EMIM 2019
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POSTER SESSION I | Exhibition | Coffee Break (Hall II)

   
Shortcut: PO 01
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

All posters are displayed during the entire duration of the EMIM. Make use of the E-POSTER STATIONS on-site: Browse through the posters, enlarge figures, leave comments, search for keywords... three digital poster stations are provided for your convinience.

DURING the DEDICATED POSTER SESSION:

The presenters will be present at their posters. All participants are invited to come along and discuss the work with the authors. Independent of this, two chairpersons are going to evaluate each poster: you can certainly also join these discussions - as you like.

One poster award per walk will be presented during the EMIM Closing Session.

Sub sessions:

Imaging Host Responses

Session chair: Manfred Kneilling (Tuebingen, Germany); Gilbert Fruhwirth (London, UK)
 
Shortcut: PW01
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

513

Imaging the Vascular Bone Marrow Niche During Inflammatory Stress (#137)

Katrien Vandoorne1, 2, David Rohde1, Hye-Yeong Kim1, Gregory Wojtkiewicz1, Filip Swirski1, Matthias Nahrendorf1

1 MGH, Harvard Medical School, Center for Systems Biology, Boston, Massachusetts, United States of America
2 Eindhoven University of Technology, Biomedical Engineering, Eindhoven, Netherlands

Introduction

Inflammatory stress induced by exposure to bacterial lipopolysaccharide causes hematopoietic stem cell expansion in the bone marrow niche, generating a cellular immune response. As an integral component of the hematopoietic stem cell niche, the bone marrow vasculature regulates the production and release of blood leukocytes, which protect the host against infection but also fuel inflammatory diseases. We aimed to develop imaging tools to explore vascular changes in the bone marrow niche during acute inflammation.

Methods

Using the TLR (Toll-like receptor) ligand lipopolysaccharide as a prototypical danger signal, we applied multiparametric, multimodality and multiscale imaging to characterize how the bone marrow vasculature adapts when hematopoiesis boosts leukocyte supply. In response to lipopolysaccharide, ex vivo flow cytometry, quantitive pcr, histology and electron microscopy showed vascular changes to the bone marrow niche. We studied these vascular changes to the bone marrow  in vivo: we tested and validated positron emission tomography (PET)/ magnetic resonance imagin (MRI) of the bone marrow vasculature, integrating PET/MR with near- infrared intravital microscopy via multimodal imaging agents that are fluorescent and radioactive or paramagnetic(Fig. 1).

Results/Discussion

In contrast to leukocytes, endothelial Itgav expression was elevated 3 hours after lipopolysaccharide treatment. While low heterogeneous staining of integrin αVβ3 in steady-state marrow was observed, integrin αVβ3 staining was increased and localized at the outer surfaces of the vessels in the marrow of lipopolysaccharide-treated mice. We detected integrin αVβ3+ Ki67+ endomucin+ endothelial cells, indicating that proliferating bone marrow endothelial cells stain for αVβ3 integrins concomitant with hypoxic conditions in response to prototypical inflammatory stress. Fluorescence and positron emission tomography integrin αVβ3 imaging signal increased during lipopolysaccharide-induced vascular remodeling (Fig. 1). Vascular leakiness, quantified by albumin-based in vivo microscopy and magnetic resonance imaging, rose when hematopoietic stem and progenitor cells (LSKs) proliferated more vigorously (Fig. 2A) and Ly6G+ neutrophils departed (Fig. 2B).

Conclusions

Introducing a tool set to image bone marrow either with cellular resolution or noninvasively within the entire skeleton, this work sheds light on angiogenic responses that accompany emergency hematopoiesis. Understanding and monitoring bone marrow vasculature during inflammatory responses may provide a key to unlock therapeutic targets regulating systemic inflammation.

References

Vandoorne K*, Rohde D*, Kim HY, Courties G, Wojtkiewicz GR, Honold L, Hoyer FF, Frodermann V, Nayar R, Herisson FE, Jung Y, Désogère P, Vinegoni C, Caravan P, Weissleder R, Sosnovik DE, Lin CP, Swirski FK, Nahrendorf M. Imaging the Vascular Bone Marrow Niche During Inflammatory Stress. Circulation Research. 2018;123:415–427 . *These authors contributed equally 

Figure 1
Transformative in vivo optical imaging towards multi-parametric PET/MRI of the bone marrow niche
Figure 2

In vivo skull microscopy. (A) Dilution of DiD-labeled LSK progenitor cells after acute inflammation by lipopolysaccharides (LPS)(Right). (B) Departure of Ly6G+ neutrophils after LPS inflammation.

Keywords: Vasculature, Hematopoietic stem cells, PET/MRI, Lipopolysaccharides
514

Dissociation of 19F and fluorescence signal upon cellular uptake of dual contrast perfluorocarbon nanoemulsions (#376)

Pascal Bouvain1, Vera Flocke1, Wolfgang Krämer2, Rolf Schubert2, Jürgen Schrader3, Ulrich Flögel1, Sebastian Temme1

1 Heinrich-Heine-University, Molecular Cardiology / Experimental Cardiovascular Imaging, Düsseldorf, North Rhine-Westphalia, Germany
2 Albert-Ludwigs-University, Pharmaceutical Technology and Biopharmacy, Freiburg, Baden-Württemberg, Germany
3 Heinrich-Heine-University, Molecular Cardiology, Düsseldorf, North Rhine-Westphalia, Germany

Introduction

Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used for tracking endogenous macrophages, ex vivo labelled stem- or T-cells [1,2].  Cellular localization of the in vivo 19F MRI signal has been performed by post mortem histology or flow cytometry. However, only limited data are available on the correlation of the fluorescence and the 19F signal of tagged PFCs after cellular uptake over time. Here, we investigated the fate of the fluorescence and the 19F signal of rhodamine (Rho) or carboxyfluorescein (Cfl) modified PFCs in vitro and in vivo.

Methods

RhoPFCs or CflPFCs were prepared by microfluidization. The fate of the 19F- and the corresponding fluorescence signal was analyzed in vitro using J774, RAW and CHO cells by flow cytometry and 19F MRI. Impact of reactive oxygen species and pH on the fluorescence and 19F signal of Cfl/RhoPFCs was also tested. RhoPFCs were intravenously applied into mice and their distribution was monitored in spleen and liver over 24 h. Furthermore and the time course of fluorescence and 19F signals were tracked in a local inflammation model making use of a subcutaneous matrigel depot doped with LPS (lipopolysaccharide) [3]. MR experiments were performed at a 9.4 T Bruker AVANCEIII Wide Bore NMR spectrometer and data were acquired using a 25 mm birdcage resonator tuneable to 1H and 19F.

Results/Discussion

Internalization of fluorescence-labelled PFCs was associated with a substantial bleaching over 24 h in macrophage cell lines while the 19F signal increased over time. Upon cellular uptake, CflPFCs were more susceptible to bleaching than RhoPFCs. Moreover, the fluorescence signal (but not the 19F signal) of CflPFCs but not RhoPFCs was susceptible to changes in pH and the exposure to H2O2. After intravenous injection of RhoPFCs the fluorescence signal in spleen and liver peaked after 30 min and 2 h, respectively, followed by a successive decrease over 24 h, whereas the 19F signal continuously increased during this observation period. Under inflammatory conditions using the matrigel/LPS model, we observed increasing 19F signals in the inflammatory area while the fluorescence signal of immune cells isolated from the matrigel depot 24 h after its implantation was only slightly higher than background.

Conclusions

In the present study, we demonstrate that cellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the 19F label over time. These results critically impact on the interpretation of long-term cell-tracking experiments which are often validated by histology or flow cytometry but is associated with a strong underestimation of the true PFC deposition at inflammatory hot spots.

References

[1] Temme S, Bönner F, Schrader J, Flögel U. 19F magnetic resonance imaging of endogenous macrophages in inflammation. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2012 May-Jun;4(3):329-43.

[2] Srinivas M, Heerschap A, Ahrens ET, Figdor CG, de Vries IJ. (19)F MRI for quantitative in vivo cell tracking. Trends Biotechnol. 2010 Jul;28(7):363-70.

[3] Temme S, Jacoby C, Ding Z, Bönner F, Borg N, Schrader J, Flögel U. Technical advance: monitoring the trafficking of neutrophil granulocytes and monocytes during the course of tissue inflammation by noninvasive 19F MRI. J Leukoc Biol. 2014 Apr;95(4):689-97.

Acknowledgement

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants ST 1209/1-1, FL 303/6-1 and the Sonderforschungsbereich SFB 1116. 

Preparation of Rho-PFCs and Cfl-PFCs

PFCs which contain perfluoro-15-crown-5 ether (PFCE) were prepared by microfluidization. To obtain dual-contrast PFCs, lipids labelled with carboxyfluorescein (Cfl) or rhodamine (Rho) were added to the lipid/PFCE mixture prior to the homogenization process. Right: Chemical structures of Cfl (upper) and Rho (lower).

Time course of fluorescence and 19F signal

(A) Decay of the fluorescence signal after cellular uptake of CflPFCs by J774, RAW and CHO cells. (B) 19F MRI of PFC uptake by J774, RAW and CHO cells incubated with PFCs for 2 h (left) or 24 h (right). Cells were pelleted (dark area, blue arrow) and analyzed by 1H/19F MRI. 19F signals are shown by hot-iron colour gradient.

Keywords: 19F MRI, Inflammation, Perfluorocarbons, PFCs
515

19F MRI of neutrophil granulocytes by targeting the cell surface receptor CD177 (#391)

Pascal Bouvain1, Vera Flocke1, Shiwa Kadir1, Zhaoping Ding3, Wolfgang Krämer2, Rolf Schubert2, Jürgen Schrader3, Ulrich Flögel1, Sebastian Temme1

1 Heinrich-Heine-Universitity, Molecular Cardiology / Experimental Cardiovascular Imaging, Düsseldorf, North Rhine-Westphalia, Germany
2 Albert-Ludwigs-University, Pharmaceutical Technology and Biopharmacy, Freiburg, Baden-Württemberg, Germany
3 Heinrich-Heine-Universitity, Molecular Cardiology, Düsseldorf, North Rhine-Westphalia, Germany

Introduction

Neutrophil granulocytes are crucial for the development and progression of inflammatory diseases and are more heterogeneous than previously thought. In humans one of the major subpopulations is characterized by expression of CD177 which has been suggested to be involved in neutrophil transmigration. However, little is known about the role of CD177+ neutrophils for inflammatory diseases in vivo. The aim of the present study was to target perfluorocarbon nanoemulsions (PFCs) to CD177 enabling the subsequent visualization of neutrophil granulocytes by 19F magnetic resonance imaging (19F MRI).

Methods

Small peptides against human (hNGP) and murine (mNGP) CD177 [1,2] as well as control peptides (NGC) were equipped with n-terminal carboxyfluorescein and c-terminal -GGG-cysteine spacer. Coupling of peptides to maleimide PFCs was performed via the c-terminal cysteine and maleimide residues on the particle surface [3]. Binding studies were performed by flow cytometry and 19F MRI using human neutrophils from healthy volunteers and patients after myocardial infarction. Murine neutrophils were obtained from blood of C57BL/6 mice or isolated from LPS-doped matrigel plugs [4]. MR experiments were performed at a 9.4 T Bruker AVANCEIII wide bore NMR spectrometer and datasets were acquired using a 25 mm birdcage resonator tuneable to 1H and 19F.

Results/Discussion

Binding studies with hNGPPFCs and mNPGPFCs revealed highly specific labelling of neutrophil granulocytes. In contrast, corresponding control PFCs showed only minor enrichment in human or mouse immune cells. Interestingly, hNGPPFCs bound to a large fraction of human neutrophils (60-80%), whereas mNGPPFCs stained all neutrophils obtained from C57BL/6 mice. Importantly, binding and internalization of NGPPFCs by neutrophils was strongly upregulated in patients suffering from myocardial infarction or after ex vivo LPS stimulation. Increased mNGPPFC binding was also found in murine neutrophils stimulated with LPS or in cells isolated from subcutaneous matrigel/LPS inflammatory hot spots. hNGPPFC-labelling did not impact on cell surface marker expression, migration or production of reactive oxygen species. Intravenous injection of mNGPFCs into matrigel/LPS mice revealed labelling of circulating neutrophils and neutrophils which have entered the inflammatory matrigel/LPS depot.

Conclusions

In this study, we show that neutrophil granulocytes can be specifically labelled with PFCs by targeting the cell surface receptor CD177. CD177 is expressed on all neutrophils in mice but only on a subpopulation of human neutrophils. CD177 is strongly upregulated under inflammatory conditions in mice and men. Therefore, m/hNGPPFCs enable the tracking of activated CD177+ neutrophils by 19F MRI in inflammatory diseases.  

References

[1] Mazzucchelli L, Burritt JB, Jesaitis AJ, Nusrat A, Liang TW, Gewirtz AT, Schnell FJ, Parkos CA. Cell-specific peptide binding by human neutrophils. Blood. 1999; 93(5):1738-48.

[2] Miettinen HM, Gripentrog JM, Lord CI, Nagy JO. CD177-mediated nanoparticle targeting of human and mouse neutrophils. PLoS One. 2018 Jul 10;13(7).

[3] Temme S, Baran P, Bouvain P, Grapentin C, Krämer W, Knebel B, Al-Hasani H, Moll JM, Floss D, Schrader J, Schubert R, Flögel U, Scheller J. Synthetic Cargo Internalization Receptor System for Nanoparticle Tracking of Individual Cell Populations by Fluorine Magnetic Resonance Imaging. ACS Nano. 2018;12(11):11178-11192.

[4] Temme S, Jacoby C, Ding Z, Bönner F, Borg N, Schrader J, Flögel U. Technical advance: monitoring the trafficking of neutrophil granulocytes and monocytes during the course of tissue inflammation by noninvasive 19F MRI. J Leukoc Biol. 2014 Apr;95(4):689-97.

Acknowledgement

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants ST 1209/1-1, FL 303/6-1
and the Sonderforschungsbereich SFB 1116. 

Targeting of neutrophil granulocytes by NGP-PFCs
(A) Neutrophils from healthy volunteers (blue) or from patients after myocardial infarction (STEMI) (red) were incubated with hNGPPFCs. (B) Murine neutrophils stimulated with LPS (red) or left untreated (blue) were labelled with mNGPPFCs. (C) 19F MRI of neutrophils from heathy subjects or STEMI patients incubated with hNGPPFCs, separated by density centrifugation and analyzed by 1H/19F MRI.
Keywords: 19F MRI, PFCs, Active Targeting, Neutrophils, Inflammation
516

In vivo Tracking of Anti-inflammatory Liposomes in a Rheumatoid Arthritis Model using PET. (#387)

Peter J. Gawne1, Fiona Clarke2, Keren Turjeman3, Andrew Cope2, Yechezkel Barenholz3, Samantha Y. A. Terry1, Rafael T. M. de Rosales1

1 King's College London, Imaging Chemistry and Biology, London, United Kingdom
2 King's College London, Centre for Inflammation Biology and Cancer Immunology, London, United Kingdom
3 The Hebrew University-Hadassah Medical School, Department of Biochemistry and Molecular Biology, Jerusalem, Israel

Introduction

Encapsulating glucocorticoids into long-circulating liposomes (LCL) is a strategy to improve the treatment of inflammatory diseases such as rheumatoid arthritis (RA). [1] LCLs exploit the well-established, but highly variable, enhanced permeation and retention (EPR) effect in inflamed tissues. We aim to develop PET labelling tools to track LCLs in vivo to support their preclinical development and potential clinical personalized nanomedicine. In this work, a methylprednisolone-LCL (LC200), was directly labelled using [89Zr]Zr-oxine, [2] and tracked in vivo in a RA mouse model.

Methods

RA was induced in 9-week old female C57Bl/6J mice (n = 5) using the K/BxN serum transfer arthritis model. [3] Control groups were injected with non-arthritogenic serum (n = 4) or PBS (n = 5). The mouse joints were measured and scored for RA on days 0, 2, 5, 7 and 9. For radiolabelling, LC200 was incubated with [89Zr]Zr-oxine for 30 min at 50oC then purified by elution through a size-exclusion column (loading efficiency >75%). [2] [89Zr]Zr-LC200 (2.5 MBq, 2 mg/kg methylprednisolone succinate) was injected 7 days post serum/PBS injection and 48 h later PET/CT images were obtained and ex vivo biodistributions carried out. All joints were fixed in 4% formalin and stained for neutrophils (anti-Ly6G) and blood vessels (anti-CD31).

