

Introduction

The objective of this project is to develop functional fluorinated bioactivatable molecules to utilize the emission of Cerenkov radiation to distinguish tumors based on metabolic markers. The metastatic potential of tumors has been correlated with several metabolic markers including acidic pH in tumor microenvironment. Deregulated cellular energetics were recently added to the Hallmarks of Cancer. [1]

 

Methods

Resazurin (RA), or Alamar Blue, is a commonly used viability dye and redox sensor. Reduction by the mitochondria converts RA into resorufin, a highly fluorescent molecule. Cold- and radio-labeled monofluorinated resazurin (MFRA) and difluorinated resazurin (DFRA) were synthesized by electrophilic fluorination.[2]  Tumor probe reduction was imaged by fluorescence and Cerenkov imaging in breast cancer cell lines and solid tumors with different metastatic potentials: MDA-MB-231 (triple-negative breast cancer) and 4175-Luc+ (MDA-MB-231-variant isolated from murine lung metastases)[3]. This was correlated with in vitro glycolytic activity as determined by glucose consumption, lactate production, and LDHA levels. In vivo pharmacokinetics were determined using PET imaging of the 18F derivatives.

Results/Discussion

Reduced and oxidized MFRA and DFRA were distinguished optically by differences in their Cerenkov emission with a ratio of 3.5 and 1.3 respectively at 640 nm. 4175-Luc+ cells showed more rapid reduction of RA, MFRA and DFRA than MDA-MB-231 cells (Fig 1). Modulation of intracellular glutathione levels had no effect on probe reduction. The 4175-Luc+ cells displayed higher consumption of glucose and production of lactate than MDA-MB-231s. PET imaging of 18F-DFRA in mice with 4175-Luc+ tumor xenografts showed 0.25-0.9% of the injected dose accumulating in the tumor. Cerenkov time course studies showed probe accumulation in the periphery of the tumor. Increased emission at 640 nm relative to 580 nm indicated increased emission of the reduced fluorescent probe via Cerenkov Radiation Energy Transfer (CRET). DFRA-treated 4175-Luc+ tumor slices ex vivo showed immediate probe reduction. IT injections into 4175-Luc+ tumors confirmed fast probe reduction in vivo (Fig 2).

 

Conclusions

The highly metastatic 4175-Luc+ cell line displayed both increased glycolytic and Krebs cycle activity relative to the MDA-MB-231 parent line. NADH dehydrogenases such as Complex 1 in the electron transport chain reduce resazurins,. Cerenkov radiation and CRET detected probe reduction in vitro and in vivo. Resazurins could be used to determine mitochondrial energy imbalance and assess metastatic potential of tumors. The use of 18F-labeled isotopomers allows for dual modality PET and Cerenkov imaging to measure probe concentration and metastatic potential simultaneously.

 

References

  1. Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011: 144, 5: 646-674
  2. Kachur AV, Arroyo AD, et al. Synthesis of F-18 labeled resazurin by direct electrophilic fluorination. J Fluor Chem. 2015: 178: 136-141
  3. Minn, AJ, et. al. Genes that mediate breast cancer metastasis to lung. Nature. 2005: 436(7050): 518–524.

 

Acknowledgement

We would like to acknowledge the Small Animal Imaging Facility and the Cyclotron Facility at the University of Pennsylvania and our funding sources, NIH grants R01 EB018645, F31 CA206453

 

Figure 1: Reduction of resazurin and fluorinated resazurins in 4175-Luc+ and MDA-MB-231.
Graphs show activation of resazurin probes in 2 cell lines: MDA-MB-231 and its metastatic variant 4175-Luc+, as measured by the time-dependent increase in fluorescence. Activation of resazurin and monofluororesazurin is more rapid and significantly higher in 4175-Luc+ cells. In the case of difluororesazurin, the difference is not as great, possibly due to its weak fluorescence.

Figure 2: Cerenkov imaging time course of 18F-DFRA in athymic nude mice
An in vivo tumor injected with 400 mCi 18F-DFRA and 18F-FDG and an ex vivo tumor injected with 400 mCi of 18F-FDG were imaged at the same time to approximate the absorption of Cerenkov radiation by tumor tissue. Using the data from the ex vivo tumor injection of FDG, we were able to calculate how much signal we could theoretically see at 640 nm, taking into account tissue scattering.

2:30 PM
PS-01-6 — Development framework for GMP translation of optical tracers cetuximab-800CW and trastuzumab-800CW (#360)

M. D. Linssen1, 2, E. J. ter Weele1, D. P. Allersma1, M. N. Lub-de Hooge1, 4, G. M. van Dam3, 4, A. Jorritsma-Smit1, W. B. Nagengast2

1 University of Groningen, University Medical Center Groningen, Department of Clinical Pharmacy and Pharmacology, Groningen, Netherlands
2 University of Groningen, University Medical Center Groningen, Department of Gastroenterology and Hepatology, Groningen, Netherlands
3 University of Groningen, University Medical Center Groningen, Department of Surgery, Groningen, Netherlands
4 University of Groningen, University Medical Center Groningen, Department of Nuclear Medicine and Molecular Imaging, Groningen, Netherlands

Introduction

Optical molecular imaging can be of significant added value to guide surgical or endoscopic procedures using fluorescently labeled tracers. Monoclonal antibodies have favorable properties for dye delivery. However, development of tracers for clinical trials is a complex process, and therefore implementation of new tracers in the clinic is slow. We present a framework for development and translation into a Good Manufacturing Process (GMP) compliant manufacturing process of monoclonal antibody tracers, illustrated by the results of cetuximab-800CW and trastuzumab-800CW.

Methods

Cetuximab and trastuzumab were conjugated under GMP conditions to IRDye 800CW according to standardized operating procedures. Optimal label ratio and formulation buffer were investigated according to our standardized framework. Best performing formulations were advanced to full-scale stability study. Tracers were analyzed for stability by SE-HPLC, pH-measurement, osmolality, visual inspection and sterility, as required by the European Pharmacopeia and GMP guidelines.

Results/Discussion

For cetuximab-800CW, 7 out of 7 formulations showed potential for long-term stability, whereas for trastuzumab, this was the case for 6 out of 10 tested formulations. Based on these results, 2 formulations for each antibody were investigated in a full-scale stability study. These formulations all performed well, showing good compliance with the acceptance criteria set for each product.

Conclusions

We designed a framework to standardize the development, formulation and GMP translation of molecular fluorescent tracers. Using our standardized approach, we developed two stable antibody-based tracers for clinical use. When developing tracers, the proposed framework can be used to efficiently develop a GMP compliant formulation and improve translation of newly developed optical tracers to first in human use.

Structured tracer development framework
The proposed framework for development and translation of a cGMP compliant manufacturing process. The framework assists the researcher by giving a bird’s eye view of the steps to be taken leading up to the first clinical-grade production, and facilitating the cooperation between the different disciplines involved.

2:40 PM
PS-01-7 — Development of near infrared probe allowing non invasive in vivo imaging of fibrosis in mouse (#392)

C. Robin-Jagerschmidt1, E. De Lemos2, D. Merciris3, E. Berrocal1, M. Pizzonero2, P. Clément-Lacroix1, R. Gosmini2

1 Galapagos, In vivo pharmacology, Romainville, France
2 Galapagos, Chemistry, Romainville, France
3 Galapagos, Histology, Romainville, France

Introduction

Currently, evaluation of fibrosis extent in preclinical rodent fibrosis models is achieved post-mortem, histologically or biochemically. Starting from a green fluorescent based collagen and elastin binding probe, replacement of the fluorescent dye and modulation of the linker moiety studies generated a new probe with near infrared (NIR) emitting properties allowing in vivo monitoring of fibrosis disease or predicting therapeutic responses in mice. The probe was evaluated in two preclinical models of CCl4-induced liver injury and BLM-induced lung injury.

Methods

Liver fibrosis was induced by repeated treatment of male Balb/C J mice with CCl4. Lung fibrosis was induced by a single instillation of Bleomycin to C57BL/6 N male mice. After 4 weeks of disease induction, the probe was injected in the retro-orbital sinus of anesthetized mice (100µL, 40µM). To determine the optimal timing for signal to background ratio, images were captured 15 min, 30min, 1h, 2h, 3h, 4h, 5h and 24h after probe injection using Bruker In-Vivo Xtreme imaging system Ex730 Em790. Confirmation of increase fibrosis extent was performed by measurement of collagen production in tissue lysate using a hydroxyproline assay (QuickZyme Bioscience), or in FFPE livers using Sirius red staining or type I collagen immunohistochemistry.

Results/Discussion

CCl4-treated mice displayed increased binding of the probe in the liver with 3 hours as the optimum timing for highest signal to background ratio. Similarly, BLM increased binding of the probe in the lungs with 2 hours as the optimum timing for highest signal to background ratio. For both models, binding was demonstrated both in vivo and ex vivo. In addition, increase collagen extent was also shown with classical histology techniques (collagen detection with immunohistochemistry or Sirius red labelling).

Conclusions

We describe the first near-infrared collagen-binding probe allowing non-invasive, specific and rapid imaging of fibrosis extend, in several preclinical models of lung and liver fibrosis. In addition to predict therapeutic responses in mice, it makes possible randomization of mice in homogeneous groups prior dosing, thus decreasing intra-group variability, leading to lower number of mice to be included in the study.

References

Biela E et al., 2013 Cytometry A, 83 : 533-9

Starkel P., 2011 Best practice & Research Clinical Gastroenterology 25: 319-333

Peng et al. 2013, PLoS One, 8: e59348.

2:50 PM
PS-01-8 — In vivo calcium imaging with photoacoustics (#484)

S. Roberts1, 2, 3, M. Seeger5, 2, Y. Jiang2, 3, A. Mishra2, 3, F. Sigmund2, 3, A. Stelzl2, 3, A. Lauri2, 3, P. Symvoulidis1, 2, 3, H. Rolbieski1, 2, 3, M. Preller6, L. Deán-Ben2, D. Razansky2, T. Orschmann3, S. Desbordes3, P. Vetschera2, T. Bach4, V. Ntziachristos5, G. G. Westmeyer1, 2, 3

1 Technical University Munich, Nuclear Medicine, Munich, Germany
2 Helmholtz Center Munich, IBMI, Oberschleißheim, Germany
3 Helmholtz Center Munich, IDG, Oberschleißheim, Germany
4 Technical University of Munich, Chair of Organic Chemistry I, Garching, Germany
5 Technical University of Munich, Chair for Biological Imaging, Munich, Germany
6 Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany

Introduction

Photoacoustic imaging has the key advantages that its resolution is insensitive to photon scattering and that it can provide volumetric data with high frame rates wihtout the need for scanning procedures. To exploit these powerful features for molecular imaging, we have synthesized the first cell-permeable photoacoustic sensor that reversibly changes its photoacoustic signal in response to specific binding of the the key second messenger calcium (Ca2+).

Methods

CaSPA_550 was synthesized via a condensation reaction between 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indol-3-ium iodide, containing an activated methyl group and the appropriate aldehyde of 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetate skeleton resulting in deep purple crystals with 85% yield. Subsequent demethylation was carried out at room temperature and the resulting product was dried under high vacuum to obtain the corresponding free acid before further esterification was carried out. CASPA_550 was characterized photophysically and biochemically in response to various metals. Combined fluorescence and photoacoustic microscopy was conducted in cell and tissue culture as well as in zebrafish larvae using custom-built microscopic or mesoscopic photoacoustic instruments.

Results/Discussion

We synthesized a selective metallochromic sensor (CaSPA_550) with high extinction coefficient, low quantum yield, and high photobleaching resistance.  Micromolar concentrations of Ca2+ lead to a robust blueshift of the absorbance and photoacoustic spectra (Figure 1 a,b) [1]. Sensitivity for calcium was in the micromolar range and selectivity towards magnesium and other metals was high due to the BAPTA chelation group. Esterification of CaSPA_550 enabled efficient cellular uptake into HEK293 and CHO cells and detection of pharmacologically induced Ca2+ influx by both fluorescence and optoacoustic microscopy (Figure 1 c,d). Calcium transients could be measured in beating heart organoids (Figure 2 a) as well as in zebrafish larval brains via simultaneous fluorescence and optoacoustic imaging (Figure 2 b) .

Conclusions

The efficient non-toxic delivery, high sensitivity and specificity of the Ca2+ sensor for photoacoustics (CaSPA) enabled us to for the first time perform molecular photoacoustic imaging of Ca2+ fluxes in genetically unmodified cells and heart organoids as well as in zebrafish larval brain. The semi-cyanine scaffold of CaSPA_550 may serve as a versatile platform to generate red-shifted variants [2] for imaging deeper in tissue as well as sensors selective for other metals or small analytes to enable molecular photoacoustic imaging with photoscattering-independent resolution in living tissue.

References

[1] Roberts, S. et al. Calcium Sensor for Photoacoustic Imaging. J Am Chem Soc jacs.7b03064 (2017). doi:10.1021/jacs.7b03064

[2] Mishra, A., Jiang, Y., Roberts, S., Ntziachristos, V. & Westmeyer, G. G. Near-Infrared Photoacoustic Imaging Probe Responsive to Calcium. Anal. Chem. 88, 10785–10789 (2016).

 

Acknowledgement

We thank Dr. Robert Pal (Durham University) for relative QY measurements, Dr. Andreas Bauer (Technical University of Munich) for absolute QY measurements. We are grateful for support from the Helmholtz Alliance ICEMED (AM, GGW), the European Research Council under grant agreements ERC-StG: 311552 (GGW), the Priority program SP1665 of the German Research Foundation (DFG), as well as the Laura Bassi Award of TUM (SR), grant agreements CRC 1123 (Z1), and Reinhard Koselleck project (NT 3/9-1).

Figure 1: Calcium Sensor for Photoaoustics (CaSPA) in vitro and in cell culture
(a) Schematic of cellular delivery of esterified CaSPA and intracellular 'unmasking' of the BAPTA chelation group (b) Absorbance and photoacoustic spectral changes in response to increasing concentrations of micromolar Ca2+ (c) Fluorescence and (d) photoacoustic microscopy of CHO cells incubated with CaSPA_550 (2 µM, 30 min.) in response to Ca2+ influx triggered by addition of a Ca2+ ionophore. 

Figure 2: Photoacoustic calcium imaging in heart organoids and zebrafish larval brain
(a) Heart organoids loaded with CaSPA_550 ester and visualized as overlay of photoacoustic image (magenta) and two-photon fluorescence volume (gray). right: Median signal difference map and photoacoustic signal time course (b) Combined fluorescence imaging (top) and multispectral photoacoustic tomography (bottom) of CaSPA-injected larval zebrafish brain and time courses after stimulation (right).

6:15 PM
emptyVal-1 — Introductory Talk Guy Bormans - Leuven, Belgium

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

6:35 PM
PS-10-2 — New Radiolabeled Exendin Analogue Shows Reduced Renal Retention (#39)

L. Joosten1, C. Frielink1, M. Gotthardt1, M. Brom1

1 Radboud University Medical Center, Radiology and Nuclear Medicine, Nijmegen, Netherlands

Introduction

A promising imaging method for detection of small insulinomas is targeting the glucagon-like peptide-1 (GLP-1) receptor by PET/CT after injection of 68Ga-labeled exendin-4[1]. High accumulation of radiolabeled exendin in the kidneys via tubular reabsorption can hamper the ability to detect small insulinomas in close proximity of the kidneys[2]. In this study, we developed a novel exendin-analogue, aiming at reducing the renal retention. We examined the renal retention and insulinoma targeting properties of this new exendin analogue in a nude mouse model bearing subcutaneous insulinomas.

Methods

NOTA was conjugated via a methionine-isoleucine-linker to the C-terminus of exendin-4 (MI-NOTA-exendin-4). Exendin with NOTA conjugated to a C-terminal lysine was used as a reference. The affinity of the peptides was determined in a competitive binding assay using GLP-1 receptor transfected CHL cells. Biodistribution of 68Ga-NOTA-exendin-4 and 68Ga-MI-NOTA-exendin-4 was performed in INS-1 tumor-bearing BALB/c nude mice to investigate the renal retention and tumor targeting properties of both peptides and to assess visualization of INS-1 tumors with PET/CT.

Results/Discussion

Labeling of NOTA-exendin-4 and MI-NOTA-exendin-4 resulted in a maximum specific activity up to 275 GBq/µmol and 242 GBq/µmol, respectively. The affinity for the GLP-1 receptor was 9.5 nM (95% confidence interval, 7.0 to 12.8 nM) for NOTA-MI-exendin-4 and 4.0 nM (95% confidence interval, 2.5 to 6.2 nM) for NOTA-exendin-4 (p<0.001). In vivo biodistribution revealed a significant lower kidney uptake of 68Ga-NOTA-MI-exendin-4 one hour post injection (102.6 ± 14.8 %ID/g), compared to 68Ga-NOTA-exendin-4 (127.2 ± 17.3 %ID/g) (p<0.0001), which was even more pronounced four hours p.i. (34.2 ± 4.2 %ID/g vs 127.7 ± 9.5 %ID/g respectively, p<0.0001). Accumulation of 68Ga-NOTA-MI-exendin-4 in the tumor was 25.0 ± 8.0 four hours p.i. and similar to that of 68Ga-NOTA-exendin-4 (24.9 ± 9.3) (p>0.05). Furthermore, INS-1 tumors were visualized using PET/CT and moreover, the reduced kidney uptake of 68Ga-MI-NOTA-exendin-4 compared to 68Ga-NOTA-exendin-4 was clearly visible.

Conclusions

In conclusion, 68Ga-MI-NOTA-exendin-4 showed reduced renal retention of more than 60% four hours p.i. when compared to 68Ga-NOTA-exendin-4, whereas tumor retention did not change significantly. Therefore, 68Ga-labeled MI-NOTA-exendin-4 is a promising improved tracer resulting in better tumor-to-kidney ratios and might allow improved detection of small insulinomas, in close proximity to the kidneys.

References

1. Antwi K, Fani M, Nicolas G, et al. Localization of Hidden Insulinomas with (6)(8)Ga-DOTA-Exendin-4 PET/CT: A Pilot Study. J Nucl Med. 2015;56:1075-1078.

2. Luo Y, Yu M, Pan Q, et al. 68Ga-NOTA-exendin-4 PET/CT in detection of occult insulinoma and evaluation of physiological uptake. Eur J Nucl Med Mol Imaging. 2015;42:531-532.

Figure 1.

Tumor and kidney uptake of 68Ga-labeled NOTA-exendin-4 and MI-NOTA-exendin-4 1, 2 and 4 hrs post injection. Values are expressed as %ID/g (n=5 mice per group), error bars SD).

6:45 PM
PS-10-3 — Human PD-L1 Nanobody for PET imaging: a site-specific radiolabelling strategy (#211)

J. Bridoux1, K. Broos2, M. Crauwels3, 1, Q. Lecocq2, C. Martin6, F. Cleeren7, G. Bormans7, S. Ballet6, V. Caveliers4, G. Raes5, K. Breckpot2, S. Muyldermans3, N. Devoogdt1, M. Keyaerts4, C. Xavier1

1 Vrije Universiteit Brussel (VUB), In Vivo Cellular and Molecular Imaging (ICMI), Jette, Brussels, Belgium
2 Vrije Universiteit Brussel (VUB), Laboratory of Molecular and Cellular Therapy (LMCT), Jette, Brussels, Belgium
3 Vrije Universiteit Brussel (VUB), Cellular and Molecular Immunology (CMIM), Elsene, Brussels, Belgium
4 UZ Brussel, Nuclear Medicine Department, Jette, Brussels, Belgium
5 VIB Inflammation Research Center, Myeloid Cell Immunology Lab, Gent, Belgium
6 Vrije Universiteit Brussel (VUB), Department of Organic Chemistry, Elsene, Brussels, Belgium
7 University of Leuven, Department of Pharmacy and Pharmacology, Radiopharmaceutical Research, Leuven, Belgium

Figure 1: Scheme of the site-specific radiolabeling

Site-specific sdAb functionalization with the NOTA or RESCA chelator via the Sortase A transpeptidation reaction, and radiolabelling with 68Ga and Al-18F respectively.

6:55 PM
PS-10-4 — On the optimal molecular design of ADAPT-based HER2 imaging probe labelled with 68Ga. (#181)

J. Garousi1, S. Lindbo2, B. Mitran3, A. Vorobyeva1, M. Oroujeni1, A. Orlova3, S. Hober2, V. Tolmachev1

1 Uppsala University, Department of Immunology, Genetics and Pathology, Uppsala, Sweden
2 KTH Royal Institute of Technology, Division of Protein Technology, Stockholm, Sweden
3 Uppsala University, Department of Medicinal Chemistry, Uppsala, Sweden

Introduction

ADAPT is a new type of engineered small (6 kDa) affinity proteins based on the scaffold of albumin-binding domain of protein G. ADAPT6 demonstrated excellent in vivo imaging of HER2 expression.1 Variants having DEAVDANS and (HE)3DANS N-terminal sequences and labelled with 111In via DOTA at N-terminus demonstrated higher tumour-to-organ ratios compared to parental molecule.2 It was shown that the placement of the DOTA chelator at C-terminus improved the tumour-to-organ ratio of 111In-labelled variants.3,4 The aim of this study was to select the best 68Ga-labelled ADAPT6 variant for PET imaging.

Methods

DEAVDANS-ADAPT6-GSSC-DOTA and (HE)3DANS-ADAPT6-C-DOTA having maleimido-DOTA conjugated at C-termini were labelled with 68Ga and 111In. Labelled proteins were purified using NAP-5 size-exclusion columns. Binding specificity and cellular processing of labelled proteins were evaluated using SKOV-3 and BT474 cell lines. Female BALB/C nu/nu mice bearing SKOV-3 xenografts with high HER2 expression (1.6 × 106 receptors/cell) and DU-145 xenografts with low HER2 expression (5.1 × 104 receptors/cell) were used as models in in vivo studies. Biodistribution of 111In/ 68Ga-DEAVDANS-ADAPT6-GSSC-DOTA and 111In/ 68Ga-(HE)3DANS-ADAPT6-C-DOTA was measured at 1 and 3 h post injection (p.i.). PET/CT imaging was performed to show discrimination between xenografts with high and low HER2 expression.

Results/Discussion

The radiochemical yields of labelling with 68Ga and 111In exceeded 90%. The radiochemical purity was over 99% for all conjugates. All variants bound specifically to HER2-expressing cells. Internalized fraction of all variants was less than 15 % after 4 h. The tumour uptake (10-12%ID/g) was appreciably higher than uptake in any normal tissue (except kidneys) already 1 h p.i. Tumour uptake of 111In-labeled variants was slightly but significantly higher than 68Ga-labeled ones, and clearance of 111In-labeled variants was rapider. All variants had several-fold higher uptake in SKOV-3 than in DU-145 xenografts. N-termini composition had no significant influence on tumour uptake, but both 111In- and 68Ga-labeled (HE)3DANS-ADAPT6 cleared rapider from blood. In mice with SKOV-3 xenografts, DEAVDANS-ADAPT6 provided ca. twice higher tumour-to-blood ratios at 3 h p.i. (208±36 for 111In and 109±17 for 68Ga) than (HE)3DANS-ADAPT6 (93±19 for 111In and 50±18 for 68Ga).

Conclusions

68Ga-labeled ADAPTs enable clear discrimination between tumours with high and low expression of HER2. 68Ga-(HE)3DANS-ADAPT6-C-DOTA is the preferable variant for imaging of HER2-expression level  using PET because of higher tumour-to-blood ratio, which might be critical for visualization of small metastases. Although the tumour-to-organ ratios are higher for 111In label, the tumour-to-organ ratios for 68Ga-(HE)3DANS-ADAPT6-C-DOTA are high enough (48±7 for liver, 104±34 for lung, and 147±70 for bone) to provide high-contrast imaging in major metastatic sites.

References

  1. Garousi et al. Cancer Res. 2015;75:4364-71.

  2. Garousi et al. Bioconjug Chem. 2016;27:2678-2688.

  3. Lindbo S et al. J Nucl Med. 2017 Sep 1. [Epub ahead of print]

  4. Garousi et al. Sci Rep. 2017;7:14780

Acknowledgement

This work was supported by grants from Swedish Research Council and Swedish Cancer Society.