Results/Discussion

PET/CT imaging showed high [89Zr]Zr-LC200 uptake (1.5-6.8 %ID; 14-30 %ID/mL) at inflamed joints with low activity present in the joints of animals in the PBS group (representative image in Fig. 1A). Ex vivo biodistribution showed [89Zr]Zr-LC200 uptake in non-target organs was similar across all groups, with high spleen uptake (48-59% ID/g) and liver uptake (29-32% ID/g) (Fig. 1B). Importantly, a clear correlation between joint swelling and high [89Zr]Zr-LC200 uptake was observed (Fig. 1C & 1D), with swelling only present in the RA mouse group. This correlation was also evident when comparing both visual inflammation scores and joint swelling measurements to in vivo imaging and ex vivo biodistribution data (Fig. 2A). Additionally, [89Zr]Zr-LC200 PET was able to detect suspected inflammation in the knee of one mice – which was only possible to confirm post-mortem from histology (Fig. 2B); highlighting the value of this imaging approach to detect and treat occult sites of inflammation.

Conclusions

We show that glucocorticoid-loaded liposomes such as LC-200 can be radiolabelled with the PET radionuclide 89Zr using [89Zr]Zr-oxine. PET imaging was used for the first time to study the biodistribution of [89Zr]Zr-LC200 and to demonstrate high uptake at visible and occult inflamed sites in an RA model. This technique has potential as a predictor of therapeutic response to treatment of inflammatory diseases with LC200 and similar nanomedicines.

References

[1] Ozbakir B. et al., J Control Release. 2014, 190, 626 - 636

[2] Edmonds S. et al., ACS Nano. 2016, 10, 10294−10307

[3] Christensen A. et al., Front Immunol. 2016, 7, 213

Figure 1: [89Zr]Zr-LC200 can be imaged and detected in arthritic joints.
(A) PET/CT maximum intensity projections of C57Bl/6J mice without and with serum induced rheumatoid arthritis. Swelling in the joints of the arthritic mice is clearly demonstrated by liposomal uptake in the wrists and ankles. (B) Ex-vivo­ biodistribution at 48 h p.i. of [89Zr]Zr-LC200. (C) Plot of %ID of [89Zr]Zr-LC200 versus swelling of ankles and (D) versus swelling of wrists at day 9.
Figure 2: [89Zr]Zr-LC200 uptake correlates with the degree of inflammation
(A) Heatmaps showing joint swelling, visual inflammation score, [89Zr]Zr-LC200 uptake (%ID) from ex-vivo biodistribution and image quantified uptake (%ID/mL) for all mice at day 9. (B) PET/CT MIP of C57Bl/6J mice with a suspected inflamed right knee along with immunohistochemistry showing increased neutrophil infiltration (anti-Ly6G) and vascularization (anti-CD31) compared to the left knee.
Keywords: PET, liposome tracking, nanomedicines, rheumatoid arthritis, personalised medicine
517

Time-lapse MRI of the brain as dynamic single cell tracking imaging technique for detection of non-CNS associated inflammation (#286)

Felix Freppon1, Max Masthoff1, Lydia Wachsmuth1, Franziska Albers1, Christian Schwarz1, Mirjam Gerwing1, Anne Helfen1, Walter Heindel1, Michel Eisenblätter1, Moritz Wildgruber1, Cornelius Faber1

1 University Hospital Muenster, Translational Research Imaging Center (TRIC), Muenster, North Rhine-Westphalia, Germany

Introduction

Time-lapse MRI enables imaging of dynamic single immune cells in the intact brain in vivo by repetitive image acquisition1. Applying this method to naïve mice and mice suffering from experimental autoimmune encephalomyelitis (EAE) has previously shown altered immune cell dynamics in EAE and could be detected before the onset of clinical symptoms2. The aim of this study is to evaluate time-lapse MRI of the brain under non-CNS associated inflammatory conditions, specifically a subcutaneous pellet model as well as in bacteremia.

Methods

In a first animal model, C57BL/6J mice were injected subcutaneously with 100µl of a polyacrylamide-gel (pellet) containing lipopolysaccharide (LPS, 10, 20, 40µg LPS/100µl PAG) to induce a sterile peripheral inflammation (n=6 each). After 24h mice were i.v. injected with iron oxide nanoparticles (Ferucarbotran, 1.9ml/kg BW). 24h later time-lapse MRI of the whole brain with 20 time-frames (scan time: 8min 12s for a single time-frame) was performed.
In a second model C57BL/6J mice (n=3) were i.v. injected with 1x105 CFU of S. aureus, followed by the same time-lapse MRI protocol.
Healthy C57BL/6J mice (n=8) injected only with Ferucarbotran were used as control.
Data was analyzed manually by counting hypointense spots in the brain (events) representing labelled cells.

Results/Discussion

In control mice overall (269±27) events were observed, which could be subcategorized in short (one or two consecutive time frames, 139±12), long (three or more consecutive time frames, 73±11) and motion (motion in three or more consecutive time frames in one or to a consecutive slice, 58±12) events (Fig. 1).

In the pellet model the overall number of events was significantly reduced to (173±16) in 10µg LPS/100µl PAG and to (163±36) in 20µg LPS/100µl PAG. Also in the subcategories short and motion significantly less events were detected in all LPS doses.
Comparing control mice and mice with bacteremia showed decreases in overall events (19±2) and in all subcategories as well (Fig. 2). Hence, time-lapse MRI enables early detection of non-CNS associated inflammation.

Depending on the increasing stimulus of inflammation, the percentage distribution within the subcategories short and motion showed decreasing trends, while the percentage of long events increased.

Conclusions

Time-lapse MRI enables to display altered immune cell dynamics in inflammatory diseases. Not only EAE2 but also peripheral inflammation and bacteremia showed altered immune cell dynamics in the brain, as detected and quantified by time-lapse MRI. Immune cell dynamics are dependent on quality and intensity of the inflammatory stimuli. Therefore, time-lapse MRI might provide a versatile tool for single cell-tracking based detection of inflammation.

References

  1. Mori et al. 2014, From cartoon to real time MRI: in vivo monitoring of phagocyte migration in mouse brain, Scientific Reports 4:6997
  2. Masthoff et al. 2018, Temporal window for detection of inflammatory disease using dynamic cell tracking with time-lapse MRI, Scientific Reports 8:9563

Acknowledgement

Felix Freppon was supported by the Medizinerkolleg of the medical faculty Muenster

Fig.1: In vivo detection of immune cells with cerebral time-lapse MRI

20 representative images of the same slice from a cerebral time-lapse MRI from the brain of a healthy control mouse. The hypointense spots were identified as immune cells and categorized in the subcategories short (red circles), long (blue circles) and motion events (green circles). The acquisition time of a single time frame was 8min 12s.

Fig. 2: Comparison of detected immune cells in peripheral inflammation and bacteremia

a) The overall events showed significant reductions between the control mice and the inflammation model with doses of 10µg and 20µg LPS/100µl PAG as well as bacteremia. In the b) short and c) motion subcategories significant decreases were observed in all groups. The d) long events showed significant reduction only between the control mice and the bacteremia group. (*p<0.05, **p<0.01, ***p<0.001)

Keywords: time-lapse MRI, single cell tracking, inflammation, bacteremia, iron oxide nanoparticle
518

Targeting activated synovial fibroblasts using photodynamic therapy in human rheumatoid arthritis synovial tissue (#162)

Daphne N. Dorst1, Mark Rijpkema1, Mijke Buitinga2, Peter Laverman1, Maarten Brom1, Desirée L. Bos1, Anne Freimoser-Grundschober3, Christian Klein3, Birgitte Walgreen1, Peter van der Kraan1, Marije I. Koenders1, Martin Gotthardt1

1 Radboudumc, Nijmegen, Netherlands
2 KULeuven, Nijmegen, Belgium
3 Roche innovation center, Schlieren, Switzerland

Introduction

Activated synovial fibroblasts (ASF) play an important role in the pathogenesis of rheumatoid arthritis (RA). They contribute to the pro-inflammatory environment in the joint and degradation of cartilage and bone. Depleting ASF could ameliorate both these hallmarks of RA. ASF are characterized by the expression of fibroblast activation protein (FAP). Here, we investigated the potential of FAP-targeted photodynamic therapy (tPDT) using the photosensitizer IRDye700DX conjugated to the anti-FAP antibody 28H1 (28H1-700DX) to selectively kill these cells.

Methods

To demonstrate the proof-of-concept of FAP-based tPDT in ex vivo human tissue, RA synovial tissue obtained during joint replacement surgery was trypsin digested and fibroblasts were cultured for at least 5 passages. Cells were incubated with or without 1 ug/well 28H1-700DX for 4h, washed with PBS and either exposed to 690nm light or not exposed. A luminescent cell viability assay (CellTiter-Glo) was used to measure cell viability. In parallel, 6 mm biopsies of synovial tissue were taken and used for FAP-based tPDT (n=8 patients). They were subjected to tPDT as described above and formalin fixed after 1h. To measure cell death, 5µm paraffin slices were stained for FAP, γH2AX and caspase-3 expression for activated fibroblasts, DNA double-strand breaks and early apoptosis, respectively.

Results/Discussion

FAP-tPDT performed on the cultured fibroblasts showed a light dose dependent increase in cell death when incubated with 28H1-700DX for 4h. Cell viability was not affected when cells were incubated with 28H1-700DX without illumination (101.37±3.9% remaining compared to 100±5.07% in the normalized control, figure 1). Radiant exposure of 17.6 J/cm2 did not significantly decrease cell viability (6.4±9.8% decrease, ns). Radiant exposures of 52.8, 105.6 and 158.4 J/cm2 significantly decreased cell viability (38.6±6.9%, 67.5±10.9% and 80.6±5.8% respectively, p<0.001 for all). After FAP-tPDT, the human synovial biopsies showed a significantly increased staining of the caspase-3 marker, but not of γH2AX (Friedman’s ANOVA, p=0.007 and p=0.810 respectively). Pairwise comparison showed that caspase-3 scores were significantly higher in biopsies treated with tPDT compared to those incubated with buffer (p=0.009).

Conclusions

Here, we demonstrated that FAP-tPDT induces cell death of FAP-positive activated fibroblasts in synovial tissue from RA patients. This is a first indication that FAP-targeted PDT can be a feasible new treatment strategy in RA.

FAP-tPDT depletes human synovial fibroblasts

Figure 1: Human synovial fibroblast cell viability after FAP-tPDT targeted photodynamic therapy subjected to varying light intensities. Results are depicted for two separate donors (donor 1: red, donor 2: blue). Mean and SD are depicted for all data combined.

Keywords: Fibroblast activation protein, photodynamic therapy, rheumatoid arthritis
519

Cathepsin Z compensates for cathepsin B deficiency during cutaneous delayed-type hypersensitivity reactions (#191)

Johannes Schwenck1, 2, Andreas Maurer1, 3, Birgit Fehrenbacher4, Roman Mehling1, Philipp Knopf1, Natalie Mucha1, Dennis Haupt1, Martin Schaller4, Thomas Reinheckel5, Hubert Kalbacher3, Bernd Pichler1, Manfred Kneilling1, 4

1 Eberhard Karls University, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Baden-Württemberg, Germany
2 Eberhard Karls University, Department of Nuclear Medicine and Clinical Molecular Imaging, Tübingen, Baden-Württemberg, Germany
3 Eberhard Karls University, Interfaculty Institute of Biochemistry, Tübingen, Baden-Württemberg, Germany
4 Eberhard Karls University, Department of Dermatology, Tübingen, Baden-Württemberg, Germany
5 Albert Ludwigs University, Institute of Molecular Medicine and Cell Research, Freiburg, Baden-Württemberg, Germany

Introduction

Activity of proteases like cysteine-type cathepsins including Cathepsin B (CathB) and Cathepsin Z (CathZ) are of importance for essential steps of inflammation such as antigen processing and angiogenesis. Protease-activatable probes for non-invasive in vivo optical imaging possess peptide sequences which are preferentially cleaved by the distinct protease. In this study, we aimed to prove the role of CathB specificity of the activatable optical imaging probe CatB680 during the effector phase of T cell driven cutaneous delayed-type hypersensitivity reactions (DTHR).

Methods

Wild-type (WT), cathepsin B-deficient (Ctsb-/-) and cathepsin Z-deficient (Ctsz-/-) mice were sensitized at the abdomen with 5% trinitrochlorobenzene (TNCB) and after seven days challenged at the right ear with 1% TNCB to induce acute cutaneous DTHR. Mice were repetitively challenged (five times) at the right ear to induce chronic DTHR. We determined CathB activity in WT and Ctsb-/- mice non-invasively in vivo using the activatable optical imaging probe CatB680. CathB expression in inflamed ears and draining lymph nodes (dLNs) was validated ex vivo by CathB fluorescence microscopy. Moreover, we conducted active site labeling and CathB and CathZ western blot (WB) analysis of in inflamed ears and dLNs as well as flow cytometry analysis (dLNs, spleens) of WT, Ctsb-/- and Ctsz-/- mice.

Results/Discussion

Non-invasive in vivo CatB680 optical imaging indicated a remarkable cysteine-type cathepsin activity in inflamed ears and dLNs of WT mice during acute and chronic DTHR.  Interestingly, Ctsb-/- mice even revealed a trend towards a stronger in vivo CatB680 signal intensity along with an enhanced ear swelling response during acute DTHR when compared to WT mice. Fluorescence microscopy of inflamed ears and dLNs of WT mice yielded CathB expression by T cells, B cells, macrophages, dendritic cells and NK cells and, as expected, a lack of CathB expression in Ctsb-/- mice. WB analysis of CathZ, exhibiting a similar activity spectrum as CathB, revealed a remarkable CathZ expression in inflamed ears and dLNs of Ctsb-/- mice, whereas CathB expression was elevated in Ctsz-/- mice, yielding similar ear swelling responses as WT mice. Flow cytometry analysis revealed an unimpaired immune cell composition and T cell activation in the dLNs and spleens of Ctsb-/- or Ctsz-/- mice.

Conclusions

The protease CathB is highly active in the effector phase of acute and chronic cutaneous DTHR. However, cathepsin Z is able to compensate CathB deficiency most probably due to a complementary spectrum of cleavable protein sequences. As proteases like cathepsins are evolving targets in different diseases, compensatory roles of proteases represent an important mechanism which potentially explains both diagnostic biases and treatment failures.

Keywords: optical imaging, inflammation, protease, cathepsin, delayed type hypersensitivity reaction

Novel synthetic and radiolabelling procedures for imaging probes

Session chair: Guy Bormans (Leuven, Belgium); Phil Blower (London, UK)
 
Shortcut: PW02
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

601

Site-directional functionalization of VHH for imaging using the C-Direct tag (#282)

Raimond Heukers1, Marta Kijanka2, Paul M. van Bergen en Henegouwen2, Theo C. Verrips1, Edward Dolk1

1 QVQ Holding BV, Utrecht, Netherlands
2 Division of Cell Biology, Department of Biology, Faculty of Science , Utrecht University, Utrecht, Netherlands

Introduction

Antibodies are popular targeting moieties for probes in imaging like PET, SPECT, CT, MRI, optical, ultrasound and PAI 1. Due to their small size (~4 x 3 x 3nm), high stability, low immunogenicity, and rapid blood clearance, the variable domains of heavy chain-only antibodies found in camelids (VHH, sdAb or nanobody) has received a lot of attention as molecular imaging tracer 2–5. As such, it is important to load the VHH with imaging probes without affecting its binding characteristics.

Methods

The C-terminal C-Direct tag was designed for optimal production yield, thermo- and enzymatic stabilty and low immunogenicity using bio-informatics, molecular modeling and wet-lab validation and a FLAG-tag as starting material. The optimal tag contained a free thiol, flanked by alanines and an optimized tag for affinity purification. Different VHH constructs were cloned into the yeast expression plasmid pYQVQ11containing the C-Direct tag and were produced in S. cerevisiae and purified using the Capture Select technology. Purified VHH were site-directionally conjugated to biotin, chelators (e.g NOTA), fluorescent dyes (HiLyte, ATTO, Alexa, IRDyes) using maleimide chemistry. Free label was removed by size exclusion chromatography. Immunogenicity and toxicity was tested in vivo.

Results/Discussion

Random conjugation to lysine-residues significantly decreased the binding affinity of the VHH 11A4 for its target HER2. In contrast, site-directional conjugation to a free thiol in a C-terminal Cys-FLAG tag did not affect its binding properties. Site-directionally conjugated anti-HER2 VHHs were tested in ELISA, IF, cryo-EM, intravital imaging 6–8. The tag was further optimized in silico for minimal immunogenicity and protease sensitivity. In addition, thermostability and the production yields of VHHs with the C-Direct tag in S. cerevisiae were evaluated and were compared to their untagged counterparts.

Conclusions

VHHs with a C-Direct tag could readily be produced in yeast and purified from yeast supernatant. Also, functionalization of such VHH did not significantly affect its binding affinity and enabled its detection using a variety of imaging modalities.

References

  1. Majumdar, D., Peng, X.-H. & Shin, D. M. The medicinal chemistry of theragnostics, multimodality imaging and applications of nanotechnology in cancer. Curr. Top. Med. Chem. (2010). doi:10.2174/156802610791384171
  2. De Groeve, K. et al. Nanobodies as tools for in vivo imaging of specific immune cell types. J Nucl Med 51, 782–789 (2010).
  3. Oliveira, S. et al. Rapid visualization of human tumor xenografts through optical imaging with a near-infrared fluorescent anti-epidermal growth factor receptor nanobody. Mol. Imaging 11, 33–46 (2012).
  4. Debie, P. et al. Effect of Dye and Conjugation Chemistry on the Biodistribution Profile of Near-Infrared-Labeled Nanobodies as Tracers for Image-Guided Surgery. Mol Pharm 14, 1145–1153 (2017).
  5. Xavier, C. et al. (18)F-nanobody for PET imaging of HER2 overexpressing tumors. Nucl Med Biol 43, 247–252 (2016).
  6. Kijanka, M. et al. Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery. Eur. J. Nucl. Med. Mol. Imaging 40, 1718–29 (2013).
  7. Kijanka, M. et al. Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery. Eur. J. Nucl. Med. Mol. Imaging (2013). doi:10.1007/s00259-013-2471-2
  8. Kijanka, M. et al. A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy. J. Struct. Biol. (2017). doi:10.1016/j.jsb.2017.05.008

Acknowledgement

Part of this work was supported by the Center for Translational Molecular Medicine (MAMMOTH).

Keywords: Variable domains of camelid heavy chain-only antibodies (VHH), site-directional conjugation, C-Direct tag
602

s-tetrazine: a “clickable” platform for the site-specific dual-labeling of proteins (#59)

Coline Canovas1, Mathieu Moreau1, Claire Bernhard1, Alexandra Oudot2, Mélanie Guillemin2, Franck Denat1, Victor Goncalves1

1 Université Bourgogne Franche-Comté, ICMUB, UMR6302, CNRS, DIJON, France
2 Georges-Francois LECLERC Cancer Center – UNICANCER, DIJON, France

Introduction

Doubly-modified proteins constitute an emerging class of drugs, which open new fields of application such as multimodal molecular imaging and theranostics. Unfortunately, the multiple modification of proteins is still a challenging task, that requires a careful control of the conjugation site(s). Here, we propose an original approach, based on the use of dichlorotetrazine as a trivalent platform, for the site-specific dual-labeling of proteins through an iEDDA reaction. A doubly-modified trastuzumab, suitable for SPECT-CT/fluorescence imaging, was prepared and evaluated in vivo.

Methods

Dichloro-s-tetrazine was functionalized, by two successive SNAr reactions, with a macrocyclic chelator (DOTAGA or NODAGA) and a fluorophore (rhodamine B, BODIPY or cyanine 5.0). The bimodal probe substituted with both a DOTAGA and a disulfonated cyanine 5.0, was selected for further study. Vectorization of the bimodal probe was performed via iEDDA reaction between the tetrazine derivative and an anti-HER2 antibody (trastuzumab) previously modified with a bicyclo-1,6-nonyne group. The bioconjugate was radiolabeled with 111In and injected in nude BALB-C mice, xenografted with BT-474 cancer cells. SPECT-CT, fluorescence images and biodistribution data were acquired 24 h post injection. Blocking experiments were performed to verify the specificity of the uptake.

Results/Discussion

The first two functionalizations of dichlorotetrazine were performed by nucleophilic substitution of the chlorine atoms of the platform. The choice of nucleophiles involved in these two reactions appeared as essential and, by choosing an appropriate combination of nucleophiles (i.e. the use of an amine for the first step and a thiol for the second), we were able to synthesize a series of doubly-substituted tetrazines. One bimodal tetrazine was then conjugated to an antibody via an IEDDA reaction to afford a targeted nuclear/optical imaging agent. The preclinical evaluation of this compound demonstrated adequate pharmacokinetics, allowing SPECT-CT and fluorescence images to be acquired 24 h p.i. The tumor/muscle ratio was calculated by gamma counting and reached a value of 28.6. The concordance between the biodistributions observed according to the two imaging modalities (gamma counting and fluorescence) showed that the bimodal imaging agent is stable in vivo.

Conclusions

This study presents a new strategy for the double modification of biomolecules via a site-specific bioorthogonal reaction. The advantage of this approach over the different ternary platforms reported in the literature is its simplicity. Indeed, the regiospecific conjugation step on the biomolecule is carried out by the platform itself, and the number of steps required to obtain the desired compound is therefore considerably reduced.

References

C. Canovas, M. Moreau, C. Bernhard, A. Oudot, M. Guillemin, F. Denat and V. Goncalves, Angewandte Chemie International Edition, 2018, 57, 10646–10650.

Acknowledgement

Financial support was provided by the French National Research Agency (ANR) under the programs Investissements d’Avenir (IMAPPI Equipex) and AAP Générique 2017 (project ZINELABEL), the CNRS and the Université de Bourgogne. C.C. was funded by the Ministry of Higher Education, Research and Innovation. This work is also part of the project Pharmaco-imagerie et agents théranostiques supported by the Conseil Régional de Bourgogne Franche-Comté through the Plan d’Action Régional pour l'Innovation (PARI) and by the European Union through the PO FEDER-FSE 2014/2020 Bourgogne program.

Dual-labeling of proteins through a iEDDA reaction
Keywords: tetrazine, dual-labeling, iEDDA, site-specific
603

Functionalisation of G-quadruplex binders to enable theranostic applications (#344)

Rainbow Y. - H. Lo1, 2, Gilbert Fruhwirth1, 3, Ramón Vilar-Compte2

1 King's College London, Imaging Cancer and Therapies Group, Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom
2 Imperial College London, Department of Chemistry, London, United Kingdom
3 King's College London and University College London, Comprehensive Cancer Imaging Centre, London, United Kingdom

Introduction

G-quadruplexes(G4) are higher-order DNA and RNA structures which contribute to regulation of gene expression, telomeric maintenance and DNA replication.1 G4 stabilisation leads to modulation of oncogene transcription and inhibition of telomerase. Consequently, there is significant interest in developing small molecules which target and stabilise G4, and in doing so act as potential anticancer therapeutics.2,3 To further explore the potential of G4 binders both in vitro and in vivo we adapted G4 binders for use in a range of applications, such as imaging or targeting, using click chemistry.

Methods

We report the synthesis and characterisation of novel nickel, zinc and platinum salphen complexes bearing “clickable” backbones. Phosphorescence properties of the platinum(II) series (λEM=582 nm) were exploited for further G4 characterisation. G4-binding was evaluated using circular dichroism melting studies and emission titrations comparing G4 DNA (i.e. oncogenic (c-Myc) and telomeric (H.Telo)) with duplex DNA (ds26). Differences in melting temperature between DNA in the presence or absence of complex (ΔTm), and the binding affinities (KA) indicate strength of DNA-ligand binding. The salphens were further functionalised to “click-on” probes suitable for optical or nuclear imaging (e.g. DOTA for radiometal labelling) or for specific targeting (Fig.1A).

Results/Discussion

Several novel metal salphen complexes functionalised with either an alkyne or azide backbone were synthesised (Fig.1B), and can be used to attach different imaging or targeting groups. This general, modular strategy enables the production of different probes for multiple applications derived from a single precursor. By utilising this synthetic route we conserve the planar ligand system necessary for G4 binding, as evaluated by various biophysical techniques. Thermal stabilisation of G4 and binding affinities for unmodified versus modified platinum(II) salphen complexes were similar (ΔTm = 12.0 ± 2.4 and 10.0 ±1.6 respectively;  Fig.2A and B. KA~1 ± 0.8 x 106 for G4 DNA; Fig.2C and D.). Therefore, these results indicated that the presence of the clickable group did not impair G4 DNA binding. DOTA was also successfully attached to nickel salphen complexes as indicated by LC-MS.

Conclusions

We successfully synthesized a series of G4 DNA binders with additional functional groups that can be used, via “click-chemistry”, to link them to contrast agents (e.g. for optical or nuclear imaging) and cancer targeting groups. Platinum(II) salphen complexes were unaffected by this type of functionalisation and retained high affinity G4 DNA binding properties and G4 DNA binding-dependent phosphorescence.

References

  1. Balasubramanian, Nat. Rev. Mol. Cell Biol, 2017 18, 279–284
  2. Cao et al., Inorg. Chem. Front. 2017, 4, 10-32.
  3. Bandeira et al, Angew.Chem. Int. Ed. 2018, 57,310 –313

Acknowledgement

We thank the EPSRC for support and funding via the CDT in Medical Imaging.

Overview of G-quadruplex (G4) binders used in this work.
(A) Proposed schematic illustration for multi-purpose G-quadruplex binders, via click chemistry.  (B) Novel metal salphen complexes synthesised in this work.
G4 binding data for unmodified (1) vs modified (10) platinum(II) salphen complexes.

(A) CD melting experiment: (i) Example sequential CD melting traces, c-Myc only (ii) melting curves for c-Myc only and c-Myc + complex 1 (5eq), (B) corresponding ΔTm values for complexes 1 and 10. (C) Example emission titration of 1 vs G4 DNA ([c-Myc] and [H.Telo]), and binding curves. (D) Corresponding KA values complex complexes 1 and 10. N= 3

Keywords: G-quadruplex, luminescent metal complexes, dual-imaging, multimodal
604

Fully automated GMP compliant synthesis of biomolecules using [18F]AlF: application to [18F]AlF-NOTA-octreotide (#298)

Terence Tshibangu Tshikondu Tshiamalu1, Frederik Cleeren1, Christophe Deroose2, Guy Bormans1

1 KULeuven, Laboratory for Radiopharmaceutical Research, Leuven, Belgium
2 UZLeuven, Nuclear Medicine department, Leuven, Belgium

Introduction

68Ga-peptides allows on-site easy production of radiopeptides by radiopharmacies not equipped with a medical cyclotron. However, these radiopeptides have limitation due to limited 68Ga yield of the generator and relatively short half-life of 68Ga. New radiofluorination methods such as [18F]AlF radiolabeling (1) became a competitive alternative for 68Ga-labelled peptides. [18F]AlF-NOTA-octreotide, shows high affinity for the SSTR2 and good tumor uptake. The translation to clinical use request a robust, reproducible, easy and efficient production process in accordance with the cGMP rules.

Methods

[18F]AlF-NOTA-octreotide was synthesized on a Trasis AllInOne ® module, with an adapted process (2,3). SPE C18 cartridge purification and reformulation was implemented. The identification and purity were determined by HPLC using a Waters XBridge® C18 column, running a gradient ammonium acetate 0.05M pH5.5/acetonitrile. Anesthetized female Wistar rats (200g) were injected through a tail vein with 1.85 MBq (1.3 µg peptide) of [18F]AlF-NOTA octreotide or [68Ga]Ga-DOTATATE for biodistribution study. 75 min post-injection, the rats were sacrificed, organs/tissues were isolated, weighed and radioactivity was measured with a gamma counter. For the blocking study, rats were co-injected with octreotide (0.65 mg/kg). The SUV gives the uptake in organ/tissue (SUV=((%ID/g) x body weight in kg) x10).

Results/Discussion

The production and QC methods were validated in accordance to cGMP. Three validation batches of [18F]AlF-NOTA-octreotide (>10GBq, apparent As=0.12 GBq/µg) were produced in conditions of clinical production. The release criteria for the quality control were adapted from the European pharmacopeia monography of [68Ga]Ga-DOTATATE. [18F]AlF-NOTA-octreotide was stable over a period of six hours post-EOS (RCP >96 %). Biodistribution of [18F]AlF-NOTA-octreotide and [68Ga]Ga-DOTATATE in control animals (figure 1) shows high tracer concentration in pancreas, adrenal glands and pituitary glands, organs known to express a high number of somatostatin receptors. For both tracers, the uptake in these organs was reduced dramatically by the co-injection of octreotide indicating that tracer uptake observed in control conditions was somatostatin receptor specific. Compare to [68Ga]Ga-DOTATATE, [18F]AlF-NOTA-octreotide shows a lower uptake in pancreas and adrenal glands.

Conclusions

The results of the preclinical experiments warrants the translation of [18F]AlF-NOTA-octreotide to human use. The cGMP procedure allows the fast production of [18F]AlF-NOTA-octreotide with the Trasis AllInOne®. The activity produced and the stability of the final product, allows shipment of the compound to remote PET centers not equipped with a GMP production facility. The method can be used for other [18F]AlF-peptides.

References

  1. Kumar, Krishan, and Arijit Ghosh. "18F-AlF Labeled Peptide and Protein Conjugates as Positron Emission Tomography Imaging Pharmaceuticals." Bioconjugate chemistry 29.4 (2018): 953-975.
  2. Laverman, Peter, et al. "Optimized labeling of NOTA-conjugated octreotide with F-18." Tumor Biology 33.2 (2012): 427-434.
  3. Allott, L., et al. "A general [18 F] AlF radiochemistry procedure on two automated synthesis platforms." Reaction Chemistry & Engineering 2.1 (2017): 68-74.

Acknowledgement

This research was funded by the project from “Kom op tegen Kanker”: “PET/MR imaging of the norepinephrine transporter and somatostatin receptor in neural crest and neuroendocrine tumors for better radionuclide therapy selection” and received support from Research Foundation – Flanders (FWO) (G0D8817N)

Figure1: Biodistribution of [68Ga]Ga-DOTATATE and [18F]AlF-NOTA-octreotide in female Wistar rats

The biodistribution of [68Ga]Ga-DOTATATE (left) and [18F]AlF-NOTA-octreotide (right) in Wistar rats: A higher organ/tissue uptake in pancreas, adrenal glands and pituitary glands is observed (bleu rods). The uptake of [68Ga]Ga-DOTATATE and [18F]AlF-NOTA-octreotide was reduced by the co-injection of octreotide (orange rods).


 
Keywords: automation, cGMP process, validation, preclinical evaluation
605

Native Chemical Ligation: general synthetic approach for development of new dual PET/OI peptide imaging probes (#449)

Ivan Hawala1, Lucia De Rosa2, Luca D. D'Andrea3, Silvio Aime1

1 Università degli studi di Torino, Dipartimento di Biotecnologie Molecolari e Scienze per la salute, Torino, Italy
2 Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy
3 Istituto di Biostrutture e Bioimmagini, CNR, Torino, Italy

Introduction

Dual Optical/PET peptide imaging probes are considered as efficient tools for disease diagnosis and/or monitoring1. Their preparation is still a challenging task due to the complicated synthetic protocols requiring fully-protected peptide segments (in solution) or the use of orthogonal protection groups (solid phase) to achieve the dual site-specific labeling. An advantageous strategy for peptide site-specific modification of unprotected peptide in solution, overcoming most of the above mentioned limitations, relies on the use of Native Chemical Ligation (NCL)2.

Methods

The linear peptide H-Cys(trt)-Asp(t-Bu)-Cha-Phe-D-Ser(t-Bu)-D-Arg(Pbf)-Tyr(t-Bu)-Leu-Trp(Boc)-Ser(t-Bu)-OH (1) (AE105, reported antagonist of uPAR3) was synthetized automatically with the support of microwaves (MW) on a Wang Resin preloaded with H-Ser(t-Bu)-OH by standard Fmoc protocol. AAZTA-C4-CO-MES (2) was synthetized in solution starting from (t-Bu)4-AAZTA-C4-COOH and 2-Mercaptoethanesulfonic acid sodium salt (MESNA). The unprotected AE105 cysteine derivative peptide and MESNA thioester activated AAZTA ligand were used for the NCL reaction in buffer at pH 7.5 (3). Last reaction between AAZTA-C4-CO-Cys-AE105 and the cyanine 5.5 maleimide to obtain a stable thioether linkage was performed in a controlled reaction buffer at pH 6.5-7.5 (4).

Results/Discussion

Targeting peptide AE105 was synthetized and functionalized with a cysteine using a solid phase peptide synthesis approach to allow the condensation with AAZTA-C4-COOH, pre-activated with MESNA. After the reaction between the free thiol of the cysteine and the AAZTA thioester derivative, a rearrangement gives a stable amide bond between the N-terminus of the same cysteine and the carboxylic group of the arm of AAZTA-C4-COOH. The transposition of the chemical bond on the amine, made the thiol accessible to the subsequent reaction with a maleimide pre-activated fluorophore (i.e. cy 5.5). After each step, the pure intermediate products were afforded after preparative HPLC purification in good chemical yields and high purity.