68Ga-labeled ADAPTs enable clear discrimination between tumours with high and low expression of HER2
Small animal PET/CT imaging of SKOV-3 (high HER2 expression) and DU-145 (lowHER2 expression) xenografts in BALB/C nu/nu mice using 68Ga- DEAVDANS-ADAPT6-GSSC-DOTA and 68Ga-(HE)3DANS-ADAPT6-C-DOTA. Images were acquired at 3 h p.i. The upper SUV threshold was set at 4.

7:05 PM
PS-10-5 — Anti-HER3 affibody molecules: negatively charged metal-chelator labeling complex facilitates imaging (#183)

A. Orlova1, S. S. Rinne1, C. D. Leitao2, B. Mitran1, S. Ståhl2, J. Löfblom2, V. Tolmachev1

1 Uppsala University, Medicinal Chemistry, Uppsala, Sweden
2 Royal Institute of Technology, Protein Technology, Stockholm, Sweden

Introduction

Introduction of HER3-targeting anti-cancer therapeutics demands development of suitable methods for patient stratification. Pre-clinically affibody molecules demonstrated to be suitable for imaging of HER3 expression1. However, low target expression in tumors and high endogenous target expression (especially in liver) remains to be a challenge in HER3-imaging. The combination of chelator and radionuclide can influence the biodistribution of affibody molecules and provides a tool for optimization of imaging sensitivity and specificity2.

Methods

Chelators NOTA, NODAGA, DOTA and DOTAGA were conjugated to affibody molecule Z08698 via C-terminal cysteine. The constructs were radiolabeled with In-111. Stability in blood plasma, binding affinity and specificity, and cellular processing were evaluated in vitro in HER3-expressing cell lines, BxPC-3 (pancreas carcinoma) and DU145 (prostate carcinoma). Biodistribution was investigated in Balb/c nu/nu mice bearing BxPC3 xenografts at 4 h and 24 h pi.

Results/Discussion

All conjugates were successfully labeled, had high purity and stability in vitro (Fig.1). DOTA conjugate had the lowest labeling yield and the highest release of free In-111, up to 5%. All conjugates bound specifically to HER3 with low picomolar affinities, DOTAGA conjugate had the best affinity of 8±6 pM. In vitro, internalization rate of conjugates was low and the majority of cell associated activity was membrane bound. In vivo, all conjugates were rapidly cleared from blood circulation via kidneys (Fig.2). No significant differences in tumor uptake were observed, but DOTAGA conjugate had the best radioactivity retention in tumor. All conjugates showed elevated initial uptake in organs with endogenous receptor expression, especially liver. Tumor-to-organ ratios did not differ significantly between NODAGA, DOTA and DOTAGA conjugates. At 24 h pi NOTA conjugate had 2-fold lower tumor-to-blood-ratio than the other conjugates due to slower blood clearance and worth retention in tumor.

Conclusions

The choice of chelator influences the biodistribution of In-labeled anti-HER3 affibody molecules: at both time points hepatic uptake was the highest for NOTA conjugate with positively charged chelator-radionuclide complex and at 4 h pi DOTAGA conjugate carrying a negatively charged complex had 2-fold lower uptake in liver than positively charged NOTA complex. Increase of negative charge of the chelator/metal complex at C-terminus facilitated blood clearance and reduced uptake in organs with endogenous receptor expression without significant influence on tumor uptake.

References

1. Rosestedt M, Andersson KG, Mitran B , Tolmachev V, Löfblom J, Orlova A, Ståhl S. Affibody-mediated PET imaging of HER3 expression in malignant tumours. Sci Rep. 2015 Oct 19;5:15226. doi: 10.1038/srep15226.

2. Strand J, Honarvar H, Perols A, Orlova A, Selvaraju RK, Eriksson Karlstrom A, Tolmachev V. Influence of Macrocyclic Chelators on the Targeting Properties of 68Ga-Labeled Synthetic Affibody Molecules: Comparison with 111In-Labeled Counterparts. PLoS One, 2013 August 1; 8(8): e70028.

Acknowledgement

This work was supported by the Swedish Cancer Society (CAN2016-463, CAN2014-474 and CAN2015-350), the Swedish Research Council (621-2012-5236, 2015-02509 and 2015-02353),  and the ESCAPE Cancer grant from VINNOVA (2016-04060).

Fifure 1

Figure 2

7:15 PM
PS-10-6 — Efficient modification of ultra-small gold nanoparticles for fluorescence & PET imaging of prostate carcinoma (#361)

M. Pretze1, A. Hien2, M. Rädle2, N. van der Meulen3, R. Schibli3, R. Schirrmacher4, C. Wängler5, B. Wängler1

1 Medical Faculty, Molecular Imaging & Radiochemistry, Mannheim, Baden-Württemberg, Germany
2 Mannheim University of Applied Sciences, Institute of Process Control and Innovative Energy Conversion, Mannheim, Baden-Württemberg, Germany
3 PSI - Paul Scherrer Institut, Laboratory of Radiochemistry (LRC)/Center of Radiopharmaceutical Sciences (CRS), Villigen, Switzerland
4 University of Alberta, Oncologic Imaging, Department of Oncology, Edmonton, Alberta, Canada
5 Medical Faculty Mannheim of Heidelberg University, Biomedical Chemistry, Mannheim, Baden-Württemberg, Germany

Introduction

The focus of this work was on the development of bimodal contrast agents based on gold nanoparticles[1,2] which allow a custom surface-modification. By simple ligand exchange it is possible to introduce chemoselectively reacting functionalities suitable for complementary particle functionalization[3]. E.g., we introduced near-infrared (NIR) dyes for fluorescence imaging and NODAGA for radiolabeling with 68Ga and 64Cu and different targeting vectors for prostate carcinoma[5].

Methods

The AuNP were synthesized by the method of Brust and Schiffrin[4] and labeled with thiol-functionalized ligands[1]. The characterization of the AuNPs were performed by NMR, TEM, UV/Vis and TGA. To evaluate the cell uptake of the functionalized AuNPs in prostate carcinoma cells, in vitro experiments were performed using LNCaP and PC3 prostate carcinoma cells[6]. Furthermore, the AuNP were tested for their imaging properties with Albira PET system in healthy mice.

Results/Discussion

AuNPs were successfully radiolabeled with 68Ga and 64Cu. After decay, AuNPs remained stable in solution. The AuNPs were stable in PBS, rat plasma and cell media for at least 2 h at 37°C. The avidity of the tumorvector-modified particles (7) towards e.g. PSMA was determined on LNCaP-cells and found to be lower compared to monomeric ethylene glycol-Lys-urea-Glu (EG-LUG) (IC50 7 = 0.524±0.2 µg/mL (~1.87 nM), IC50 EG-LUG = 0.052±0.01 µg/mL, (~111 nM)). Further cell studies with AuNP 6 revealed a higher accumulation within 3 hours at PC3 compared to A431 cells. The cellular uptake of the dually modified particles could partly be blocked up to 65% by the respective monomeric targeting vector.

Conclusions

These initial results show that the dually modified particles show potential as bimodal imaging tools for PSMA- or GRPR-expressing cells in vitro. Further in vivo PET studies at tumor-bearing mice are planned and will show the biodistribution of the AuNP and prove a possible tumor accumulation.

References

[1] J. Zhu, C. Waengler, R. B. Lennox, R. Schirrmacher, Langmuir 2012, 28, 5508-5512

[2] P. Gobbo, M. S. Workentin, Langmuir 2012, 28, 12357-12363

[3] J. Zhu, J. Chin, C. Wängler, B. Wängler, R. B. Lennox, R. Schirrmacher, Bioconjugate Chem. 2014, 25, 1143-1150

[4] M. Brust, M. Walker, D. Bethell, D. J. Schiffrin, R. Whyman, J. Chem. Soc., Chem. Commun. 1994, 801-802

[5] M. Eder, O. Neels, M. Müller, U. Bauder-Wüst et al. Pharmaceuticals 2014, 7, 779-796

[6] Y. S. Yang, X. Z. Zhang, Z. M. Xiong, X. Y. Chen, Nucl. Med. Biol. 2006, 33, 371-380

Scheme 1
Syntheses pathways for NODAGA-modified 6, 7, 8 and fluorescent AuNPs 4.

Figure 1
Binding curves for the determination of avidity of AuNP 4 (blue line), AuNP 7 (green line) and EG-Lys-urea-Glu (red line) against [177Lu]PSMA-617.

7:25 PM
PS-10-7 — Evaluation of 18F-PI-2620 – a next generation tau PET agent for the use in Alzheimer’s disease (AD) and Progressive Supranuclear Palsy (PSP) (#368)

F. Oden1, H. Kroth2, H. Schieferstein1, M. Berndt1, A. Mueller1, F. Capotosti2, J. Molette2, J. Seibyl3, H. Schmitt-Willich1, D. Hickman2, G. Tamagnan3, A. Pfeifer2, L. Dinkelborg1, A. Muhs2, K. Marek3, A. Stephens1

1 Piramal Imaging, Berlin, Germany
2 AC Immune, Lausanne, Switzerland
3 Invicro, New Haven, United States of America

Introduction

Tau deposition is a key pathologic feature of AD and other neurodegenerative disorders. Several PET probes have been developed for in vivo detection of brain tau load. Several tau radiotracers have limitations in regard to off-target binding and tau detection in non-AD tauopathies like PSP. 18F-PI-2620 is a novel tracer with high affinity for aggregated tau. It binds specifically to tau deposits in brain sections from AD, Pick’s, and PSP patients. The first-in-human study shows its potential to visualize tau deposition in subjects with AD and PSP, compared to non-demented controls (NDCs).

Methods

Pre-clinically 18F-PI-2620 was tested for its affinity to aggregated tau using human brain homogenates, isolated paired helical filaments, and recombinant tau fibrils. Off-target binding was evaluated in competition assays with radiolabeled MAO A/B and beta-amyloid binders. Using AD, non-AD and NDC brain tissues in both, autoradiography (ARG) on human brain sections and competition experiments with brain homogenates, specific binding to tau aggregates was confirmed. The pharmacokinetic profile of the 18F-PI-2620 was assessed in mice and non-human primates. Currently, 18F-PI-2620 is clinically evaluated for its use in AD and PSP.

Results/Discussion

18F-PI-2620 displays high affinity for tau in AD brain homogenate competition-assays (IC50: 1.8 nM). Specific binding to tau deposits was further demonstrated by ARG on AD brain sections (Braak I-VI), Pick’s and PSP pathology, whereas no specific tracer binding was detected on brain slices from non-demented donors. In addition to its high affinity to tau, the compound shows excellent selectivity with no off-target binding to beta-amyloid or MAO A/B. Good brain uptake and fast wash-out was observed in healthy mice and non-human primates. Initial human imaging data show robust brain uptake and fast wash-out in non-target regions with peak SUV = 4-4.5. There was no increased uptake seen in choroid plexus, basal ganglia, striatum, amygdala, meninges or other regions noted in first generation tau agents.

Conclusions

A promising new tau PET imaging tracer with improved characteristics has been discovered with high affinity, good selectivity and excellent PK properties. Initial 18F-PI-2620 PET FIH data in AD, PSP and NDC subjects demonstrate excellent brain penetrance, favorable kinetics, and high target specificity with low nonspecific binding and high signal in regions of expected tau pathology.

7:35 PM
PS-10-8 — 68Ga-metal nanoparticle radiolabeling for PET/Cerenkov imaging and therapeutic drug loading for colorectal carcinomas and metastases (#421)

E. C. Pratt1, 2, C. L. Machado1, T. M. Shaffer1, 3, 4, K. P. O'Rourke5, 11, 10, V. Longo12, K. Ganesh8, 11, M. Marco6, 7, N. E. Kemeny8, C. Wu6, 7, J. Garcia-Aguilar7, C. L. Sawyers6, S. W. Lowe11, 9, J. J. Smith6, 7, J. Grimm1, 2, 13

1 Memorial Sloan Kettering Cancer Center, Molecular Pharmacology, New York, New York, United States of America
2 Weill Cornell Graduate School, Pharmacology, New York, New York, United States of America
3 Stanford, Radiology, California, California, United States of America
4 Hunter College, Chemistry, New York, New York, United States of America
5 Weill Cornell/Rockefeller University/Memorial Sloan Kettering Cancer Center, Tri-Institutional MD-PhD Program, New York, New York, United States of America
6 Memorial Sloan Kettering Cancer Center, Human Oncology and Pathogenesis Program, New York, New York, United States of America
7 Memorial Sloan Kettering Cancer Center, Colorectal Service, Department of Surgery, New York, New York, United States of America
8 Memorial Sloan Kettering Cancer Center, Gastrointestinal Oncology service, Department of Surgery, New York, New York, United States of America
9 Memorial Sloan Kettering Cancer Center, Howard Hughes Medial Institute, New York, New York, United States of America
10 Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute, New York, New York, United States of America
11 Memorial Sloan Kettering Cancer Center, Cancer Biology and Genetics, New York, New York, United States of America
12 Memorial Sloan Kettering Cancer Center, Radiology, New York, New York, United States of America
13 Weill Cornell Medical College, Radiology, New York, New York, United States of America

Introduction

Colorectal cancer (CRC) represents 8% of new cancer cases in the US with high metastasis incidence. Herein the metal oxide nanoparticle (MNP), was chelator-free radiolabeled with 68Ga for imaging primary CRC tumors and lung metastases based on murine CRC organoids. The 68Ga-MNP tracer was used to image CRC progression by PET and ex-vivo using Cerenkov Luminescence Imaging. The MNP can additionally be loaded with small molecule drugs. Herein we additionally report the loading of select inhibitors such as MEK and BRAF inhibitors for the localized treatment of CRC.

Methods

C57black6 mice were implanted for syngeneic orthotropic and metastatic CRC models1-2 initiated with dextran sodium sulfate (DSS) while NOD-SCID mice were used for PDX models. MNP (15-40nm) was incubated with 68Ga at 95OC, purified within 1h for IV injection using methods published previously3, and subsequently imaged 3h later by both PET and Cerenkov luminescence. Mice bearing lung metastasis were also tracked by CT for tumor burden. Ex vivo, tissues were imaged by Cerenkov luminescence or preserved in OCT for iron, immune populations and H&E staining. Drug loading of MNP was performed using a modified solvent diffusion method whereby MEK and BRAF inhibitors were loaded individually onto the MNP, assayed by HPLC and compared in in-vitro assays to stand alone monotherapy.

Results/Discussion

Primary colorectal tumors showed by PET diffuse uptake in the colon 3h p.i. with predominant uptake in liver and spleen (Fig 1A). Cerenkov luminescence ex-vivo indicate tumor uptake with reduced Cerenkov signal from normal tissue (Fig. 1B). Biodistribution of 68Ga-MNP in primary CRC tumors revealed 7.9% ID/g at 3h post injection in tumors with 12.6%, and 9.2% ID/g in the liver and spleen respectively (Fig. 1C) and confirmed in an orthogonal study with 89Zr-Ferumoxytol showing 7.7 %ID/g in the tumors at 24h. Histology shows uptake of MNP-Cy5.5 in lymph node regions beyond CD68 positive cells. 68Ga-MNP CT images identified lung metastases and confirmed presence of Cerenkov luminescence ex vivo showing increased radiance of tumors compared to normal tissues. In vitro drug administration of the MEK inhibitor loaded onto the MNP affects CRC cell viability as low as 3µM with an IC50 of ~200µM (Fig. 2). The MNP-MEK complex had a higher killing ability compared to the MEK inhibitor alone.

Conclusions

We have shown a facile method for chelator free radiolabeling this MNP, with 68Ga and report high uptake in a model of colorectal tumors. We saw appreciable tumor uptake in this colorectal model suggesting a new use of this MNP as a putative tracer for preoperative imaging by PET and CLI during operation for the confirmation of specimen removal. In addition the combination of MEK or BRAF inhibitors onto the MNP also highlights the use as a therapeutic alongside imaging. We show that loading of the MEK inhibitor onto this MNP is superior compared to co dosing of the inhibitor and MNP.

References

1. O'Rourke KP, Ackerman S, Dow LE, Lowe SW. Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells. Bio Protoc. 2016 Feb 20;6(4).

2. O'Rourke KP, Lowe SW. Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer. Nature Biotechnology 35, 577–582 (2017)

3. Boros E, Bowen AM, Josephson L, Vasdev N, Holland JP. Chelate-free metal ion binding and heat-induced radiolabeling of iron oxide nanoparticles. Chem Sci 2015, 6(1): 225-236.

Acknowledgement

We gratefully acknowledge funding from the National Institutes of Health (NIH) R01EB014944, R01CA183953, and R01CA215700 and National Science Foundation Integrative Graduate Education and Research Traineeship Grant (NSF) IGERT 0965983

68Ga labeled metal nanoparticle can visualize murine colorectal tumors
A) Colonoscopy of a mouse implanted with AKP organoids 10 weeks prior showing a well-defined tumor in the distal rectal region. B) Cerenkov luminescence showing photograph of colorectal tumor in A (red arrow) and Cerenkov image of 68Ga MNP 3h post injection. C) biodistribution of healthy mice and mice with similar colorectal tumor burden (n=3 per group, SD shown) 3hr post injection of 68Ga-MNP.

Improved therapeutic response with MEK loaded metal nanoparticle
Metal nanoparticle (MNP) alone did not produce any marked change in viability. Addition of MEK inhibitor alone reduced viability with an IC50 of 766µM. Co-dosing MNP with MEK inhibitor yielded no therapeutic benefit, yet MEK inhibitor loaded MNP yielded an increased IC50 of nearly 200µM.

4:00 PM
emptyVal-1 — Introductory Talk by Eric Ahrens - La Jolla, USA

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

4:20 PM
PS-19-2 — General in situ activation of [11C]CH3I to form 11C–C bonds in Negishi coupling reaction – proof of concept (#158)

L. Rejc1, N. Alonso2, V. Gómez-Vallejo1, J. Alcazar2, F. P. Cossio3, J. I. Andres2, J. Llop1

1 CIC biomaGUNE, Radichemistry and Nuclear Imaging, San Sebastian, Gipuzkoa, Spain
2 Janssen Research & Development, Lead Discovery Chemistry, Toledo, Spain
3 University of Basque Country, Department of Organic Chemistry I, San Sebastian, Gipuzkoa, Spain

Introduction

The most common method for labelling with carbon-11 (11C), the use of [11C]CH3I, is limited to the production of [11C]methoxides, [11C]methylamines, and [11C]methylthioesters. The development of metal mediated reactions is expanding 11C-labelling options,1 however, the main limitation remains the need for specific precursors, which are challenging to prepare, require careful handling, and are often toxic. To avoid these limitations, we designed a general and efficient one-pot Negishi reaction via in situ formation of [11C]CH3ZnI to produce a variety of [11C]methylaryls and [11C]thymidine.

Methods

A series of aryl halides, obtained from Sigma Aldrich, was tested to investigate the generality of one-pot two-step Negishi reaction. [11C]CH3I was produced by iodination of cyclotron-produced [11C]CH4 using a recirculation system. [11C]CH3I was distilled into a zinc-filled cartridge,2 preloaded with a solution of an aryl halide and a mediator tetrakistriphenylphosphine palladium (0). The success of the reaction was evaluated by radio-HPLC analysis of the reaction mixture immediately after reaction. In case of [11C]thymidine, commercially available 5-iodo-2-deoxyuridine was used as the substrate and the crude reaction mixture was submitted to purification by semi-preparative HPLC, to produce pure [11C]thymidine, appropriate for use in animal studies.

Results/Discussion

Close-to-quantitative (>90%) trapping of [11C]CH3I in the zinc cartridge was achieved. [11C]CH3I underwent fast and quantitative reaction with zinc to form [11C]CH3ZnI. The latter was indirectly identified by the detection of [11C]CH4 in the radio-HPLC chromatogram and reaction kinetics studies. Subsequent Negishi coupling reactions in the presence of transition metal mediator were successful with a variety of aryl halides and 5-iodo-2-deoxyuridine (Figure 1, Table 1). The electron donating and electron withdrawing effects of aryl substituents narrate the reaction rate according to their reactivity;3 hence higher temperatures or the use of different metal complexes might be required in specific cases to achieve close-to-quantitative conversion of [11C]CH3I into the product

Conclusions

[11C]CH3I can efficiently be activated with zinc in situ followed by a fast Negishi coupling reaction with a wide variety of substrates. As a proof of concept, unprecedented synthesis of [11C]thymidine was chosen. Successful radiolabelling and isolation of this proliferation biomarker candidate proves the potential of this easy, general, and fast method to become an alternative for the production of new 11C-labelled imaging probes and further expand the possibilities for evolution of PET imaging.

References

  1. a) S. Kealey, J. Passchier, M. Huiban, Chem. Commun. 2013, 49, 11326-11328; b) K. Dahl, C. Halldin, M. Schou, Clinical and Translational Imaging 2017, 5, 275-289.
  2. N. Alonso, L. Z. Miller, J. M. Muñoz, J. Alcazar, D. T. McQuade, Adv. Synth. Catal. 2014, 356, 3737-3741.
  3. Z.-B. Dong, G. Manolikakes, L. Shi, P. Knochel, H. Mayr, Chem. Eur. J. 2010, 16, 248–253

Figure 1
Reaction for [11C]methylaryl synthesis using Negishi cross-coupling reaction, via in situ formation of 11CH3ZnI.

Table 1

Conversion values obtained for different aryl halides and triflates and 5-iodo-3-deoxyuridine. In all cases, T= 70°C, t=5 min; conversion calculated as the ratio between the area of the peak corresponding to 11C-labelled compound and the sum of the areas of all the peaks in the radiochromatogram.

4:30 PM
PS-19-3 — A new generic platform for PET imaging based on 68Ga-labeled Pept-insTM: from infectious disease to cancer imaging (#166)

M. Siemons1, 2, L. Khodaparast2, L. Khodaparast2, J. Lecina1, F. Claes2, R. Gallardo2, M. Ramakers2, F. Rousseau2, J. Schymkowitz2, G. Bormans1

1 Laboratory for Radiopharmaceutical Research, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium
2 Switch Laboratory, VIB‐KU Leuven Center for Brain & Disease Research, Leuven, Belgium

Introduction

The Pept-in technology is based on a highly specific beta-sheet aggregation of a target protein, induced by short amyloidogenic peptides called Pept-ins (7-20 AA). Pept-In sequences are derived from aggregation-prone regions present in the target protein. They provide a generic platform for diagnostics similar to antibodies and derivatives. However, the rapid production by solid-phase peptide synthesis results in lower cost for production and a higher production throughput with lower batch-to-batch variation compared to antibodies and derivatives.

Methods

Gallardo et al. (2016, Science) designed vascin which inhibits VEGFR2 function by inducing aggregation of this receptor with high specificity. Furthermore, Khodaparast et al. (unpublished data) showed that Colpeptin1 induced proteostatic collapse in E.coli leading to bacterial cell death. Vascin and Colpeptin1 were modified for 68Ga-labeling by synthesis of [68Ga]Ga-NODAGA-PEG4-vascin and [68Ga]Ga-NODAGA-PEG2-Colpeptin1. Since proline is a known beta-sheet structure breaker, proline-mutated variants were synthesized as controls to obtain [68Ga]Ga-NODAGA-PEG4-vascin(Pro) and [68Ga]Ga-NODAGA-PEG2-Colpeptin1(Pro). Dynamic µPET imaging and biodistribution studies were performed in a mouse melanoma tumor model for vascin and an E. coli muscle infection model for radiolabeled Colpeptin1.

Results/Discussion

Radiolabeled vascin and Colpeptin1 provided good target-to-background contrast in PET images with high specificity, since for vascin tumor-to-muscle SUV ratios (T/M SUVR) were significantly higher than for its proline-mutated variant (Fig. 1a) and the same observation was made for Colpeptin1 muscle-to-muscle SUV ratios (muscle SUVR) compared to its proline-mutated variant (Fig. 1b). The possibility of the aspecific EPR effect being responsible for in vivo accumulation through self-aggregates, was countered by injection of radiolabeled Colpeptin1 into the melanoma tumor model (Fig. 1a), confirming that the presence of the target protein is required for retention of the radiolabeled Pept-in. Furthermore, radiolabeled Colpeptin1 did not show accumulation at sites of LPS-induced sterile inflammation nor in inactive E. coli infected muscle. Biodistribution studies proved favorable pharmacokinetic properties for both radiolabeled vascin and Colpeptin1.