We combine the ease of stepwise solid phase synthesis with Native Chemical Ligation, which only requires the introduction of a Cys residue, in order to make the most efficient use of those peptide segment starting materials.

Conclusions

Herein we developed a standard synthetic strategy to easily obtain a targeting molecule functionalized with a fluorophore to be employed in optical imaging applications, and a cage, to allow the complexation with PETs radionuclides. In conclusion, the reported synthesis procedure can be generally used to obtain dual imaging probes using fully unprotected moieties with a selective and rapid chemistry based on the Native Chemical Ligation.

References

1 Louie et al, Chem Rev, 2010, 110: 3146-3195

2 D’Andrea et al, Org Biomol Chem 2012 January 14; 10(2): 273–280

3 Li ZB at al, Carcer Res2008, 14: 4758-66.

Figure 1. Synthesis of AAZTA-C4-CO-Cys(Cy 5.5)-AE105
(1) uPAR targeting peptide: AE105-Cys; (2) Preactivated AAZTA thioester derivative: AAZTA-C4-CO-MES; (3) AAZTA-C4-CO-Cys-AE105; (4) AAZTA-C4-CO-Cys(Cy 5.5)-AE105
Keywords: Native Chemical Ligation, Solid Phase Peptide Synthesis, PET, Optical Imaging, AAZTA
606

The added value of cell-penetrating peptides in nanobody-based molecular imaging and theranostics (#231)

Estel Collado Camps1, 2, Sanne van Lith1, Maarten Brom1, Martin Gotthardt1, Roland Brock2

1 Radboudumc, Department of Radiology and Nuclear Medicine, Nijmegen, Netherlands
2 Radboudumc, Department of Biochemistry, Nijmegen, Netherlands

Introduction

Nanobodies are promising probes for molecular imaging and theranostics. This project aims at improving the tumour targeting abilities of an anti-Epidermal Growth Factor Receptor (EGFR) nanobody using cell-penetrating peptides (CPPs). CPPs are short peptides that induce cellular uptake of themselves and their cargoes in a receptor independent manner. We previously showed that the CPP hLF (a CPP derived from human lactoferrin) improves nanobody internalisation1. Here, we compared different CPPs with respect to their influence on the targeting characteristics and functionality of the nanobody.

Methods

Nanobodies were conjugated to the different CPPs via sortase-mediated coupling, followed by site-specific fluorescent labelling. Conjugates were then investigated with respect to binding, uptake, ability to compete with EGF and inhibition of EGFR activation. Lysotracker staining was used to quantify endocytosis by colocalization analysis. EGF binding was detected using streptavidin labelling of biotinylated EGF. EGFR activation was assessed by immunostaining of phospho-tyrosine 1068. Furthermore, we investigated how differences in 2D uptake and cell activation were reflected in a 3D context. To this end we used cell-spheroids, which we fixed and cleared after incubation. All imaging was done by laser scanning confocal microscopy.

Results/Discussion

The presence of a CPP enhanced uptake in all cases. Furthermore, all constructs showed a slower dissociation than the nanobody alone, suggesting that the CPP also decreases the dissociation constant of the nanobody. The R9 construct showed 57% stronger binding than the nanobody alone. Nanobodies coupled with D-enantiomer variants of R9 and Pen showed the same binding as their L-enantiomer coupled counterparts, providing the option for proteolytically stabilized conjugates. When co-incubated with EGF there was no difference between constructs and nanobody in binding competition or in reduction of EGFR activation. In the 3D context, coupling the D-enantiomer of R9 to the nanobody led to 59% higher intensity inside the spheroid, after 3h incubation. Penetration into the core of the spheroid was complete after 1h incubation. This is promising, as good 3D penetration and retention are key to successful tumour targeting.

Conclusions

Overall, CPPs improve the capacity of the nanobody to compete for EGF and block EGFR activation by enhancing binding and endocytosis. These results stimulate further research into how enhanced internalisation and reduced activation of the EGFR dampen its tumour promoting effects. Addition of an imaging and cytotoxic agent to the conjugate, provides a perspective for a highly powerful theranostic agent.

References

1- van Lith et al, A Conjugate of an Anti-Epidermal Growth Factor Receptor (EGFR) VHH and a Cell-Penetrating Peptide Drives Receptor Internalization and Blocks EGFR Activation. Chembiochem, 2017; 18(24):2390-2394

Acknowledgement

This work has been supported by a PhD project granted to Martin Gotthardt and Roland Brock by the Radboudumc.

Preliminary results showing the uptake of r9 construct and uncoupled nanobody in spheroids of SKOV-3
SKOV3 cells were stably transfected with GFP. A: Construct channel.The mean intensity of the spheroid incubated with r9 construct is 59% higher than that of the spheroid incubated with uncoupled nanobody. Both images were contrast adjusted for visualisation purposes. Mean intensities were measured in the original images using a circular ROI around the z-slice used in the figure. B: GFP channel.
Keywords: nanomedicine, nanobodies, tumour targeting, theranostics, cell-penetrating peptides
607

Detection limit and quantification of 89Zr-labelled cells for cellular tracking: An in vitro approach using a human PET system (#68)

Laura M. T. Lechermann1, Roie Manavaki1, Tim Fryer3, Bala Attili1, Doreen Lau1, Lorna Jarvis4, Luigi Aloj1, Bristi Basu2, Franklin Aigbirhio3, Matthew Cleveland5, Neel Patel5, Ferdia A. Gallagher1

1 Department of Radiology, University of Cambridge, Cambridge, United Kingdom
2 Department of Oncology, University of Cambridge, Cambridge, United Kingdom
3 Wolfson Brain Imaging Centre, University of Cambridge, Cambridge, United Kingdom
4 Department of Clinical Neurosciences, School of Clinical Medicine, University of Cambridge, Cambridge, United Kingdom
5 GSK Medicines Research Centre, Stevenage, United Kingdom

Introduction

Imaging and in vivo tracking of cells can provide much needed information and help non-invasively to improve the accuracy, efficacy and safety of cell and novel immune- modulator therapies. Zirconium-89 (t1/2 = 78.4 h) has been used recently to synthesize [89Zr]Zr(oxinate)41 (Fig.1), a suitable radiotracer for cellular tracking and imaging using Positron Emission Tomography (PET)2-4. This work presents an in vitro approach to quantify and determine the detection limit of T cells labeled with [89Zr]Zr(oxinate)4 utilizing a human PET/MR system.

Methods

[89Zr]Zr(oxinate)4 was synthesized according to published methods4. Jurkat cells were labeled with different amounts of radiotracer to generate different specific activities on cells (kBq/106 cells). Cell suspensions of 104, 105 and 106 cells per well were seeded on 6-well plates (Fig.1) and labelled with [89Zr]Zr(oxinate)4 by a 30 min incubation in both PBS and complete medium at 37ºC. Plates were imaged on a GE Signa PET/MR scanner for 30 min and reconstructed using parameters from a standard clinical protocol. The total activity in each well was determined by drawing regions of interest over each well on coronal images using OsiriX™. Cell suspensions were transferred into 1.5 mL Eppendorf tubes after the scan, measured in a well counter and correlated with imaging data.

Results/Discussion

[89Zr]Zr(oxinate)4 was synthesized at a yield of >95% in an aqueous solution. Total activities in each well ranged from 0.1-10.6 kBq. The specific activity (SA) of labeled cells ranged from 0.6 kBq/106 cells to 12.8 kBq/106 cells. Cells with a SA of 3.0 kBq/106 and above, could be visualized and quantified on PET images in a volume of 1.5 mL. At a SA below 1.0 kBq/106 no cells were detectable (Fig.2). There was a significant correlation between the total activity measured in wells on PET images and well counter measurements (r=0.995, p<0.001). Cells maintained their viability and stably retained 89Zr throughout and after the scan.

Conclusions

The presented results show the feasibility of quantifying and detecting Jurkat T cells labeled with [89Zr]Zr(oxinate)4 at different specific cellular activities. In an in vitro model approach we showed that a SA of 3.0 kBq/106 cells allowed visualization of cell suspensions on PET images. This has important implications for cell labeling of radiosensitive cell types (e.g. T-cells) that can only be labelled with a certain maximum activity.

References

1 Dalton Trans. 2014; 43(39): 14851–14857

2 Clin Cancer Res. 2017; 1; 23(11): 2759-2768

3 Mol. Ther. 2018; doi:https://doi.org/10.1016/j.ymthe.2018.10.006

4 J Nucl Med. 2018; 59(10):1531-1537

Acknowledgement

To Nick Bird and the Nuclear Medicine team for collaboration on the PET/CT scanner. This work is funded and supported by Cancer Research UK (CRUK) and GlaxoSmithKline (GSK).

Keywords: PET imaging, Direct cell labelling, Cell tracking, Zirconium-89
608

Lower temperature radiofluorination can be achieved by Al-18F chelation (#150)

Phuong T. Huynh1, Nisarg Soni1, Rammyani Pal1, Swarbhanu Sarkar1, Jeongsoo Yoo1

1 School of Medicine, Kyungpook National University, Molecular Medicine, Daegu, Republic of Korea

Introduction

18F is the most widely used radionuclide for PET imaging. However, peptides and antibodies cannot be radiolabeled with fluorine-18 without extra attachment of an auxiliary group, and are typically radiolabeled indirectly through two reaction steps. Recently, Al-18F complex has been developed as a new radiolabeling technique; however, the harsh conditions required for Al-18F complexation have restricted the widespread application of this technique to heat-sensitive biomolecules. Here we report optimized Al-18F radiofluorination enabling a successful radiolabeling at mild conditions.

Methods

Six different macrocyclic chelators having different backbone (NOTA, DOTA, NODA) as well as pendant arms were tested for efficient radiolabeling using Al-18F complex. We also optimized radiolabeling conditions to achieve radiofluorination at lower temperature by changing radioactive fluorine source, labeling buffer, pH and heating method.

Results/Discussion

By employing the chelator of NODA backbone bearing two acetate pendant arms and one benzyl-NCS moiety, more than 60% of Al-18F complexation was achieved at 30 °C in ammonium acetate buffer (pH 5.5).

Conclusions

These results represent an important step toward efficient radiofluorination of biomolecules at lower temperature. This optimized radiolabeling conditions might facilitate the application of Al-18F complexation in radiolabeling of heat-sensitive biomolecules.

References

  1. Laverman P, McBride WJ, Sharkey RM, Eek A, Joosten L, Oyen WJG, et al. A novel facile method of labeling octreotide with 18F-fluorine. J Nucl Med. 2010;51:454-61.
  2. McBride WJ, Sharkey RM, Karacay H, D'Souza CA, Rossi EA, Laverman P, et al. A novel method of 18F radiolabeling for PET. J Nucl Med. 2009;50:991-8.

Acknowledgement

This work was supported by an R&D program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (No. 2016R1A2B4011546, 2017M2C2A1014006, 2017R1D1A1B03033974, and 2017M2A2A6A02018506) and supported by a grant of the Korea Health Technology R&D Project funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C0221).

Keywords: Radiofluorination, fluorine-18, chelator, Al-F chelation
609

Laponite nanocrystal radiolabelling: characterisation of potential in drug delivery applications using PET and SPECT imaging probes (#465)

Gonçalo S. Clemente1, Benjamin Burke1, David Roberts1, Juozas Domarkas1, Christopher Cawthorne3, Christopher Wilson2, Steve J. Archibald1

1 University of Hull, PET Research Centre, Hull, United Kingdom
2 Listerdale Life Sciences, Nijmegen, Netherlands
3 KU Leuven, Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, and MoSAIC- Molecular Small Animal Imaging Centre, Leuven, Belgium

Introduction

Laponite is a commercially available synthetic nanosilicate clay, that forms small uniform nanodiscs. Laponite nanodiscs can electrostatically form small stacked arrays by sharing adsorbed sodium ions within interlayer regions. These stacks display formulation modifiable swelling characteristics upon hydration with a solute. The modifiable properties and low toxicity have driven the exploration of Laponites in biomedical applications.[1] In this work we demonstrate the use of short lived radioisotopes to characterise Laponite nanosystems in a robust and sensitive manner.

Methods

Laponite radiolabelling was attempted based on previous work from the group that showed the viability of chelator free, stable radiolabelling of silica surfaces.[2] Gallium is ideal, because it is cationic & strong Lewis acid and therefore acts as a direct replacement for sodium in the interlayer regions of the Laponite stacks. Additionally, this method doesn’t require prior modification of Laponite surfaces. Laponite was dispersed in ammonium acetate and added to processed & dried [68Ga]GaCl3. Labelling time determined by radio-TLC.

Results/Discussion

The core Laponite structure is not significantly affected by temperature, ionic strength or pH changes but these factors do influence the swelling, and hence physical characteristics and radiolabelling efficiency.[3,4] Radiolabelling incorporation increases with temperature, while for pH, maximum swelling and subsequently high 68Ga incorporation is reached by around pH 4. Above pH 5, formation of GaOH species hinder incorporation of 68Ga into Laponite. Intermediate ionic strengths offer the optimal radiolabelling efficiency. The [68Ga]Ga-Laponite has good stability, with >80% radiometal adsorbed to the nanoclay after 3h in 95% PBS and under incubation with apo-transferrin. 68Ga is relatively strongly bound to Laponite, with ~50% remaining attached to the nanoclay even in competition with EDTA. The PET tracer [68Ga]Ga-transferrin and SPECT tracer [99mTc]Tc-sestamibi are encapsulated in Laponite with adsorption efficiency, stability in competition and release capability investigated.

Conclusions

Laponite was radiolabelled with gallium-68 granting an understanding of how formulation/modification affects biodistribution. Mimicking of in vivo conditions to model aggregate disruption & therapeutic release in drug delivery applications. Encapsulation of bioactive species was studied to show the potential for using these radiolabelled species to understand uptake/release behaviour in drug delivery applications. 

References

[1] Guimarães, A. d. M. F.; Ciminelli, V. S. T.; Vasconcelos,W. L. Mater. Res. 2007, 10, 37-41.


[2] Burke, B. P.; Baghdadi, N.; Kownacka, A. E.; Nigam, S.; Clemente, G. S.; Al-Yassiry, M. M.; Domarkas, J.; Lorch, M.; Pickles, M.; Gibbs, P.; Tripier, R.; Cawthorne, C.; Archibald, S. J. Nanoscale 2015, 7 (36), 14889-14896.

[3] Hansen, E. L.; Hemmen, H.; Fonseca, D. M.; Coutant, C.; Knudsen, K. D.; Plivelic, T. S.; Bonn, D.; Fossum, J. O. Sci. Rep. 2012, 2, 1-4.


[4]Li, P.; Kim, N. H.; Siddaramaiah; Lee, J. H. Compos. part B-Eng. 2009, 40 (4), 275-283.

Acknowledgement

Daisy Appeal Charity for funding (Grant: DAhul0211, Registered Charity No. 1095652) and Dr Assem Allam for his generous donation and ongoing support to the PET Research Centre at the University of Hull.

Structure of Laponite & in-vivo imaging
(A) Structure of Laponite nanocrystals and their swelling behaviour (B) In vivo mouse image of [68Ga]Ga-laponite fused PET-CT coronal slice image at 80-90 minutes post-injection.
Keywords: Gallium-68, Laponite, PET, SPECT, nanoparticle
610

Practical Experiences on the Radiosynthesis of [18F]PSMA-1007 for PET Imaging of Prostate Cancer in Thailand (#21)

Jiradanai Sarasamkan1, Natphimol Boonkawin1, Sukanya Yaset1, Jiratthakorn Keeratipiriyakul1, Pannawattana Lertveerakun1, Pattanapong Kongsakorn1, Nuttiya Usaha1, Chanisa Chotipanich1

1 Chulabhorn Royal Academy, Chulabhorn Hospital, National Cyclotron and PET Centre, Bangkok, Thailand

Introduction

Prostate cancer is one of the most common cancers among men worldwide. Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein that overly expressed in prostate cancer which is a promising target for diagnostic and therapeutic purposes. Currently, [18F]PSMA-1007 has been drawing attention among the other PSMA candidates due to its several advantages such as high spatial resolution, high tumor uptake, lower renal clearance, and large-scale production.1 Therefore, our objective was to optimize one-step radiosynthesis of [18F]PSMA-1007 using a non-cassette based synthesizer.

Methods

The radiosynthesis of [18F]PSMA-1007 was carried out on the Synthra RNplus synthesizer using a non-cassette based technique as previously reported.2 [18F]-fluoride was trapped onto the QMA cartridge and eluted by the TBAHCO3 solution. Azeotropic drying was carried out at 98° C for 10 min followed by the addition of deprotected [18F]PSMA-1007 precursor in DMSO, and radiofluorination was performed at 85° C for 5 min. The reaction mixture was diluted with 8% ethanol and purified by passing and trapping through a series of PS-H+ and C18ec cartridges which were then washed with an additional 8% ethanol. The eluted final [18F]PSMA-1007 in 40% ethanol was passed through 0.22 μm sterile filter into the product vial containing a saline solution with the addition of sodium ascorbate as a stabilizer.

Results/Discussion

The radiosynthesis of [18F]PSMA-1007 using a non-cassette based technique was achieved in 40 min with the radiochemical yield between 10-30% (n.d.c., n = 23), the radiochemical purity > 99% and molar activity > 300 GBq/μmol at the end of synthesis. By using this method resulted in slightly lower radiochemical yield but good radiochemical purity compared to the method using a disposable cassette. After synthesis, the tracer remained intact for at least 8 hr (RCP > 95%). In addition, only a minute amount of residual solvents including acetonitrile, acetone, and DMSO were detected in the final solution, however, residual TBA could not be observed. Ethanol portion of injection solution was less than 6%.

Conclusions

Radiosynthesis of [18F]PSMA-1007 was accomplished by a one-step reaction using a non-cassette based synthesis module with good and reliable radiochemical yield and radiochemical purity.

References

  1. Giesel FL, Hadaschik B, Cardinale J, Radtke J, Vinsensia M, et al. F-18 labeled PSMA-1007: biodistribution, radiation dosimetry and histopathological validation of tumor lesions in prostate cancer patients. Eur J Nucl Med Mol Imaging 2017; 44: 678-688.
  2. Cardinale J, Martin R, Remde Y, Schafer M, Hienzsch A, et al. Procedures for the GMP-compliant production and quality control of [18F]PSMA-1007: A next generation radiofluorinated tracer for the detection of prostate cancer. Pharmaceuticals (Basel) 2017; 10. pii: E77. doi: 10.3390/ph10040077.

Acknowledgement

Our gratitude and most appreciation are especially extended to Prof. Eyal Mishani for technical assistance on radiotracer production.

Radiosynthesis of [18F]PSMA-1007

The general setup of a non-cassette based Synthra RNplus synthesizer for one-step radiosynthesis of [18F]PSMA-1007

[18F]PSMA-1007 PET/CT images of a patient with prostate cancer

[18F]PSMA-1007 PET/CT images of a patient with prostate cancer demonstrate strong enhanced PSMA-uptake in the right lobe of the prostate gland (red arrows) and bone metastases (green arrows).

Keywords: [18F]PSMA-1007, PET imaging, prostate cancer
611

Bismuth based Image-able Microspheres: First Application for use in Dual Energy Computed Tomography for Image-Guided Therapuetic Embolization  (#122)

Ayele H. Negussie1, 2, Quirina De Ruiter1, Hugh Britton3, Chrit Moonen4, Gert Storm2, 5, William Pritchard1, Andrew Lewis3, Bradford Wood1

1 National Institutes of Health, Radiology & Imaging Sciences, Bethesda, Maryland, United States of America
2 Universtiy of Twente, Institute for Biomedical Technology and Technical Medicine, Twente, Netherlands
3 Biocompatibles UK Ltd, Camberley, United Kingdom
4 Utrecht University, Center for Imaging Sciences, Utrecht, Netherlands
5 Utrecht University, Department of Pharmaceutics, Utrecht, Netherlands

Introduction

Image-able embolizing bead by covalent attachment of iodinated groups (LUMI) reported for treatment of human hepatocellular carcinoma. This product is proving useful for identification of tumor at risk of under treatment and confirmation of tumor targeting [1-3]. Such beads suspended in and delivered using iodinated contrast agent (CA). This make differentiation of bead from CA is impossible with clinical Cone Beam CT. Therefore, microsphere were fabricated with covalently attached a chelated bismuth compounds, with potential to differentiate the bead from suspension CA using Dual energy CT.

Methods

Synthesis of Bismuth-Based Embolic microspheres

Bifunctional linker consisting of aromatic or aliphatic moieties were conjugated with a macrocycle (1) and acetalated on to PVA-beads (BTG Int. group) sieved into different sizes as reported previously[4, 5]. Bismuth chelation was performed using Bi(OTf)3in water at pH 8-9 or organic solvent with pyridine at 55°C. The resulting radiopaque beads were thoroughly washed with water or 1% (w/v) K2HPO4 in NMP (250 mL). MicroCT imaging for different bead sizes were performed as reported previously[3]and material decomposition of different concentration of bismuth from iodine CA (mixed in 1% agarose phantom) was performed using two x-ray sources operating at 80 kVp and 150 kVP and two detectors (Siemens Healthcare) as described in [6].

Results/Discussion

Bismuth chelating agents were synthesized by alkylating 1with bifunctional linkers (Scheme 1). The resulting linker containing macrocycle acetalated on to the beads as reported previously [5]and chelated with Bi(CF3SO3)3. The radiopacity of the beads is uniform across bead sizes (3310-4081HU) but slightly less than reported for LUMI (4178-4553HU). There is however, a significantly greater amount of I2on an atom basis in LUMI (150 mg/mL) compared to Bi in the current formulation (50-70 mg/mL), demonstrating that bismuth is sufficiently radiodense to provide useful levels of opacity at lower levels of incorporation (3.5 times more iodine for LUMI at 1.2 mole of I2versus 0.34 mole of Bi). This level of radiopacity therefore, is more than adequate for visualization under clinical CT imaging. Material decomposition of different concentration of iodinated CA and of bismuth beads were evaluated with clinical DECT scanner. We have shown here that DECT can differentiate both components (Fig.1)

Conclusions

Methods for the synthesis of bismuth microspheres from clinically approved embolizing agent presented. The radiopacity was confirmed by Micro-CT while material decomposition from iodinated CA performed using DECT. This technology may further improve IGTE by providing real time information on the accuracy of treatment, therapeutic dose map in a given location as well as post treatment therapeutic needs on liver tumor with risk of under-treatment.

References

  1. Levy, E.B., et al., First Human Experience with Directly Image-able Iodinated Embolization Microbeads.Cardiovascular and Interventional Radiology, 2016. 39(8): p. 1177-1186.
  2. Duran, R., et al., A Novel Inherently Radiopaque Bead for Transarterial Embolization to Treat Liver Cancer - A Pre-clinical Study.Theranostics, 2016. 6(1): p. 28-39.
  3. Negussie, A.H., et al., Synthesis and characterization of image-able polyvinyl alcohol microspheres for image-guided chemoembolization.Journal of Materials Science-Materials in Medicine, 2015. 26(6).
  4. Mondal, R. and A.K. Mallik, Recent Applications of Potassium Carbonate in Organic Synthesis.Organic Preparations and Procedures International, 201 46(5): p. 391-43
  5. Ashrafi, K., et al., Characterization of a novel intrinsically radiopaque Drug-eluting Bead for image-guided therapy: DC Bead LUMI™.Journal of Controlled Release, 2017. 250: p. 36-47.
  6. Danad, I., et al., New Applications of Cardiac Computed Tomography Dual-Energy, Spectral, and Molecular CT Imaging.Jacc-Cardiovascular Imaging, 2015. 8(6): p. 710-723.

Acknowledgement

This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) and a Cooperative Research and Development agreement between the NIH and Biocompatibles - BTG International group. NIH and BTG have intellectual property in the field.

Synthesis of Bi-Based Embolic Beads

Scheme 1. Synthesis of bismuth beads with aromatic or aliphatic linker between the bead and the macrocycle. 

Reagent and condition: a) K2CO3/DMF, b) HCl/dioxane, c) MeSO3 in DMF or DMSO, 50 C, d) Bi(CF3SO3)3, pH 8-9.

Linear Attenuation Coefficients of Bismuth and iodine

Figure 1. Relation in attenuation coefficients between the low energy acquisition (80 kV) and high energy acquisition (150 kV) for iodine contrast media, Bismuth beads and a mixture of both at various relative concentrations, 100% iodine (I) corresponds with 40 mg/mL and 500 microL Bismuth Beads (Bi) with 500 microL imbedded in 1% agarose.

Keywords: HCC, Image-guided TACE, DECT, Bismuth, CT

PET/SPECT/CT & Multimodal | New Probes I

Session chair: Franck Denat (Bourgogne, France); Renata Mikolajczak (Otwock, Poland)
 
Shortcut: PW03
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

635

The uptake of a new protein tau radiomarker in cortex of P301S mice corresponds to increase in oxidative stress and loss in mice condition (#185)

Luka Rejc1, Vanessa Gómez-Vallejo1, Ana Joya1, 2, Aitor Lekuona1, Xabier Ríos-Anglada1, Zuriñe Baz1, Abraham Martin2, Jordi Llop1

1 CIC biomaGUNE, Radiochemistry and Nuclear Imaging, San Sebastian, Spain
2 Achucarro, Laboratory of Neuroimaging and biomarkers of inflammation, Leioa, Spain

Introduction

The real effect of different biomarkers such as hyperphosphorylated tau protein (pTau) deposition, inflammation, oxidative stress, etc. on development of neurodegenerative diseases (NDs) is not yet well determined. This is important to develop reliable tools that would be able to follow the disease progression. In this work, the possibility of detection of pTau in P301S mice with a new PET imaging probe [18F]1is examined. pTau deposition was compared to inflammation and oxidative stress in brains of these mice, using [18F]DPA-714 and [18F]FSPG, respectively.

Methods

A recently reported radiotracer for the detection of pTau (Figure 1A) was synthesized as previously described.1Metabolite studies were performed on blood plasma and brain homogenates of wild type (WT) mice using radio-HPLC. The biodistribution of the radiotracer in major organs was assessed by PET imaging. Longitudinal PET imaging studies were conducted with [18F]1, [18F]DPA-714 and [18F]FSPG on P301S Tg mice that overexpress pTau in brain from 6 months of age, and the results compared to age-matched healthy control mice (B6C3F1/J). In vitroimmunohistochemistry staining was performed to confirm the results observed in vivo.The loss in locomotor activity and muscle tone was assessed in P301S and WT mice as previously described2to determine the disease progression. 

Results/Discussion

The compound [18F]1 (Figure 1A) readily crossed blood brain barrier (BBB) and underwent metabolic decay, resulting in 14% of the parent compound left in blood plasma 30 minutes after injection. Rapid clearance from blood plasma promised high potential for the use of selected probe in vivo. PET imaging studies with [18F]1 showed a steady increase of the ratio between the relative uptake in P301S mice cortex compared to healthy controls (Figure 1B). Similar trend was observed for [18F]FSPG, which corresponds to oxidative stress-induced increase in activity of cysteine-glutamate pathway, while TSPO radiomarker [18F]DPA-714 showed less prominent difference between P301S and WT mice, probably due to age-related inflammation in healthy mice. The increase in pTau deposition and oxidative stress corresponded well with the loss in locomotor activity and muscle tone in diseased mice (Figure 1C).

Conclusions

Radiotracer [18F]1 possesses favorable bio-chemical properties for the use in PET imaging. It binds to pTau and crosses the BBB. Comparison between P301S and WT mice show accumulation of the radiotracer in cortex over time. The pTau deposition seems to follow the same time-trend as oxidative stress and loss of condition of the diseased mice, while overexpression of TSPO with age seems to be common for both mouse models.

References

  1. L. Rejc et al. J. Med. Chem. 60, 21, 8741-8757.
  2. S. J. Guyenet et al. J. Vis. Exp.2010, 39, 1787–1790.

Acknowledgement

The work has been partially funded by the Spanish Ministry of Economy and Competitiveness, grant number CTQ2017-87637-R.

Figure 1
(A) The structure of the labelled diphenylacetylene analogue 1; (B) a comparison of cortex uptake ratios between [18F]1, [18F]FSPG and [18F]DPA-714 radiotracers;(C) behavioural score of diseased (pTau) and control (WT) mice
Keywords: protein Tau, P301S, inflammation, oxidative stress
636

Gallium-68-radiolabeling performance of azamacrocycle-modified peptides: a conception for improving the design of future peptide-based PET tracers (#414)

Thomas Ebenhan1, 2, Amanda H. Mdlophane1, 2, Janine Suthiram1, 2, Werner du Toit3, Judith Wagener2, Biljana Marjanovic-Painter2, Hendrik G. Kruger4, Thavendran Govender4, Mike M. Sathekge5, Jan R. Zeevaart6, 2

1 University of Pretoria, Nuclear Medicine, Gezina/ Pretoria, South Africa
2 NECSA, Radiochemistry, Pelindaba/Brits, South Africa
3 Sefako Makgatho Health Sciences University, School of Pharmacy, Medunsa, South Africa
4 University of KwaZulu-Natal, CPRU, School of Health Sciences and School of Chemistry and Physics, Durban, South Africa
5 University of Pretoria and Steve Biko Academic Hospital, Nuclear Medicine, Gezina/ Pretoria, South Africa
6 North West University, Department of Science and Technology, Preclinical Drug Development Platform, Potcheftroom, South Africa

Introduction

For the design of novel radiometal-based tracers in nuclear imaging the azamacrocyclic chelators (AMC) 1,4,7,10-tetraaza-cyclododecane-tetraacetic acid (DOTA), 1,4,7-triaza-cyclononane-1,4-7-triacetic acid (NOTA) or 1,4,7-triaza-cyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) are favorable vectors. AMC may warrant a robust and simple complexation of diagnostic (gallium-68 (68Ga)) or therapeutic (lutetium-177) isotopes and the uncompromising conjugation of targeting vehicles (i.e. peptides) (1). The promiscuity of 68Ga-radiolabeling to changes of vector or peptide moieties is presented.

Methods

Ten different peptides (RP1-RP10) were modified with A) DOTA B) NODAGA or C) NOTA. RP9 was functionalized with all chelators (A-C); RP10 was modified with A/ B. Routine fractionated 68Ge-68Ga-generator elution was followed by 68Ga-radiolabeling (sodium acetate buffer) (2). Incubation (10-15 min at 80-95°C) was followed by HPLC/ITLC analyses. 68Ga-citrate and 68Ga-PSMA11 were used as references. Peptide characterization compared AMC-peptide size, net charge and number of amino acids (AA) with the radiolabeling performances by way of optimal pH and crucial precursor molarity were determined (Table 1). The analyses of 68Ga-AMC-peptides further included: tracer concentration to attain 50% labeling efficiency (LEF50), optimal LE%,specific activity (SA) and the maximal achievable SA(SAmax).

Results/Discussion

All AMC-peptides showed moderate-high labeling (85.7±6.5, N=13), despite of peptide size, net charge, number of AA or labeling pH; however, larger DOTA-peptides labeled better (8-37 AA; 86.9±5.2, N=4; P=0.02) than smaller DOTA-peptides (3-7 AA; 76.7±0.9, N=3). Contrary to the larger AMC-peptides, smaller AMC-peptides (3-8 AA; N=7) required structural enhancements and linkers for a successful 68Ga-radiolabeling. 68Ga-NOTA/NODAGA-peptides (N=6) showed significantly better specific activities (2.0±0.6 mCi/µmol; P=0.001) due lower procurer amounts (15.5±2.7 µM; P=0.01) required and lower tracer molarity to yield a LEF50 threshold (10.1.3±1.4 µM; P=0.01) compared to 68Ga-DOTA-peptides (N=7; SA= 0.9±0.2 mCi/µmol; precursor= 29.3±9.9 µM; LEF50= 20.7±7.5 µM). The overall AMC performance was A< B and A< C< B for PR9 and PR10, respectively. The SAmax (0.56-2.63) correlated with the number of AA (r2=0.79). 68Ga-NODAGA-PR10 showed instant (>95%) labeling similar to 68Ga-citrate and 68Ga-PSMA11.

Conclusions

This original investigation across a panel of different AMC-peptides demonstrated high 68Ga-radiolabeling regardless of peptide size, charge or structural amendments; however, crucial differences in the 68Ga-radiolabeling performance can be made by using NODAGA or NOTA as alternatives to DOTA.