Conclusions

The excellent in vivo specificity of Pept-ins observed in two totally different disease models, i.e. cancer and infectious disease, highlights the immense potential of a platform based on this technology for diagnostic and therapeutic applications.

Specific in vivo accumulation of 68Ga-labeled Pept-ins at their target site

(a) PET image in a mouse melanoma tumor model. PET T/M SUVR bar plots of radiolabeled vascin (n= 8), vascin(Pro) (n= 5) and Colpeptin1 (n= 4). (b) PET image in a foreleg muscle infection model. PET muscle SUVR of radiolabeled Colpeptin1 (n= 5) and Colpeptin1(Pro) (n= 6). Data are expressed as mean ± SD. Statistical significance was calculated by using one-way ANOVA or unpaired student’s t-test.

4:40 PM
PS-19-4 — Small CaF2 nanocrystals as nano-sized tracers for in vivo 19F-MRI (#179)

I. Ashur1, H. Allouche-Arnon1, A. Bar-Shir1

1 The Weizmann Institute of Science, Department of Organic Chemistry, Rehovot, Israel

Introduction

Fluorine-19 MRI agents are widely used as tracers for cellular imaging. In recent years, perfluorocarbon (PFC) nanoemulsions have been used successfully as 19F-tracers in various applications1-4 for preclinical studies and clinical practices. However, PFCs lack the merits of inorganic nanocrystals (small size, dense material, high crystallinity, surface modifiability, etc.). Here we show that small (8 nm) water-soluble CaFnanoparticles (NPs) allow to obtain high-resolution 19F-NMR signals. This makes them suitable as 19F-MRI tracers and allows a "hot-spot" display. 

Methods

Synthesis and characterization: PEGylated CaF2 nanofluorides (CaF2-PEG, Fig. 1) were synthesized using a solvothermal method. The PEGylated NPs were fluorescently labeled by FITC and SCY3-based fluorophores and fully characterized.

19F-NMR and 19F-MRI: High-resolution 19F-NMR spectra collected with a 9.4 T NMR spectrometer. T1 and T2 relaxation times of the NPs 19F were determined. In vitro and in vivo MRI performed on vertical 9.4 T MRI scanner. 1H-MRI: A FLASH sequence with TR/TE=360/4 ms. 19F-MRI: 3D ultrashort TE protocol  (3D-UTE) with a flip angle of 10o, TR/TE=150/0.02 ms, FOV=3.2×3.2×3.2 cm3, matrix=32×32×32, and NA=8. Mice were immunized by footpad injection of immunogenic emulsion. MRI data were collected 10 days post immunization and after NPs injection to the footpad.

Results/Discussion

Figure 1 shows that PEGylated CaF2 NPs (Fig. 1a) are crystalline and small (Fig. 1b) with overtime colloidal stability (Fig. 1c). Figure 1d shows that small CaF2 NPs could be detected with high-resolution 19F-NMR. The ability to monitor the NPs with 19F-MRI was demonstrated on a phantom (Fig. 1e). A clear 19F-MR signal of NPs was acquired by using UTE sequence, overcoming their relative short T2 and allowing a “hot-spot” display. The performance of our newly proposed NPs as imaging tracers for in vivo 19F-MRI was tested in mice model of inflammation. The immunized mice were subjected to 1H- and 19F-MRI that acquired pre- and post- injection of fluorescently labeled PEGylated CaF2 NPs that preserved their 19F-NMR properties (Fig 2a). By using 19F-UTE-MRI, a clear 19F-signal was observed at the popliteal lymph node ROI (LN, Fig 2b-c, 2 repsentative mice). FACS analysis of excised cells from the LN revealed that the majority of the NPs were accumulated in macrophages and dendritic cells.

Conclusions

We demonstrated, for the first time, that small fluoride-based nanocrystals (specifically, CaF2 NPs) freely tumbling in solution can be studied while in solution with high-resolution 19F-NMR and be used as nano-tracers for 19F-MRI. The proposed nanocrystals elucidate a novel type of 19F-tracers that combine the advantages of using nanocrystals (small, high 19F-equivalency, maximal 19F-density, and surface modifiability) with the merits of 19F-MRI tracers.

References

  1. Ahrens, E.T., Flores, R., Xu, H. & Morel, P.A. In vivo imaging platform for tracking immunotherapeutic cells. Nat Biotechnol 23, 983-987 (2005).
  2. Flogel, U. et al. In vivo monitoring of inflammation after cardiac and cerebral ischemia by fluorine magnetic resonance imaging. Circulation 118, 140-148 (2008).
  3. Janjic, J.M., Srinivas, M., Kadayakkara, D.K. & Ahrens, E.T. Self-delivering nanoemulsions for dual fluorine-19 MRI and fluorescence detection. J Am Chem Soc 130, 2832-2841 (2008).
  4. Ruiz-Cabello, J. et al. In vivo "hot spot" MR imaging of neural stem cells using fluorinated nanoparticles. Magn Reson Med 60, 1506-1511 (2008).
  5. Ahrens, E.T., Helfer, B.M., O'Hanlon, C.F. & Schirda, C. Clinical cell therapy imaging using a perfluorocarbon tracer and fluorine-19 MRI. Magn Reson Med 72, 1696-1701 (2014).

Water soluble PEGylated CaF2 NPs.

a, Schematics of PEGylated CaF2 NPs. b, TEM images of the NPs. c, DLS of the NPs after purification (red) and 40 days later (blue). d, High-resolution 19F-NMR of the NPs. e, MRI of phantom containing solutions with or without the NPs in two concentrations (1H-MRI, top panel). Middle panel: 19F-MRI acquired by 3D-UTE sequence (TR/TE=150/0.02 ms). Bottom panel: “hot spot” display of the 19F-data.

In vivo imaging of fluorescently-labeled PEGylated CaF2 NPs in a model of inflammation.
a, Fluorescently labeled PEGylated NPs, their fluorescent properties, and high-resolution 19F-NMR. b, Anatomical 1H-MRI of studied mice (left); matched 19F-MRI (middle) and pseudo-color maps of 19F-MRI data on the 1H-MRI (right). c, FACS analysis for specific cell populations (NPs in red, and PBS in black). 
 

4:50 PM
PS-19-5 — A Design of Experiments (DoE) Approach Towards the Optimization of Copper-Mediated Radiofluorination Reactions of Arylstannanes. (#227)

G. D. Bowden1, A. Maurer1, B. J. Pichler1

1 Eberhard Karls University of Tübingen, Werner Siemens Imaging Center, Tübingen, Baden-Württemberg, Germany

Introduction

Copper-mediated radiofluorination reactions (CMRFs) are multicomponent reactions that provide access to previously hard to synthesize tracers.1 They involve many factors that affect radiochemical conversion (RCC), which makes reaction optimization difficult and time-consuming. Unlike the traditional “One Variable at a Time” method, DoE is a statistical approach to reaction optimization that models and maps reaction space across multiple factors simultaneously.2 This study aimed to determine the usefulness of DoE to radiochemical reaction development and optimization using a model CMRF. (Fig 1)

Methods

A fractional factorial (Res V+) design was constructed using MODDE Go to model the effects of temperature, solvent volume, catalyst loading, ligand loading, and atmosphere on RCC. 24 runs were performed in 4 blocks of 6, which were included as blocking factors to account for variances in 18F processing. [18F]Fluoride was trapped on QMA resin and eluted with 50 µg K2CO3, 10 mg KOTf in 550 µl H2O.1 Aliquots (250-300 MBq) were transferred to 6 vials and dried azeotropically with acetonitrile under a stream of argon. Pre-made reaction mixtures with BiPhSnBu3 (4,5 nmol) were then added and left to stir under the required atmospheric and temperature conditions for 10 min. Reactions were quenched with 1 ml of H2O. RCCs were assessed using radioTLC and product identity was confirmed using HPLC.

Results/Discussion

The obtained RCCs were fitted to a model in MODDE Go using multiple linear regression (MLR). The summary of fit statistics (R2 = 0,88 (observed % variance), Q2 = 0,65 (predicted % variance)) indicated a valid model. The factor screening study indicated that temperature and solvent volume did not have a significant effect on RCC, while catalyst and ligand loading were significant factors. Interestingly, argon atmospheres had a slightly positive yet non-significant influence on RCC, a result contrary to the literature-published protocols which perform these reactions under air. There were no significant differences between experimental blocks. These results were used to design a more detailed Response Surface Optimization (RSO) study for a new tracer currently in development. RSO modelling of the results of this study (17 runs) showed a quadratic function for catalyst and ligand loading factors as well as an interaction between ligand loading and substrate concentration. (Fig 2)

Conclusions

DoE is a powerful approach to radiochemical reaction optimization that has the potential to reveal new insights into the behaviour of new 18F chemistry. It was used to show which experimental factors were most important to a model CMRF. It was also used to optimize and predict the experimental factors that will aid in the development of an improved synthesis for a novel tracer currently under development. Further work is currently being done to apply this approach to a number of novel tracers that have up until now suffered from low yields and poor synthesis performance.

References

1.        Makaravage, K. J., Brooks, A. F., Mossine, A. V., Sanford, M. S. & Scott, P. J. H. H. Copper-Mediated Radiofluorination of Arylstannanes with [18F]KF. Org. Lett. 18, 5440–5443 (2016).

2.        Murray, P. M. et al. The application of design of experiments (DoE) reaction optimisation and solvent selection in the development of new synthetic chemistry. Org. Biomol. Chem. 14, 2373–2384 (2016).

Figure 1:
The general reaction scheme explored by DoE. The factors below the arrow were investigated in the initial factor screening study: Catalyst loading, 2) Ligand loading, 3) Solvent volume, 4) Temperature and 5) Atmosphere. The range of the investigated factors are displayed in brackets.

Figure 2:
The reaction response surface generated by the Response Surface Optimization study revealed a quadratic relationship between catalyst and ligand loading. From this model, optimal reaction conditions can be estimated.

5:00 PM
PS-19-6 — Bioengineered bacterial encapsulins for multiscale in vivo molecular imaging (#295)

F. Sigmund1, 2, C. Massner1, 2, 4, P. Erdmann6, A. Stelzl1, 2, H. Rolbieski1, 2, H. Fuchs3, M. Hrabe de Angelis3, M. Desai7, S. Bricault7, A. Jasanoff7, V. Ntziachristos1, 5, J. Plitzko6, G. G. Westmeyer1, 2, 4

1 Helmholtz Zentrum München, Institute of Biological and Medical Imaging, Neuherberg, Bavaria, Germany
2 Helmholtz Zentrum München, Institute of Developmental Genetics, Neuherberg, Bavaria, Germany
3 Helmholtz Zentrum München, Institute of Experimental Genetics, Neuherberg, Bavaria, Germany
4 Technical University of Munich, Department of Nuclear Medicine, Munich, Bavaria, Germany
5 Technical University of Munich, Chair for Biological Imaging, Munich, Bavaria, Germany
6 Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Martinsried, Bavaria, Germany
7 Massachusetts Institute of Technology, Department of Biological Engineering, Camebridge, Massachusetts, United States of America

Introduction

Compartmentalization, the spatial separation of processes into closed subspaces, is an important biological principle that enables the maintenance of specific local conditions which facilitate reactions and interactions in confined environments [1]. Whereas eukaryotic organisms usually employ membrane-enclosed organelles to achieve compartmentalization, bacteria have evolved proteinaceous nanocompartments that can spatially confine chemical reactions and sequester metals. Herein, we express bacterial encapsulins in mammalian systems for multimodal molecular imaging across different scales.

Methods

We expressed the encapsulin shell protein of Myxococcus xanthus [2], its native cargo proteins and a set of engineered cargo molecules in HEK293T cells and murine brains. We characterized their expression, self-assembly and cargo loading of the shell using co-Immunoprecipitation (Co-IP), Blue-Native-PAGE (BN-PAGE), and gel-based staining methods as well as fluorescence and bioluminescence imaging. Furthermore, we imaged HEK293T cells expressing melanin-producing encapsulins using multispectral optoacoustic tomography (MSOT). We conducted relaxometry measurements of cells expressing iron-loaded encapsulins and in vivo MR imaging in rat brains of xenografted cells at 7 Tesla. Finally, we imaged encapsulins in HEK293 cells using cryo-electron tomography (cryo-ET).