References

1) I. Velikyan, Theranostics (2014), 4(1):47-80

2) T. Ebenhan et al., Nucl Med Biol (2014), 41(5):390-400

Acknowledgement

none

Tabulated overview of results
Summary of results achived for peptide characterization and 68Ga-radiolabeling performance for references and research peptides (RP1-RP10).
Keywords: 68-gallium radiolabeling, functionalized peptides, DOTA, NOTA, NODAGA
637

Development of 68Ga and 18F labelled CXCR4 targeting Pentixafor derivatives: Increasing simplicity to increase widespread availability (#473)

Joazas Domarkas1, Rhiannon Lee1, Jarrad Plater1, Isaline Renard1, Ben Burke1, Michelle Ma2, Philip Blower2, Chris Cawthorne1, Steve Archibald1

1 University of Hull, Positron Emission Tomography Research Centre, Hull, United Kingdom
2 King's College London, Division of Imaging Sciences and Biomedical Engineering, London, United Kingdom

Introduction

68Ga-Pentixafor has been successfully used to image chemokine receptor CXCR4 expression in multiple cancers1, however, its clinical use is limited by the existing production methods because of the DOTA chelator.2 Here, we present the development of novel Pentixafor derivatives which may be more appropriate and facilitate wider availability; a 68Ga-THP derivative would allow efficient kit like production in a radiopharmacy at room temperature whilst an 18F derivative would allow remote supply of the tracer from a single production site.

Methods

The core cyclic pentapeptide (Fig 1) was prepared by solid phase peptide synthesis and cyclised / functionalised with DO3A / THP chelator3 in solution. 68Ga3+ was introduced either by heating ([DO3A]-Pentixafor, HEPES pH 3.5, 90oC, 10 min) or at room temperature ([THP]-Pentixafor, NaOAc pH 6-7, RT, 5 min.). 18F label was introduced via Cu(I) catalysed “click” cycloaddition using azide functionalised precursor and 18F labelled HPLC purified prosthetic prepared from a tosylated precursor and K222/K18F. Lipophilicity (LogD4.7) was measured by shaken flask method. Tracers were purified by HPLC, formulated in PBS and administered to U78CXCR4 xenograft bearing mice imaged by PET/CT (Fig 2).

Results/Discussion

68Ga-Pentixafor was radiolabelled with 22 ± 13% (n=5, decay corrected) yield with the preparation yield of 5 ± 2% (n=5). [68Ga-THP]-Pentixafor was prepared with 54% decay corrected yield with preparation yield of 17%. Both preparations started from 100-130 MBq of 68GaCl3 and were carried within 60 min from start of labelling. 18F prosthetic group was radiolabelled with 36% decay corrected yield (starting from 2 GBq of K222/K18F) and clicked 18F-Pentixafor obtained with 7% non-decay corrected yield (calculated from 18F-prosthetic). 18F-Pentixafor was found to be highly lipophilic compared to 68Ga-Pentixafor (LogD4.7 = - 0.14 ± 0.02 vs LogP = - 2.90 ± 0.03).2 PET images (Fig 2) showed high tumour and low liver / gut uptake for 68Ga-Pentixafor with mainly renal excretion attested by accumulation in the bladder. For [68Ga-THP]- and 18F-Pentixafor, tumour uptakes were significantly reduced, with excretion switching from renal towards biliary (reduced bladder / increased liver uptake).

Conclusions

Introducing a THP chelator increases tracer lipophilicity which is detrimental to tracer’s tumour accumulation, leading to a nonspecific liver and gut uptake. Initial efforts towards fluorine-18 derivatives results in similar suboptimal clearance. Introduction of more hydrophilic linkers in tracer design reducing overall lipophilicity currently under investigation is expected to improve biodistribution while retaining CXCR4 targeting properties.

References

1K. Philipp-Abbrederis, K. Herrmann, S. Knop, M. Schottelius, M. Eiber, K. Luckerath, E. Pietschmann, S. Habringer, C. Gerngross, K. Franke, M. Rudelius, A. Schirbel, C. Lapa, K. Schwamborn, S. Steidle, E. Hartmann, A. Rosenwald, S. Kropf, A. J. Beer, C. Peschel, H. Einsele, A. K. Buck, M. Schwaiger, K. Gotze, H. J. Wester and U. Keller, EMBO Mol. Med., 2015, 7, 477-487.

2E. Gourni, O. Demmer, M. Schottelius, C. D'Alessandria, S. Schulz, I. Dijkgraaf, U. Schumacher, M. Schwaiger, H. Kessler and H.-J. Wester, J. Nucl. Med., 2011, 52, 11, 1803-1810.

3M. T. Ma, C. Cullinane, C. Imberti, J. Baguna Torres, S. Y. A. Terry, P. Roselt, R. J. Hicks and P. J. Blower, Bioconjugate Chem., 2016, 27, 309-318.

Acknowledgement

Authors would like to thank Dr Assem Allam for his generous contribution to University of Hull PET research centre.

Fig 2. Dynamic PET
Coronary maximum intensity projections, 40-66 min post administration, U87CXCR4 xenograft mouse model (arrow colour code: red – tumour, white – liver, green – gut, yellow – bladder).
Fig 1. Structure of core peptide functionalised with respective chelators / 18F label.
Keywords: Pentixafor, CXCR4, Nuclear Imaging
638

A Tris(hydroxypyridinone)-phospholipid Conjugate for Pretargeted PET Imaging of Liposomal Nanomedicines (#314)

Aishwarya Mishra1, James Choi2, Rafael T. M. de Rosales1

1 King's College London, School of Biomedical Engineering & Imaging Sciences, London, United Kingdom
2 Imperial College London, Faculty of Engineering, Department of Bioengineering, Royal School of Mines, South Kensington Campus, London, United Kingdom

Introduction

Liposomal nanomedicines have emerged as efficient drug carriers, which can passively accumulate in tumours1. However, the variable uptake among tumour types and patients may results in variable clinical efficacies.2 Nuclear imaging can provide pre-therapy information on their whole-body biodistribution of individual patients, at the cost of relatively high radiation doses. A pretargeted imaging approach (Fig. 1) could further improve our ability to study nanomedicine biodistribution and efficacy over time with the advantage of lower radiation doses compared to conventional imaging.

Methods

A THP-phospholipid (THP-PL) conjugate was synthesized via conjugation of DSPE-PEG(2k)-NH2 (PL) to THP-NCS in DMSO/DIPEA, RT for 24 h. THP-PL was purified using dialysis against saline followed by deionized water overnight and characterized using NMR. THP-PL was radiolabelled by reacting THP-PL (10 µL, 26 µM in EtOH) to 68GaCl3 (90 μL, 3.4 M Na2CO3 pH 6.5) at RT for 30 min. Radiolabelling was analysed by radio-TLC and size exclusion chromatography. THP-PL was inserted into thephospholipid bilayer of empty PEGylated liposomes (EPLs) by incubation of THP-PL (5 mol%, 100 µL 20% EtOH/H2O) with EPLs (100 µL) at 50oC/30 min to give THP-PL-EPLs. THP-PL-EPLs were radiolabelled with 68Ga and purified using size exclusion chromatography followed by characterization by dynamic light scattering (DLS).

Results/Discussion

The desired THP-phospholipid conjugate (THP-PL, Fig. 2A) was successfully synthesized via electrophilic acylation of DSPE-PEG NH2 (PL) with isothiocyanate derivative of THP. The conjugate was purified by dialysis giving a yield of 76%. The NMR of the purified conjugate shows the formation of a thiourea linkage and disappearance of the amine resonance from PL (Fig. 2B). Unlike PL, THP-PL shows a high affinity towards 68Ga with 85 % radiochemical yield at 30 μM concentrations, further demonstrating successful THP conjugation. The incubation of 3 molar % of THP-PL conjugate with EPLs resulted in its incorporation into the lipid bilayer (THP-PL-EPLs). THP-PL-EPLs showed efficient 68Ga radiolabelling (84 ± 3%), in contrast to EPLs (3.0  ± 0.2 %) (Fig. 2C).

Conclusions

The primary component of a liposome pretargeting system (THP-PL), including the efficient 68Ga chelator THP,3 for PET imaging has been successfully synthesized and characterized. THP-PL can be incorporated into preformed liposomes, providing them with efficient radiolabelling properties. Ongoing work includes studies evaluating the possibility of pretargeted PET imaging of THP-PL modified liposomes in vitro and in vivo.

References

1.         Shi, J. et al. Nature Reviews Cancer (2017). doi:10.1038/nrc.2016.108

2.         Man, F. et al.  Mol. Imaging Biol. 1–13 (2018). doi:10.1007/s11307-018-1255-2

3.         Berry, D. J. et al. Chem. Commun. (Camb). 47, 7068–70 (2011).

Acknowledgement

Funding: EPSRC, King's College London and Imperial College London

Proposed strategy for pretargeted imaging with 68Ga

Schematic representation of the proposed pretargeting strategy showing the the THP-phospholipid modified liposomes as the primary vector followed by 'free' 68Ga as the secondary vector targeting primary vector in vivo.

Structure and characterisation of THP-PL and radiolabelling
(A) Structure of THP-PL; (B) 1H-NMR in CDCl3 of PL (blue) and THP-PL (red) showing presence of THP; (C) Comparison of 68Ga-radiolabelling yields of liposomes where THP-PL has been incorporated (THP-PL-EPLs) with unmodified, empty PEGylated liposomes (EPLs); demonstrating that THP-PL can be incorporated into PEGylated liposomes providing them with radiolabelling properties.
Keywords: liposome, pretargeting, PET, nanomedicine, Gallium-68
639

Synthesis and evaluation of [68Ga]Ga-THP-Pam: A bone-seeking PET agent with rapid, facile radiolabelling (#222)

George P. Keeling1, Belinda San Juan1, Philip J. Blower1, Gareth Smith2, Samantha Y. A. Terry1, Rafael T. M. de Rosales1

1 King's College London, Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom
2 Theragnostics, UK Office, Bracknell, United Kingdom

Introduction

Bone metastases and vascular calcification can be detected in vivo using imaging agents such as fluoride (F-) and bisphosphonates (BP).1 [18F]F- is the standard clinical radiotracer to image these lesions with PET but its use is limited to areas close to a cyclotron. With the aim of developing a cyclotron-independent kit-based PET agent for bone disease/vascular calcification, we report the synthesis, 68Ga radiolabelling and preliminary in vitro/vivo evaluation of a conjugate of tris(hydroxypyridinone) with the aminobisphosphonate pamidronate (THP-Pam, Fig. 1A).

Methods

Pamidronate (Pam) was synthesised following a published method.2 Pam was conjugated to THP-NCS in H2O and TEA, 90°C, pH 9. THP-Pam was purified using reverse-phase chromatography, and analysed using HPLC/MS/NMR. [68Ga]Ga-THP-Pam was radiolabelled by reacting THP-Pam (5 μL, 0.8 mM in H2O) with 68GaCl3 (250 μL in 0.1 M HCl) in the presence sodium bicarbonate (26 μL, 1 M in H2O) for 5 min at rt. Radiolabelling yield was assessed by radio-HPLC/ITLC and serum stability by size exclusion chromatography. In vitro binding of [68Ga]Ga-THP-Pam to calcium materials was tested and compared with two bone-seeking radiotracers ([68Ga]Ga-BPAMD; [18F]F-). In vivo imaging was performed in a Mediso nanoScan PET/CT 15 min after i.v. injection of 22 MBq of [68Ga]Ga-THP-Pam in one healthy female rat (2 months).

Results/Discussion

THP-Pam was synthesised after reaction of excess Pam with THP-NCS. Excess reactants/side-products were removed by reverse-phase chromatography, leading to the pure compound (MS/NMR/HPLC - Fig. 1B-D) as a white solid (1.3 mg, 53%). Radiolabelling of THP-Pam with 68Ga was fast (5 min) and mild (room temperature), leading to 97% radiochemical yields (Fig. 1 E-F) (non-optimised specific activity 6.44 TBq/g). In vitro testing of binding to calcium minerals (Fig. 2A) indicated strong binding to hydroxyapatite (HAp; main bone mineral) that was fully blocked in the presence of excess non-labelled BP. While HAp binding levels were similar to those of [68Ga]Ga-BPAMD and [18F]F-, binding of [68Ga]Ga-THP-Pam was higher in other tested calcium minerals. Serum stability studies indicate no transchelation of 68Ga for at least 2 h (Fig. 2B). In vivo data from a proof-of-concept study showed high bone uptake (10.3% ID/g), a bone-to-blood ratio of 23.60 and renal clearance/urinary excretion (Fig. 2C-D).

Conclusions

THP-Pam - a novel bisphosphonate based on the 68Ga chelator THP - has been synthesised, radiolabelled and assessed in vitro/vivo. The simple, mild and efficient radiolabelling, as well as the high binding to calcium minerals (in vitro) and bone (in vivo) indicate that [68Ga]Ga-THP-Pam could provide for the unmet clinical need for a kit-based, generator-produced PET imaging agent for bone diseases and arterial calcification.

References

  1. L. E. Cole et al., Adv. Drug Del. Rev., 2016, 99, 12-27.
  2. US. Pat., 7411087, 2008.

Acknowledgement

Funding: EPSRC and Theragnostics Ltd

THP-Pam Analysis and Radioanalysis
A: Structure of THP-Pam. B: ESI-MS of THP-Pam. C: 31P NMR of THP-Pam. D: RP-HPLC of THP-Pam. E: Radio-HPLC of [68Ga]Ga-THP-Pam in comparison to free 68Ga. F: Radio-ITLC of [68Ga]Ga-THP-Pam in comparison to free 68Ga.
Biological Evaluation of gallium-68 labelled THP-Pam
A: Binding of [68Ga]Ga-THP-Pam, [68Ga]Ga-BPAMD, [18F]F- to an array of calcium minerals in saline and in the presence of blocking agents. B: Serum stability of [68Ga]Ga-THP-Pam analysed by size exclusion chromatography. C: Biodistribution of [68Ga]Ga-THP-Pam in a healthy rat (n = 1). D: PET/CT image acquired over 1 h of a healthy rat injected with 22 MBq [68Ga]Ga-THP-Pam, 15 min post-injection.
Keywords: PET, gallium-68, bones, bisphosphonates, THP
640

Dual mode SPECT/optical bioimaging with mitochondria targeting 99mTc(I) radiolabelled naphthalimide conjugate (#484)

Juozas Domarkas1, Adam Day2, Shubhanchi Nigam1, Isaline Renard1, James Thompson1, Ian Fallis2, Gurmit Bahra3, Petra Oyston3, Christopher Cawthorne1, Benjamin Burke1, Stephen Archibald1, Simon Pope2

1 University of Hull, PET Research Centre, Hull, United Kingdom
2 Cardiff University, School of Chemistry, Cardiff , United Kingdom
3 Defence Science and Technology Laboratory, Salisbury, United Kingdom

Introduction

Multimodal imaging agents are designed to combine strengths of individual modalities, offering clinically useful information not available by a single method. Combined SPECT / optical imaging (OI) agents can provide early phase high sensitivity biodistribution data using SPECT complemented by OI used as a sensitive visual guide during surgery. Here, we report the development of dual SPECT/OI mitochondria targeted agent, based on naphthalimide fluorophore1 functionalised with dipicolylamine Tc(I) chelator2.

Methods

1,8-Naphthalimide pharmacophore was functionalised with dipicolylamine chelator (Fig 1). Rhenium complexes were formed using fac-[Re(CO)3(MeCN)3]BF4 and their optical properties verified by luminescence spectroscopy. Mitochondrial localisation was confirmed by CFM in MCF-7 and H9c2 cells using Mitotracker Deep Red (MDR) dye. Generator eluted [99mTc][TcO4]- was reduced to fac-[99mTc][Tc(CO)3(H2O)3]Cl following the published procedure3 and radiolabelling conditions, regarding the amount of precursor, pH, temperature and time, were optimised. Lipophilicity of the selected radiotracer was measured and its mitochondrial uptake was investigated by hot ligand uptake assay on freshly isolated rat heart mitochondria. The in vivo biodistribution in naïve mice was assessed by SPECT/CT imaging.

Results/Discussion

Six functionalised naphthalamide ligands and corresponding Re(I) complexes were prepared, purified and characterised by NMR and MS (Fig 1). All complexes luminesce at 505-550 nm with the position of the emission maximum moderately affected by the nature of the linker and by solvent polarity. Lifetimes were measured at < 15 ns and quantum yields up to 67%. All, except L5, ligands suffered from poor water solubility compromising their radiolabelling in aqueous conditions, thus, only the most hydrophilic tracer, L5, was radiolabelled and investigated further. The tracer was found to be moderately lipophilic (logD7.4 0.67 ± 0.06) and accumulated in isolated mitochondria in an MMP dependent manner (56 ± 6% admin. dose). Confocal microscopy confirmed mitochondrial localisation (66% overlap with MDR). Cardiac uptake was not observed in the in vivo imaging experiments, which showed rapid, predominantly biliary, clearance (Fig 2).

Conclusions

Water soluble fac-[99mTc(CO)3(L5)] possesses optical characteristics required for OI tracer (visible fluorescence, good Stokes’ shift, high quantum yield) and can be effectively radiolabelled with 99mTc(I) for high sensitivity imaging by SPECT. Further structural optimisation would be required for either cardiac or tumour imaging in order to increase circulation time and improved organ specific uptake.