Results/Discussion

We demonstrate that eukaryotically expressed encapsulins not only auto-assemble into 32 nm large nanospheres at high density and without toxic effects but that self-targeting and encapsulation of cargo molecules still efficiently occur in mammalian cells (Figure 1 a). We furthermore show localized enzymatic reactions inside the nanocompartment useful for optical and optoacoustic imaging (Figure 1 b), as well as confined iron biomineralization that labels cells for detection by MRI in vivo in mouse brains (Figure 1 c) and electron microscopy (EM) (Figure 1 d), demonstrating the potential of encapsulins as genetic markers across modalities. In addition, the iron-sequestration inside the nanoshells affords magnetic manipulation of cells genetically labelled with encapsulins (Figure 1 e).

Conclusions

In conclusion, we have introduced encapsulins as a versatile method to genetically engineer orthogonal compartments in mammalian cells that can self-convert into nanomaterials with attractive properties. These features offer applications in molecular imaging across multiple scales from subcellular resolution by EM, via fluorescence to non-invasive optoacoustic imaging and in vivo MRI. Genetically controlled encapsulation of multi-component processes in eukaryotic cells is a fundamental biotechnological capability with important implications for mammalian cell engineering and cellular therapy.

References

[1] DeLoache, W. C. & Dueber, J. E. Compartmentalizing metabolic pathways in organelles. Nat. Biotechnol. 31, 320–321 (2013).

[2] McHugh, C. A. et al. A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. EMBO J. 33,​ 1896–1911 (2014).

Acknowledgement

We are grateful for support from the European Research Council under grant agreements ERC-StG: 311552 (F.S., A.S., H.R., G.G.W.).

Figure 1 a-b
(a) Encapsulins express and auto-assemble in mammalian cells as shown by BN-PAGE and electron microscopy. Cargo targeting was confirmed via Co-IP and subsequent silver-stained SDS-PAGE. Encapsulin over-expression did not cause reduced cell viability. (b) Targeting of engineered cargo enzymes as shown on BN-PAGE. Targeting of tyrosinase yields nanomelanosomes that can be dected via optoacoustics. 

Figure 1 c-e
(c) Encapsulins can effectively load iron inside mammalian cells as shown by Prussian-Blue staining on BN-PAGE and can be detected via MRI after xenografting into rat brains. (d) Encapsulins can serve as markers for cryo electron tomography (cryoET). (e) Cells expressing iron-loaded encapsulins can be magnetically sorted. 

5:10 PM
PS-19-7 — Using Magnetic Relaxation for Color Magnetic Particle Imaging: First In Vivo Experimental Results (#410)

D. Hensley1, 2, Z. W. Tay1, X. Y. Zhou1, P. Chandrasekharan1, B. Zheng1, P. W. Goodwill2, S. M. Conolly1, 3

1 University of California, Berkeley, Bioengineering, Berkeley, California, United States of America
2 Magnetic Insight, Inc., Alameda, California, United States of America
3 University of California, Berkeley, Electrical Engineering and Computer Sciences, Berkeley, California, United States of America

Introduction

Reporting physiologic contrast is of great interest to magnetic particle imaging (MPI) [1, 2], especially as the field explores molecular imaging applications. One avenue to such contrast is through the rich dynamic magnetic physics of the tracers used in MPI [3]. Signal is generated in MPI when magnetic tracers rotate in response to movement of a sensitive magnetic field region. The non-instantaneous response of the tracers is termed magnetic relaxation. A canonical way of reporting such information is via color images [4,5]. Here we show the first in vivo color MPI results.

Methods

0.229 mg of Chemicell tracer was administered to a 0.2 kg female Fisher rat via tail vein injection. This was followed by an injection of 875,000 (0.08 mg Fe) macroaggregated albumin and MPI tracer conjugates [6] using the Perimag MPI tracer (MAA-perimag). Two reference markers (2 uL each) were placed above the rat’s chest. The rat was then scanned at four excitation amplitudes of 10, 15, 17.5, and 20 mT using our in house MPI scanner [7] (4 x 3.75 x 10 cm FOV with respiratory gating). X-ray imaging was performed with a Kubtec Xpert 40. The Chemicell tracer clears to the liver while the MAA-perimag gets stuck in the lungs, allowing us to test our color algorithm’s ability to disambiguate the two organs. Fig. 1 describes the color MPI algorithm we applied to the data.

Results/Discussion

Fig. 1 (f) shows imaging results and ROI quantification of our color algorithm applied to a known vial phantom, demonstrating that our algorithm is able to identify, disambiguate, and quantify co-localized tracers. Fig. 2 is the first demonstration of color MPI in vivo with Fig. 2 (a) showing MPI coronal slices, including a standard x-space MPI image, separate MAA-perimag (Tracer 1) and Chemicell (Tracer 2) images provides by the color algorithm, and combined colorized images overlaid with an X-ray reference. Fig. 2 (b—e) show different slices of the 3D colorized data set, further demonstrating the ability of the algorithm to parse the lungs and liver, each tagged with a different tracer. We see that the color MPI algorithm successfully parses the MAA-perimag tracer in the lungs from the Chemicell tracer in the liver, converting a standard MPI image with no discernible distinction between the lungs and liver into a colorized image with clear distinction between these organs.

Conclusions

We have reported the first in vivo color MPI results. These and other recent results in the field are beginning to paint a clear picture of how the rich relaxation physics of MPI tracers can enable new molecular imaging applications. More fully exploiting these physics will require improvements in areas such as tailored tracer design, how we encode relaxation information, and how we formulate reconstruction algorithms. Longer term, these approaches may enable applications such as monitoring of cell metabolism, targeted binding contrast, and imaging local micro-environmental conditions.

References

[1]       B. Gleich and J. Weizenecker. Tomographic imaging using the nonlinear response of magnetic particles. Nature, 435(7046):1214-1217, 2005. doi: 10.1038/nature03808.

[2]       P.W. Goodwill and S. M. Conolly. The X-space formulation of the magnetic particle imaging process: 1-D signal, resolution, bandwidth, SNR, SAR, and magnetostimulation. IEEE transactions on medical imaging, 29.11: 1851–1859, 2010.

[3]       L. R. Croft, et al. Low drive field amplitude for improved image resolution in magnetic particle imaging. Medical physics, 43.1: 424–435, 2016.

[4]       J. Rahmer, A. Halkola, B. Gleich, I. Schmale, and J. Borgert. First experimental evidence of the feasibility of multi-color magnetic particle imaging. Physics in medicine and biology, 60(5), 1775, 2015.

[5]       D. Hensley, P. Goodwill, L. Croft, and S. Conolly. Preliminary experimental x-space color MPI. Magnetic particle imaging (IWMPI), 2015 5th international workshop on, 2015.

[6]       X.Y. Zhou, et al. First in vivo magnetic particle imaging of lung perfusion in rats. Physics in Medicine and Biology, 62(9), 3510, 2017.

[7]       P. Goodwill, K. Lu, B. Zheng, and S. Conolly An x-space magnetic particle imaging scanner. Review of Scientific Instruments, 83(3), 033708, 2012.

Acknowledgement

We would like to acknowledge funding support from NIH 5R01EB019458-03, NIH 5R24MH106053-03, UC Discovery Grant 29623, W. M. Keck Foundation Grant 009323, and NSF GRFP.

Color Magnetic Particle Imaging Approach

(a) Multiple MPI scans are taken, each with a different excitation amplitude. (b,c) Tracers exhibit different image domain behavior at different excitation amplitudes per their relaxation properties. (d,e) Colorized images are produced by solving a pixel-wise inverse problem. (f) Initial vial phantom data and ROI quantification to test performance, especially unmixing of co-localized tracer.

First In Vivo Color MPI Results

(a) Standard and Color MPI coronal slice images. Separate MAA-perimag (Tracer 1) and Chemicell (Tracer 2) images obtained from the color algorithm are shown along with a combined image overlaid with X-ray reference. (b—e) Color images from different slices of the 3D dataset further demonstrating the separation of the lungs from the liver, which is not possible in the standard MPI images.

5:20 PM
PS-19-8 — Chemical design of innovative LnIII-chelates for T1 and CEST MRI applications (#481)

L. Tei2, L. Leone2, G. Ferrauto1, 4, D. Delli Castelli1, 4, Z. Baranyai3, M. Botta2

1 Università di Torino, Department of Molecular Biotechnology for Health Sciences, Torino, Italy
2 Università del Piemonte Orientale, Dipartimento di Scienze e Innovazione Tecnologica, Alessandria, Italy
3 University of Debrecen, Department of Inorganic and Analytical Chemistry, Debrecen, Hungary
4 University of Torino, Molecular Imaging Center, Torino, Italy

Introduction

Ln(HPDO3A) complexes (Fig 1A) have been useful MRI diagnostic tools for several years: the Gd-complex is the MRI agent ProHance, whereas Yb(HPDO3A) has been used as paraCEST agent for cell labelling and for pH and temperature mapping.[1] The presence of different exchanging isomers in solution (square, SAP, and twisted square antiprismatic, TSAP) explains the relaxometric and CEST behaviour of the Gd and Yb complexes, respectively.[2] The two coordination isomers of Yb(HPDO3A) allow a ratiometric method for pH mapping but reduce the signal achievable thus hampering its use for cell labelling.

Methods

HPDO3A was modified with the aim to increase the population of the fast (water) exchanging coordination isomer (TSAP). Thus, we designed a new polyaminocarboxylic macrocyclic ligand where the three acetic pendant arms of HPDO3A were substituted by three methyl acetic arms (HPDO3MA, Fig 1A). A complete 1H and 17O NMR relaxometric study on the GdIII complex, a detailed CEST characterization and a dissociation kinetic study on the YbIII complex were carried out. Finally, cell labelling experiments with the YbIII complex were performed and compared to Yb(HPDO3A) to show the better performances of the novel CEST agent.

Results/Discussion

The synthesis started by the preparation of the tris methyl acetic substituted macrocycle followed by the insertion of the hydroxypropyl pendant arm by reaction with 2-(R)-propylene oxide. 1H HR-NMR analyses revealed the presence of different isomeric species in solution for the EuIII complex whereas the YbIII complex showed only the TSAP isomer. Thus, Yb(HPDO3MA) shows only one CEST peak at 126 ppm with ST of 17.2%, corresponding to the sum of the ST effects of Yb(HPDO3A) SAP and TSAP isomers (Fig 1B). This allowed the labelling of TS/A cells with a CEST effect of 15%, almost double than that obtained for Yb(HPDO3A). Moreover, kinetic studies on Yb(HPDO3MA) showed that its kinetic inertness is about twice as high that of Yb(HPDO3A) (t1/2 = 2.2×107 h vs 1.2×107 h). Finally, the relaxivity of Gd(HPDO3MA) at 20 MHZ and 298 K is 5.1 mM-1 s-1 and analysis of NMRD profiles and 17O NMR data showed the occurrence of a faster water exchange rate than for Gd(HPDO3A) (kex = 3.3×107 s-1).

Conclusions

We have designed and characterized a new macrocyclic ligand able to form LnIII complexes with improved performance than LnHPDO3A. In case of the Gd-complex, a faster water exchange rate was determined, whereas for Yb(HPDO3MA) the presence of only one isomer in solution allowed to double the CEST effect in cell labelling experiments. Finally, the kinetic inertness of the latter complex, higher than Yb(HPDO3A), is promising for further application in molecular imaging protocols.

References

1. Magn. Reson. Med. 2013, 69, 1703; Angew. Chem., Int. Ed. 2011, 50, 1798; Magn. Reson. Med. 2014, 71, 326.

2. Inorg. Chem. 2013, 52, 7130

Figure 1

A: chemical structures of Ln(HPDO3A) and Ln(HPDO3MA); B and C: Z- and ST%-spectra of Yb(HPDO3A) and Yb(HPDO3MA)

1:30 PM
emptyVal-1 — Introductory Talk Emmet Mc Cormack - Bergen, Norway

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

1:50 PM
PS-02-2 — Preclinical Evaluation of a Novel Radiolabeled Molecule (FPy-Gal) as an Emerging Tumor Senescence PET Tracer (#91)

B. Zhou1, J. Cotton1, K. Wolter2, A. Kuehn1, K. Fuchs1, A. Maurer1, L. Zender2, M. A. Krueger1, B. J. Pichler1

1 Eberhard Karls University of Tübingen, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Baden-Württemberg, Germany
2 University Hospital Tübingen, Department of Internal Medicine VIII, Tübingen, Baden-Württemberg, Germany

Introduction

Cellular senescence is a process of stable cell-cycle arrest that has an outstanding impact on tumor suppression. However, technologies for senescence detection are limited to ex vivo methods.
We have designed and synthesized a novel PET tracer, which has the potential to be used as a non-invasive in vivo imaging tool for both basic research and clinical routine. In addition to our preliminary work with a therapy induced senescence model, we report here on the further evaluation of the specificity of our tracer in advanced preclinical tumor models, representing different types of senescence.

Methods

In vitro, senescence was induced in a HRas driven liver progenitor carcinoma cell line by p53-reactivation and in a liver carcinoma cell line by ribosomal checkpoint inhibition. After 40 min tracer incubation, cells were washed and the activity retained in the cells was measured with a gamma counter.
For in vivo studies, two murine tumor models were employed. After tumor progression, senescence was induced in subcutaneous HRas driven liver tumors by p53-reactivation and in subcutaneous liver tumors by ribosomal checkpoint inhibition. Sequential PET/MRI was performed with our tracer and the uptake in the tumors was quantified.
Tumors were dissected and cryo sectioned for autoradiography, H&E staining and X-gal assay.

Results/Discussion

In vitro, tracer uptake increased 1.6 fold for HRas cells and 3.7 fold for liver carcinoma cells, upon senescence induction.
In both animal models, the tracer uptake was significantly increased in the senescent tumors compare to control tumors. 2.0 ± 0.5 (n = 7) v.s. 1.0 ± 0.3 (n = 6) ID%/cc in the HRas model (P = 0.0023) and 1.1 ± 0.4 (n = 12) v.s. 0.6 ± 0.2 (n = 10) ID%/cc in the subcutaneous liver carcinoma model (P = 0.0240). ß-galactosidase activity staining and autoradiography further supported the tracer performance in vivo.

Conclusions

Our preclinical in vitro and in vivo studies showed clearly increased uptake of our novel PET tracer in senescent cells and tumors. Therefore our tracer could be of great value for diagnosis and therapy monitoring of cancer patients before and during senescence inducing therapies. Especially with the development and advent of senolytic compounds, our tracer has a remarkable potential for clinical use. The compound is currently in GMP-grade production and toxicity studies are performed. First-in-man studies are currently under preparation.

2:00 PM
PS-02-3 — Activated leukocyte cell adhesion molecule (ALCAM) as a potential MRI biomarker for detection of brain micrometastases (#117)

N. Zarghami1, M. Sarmiento Soto1, F. Perez-Balderas1, A. A. Khrapitchev1, N. R. Sibson1

1 University of Oxford, Oncology, Oxford, United Kingdom

Introduction

The incidence of brain metastasis from primary cancers such as breast, lung and melanoma is increasing. Clinically, diagnosis and treatment of these tumours in the early stages are challenging since the intact blood-brain barrier (BBB) limits access for both imaging contrast agents and systemic therapies. The aims of this study are: (1) to evaluate activated leukocyte cell adhesion molecule (ALCAM) as a potential target for brain micrometastasis detection; and (2) to develop a new MRI contrast agent based on microparticles of iron oxide (MPIO) and anti-ALCAM antibodies.

Methods

Human and mouse brain metastasis samples (breast, lung and melanoma primaries) were stained for ALCAM and an endothelial cell marker. Antibodies against ALCAM were conjugated to MPIO (ALCAM-MPIO) and antibody loading assesed using flow cytometry. ALCAM-MPIO binding to ALCAM was verified on mouse endothelial cells treated with TNF-a, and also under flow conditions in capillaries coated with mouse recombinant ALCAM protein. Mice (SCID, n=12) were injected intracardially with human brain tropic breast cancer (MDA-MB-231-Br) or melanoma (H1-DL2). On imaging day, mice were injected with ALCAM-MPIO and scanned using a T2*-weighted multi-gradient echo sequence. Pre- and post-gadolinium T1-weighted images were also acquired. Mice were perfused and brains taken for immunohistochemistry.

Results/Discussion

Immunohistochemistry of both human and mouse brain metastasis tissue from all origins showed presence of ALCAM within the tumour microenvironment and on associated vessels. No ALCAM was detected on healthy brain samples. These results suggest that ALCAM may be a suitable biomarker for detection of brain micrometastases, when the BBB is intact. Successful coupling of the ALCAM-MPIO conjugate was confirmed (22000 Ab/MPIO). ALCAM-MPIO showed significantly higher binding to pre-activated endothelial cells and capillaries (t-test, P < 0.001) compared to MPIO conjugated to an equivalent, but non-specific IgG antibody (Figure 1). In vivo MRI results for both tumour models showed focal hypointensities on T2*-weighted images indicating presence of ALCAM-MPIO. No gadolinium enhancement was detected at these sites. Co-localisation of MRI hypointense signals with micrometastases and bound ALCAM-MPIO was verified histologically (Figure 2).

Conclusions

The results of this study suggest that ALCAM may be a potential target for early detection of brain micrometastases. We have demonstrated ALCAM-specific binding of a new molecular MRI contrast agent (ALCAM-MPIO) both in vitro and in vivo, in two different models of brain metastasis. These studies suggest that ALCAM-MPIO in combination with MRI may provide a new approach for the early detection of brain metastases, prior to blood-brain barrier breakdown, and could substantially improve diganosis for patients at risk of secondary progression to the brain.

Acknowledgement

This work is supported by Cancer Research UK and the CRUK/EPSRC Cancer Imaging Centre.  

Figure1: ALCAM-MPIO compound.

Monoclonal antibodies against ALCAM were conjugated to MPIO (ALCAM-MPIO). ALCAM-MPIO binding to ALCAM was verified under static conditions on mouse endothelial cells treated with TNF-a in vitro. ALCAM-MPIO (red) showed significantly higher binding than IgG-MPIO.

Figure 2: MRI and histological detection of ALCAM-MPIO in MDA-MB-231 model on day 21.

(a)(b) No tumour gadolinium enhancement was detected on T1-weighted images, (c) intense dark focal spots (green arrow) on MGE3D data set correspond to MPIO binding, (d) co-localisation of MRI hypointensities with histological detection of brain metastases, (e) co-localisation of ALCAM (brown) and ALCAM-MPIO (arrows).

2:10 PM
PS-02-4 — cCPE Peptides for SPECT Imaging of Claudin-4 Overexpression in Pancreatic Cancer (#312)

J. Baguña Torres1, M. Mosley1, S. Koustoulidou1, S. Hopkins1, S. Knapp2, A. Chaikuad2, V. Kersemans1, B. Cornelissen1

1 Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oncology, Oxford, United Kingdom
2 Structural Genomics, University of Oxford, Medicine, Oxford, United Kingdom

Introduction

Overexpression of tight junction protein claudin-4 has been detected in primary and metastatic pancreatic cancer tissue and is associated with better prognosis in patients1,2. Non-invasive measurement of claudin-4 levels by imaging methods could provide a means for accelerating disease detection and stratifying patients into risk groups. A GST-tagged version of the C-terminus of Clostridium perfringens enterotoxin (cCPE), a natural ligand for claudin-4, was previously used to measure claudin-4 overexpression by SPECT, but showed only modest binding affinity and slow blood clearance in vivo3.

Methods

Based on the crystal structure of cCPE, a series of smaller-sized variants of cCPE194-319 (S313A, S307A+N309A+S313A, L254F+K257D and D284A) with putatively improved binding affinity for claudin-4 were developed by site-directed mutagenesis4. Wildtype cCPE194-319, S313A and S307A+N309A+S313A were conjugated site-specifically on a C-terminal cysteine using maleimide-DTPA to allow radiolabelling with 111In. The binding affinity of all three 111In-radiolabelled peptides was evaluated in claudin-4-overexpressing Panc-1 cells and HT1080 negative controls. In vivo SPECT imaging was performed using BALB/c nude mice bearing Panc-1 or HT1080 tumour xenografts, and genetically engineered KPC mice, a clinically relevant model of pancreatic ductal adenocarcinoma.

Results/Discussion

Uptake of radiolabelled cCPE mutants was considerably higher in Panc-1 cells (claudin-4-positive) than HT1080 cells (claudin-4-negative). All peptides showed a marked improvement in affinity for claudin-4 in vitro when compared to 111In-cCPE.GST (Kd values of 2.9±0.4, 3±0.5 and 6.1±0.6 vs. 14.8±1.3 nM; Figure 1). Blood clearance of the novel cCPE peptides, as measured by SPECT imaging, was significantly faster when compared to that of 111In-cCPE.GST. Although moderate, uptake of all three 111In-radiolabelled cCPE peptides was significantly higher in Panc-1 xenografts than HT1080 tumours at 90 min post-injection (2.7±0.7, 2.3±0.9 and 2±0.4 vs. 0.4±0.1, 0.5±0.1 and 0.3±0.06 %ID/g; P<0.01, P<0.01 and P<0.05, respectively; Figure 2). Preliminary in vivo evaluation of wildtype cCPE radiotracer in the KPC model by SPECT revealed prominent pancreatic uptake (10.7±1.9 %ID/g), which was associated with the presence of precancerous lesions.

Conclusions

These optimised cCPE-based SPECT imaging agents show great promise as claudin-4-targeting vectors for in vivo imaging of claudin-4 overexpression in pancreatic cancer.

References

  1. Nichols et al, 2004, Am J Clin Pathol 121:226–30.
  2. Tsutsumi et al, 2012, Ann Surg Oncol 19:491-99.
  3. Mosley et al, 2015, J Nucl Med 56:745-51.
  4. Van Itallie et al, 2008, J Biol Chem 283(1):268-74.

Acknowledgement

The authors acknowledge financial support from CRUK/MRC Oxford Institute for Radiation Oncology, Pancreatic Cancer UK and Pancreatic Cancer Research Fund.

Figure 1: Determination of binding affinity of cCPE probes for claudin-4 in vitro
Panc-1 and HT1080 cells were exposed to increasing concentrations of cCPE radioconjugates and the extent of cell binding was compared to that of cCPE.GST. Affinity of cCPE probes for claudin-4 was confirmed by binding to Panc-1 cells, but not to HT1080 cells. Binding affinity for claudin-4 was similar among cCPE mutants (KD~5 nM), while cCPE.GST showed lower affinity for the target (KD~15 nM).

Figure 2: SPECT imaging of cCPE mutants in PDAC tumour xenograft models
A) Representative SPECT/CT images of mice carrying Panc-1 and HT1080 xenografts 90 min after injection of 111In-radiolabelled S313A. B) Although tumour uptake was modest, a clear difference in cCPE accumulation was observed between the two cell lines by ex vivo gamma-counting . Statistically significant differences are indicated by * (P<0.05) and ** (P<0.01). Data are mean (n =3/group) ± SD.

2:20 PM
PS-02-5 — Preclinical evaluation of novel mesothelin-specific ligands for SPECT imaging of Triple Negative Breast Cancer. (#283)

C. Montemagno1, S. Bacot1, M. Ahmadi1, B. Kerfelec2, D. Baty2, M. Debiossat1, A. Soubies1, P. Perret1, L. Riou1, D. Fagret1, A. Broisat1, C. Ghezzi1

1 UMR INSERM 1039, Laboratoire Radiopharmaceutiques Biocliniques, Grenoble, France
2 Aix Marseille Univ, CNRS, INSERM, Intitut Paoli-Calmettes, CRCM, Marseille, France

Introduction

Mesothelin is a cell-surface glycoprotein restricted to mesothelial cells overexpressed in several cancers, including triple negative breast cancers (TNBC) not responding to trastuzumab and hormonal-based therapies (1,2). Mesothelin-targeting therapies are currently being developed (3). However, the identification of patients potentially eligible to such therapeutic strategy remains challenging. The objective of this study was to perform the radiolabeling and pre-clinical evaluation of 99mTc-A1 and 99mTc-C6, two anti-mesothelin single domain antibodies (sdAb)-derived imaging agents.

Methods

A1 and C6 were radiolabeled with 99mTc and evaluated in vitro on recombinant protein and cells, as well as in vivo in xenograft mice models of TNBC HCC70 (mesothelin-positive, n=22) and MDA-MB-231 (mesothelin-negative, n=5) cell lines.