References

1E.E. Langdon-Jones, D. Lloyd, A.J. Hayes, S.D. Wainwright, H.J. Mottram, S.J. Coles, P.N. Horton, S.J.A. Pope, Inorg. Chem., 2015, 54, 6606.

2S. Imstepf, V. Pierroz, P. Raposinho, M. Felber, T. Fox, C. Fernandes, G. Gasser, I. R. Santos and R. Alberto, Dalton Trans., 2016, 45, 13025–13033.

3R. Alberto, R Schibli, A. Egli, P.A. Schubiger, T.A. Kaden, J. Am. Chem. Soc., 1998, 120, 7987.

Acknowledgement

We thank Dr Assem Allam for his generous contribution to University of Hull PET Research Centre and the staff of the EPSRC Mass Spectrometry National Service (at Swansea University) for MS analysis. AD, IF and SP thank Defence Science and Technology Laboratory for financial support.

Fig 1. Naphthalimide - dipicolylamine conjugates
Fig 2. fac-[99mTc(CO)3(L5)] SPECT/CT static scan

Maximum intensity projections coronal and sagittal views. Arrow colour code: red – gall bladder, blue – kidney, yellow – gut, green – bladder.

Keywords: multimodal SPECT/OI, Tc(I), mitochondrial imaging
641

The synthesis and validation of a multimodal PET/fluorescence zinc sensing probes as potential imaging agents for prostate cancer (#224)

George Firth1, Thomas W. Price1, Graeme J. Stasiuk1

1 University of Hull, Department of Biomedical Sciences, Hull, United Kingdom

Introduction

Prostate cancer (PCa) is the 2nd most common cancer worldwide for males, and the 4th most common cancer overall, accounting for 15% of all male cancers in 20121. PCa is asymptomatic in its early stages and advances in medical imaging are improving diagnosis, however novel simple non-invasive diagnostic tests are needed to catch the disease early. Zinc has emerged as a promising diagnostic target in PCa progression as it’s accumulation in PCa cells is significantly lower compared to normal prostate2.

Methods

2-((2-(2-fluoroethoxy)ethyl)amino)-N-(quinolin-8-yl)acetamide was synthesised in seven step to produce AQA-F which was validated for zinc binding using UV-vis and fluorescence. its toxicolgy was studied in PC-3 and RWPE1 cell lines, fluorescent micrscopy studies in PC-3 and RWPE1 cells where undertaken , with and without zinc. radiolabelling of AQA-Z to prodice 18F-AQA-f was undetaken in a two step process.

Results/Discussion

We herein propose a fluorescent imaging probe, 2-((2-(2-fluoroethoxy)ethyl)amino)-N-(quinolin-8-yl)acetamide (AQA-F) shown in figure 1, that changes emission profile when binding the metal zinc. A stokes shift of 90 nm can be observed upon zinc binding, enhancing the emission wavelength from 410 to 500 nm, with a Kd of 15.2x10-6 M. A 6-fold increase in fluorescence intensity is observed upon the addition of one equivalent of zinc. This significant increase in fluorescence intensity is specific to zinc and is not observed with the other transition metals that were tested. AQA-F can therefore be translated into in vitro studies where the endogenous localisation and uptake of the probe can be assessed. This research sets the foundation for a zinc sensing probe that has the ability to be radiolabelled with 18F in 8.6% radiochemical yield with a 97% radiochemical purity and can be utilised for the diagnosis of prostate cancer.

Conclusions

We have developed the first dual modal PET/fluorescent zinc sensor, with 8.6% radiochemical yield. Which could be used as a tool for the detection of prostate cancer.

References

1. Hassanipour-Azgomi, S., et al. (2016). Incidence and mortality of prostate cancer and their relationship with the Human Development Index worldwide. Prostate International, 4(3):118–124.

2. Franklin, R. B., et al. (2005) hZIP1 zinc uptake transporter down regulation and zinc depletion in prostate cancer. Molecular Cancer, 4(32):1-13.

3. G. Firth, T. W. Price, M. Kinnon, C. J. Eling, N. J. Long, J. Sturge and G. J. Stasiuk “A 18F Radiolabelled Zn(II) Sensing Fluorescent Probe”, Chem. Commun.,, 2018, 54, 3227-3230.

Figure 1
 Cellular studies in prostate cells Fluorescence spectra for AQA-F (0.1 mM) in HEPES buffer (10 mM, pH = 7.68) with increasing concentrations of zinc (0-3 equivalences) and radiotrace of [18F]AQA-F.
Keywords: PET, Zinc, Prostate cancer, dual modal, fluorescence
642

Molecular PET-CT imaging for evaluation of liver function and volume in a hepatectomy mouse model (#220)

Freya Svedberg1, 2, Benedicte Descamps3, Sara Neyt4, Stef De Lombaerde5, Bert Vandeghinste4, Pieter Mollet4, Roel Van Holen4, Christian Vanhove3, Martin Guilliams1, 2

1 VIB, Center for Inflammation Research, Laboratory of Myeloid Cell Ontogeny and Functional Specialisation, Gent, Belgium
2 Ghent University, Department of Biomedical Molecular Biology, Gent, Belgium
3 Ghent University, IBiTech-MEDISIP-INFINITY, Gent, Belgium
4 MOLECUBES NV, Gent, Belgium
5 Ghent University, Laboratory of Radiopharmacy, Gent, Belgium

Introduction

The liver has an amazing regenerative capacity after injury and cell tissue damage, this unique function can be demonstrated in a partial hepatectomy (PHx) mouse model. Here, molecular imaging was used to evaluate liver volume and liver function in a PHx mouse model. Contrast enhanced µCT with Exitron 6000, a preclinical contrast agent, was used to determine liver volume and 3β-[18F]-fluorocholic acid (3β-[18F]-FCA), a bile acid analogue, was used to visualize and quantify liver function with PET as previously described (1).

Methods

100 µL Exitron 6000 was injected 6 hours prior to imaging (n=3 sham, n=3 PHx). High resolution µCT data were acquired and reconstructed with a voxel size of 100 µm using an iterative reconstruction algorithm (ISRA) in order to calculate liver volume automatically using PMOD v3.9. To evaluate liver function, mice (n=3 sham, n=3 PHx) were injected with 5.07 ± 0.74 MBq 18F-FCA. A dynamic 60 minute PET acquisition was performed, followed by µCT for anatomical correlation. The dynamic PET data were reconstructed in 54 frames (OSEM, 30 iterations, 400 µm voxel size, CT based attenuation correction) of 15-120 seconds. Regions of interest were drawn around the liver, left ventricle, gallbladder and intestines using PMOD and the corresponding time activity curves (TACs) were obtained.

Results/Discussion

A significant decrease in liver volume was observed when comparing µCT data of control and PHx mice. Functional PET-CT imaging in control mice was reproducible and was also demonstrated by De Lombaerde et al. (1). When comparing PHx and control mice, functional differences  in both liver uptake of 3β-[18F]-FCA and efflux to gallbladder and intestines were observed. In addition, differences between the PHx mice were also observed, ranging from normal to heavily disturbed transport, as shown in Figure 1. Surgery_L0R2 closely resembles the TACs of control mice, while Surgery_L0R0 displays decreased liver uptake and efflux to gallbladder and intestines. Lastly, Surgery_L0R1 shows decreased uptake and no efflux to gallbladder and intestines, combined with an increased blood pool concentration. Further research with a larger sample size is needed to correlate the disturbed transport with liver volume, resection of gallbladder, etc.

Conclusions

Our study demonstrates that it is possible to evaluate liver function and liver volume with molecular imaging using respectively dynamic 3β-[18F]-FCA PET-CT and Exitron 6000 enhanced µCT. This technique could be used in the future to non-invasively monitor liver function and liver volume longitudinally during regeneration in PHx mice.

References

  1. De Lombaerde et al. Plos One, 2017;12(3).
Time activity curves and static PET-CT reconstruction.

TACs in blood (A), liver (B) and gallbladder&intestines (C) in sham (control) and PHx (surgery) mice. Functional differences  in both liver uptake of 3β-[18F]-FCA and efflux can be observed when comparing sham and PHx mice. In addition, differences between the individual PHx mice are also observed. A static reconstruction (D) also visualizes the functional difference between sham and PHx mice.

Keywords: PET, CT, functional liver imaging
643

Pharmacokinetics evaluation of new drugs with potential application in Duchenne muscular dystrophy (#310)

Rossana Passannante1, Vanessa Gómez-Vallejo1, Abraham Martín1, Ainara Vallejo3, Pablo A. Fernandez4, Maite Eceiza2, Ana Joya Villanua1, Jesus M. Aizpurua2, Jordi Llop1

1 Parque Científico y Tecnológico de Gipuzkoa, CICbiomaGUNE, Donostia/San Sebastián, Spain
2 Departamento de Química Orgánica-I, UPV/EHU, University of the Basque Country, Donostia/San Sebastián, Spain
3 Biodonostia Health Research Institute, Donostia/San Sebastián, Spain
4 Universidad de Santiago de Compostela, Santiago de Compostela, Spain

Introduction

Duchenne muscular dystrophy (DMD) is a severe type of muscular dystrophy with no cure. It has been shown that stabilization of Ryanodine receptors (RyRs), which are responsible of long-term alteration of Ca2+ homeostasis, could be an efficient therapeutic alternative1,2,3. In this work, we evaluate the pharmacokinetics properties (biodistribution and metabolism) of new triazole-based RyR calcium release channel stabilizers using radiolabelling and positron emission tomography (PET) imaging in combination with other in vivo/ex vivo studies.

Methods

The drug candidate (Figure 1) was labelled by 11C-methylation using [11C]CH3I. For animal studies healthy female Sprague-Dawley rats weighing ca. 300 g were used. PET-CT dynamic imaging studies after intravenous administration of the radiotracer at different doses (1µg/Kg and 5mg/Kg) were combined with determination of arterial blood time-activity curves (TACs) and analysis of plasma samples by HPLC, the latter to investigate the presence of radiolabelled metabolites. Arterial TACs were obtained in continuous mode by using an in-house developed system which measured the concentration of radioactivity in extracorporeal circulated blood, obtained by connecting the femoral artery to the femoral vein.

Results/Discussion

The11C-labelled compound was obtained in average production time of 30 min with decay corrected radiochemical yield of 60.1±13.1% and molar activity values of 134±90.2 GBq/μmol. Radiochemical purity was >95% at injection time. The compound showed a fast metabolism in blood under no carrier added conditions, with only 33.3±11.6% of the radioactivity in blood corresponding to the parent compound at t=4-5 min. Metabolism was slower at higher administered doses (69.1±13.2% at 5mg/Kg at the same time point). Time activity curves showed a fast blood clearance (Figure 2a and 2b). PET images showed accumulation of radioactivity in the liver and kidneys at early time points (0-9 min), and progressive accumulation in the gastrointestinal tract at longer times (Figure 2c).

Conclusions

In vivo and ex vivo studies performed with the 11C-labelled calcium release channel stabilizer suggest a fast blood clearance and rapid metabolism. The metabolism is retarded with increased administered doses. Experiments tackling the radiolabelling in different positions to identify the metabolites are currently on-going.

References

1) Andrew M Bellinger et al., Nature Medicine,2009, 15, 3, (325-330).

2) Jérémy Fauconnier et al., PNAS, 2010, 107, 4, (1559-1564).

3) Steven O. Marxa and Andrew R. Marks, J Mol Cell Cardiol. 2013, 58, (225–231).

Acknowledgement

This project has received funding from the European Union’s H2020-MSCA-ITN Framework Programme, project reference 675417, and from the Spanish Ministry of Economy and Competitiveness, grant number CTQ2017-87637-R.

Figure 1
Radiolabelling of the triazole-based RyR calcium release channel stabilizer AHK-2.1.
Figure 2
a) Time activity curves in no carried added (NCA) conditions (total dose ca. 1µg/Kg); b) Time activity curves in carried added (CA) conditions (total dose ca. 5mg/Kg); c) PET images at different time points after intravenous administration of 11C-labelled AHK-2.1. Images correspond to coronal projections corresponding to CT representative slices for anatomical localization of the radioactivity.
Keywords: [11C]CH3I, Arterial TACs, PET-CT, Duchenne muscolar distrophy, RyRs stabilizers

MRI & MRS Technologies

Session chair: Georgios Keliris (Antwerp, Belgium); Graham Kemp (Liverpool, UK)
 
Shortcut: PW04
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

728

3D quantitative MRI of aerosolized Gd_based AGuIX nanoparticles in isolated ventilated pig lungs (#535)

Yannick Crémillieux1, Yoann Montigaud2, Clémence Bal3, Noël Pinaud1, Vi Pham1, François Lux4, Olivier Tillement4, Bei Zhang5, Jérémie Pourchez2

1 Université de Bordeaux, CNRS, Institut des Sciences Moléculaires, Bordeaux, France
2 Mines Saint-Etienne, Univ Lyon, Univ Jean Monnet, Inserm, Sainbiose, Centre CIS, Saint-Etienne, France
3 Université de Bordeaux, Bordeaux, France
4 Université de Lyon, Institut Matière Lumière, Villeurbanne, France
5 Canon Medical Systems Europe, Zoetermeer, Netherlands

Introduction

Aerosol therapy represents an attractive and efficient administration route for delivering therapeutic compounds either locally or systemically (1). Indeed, the lungs present a large surface area for drug absorption and an extensive vasculature with a weak anatomical barrier that does not limit access to the body (2). MR imaging of nebulized theranostic nanoparticles (NP) in the lung of small animal was demonstrated previously (3). Here we report MR imaging of the distribution of aerosolized Gd_based theranostic AGuIX NP, already used in patients, in a model of isolated ventilated pig lungs.

Methods

Aerosol was administered to ex vivo porcine lung connected to a 3D-printed human head replica and respiratory tract (4). The lungs were ventilated by passive expansion, simulating pleural depressions and was synchonized to aerosol administration generated with a medical jet nebulizer (Fig.1). Solutions (6 ml) of AGuIX (NHTheAguix, France) nanoparticles were aerosolized during 15 minutes.  AGuIX NP is composed of a polysiloxane core with 10 DOTA cyclic ligands chelating Gd ion (5). Its diameter is 3 ± 0.1 nm, its mass 8.5 ± 1 kDa and its relaxivity r1 at 3 Tesla is equal to 8.9 mM-1.s-1 per Gd ion. MRI acquisitions were performed at 3 Tesla (Vantage Galan 3T ZGO, Canon Medical Systems Corporation, Japan) using a 3D UTE sequence with a total acquisition time of 3.5 min (TR=3.7ms, TE=96μs).

Results/Discussion

In this lung model, large signal enhancement (SE) of the lung was observed after aerosolization of 6 ml solution containing 1.8 mmol of Gd3+. As a point of comparison, the dose of i.v. administered Gd3+ with clinical contrast agent in patient is in the order of 8 mmol. The SE is conspicuous for the whole lung up to the distal regions of the lung (distribution similar to that previously reported with SPECT techniques in ref. 4) with a more pronounced SE in the airways. The large SE can be appraised in Fig. 2 where the left lung was clamped during aerosolization. The concentration of Gd3+, assessed from 4 consecutive 3D UTE acquisitions at different flip angles, was evaluated, in regions excluding large airways, to be 130 μM. Considering a total lung tissue volume of 1 liter, it can be estimated that 7 % of Gd3+ ion have diffused into the lung tissue and contribute to the SE in our experimental set-up, the missing fraction being located in the upper respiratory tract and large airways.

Conclusions

We demonstrate In this study that the distribution of aerosolized Gd_based nanoparticles can be vizualized and quantified using MRI in large animal ventilated lung model. This protocol can be used for assessing aerosol deposition with high spatial resolution (1 mm 3D isotropic) without ionizing radiation. Moreover, the AGuIX NPs have well known radiosentitizing properties (6, 7) that can be applied for enhancing radiotherapy to treat lung cancer.