Results/Discussion

Both 99mTc-A1 and 99mTc-C6 bound mesothelin with high affinity in vitro, with 99mTc-A1 affinity being 2.4-fold higher than that of 99mTc-C6 (KD= 43.9 ± 4.0 vs 107 ± 16 nM, P<0.05). 99mTc-A1 and 99mTc-C6 remained stable in vivo in murine blood (>80 % at 2h) as well as ex vivo in human blood (>90 % at 6h). In vivo 99mTc-A1 uptake in HCC70 tumors was 5-fold higher than in MDA-MB-231 tumors and 1.5-fold higher than that of 99mTc-C6 (2.34 ± 0.36 vs 0.48 ± 0.18 and 1.56 ± 0.43 %ID/g respectively, P<0.01) and resulted in elevated tumor-to-background ratios. In vivo competition experiments demonstrated the specificity of 99mTc-A1 uptake in HCC70 tumors.

 

Conclusions

Mesothelin-positive tumors were successfully identified by single-photon emission computed tomography (SPECT) using 99mTc-A1 and 99mTc-C6. Considering its superior characteristics, 99mTc-A1 was selected as the most suitable tool for further clinical translation.

References

1.         Tchou J, Wang L-C, Selven B, et al. Mesothelin, a novel immunotherapy target for triple negative breast cancer. Breast Cancer Res Treat. 2012;133:799-804.

2.         Ordóñez NG. Application of mesothelin immunostaining in tumor diagnosis. Am J Surg Pathol. 2003;27:1418–1428.

3.         Hassan R, Thomas A, Alewine C, Le DT, Jaffee EM, Pastan I. Mesothelin Immunotherapy for Cancer: Ready for Prime Time? J Clin Oncol. 2016;34:4171-4179.

1. In vivo imaging of MSLN-expressing tumors with 99mTc-sdAbs

(A) Sagittal, coronal and transversal views of fused SPECT/CT images of HCC70 and MDA-MB-231 tumor-bearing mice 1h after i.v. injection of 99mTc-A1 or 99mTc-C6. K: kidneys; B: bladder and L: liver. In vivo (B) or ex vivo (C) quantification of 99mTc-A1 and 99mTc-C6 tumor uptake. ## P<0.01, ### P<0.001 vs MDA-MB-231 A1, ** P<0.01, *** P<0.001 vs HCC70 Control sdAb, †† P<0.01 vs HCC-70 A1.

2. In vivo competition

In vivo competition study. (A) Representative SPECT/CT images of HCC70 tumor-bearing mice injected with 99mTc-A1 alone (left) or with a 150-fold excess of unlabeled A1 (right). White arrows indicated tumor localization (B) SPECT image quantification. Results were expressed as % ID/cm3 of tissue. ** P<0.01 vs HCC70 A1 + competition.

2:30 PM
PS-02-6 — An 131I-labeled Camelid single-domain antibody fragment to treat HER2-overexpressing cancer (#200)

M. D'Huyvetter1, J. De Vos1, 2, C. Xavier1, M. Pruszynski3, Y. G. J. Sterckx4, S. Massa4, 5, G. Raes4, 5, V. Caveliers1, 6, M. R. Zalutsky7, T. Lahoutte1, 6, N. Devoogdt1

1 Vrije Universiteit Brussel, In Vivo Cellular and Molecular Imaging Lab, Brussels, Belgium
2 Camel-IDS NV, Brussels, Belgium
3 Institute of Nuclear Chemistry and Technology, Warsaw, Poland
4 Vrije Universiteit Brussel, Cellular and Molecular Immunology, Brussels, Belgium
5 VIB-UGent Center for Inflammation Research, Myeloid Cell Immunology Laboratory, Brussels, Belgium
6 UZ Brussel, Nuclear Medicine Department, Brussels, Belgium
7 Duke University Medical Center, Department of Radiology, Durham, United States of America

Introduction

Camelid single-domain antibody-fragments (sdAbs) have beneficial pharmacokinetic properties, and those targeted to HER2 can be used for imaging of HER2-overexpressing cancer. Labeled with a therapeutic radionuclide, they may be used for HER2-targeted therapy. Here we describe the generation of a 131I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer.

Methods

Anti-HER2 sdAb 2Rs15d was labeled with 131I using [131I]SGMIB and evaluated in vitro. Biodistribution was evaluated in two HER2+ murine xenograft models by micro-SPECT/CT imaging and at necropsy, and under challenge with trastuzumab and pertuzumab. The therapeutic potential of [131I]SGMIB-2Rs15d was investigated in two HER2+ tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [127I]SGMIB-sdAb at 1.4mg/kg. The structure of the 2Rs15d-HER2 complex was determined by X-ray crystallography.

Results/Discussion

[131I]SGMIB-2Rs15d bound specifically to HER2+ cells (KD=4.74±0.39nM). High and specific tumor uptake was observed in both BT474/M1 and SKOV-3 tumor xenografted mice and surpassed kidney levels by 3h. Extremely low uptake values were observed in other normal tissues at all time points. The crystal structure revealed that 2Rs15d recognizes HER2 Domain 1, consistent with the lack of competition with trastuzumab and pertuzumab observed in vivo. [131I]SGMIB-2Rs15d alone, or in combination with trastuzumab extended median survival significantly. No toxicity was observed after injecting [127I]SGMIB-2Rs15d.

Conclusions

These findings demonstrate the theranostic potential of [131I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [131I]SGMIB-2Rs15d, and could thereby impact therapy outcome on HER2+ BC patients. A first-in-human study evaluating [131I]SGMIB-2Rs15d in healthy volunteers and HER2+ BC patients is currently ongoing (NCT02683083).

Acknowledgement

This work was supported by Innoviris.Brussels, Stichting Tegen Kanker and in part by the National Cancer Institute Grant CA42324. M. D’Huyvetter is a postdoctoral fellow of the Research Foundation-Flanders, Belgium (FWO) and was supported by the Belgian American Education Foundation (BAEF) and the Germaine Eisendrath-Dubois Foundation. The authors thank Cindy Peleman, Jan De Jonge and Claudia Mebis for technical assistance, 

In Vivo evaluation of 131I-labeled anti-HER2 2Rs15d
131I-labeled anti-HER2 2Rs15d was cleared through kidneys, mainly within the first 2h after injection (A). High tumor uptake was observed, especially at early timepoints. Surprisingly, the amount in tumor exceeded uptake in kidneys by 3h (B). Animals treated with 131I-labeled 2Rs15d had a median survival of 137.5 days versus only 93.5 and 78 days for animals in the control groups (C).  

2:40 PM
PS-02-7 — Optoacoustic imaging of multi-scale dynamics in solid tumors with spiral volumetric optoacoustic tomography (#507)

A. Ron1, 2, X. L. Deán-Ben1, D. Razansky1, 2

1 Helmholtz Center Munich, . Institute for Biological and Medical Imaging (IBMI), Neuherberg, Bavaria, Germany
2 Technical University of Munich, Faculty of Medicine, Munich, Bavaria, Germany

Introduction

Deep-tissue longitudinal visualization of tumor development, neovascularization, its perfusion and oxygenation profiles is essential for the study of cancer in preclinical models. To this end, various optoacoustic tomography and microscopy methods have offered excellent optical contrast, fast imaging speed and high resolution in deep-tissue observations. However, efficient visualization of multi-scale tumor dynamics remained difficult with state-of-the-art systems due to inefficient trade-offs between image acquisition time and effective field of view.

Methods

Here we used a multi-scale imaging approach based on the spiral volumetric optoacoustic tomography (SVOT) technique. The imaging system offers high quality visualization of the intricate anatomy at the whole-body scale along with the ability for fast tracking of kinetics and functional changes at the whole tumor level rendered at a volumetric frame rate of 100Hz. This is further combined with the multispectral imaging capability which allows the extraction of specific chromophores and contrast agents. In our study, female hairless NOD.SCID mice bearing orthotopic MDA-MB231 breast cancer tumors were imaged longitudinally once a week, for a time period of three to four weeks. In addition, oxygen challenges were introduced by changing the anesthesia between room air and 100% oxygen.

Results/Discussion

Longitudinal tracking of tumor development, angiogenesis and necrosis was achieved with 3D spatial resolution of 70μm and penetration of up to 10mm (Fig. 1). Unmixing of the multispectral image data further allowed to observe oxygenation gradients across the entire tumor mass. By exploiting fast volumetric imaging rates of 100Hz we were able to monitor the rapid changes in the SO2 values which were caused by oxygen challenges, showing differential responses across the tumor (Fig. 2).

Conclusions

The excellent optical contrast, deep tissue imaging capability and spatio-temporal resolution performance of SVOT system has enabled here the visualization of tumor development at multiple spatial and temporal scales not attainable with other imaging modalities. Combined with its capacity for rapid 3D assessment of the SO2 gradients, the technique offers new level of performance in preclinical studies of tumor development as well as other complex dynamics, such as vessels permeability and vasoactivity.

Acknowledgement

The authors would like to acknowledge grant support from the Human Frontier Science Program (RGY0070/2016) and Deutsche Forschungsgemeinschaft (RA1848/5-1).

 

Figure 1.
(Left) Schematic of the SVOT system. Pulse light illuminates the mouse, resulting in optoacoustic responses that are recorded by a spherical matrix transducer array. The array and the light source are scanning the object in a Raster motion. (Right) The yielded 3D image of the whole-body mouse anatomy in vivo. Presented here is a maximal intensity projection (MIP) of the coronal plane.

Figure 2.
Changes in the SO2 level during oxygen challenge in two different areas within the tumor representing a major feeding vessel (green) and neovasculature (purple).

2:50 PM
PS-02-8 — Immunotargeting of Galectin-3 in thyroid orthotopic tumors allows specific imaging and characterization of thyroid cancer (#49)

F. De Rose1, M. Braeuer1, M. T. Kuhlmann2, S. Reder1, S. Braesch-Andersen3, A. Otto4, K. Steiger5, 6, S. Mall7, A. Bartolazzi8, 9, M. Schwaiger1, C. D'Alessandria1

1 Klinikum rechts der Isar, Nuklearmedizinische Klinik und Poliklinik, München, Germany
2 European Institute for Molecular Imaging (EIMI), Münster, Germany
3 Mabtech AB Research Laboratory, Stockholm, Sweden
4 Technische Universität München, Munich School of Biomedical Engineering, Garching, Germany
5 Klinikum rechts der Isar, Institute of Pathology, München, Germany
6 Klinikum rechts der Isar, Comparative Experimental Pathology, München, Germany
7 Klinikum rechts der Isar, III. Medical Department, Ismaninger Str. 22, Germany
8 Cancer Center Karolinska, CCK R8:04, Karolinska Hospital, Pathology Research Laboratory, Stockholm, Sweden
9 Sant'Andrea University Hospital, Pathology Research Laboratory, Rome, Italy

Introduction

Thyroid nodules present a challenging preoperative characterization. The routine thyroid scan via radio-iodine scintigraphy provides functional information based on radio-iodine uptake mediated by sodium/iodide symporter (NIS), but fails to distinguish among benign and malignant lesions. Galectine-3 (Gal-3) expression in the thyroid is restricted to well-differentiated and undifferentiated carcinomas. In this work we aimed to demonstrate the specificity of Gal-3 immuno-targeting compared to radio-iodine in imaging thyroid cancer in orthotopic tumor models, characterized by low NIS expression.

Methods

A papillar (BcPAP) and two anaplastic (FRO82-1 and CAL62) thyroid carcinoma cells lines were characterized for Gal-3 and NIS expression via WB and PCR. An anti-Gal3 F(ab’)2 fragment was generated from a rat mAb via pepsin digestion, functionalized with DFO-NCS, labeled with 89Zr and characterized for binding and immunoreactivity on 2D and 3D cell cultures. A thyroid orthotopic murine model were established by inoculation of carcinoma cells into the left thyroid lobe of athymic nude mice and tumor growth was monitored weekly via US scans and fluorescence molecular tomography (FMT). Mice were imaged by PET/CT after i.v. injection with Iodine-124 and later with 89Zr-DFO-F(ab’)2 anti-Gal3, followed by biodistribution studies and immunohistochemistry analysis for Gal3 and NIS expression.

 

Results/Discussion

Thyroid carcinoma cells investigated were invariably Gal-3 positive, while presenting low/lost NIS expression. 89Zr-DFO-F(ab’)2 anti-Gal3 tracer showed high stabil