References

  1. Dolovich MB et al. Device selection and outcomes of aerosol therapy: Evidence-based guidelines: American College of Chest Physicians/American College of Asthma, Allergy, and Immunology. Chest. 2005 Jan;127(1):335-71.
  2. Choi HS, Ashitate Y, Lee JH, et al. Rapid translocation of nanoparticles from the lung airspaces to the body. Nat Biotech 2010;28:1300–1303
  3. Bianchi A, Lux F, Tillement O, Crémillieux Y. Contrast enhanced lung MRI in mice using ultra-short echo time radial imaging and intratracheally administrated Gd-DOTA-based nanoparticles. Magnetic Resonance in Medicine. 2013;70:1419-26
  4. Perinel S, Pourchez et al. Development of an ex vivo human-porcine respiratory model for preclinica studies.Sci Rep. 2017 Feb 24;7:43121.
  5. Lux F et al. Ultrasmall rigid particles as multimodal probes for medical applications. Angew Chem Int Ed Engl. 2011 Dec 16;50(51):12299-303.
  6. A. Bianchi, S. Dufort, F. Lux F, P.Y. Fortin, N. Tassali, O. Tillement, J.L. Coll, Y. Crémillieux. Targeting and in vivo imaging of non-small-cell lung cancer using nebulized multimodal contrast agents. Proc Natl Acad Sci U S A. 111 9247-9252 (2014).
  7. Lux F. et al. AGuIX(®) from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine. Br J Radiol. 18:20180365 (2018).

Acknowledgement

The authors are grateful to the company NH TherAguix for providing AGuIX nanoparticles. This study was carried out within the IBIO (Institut de Bio-Imagerie de Bordeaux).

Figure 1
Overview of the experimental set-up with the ENT (ear, nose, throat) replica and the pig lung in the sealed enclosure. The pump for lung ventilation and aerosol device are not shown in the picture.
Figure 2

Coronal slice from a UTE 3D data set acquired after aerosolization of 6 ml solution of AGuIX nanoparticles. Left bronchus was clamped during the aerosolization.

Keywords: Gd_based nanoparticle, aerosol, MRI, Lung, theranostic nanoparticle
729

Magnetic Resonance Imaging-observable Phenotypic and Functional Changes associated with Steatosis Development in the Mouse Liver  (#15)

Frauke Conny Waschkies1, 2, Lukas Frick1, Bostjan Humar1, Udo Ungethuem1, Rolf Graf1, Pierre-Alain Clavien1

1 University Hospital Zurich, Visceral- and Transplantation Surgery, Zurich, Switzerland
2 ETH and University of Zurich, Institute for Biomedical Engineering, Zurich, Switzerland

Introduction

Capacity of the liver for regeneration is adversely affected by preconditions such as steatosis or fibrosis – a risk factor for postoperative liver failure. For this purpose it is important to monitor hepatic functional reserve and liver regeneration. BOLD MRI may assess early changes pertaining to microenvironmental changes in tissue oxygenation, perfusion & metabolism, yet studies in liver are scarce [1,2]. Here we report on response of the liver to vasodilatory challenges in three animal groups, under control diet (CD), high-fat diet (HFD) and HFD substituted with Ω3 (HFD+Ω3).

Methods

MR imaging was performed on a 4.7T Bruker Pharmascan unit. Mice were intubated, mechanically ventilated and maintained under 2% isoflurane anesthesia. 4-5 male C57/B6 mice were studied in each group. T1w, ip/op FLASH images with and w/o Magnetization Transfer (MT) prepulse and multi-echo gradient-echo images for T2* maps (qT2*) were recorded and BOLD response to hypercapnic and pharmacological (acetazolamide, Diamox™ parenteral, Goldschield Pharm.) stimulus quantified. Signal fat fraction, MT ratio, basal qT2* and BOLD response were computed; graphs & statics were generated with R v3.5.0.

Results/Discussion

Abdominal MR images were obtained from all three animal groups (Fig. 1), depicting different contrasts pertaining to changes in water-fat content (signal fat fraction), altered protein-water interaction (MT ratio), alterations in basal oxygenation/perfusion and/or iron load (qT2*) and the ability to respond to a functional challenge (BOLD). ROI analysis was performed for quantitative assessment of these changes, shown here for the left lateral liver lobe (Fig. 2). HFD group displayed the highest fat fraction and lowest MT ratio, however, MTR may be affected by presence of fat. Interestingly, basal qT2* was lowest in this group, and is suggestive for less efficient perfusion and/or lower oxygenation status, but may also relate to iron accumulation, as implicated for NAFLD [3]. Increased (rather than the expected decreased) BOLD response might relate to the drop in baseline. HFD+Ω3 group was similarly affected, though to a much lesser extent.

Conclusions

BOLD MRI in response to vasodilatory challenge assesses microenvironmental changes in tissue oxygenation, perfusion and metabolism and combination with structural MRI markers allows a comprehensive characterization of pathological changes during steatosis development in mouse liver. However, our preliminary findings require substantiation in larger animal cohorts and by corroborative histology. 

References

[1] Ganesh et al. J Magn Reson Imaging 2016. 44(2): 305-16.

[2] Jin et al. Radiology 2010 254(1): 129-37.

[3] Britton et al. World J Gastroenterol. 2016 22(36): 8112-8122

Acknowledgement

 

 

Fig. 1: MRI-based characterization of steatosis-associated changes in the mouse liver.

Sample liver images of each a mouse under control diet (CD, top), HFD (HFD, middle) and Omega-3-based HFD for two weeks (HFD+Ω3, bottom row). Standard anatomical T1w images are shown with corresponding color-coded quantitative maps of the signal fat-fraction, magnetization transfer ratio (MTR), basal qT2* and BOLD signal change (∆T2*) in response to hypercapnia and acetazolamide, respectively. Map colour scales indicated under each map. Scalebar 500 um.

Fig. 2: Quantitative analysis of MRI markers in the left lateral lobe of the mouse liver.

Comparison between CD (blue), HFD (yellow) and HFD+Ω3 groups (gray) as characterized by signal-fat-fraction, MTR as a marker for macromolecular content, basal qT2* and BOLD signal in response to the hypercapnic and pharmacological stimulus acetazolamide as a marker for liver vascular function.

Keywords: murine liver steatosis, vascular dysfunction, Blood Oxygenation Level Dependent (BOLD) MRI
730

MRI Phenotypization, Vasoreactivity and Hypoxia in MC-38 colon and A549 lung adeno-carcinoma cell grafts grown on the Chorioallantoic Membrane of the Chick Embryo in ovo  (#29)

Frauke Conny Waschkies1, 2, Fatma Kivrak Pfiffner3, Dorothea M. Heuberger4, Petra Wolint5, Marcel Andre Schneider1, Yinghua Tian1, Maurizio Calcagni6, Pietro Giovanoli6, Johanna Buschmann5, 6

1 University Hospital Zurich, Visceral- and Transplantation Surgery, Zurich, Switzerland
2 ETH and University of Zurich, Institute for Biomedical Engineering, Zurich, Switzerland
3 University of Zurich, Institute of Medical Molecular Genetics, Schlieren, Switzerland
4 University Hospital Zurich, Clinic of Intensive Care, Zurich, Switzerland
5 University Hospital Zurich, Surgical Research Division, Zurich, Switzerland
6 University Hospital Zurich, Plastic Surgery and Hand Surgery, Zurich, Switzerland

Introduction

Recently, a tumor model based on the chorioallantoic membrane (CAM) was characterized structurally with MRI [1]. Yet, vascular functional reserve and oxygenation-sensitive MRI measures [2-3] remain largely unexplored in this model. In our preliminary experiments we compare MC-38 colon and A549 adeno-carcinoma cell grafts with regards to their vascular and oxygenation phenotypes. We demonstrate that a functional gas challenge with carbogen is feasible through gas exchange on the CAM, allowing to access vascular function and oxygenation status of the tumor graft in this experimental model.

Methods

Fertilized Lohman white LSL chick eggs were opened on incubation day (ID) 3.5 and on ID7 5 x 105 matrigel embedded MC-38 colon or A549 lung adenocarcinoma cells were planted onto the CAM. MRI was performed in ovo on ID 14 in 5 samples for each graft type on a 4.7T Bruker PharmaScan system, with chicken embryos sedated (0.3 mg/kg medetomidine). T1w and T2w anatomical reference images obtained and quantitative T1 and T2* maps were compared between periods of air and carbogen (95% O2, 5% CO2). Gases were delivered through a plastic tubing to the CAM. T1w scans were repeated in selected samples after i.v. injection Gd-DOTA (Dotarem®, Guerbet S.A., Switzerland) to study enhancement in the tumor graft. Corroborative histology was obtained from H&E and Ki-67 staining. 

Results/Discussion

MC-38 colon and A549 lung adenocarcinoma cell grafts were compared using quantitative T1 and T2* MRI, readouts associated with vascular responsivity and oxygenation status, when compared between periods of air and carbogen. Since the CAM serves as a breathing organ during chick embryo development, these markers might be also applicable for the grafts on the CAM (Fig. 1). Our preliminary data show that in A549 lung adenocarcinoma cell grafts T2* values increased upon carbogen exposure, while MC-38 grafts displayed a decreasing trend in T1 (Fig. 2). Qualitative assessment of Gd-enhancement, suggests that A549 grafts display a more prominent enhancement compared to MC-38 grafts (not shown). Furthermore, it will be of interest to explore if such enhancement patterns might be supportive for our vague notion that A549 grafts might display a better vascular response, while only MC-38 show oxygen-induced T1 shortening as observed in normoxia.

Conclusions

Our preliminary experiment shows that different tumor grafts planted on the CAM can be distinguished non-destructively in ovo using MRI. We show that a functional gas challenge is feasible through the CAM, and affects MRI signals associated with vascular reactivity and oxygenation status of the graft. The CAM assay may thus help qualifying such MRI markers to discern distinct vascular functional and oxygenation phenotypes.

References

[1] Zuo et al. (2014) NMR Biomed 28: 440-447
[2] Baudelet et al. (2006) NMR Biomed 19: 69-76
[3] O’Connor et al. (2015) Cancer Res 76(4) : 787-95

 

Representative MRI images of MC-38 colon carcinoma cell graft in ovo.
A. Tumor cells planted into the middle of a supportive plastic ring on top of the CAM, shown 7 days after grafting. B. Experimental setup with gas challenge delivery tube and surface coil placed over the egg shell window. C. Graft outlined on T1w and T2w anatomical reference images and on quantitative color-coded T1 and T2* maps obtained while the graft was exposed to medical air and carbogen, respectively.
Fig. 2: Quantitative analyses of changes in T1 and T2* upon carbogen gas challenge
in A549 lung adenocarcinoma cell grafts and in MC-38 colon carcinoma cell grafts. While no significant response was observed in neither, qT1 and qT2* in both graft types, in A549 lung adenocarcinoma cell grafts T2* values almost doubled upon carbogen exposure (p < 0.06, Wilcoxon test), while T1 displayed a decreasing trend in MC-38 colon carcinoma cell grafts (p < 0.11, Wilcoxon test). No consistent trend was observed in T1 for A549 lung adenocarcinoma grafts and in T2* in MC-38 colon carcinoma grafts.
Keywords: Chorioallantoic membrane (CAM), magnetic resonance imaging (MRI), MC-38 colon carcinoma cells graft, A549 lung adeno-carcinoma cell graft, oxygenation-sensitive MRI
731

An Extracorporeal Circulation Mouse Model for Simultaneous Measurements of DCE-MRI Arterial Input Functions and Radiotracer Blood Concentrations (#321)

Philipp Backhaus1, 2, 3, Florian Büther1, 2, Lydia Wachsmuth3, Lynn Frohwein1, 2, Klaus Schäfers1, 2, Sven Hermann1, 2, Michael Schäfers1, 2, Cornelius Faber3

1 University Hospital Münster, Department of Nuclear Medicine, Münster, North Rhine-Westphalia, Germany
2 University of Münster, European Institute for Molecular Imaging - EIMI, Münster, North Rhine-Westphalia, Germany
3 University of Münster, Translational Research Imaging Center - TRIC, Münster, North Rhine-Westphalia, Germany

Introduction

DCE-MRI can provide quantitative estimates of blood-brain barrier integrity. Dynamic PET measurements allow to quantify molecular processes. The calculation is based on contrast agent (CA) / radiotracer dynamics in tissue and in blood (AIF). Measurement of the AIF however is challenging in both small animal MRI and PET. Only few examples of direct AIF-measurements of MR CA in mice are published in the literature1-4, each featuring significant limitations.

Methods

Intracranial tumor-bearing mice received an extracorporeal shunt from the femoral artery to the tail vein. MRI scanning was performed using a 9.4 T MRI (Bruker BioSpec) and a cryo-cooled surface coil. The extracorporeal line featured two reservoirs which resided in the MRI field of view. A MRI-compatible measuring chamber for a β-Microprobe (biospace lab) was included in the circulation. Dynamic MRI scanning of the head was performed for 15 minutes using a 3D FLASH with a spatial resolution of 0.175 x 0.175 x 1 mm and a temporal resolution of 4.015 s. A 100 µl solution containing 10-20 MBq F-18-PSMA-1007 and CA (Gadovist, 35 mM) was injected intravenously at 1 ml/min. Dispersion correction for MRI CA was performed based on the recorded dispersion effect at the two interspaced reservoirs.

Results/Discussion

The CA AIFs of nine recorded mice show little noise and typical AIF curve shapes after dispersion correction (Figure 2, A & C). Eight of nine mice show a close range of peak concentrations (0.55 – 0.85 µmol/ml) and shunt flow velocities (34-58 µl/min). β-emitting radioactive tracer AIFs can be simultaneously recorded using a MR-compatible β-Microprobe (Figure 2 A) and mice were transferred into the PET-scanner immediately after DCE-MRI (Figure 2 B). Significant inverse correlation between AIF maxima and the delays between the CA-influx into the two reservoirs was observed (r = -0.84). The time constant τ for monoexponential deconvolution was significantly positively correlated with the delay (r = 0.98). The results of mass spectrometry (MS) validation show a systematic and consistent underestimation of the image-derived concentrations (MS-quantification / MR-quantfication: 1.57-1.8). 

Conclusions

We present a novel approach for DCE-measurements of the AIF in mice with conceivable potential compared to so far published methods. Moreover, we present the first dual recordings of AIFs of a MR CA and a PET tracer in mice. This supports evaluation approaches to deduce the CA/PET tracer AIF from one another. Further, it might provide the basis for simultaneous and integrated modeling of PET tracer and CA kinetics in mice,

References

1.Loveless ME, Halliday J, Liess C, et al. A quantitative comparison of the influence of individual versus population-derived vascular input functions on dynamic contrast enhanced-MRI in small animals. Magn Reson Med. 2012;67:226–236.

2.    Moroz J, Wong CL, Yung AC, et al. Rapid measurement of arterial input function in mouse tail from projection phases. Magn Reson Med. 2014;71:238–245.

3.    Kim J-H, Im GH, Yang J, et al. Quantitative dynamic contrast-enhanced MRI for mouse models using automatic detection of the arterial input function. NMR Biomed. 2012;25:674–684.

4.    Barnes SL, Whisenant JG, Loveless ME, et al. Practical Dynamic Contrast Enhanced MRI in Small Animal Models of Cancer: Data Acquisition, Data Analysis, and Interpretation. Pharmaceutics. 2012;4:442–478.

Extracorporeal shunt imaged in DCE-MRI
A) The extracorporeal volume between artery, 1st reservoir (S1), 2nd reservoir (S2) and β-microprobe are of equal volume. A tube (“Fix”) filled with defined concentration of CA (1 µmol/ml) and F-18 is centrally above the head. DCE images (same animal as in Figure 2 A & B) before Gd-injection (B) at 40 s p.i. (C, S1 filled with contrast) and 101 s p.i. (D, S1 and S2 filled). (E) Corresponding T2wi.
Parallel dynamics of CA and radiotracer
A) Dynamic cGd and CF-18-PSMA-1007 curves after simultaneous CA and tracer injection at 60 s. The green curve shows the deconvolved S1 curve with τ derived from the estimated convolution between S1 and S2. (B) Subsequent PET-image 23-40 minutes after injection fused to T2wi. (C) Dynamic cGd blood curves of another animal with plotted results of mass spectrometry validation measurements.
Keywords: DCE-MRI, PET/MRI, Arterial Input Function
732

Assessment of tumor hypoxia with Blood (BOLD) and tissue oxygenation level-dependent (TOLD) MRI measurements as prognostic biomarkers for breast cancer aggressiveness (#256)

Viktoria Ehret1, Joachim Friske1, Lubos Budinsky1, Katja Pinker-Domenig1, Vanessa Fröhlich1, Daniela Laimer-Gruber1, Thomas Helbich1

1 Medical University Vienna, Department of Biomedical Imaging and Image-guided Therapy, Division of Gender and Molecular Imaging, Preclinical Imaging Laboratory, Vienna, Wien, Austria

Introduction

In tumors, hypoxia reflects an imbalance of oxygen delivery and consumption [1] and is therefore a prognostic biomarker of aggressiveness, local recurrence and metastasis [2‐4]. To quantify the oxygenation level within a tumor, two different techniques namely blood- (BOLD) [5‐6] and tissue oxygenation level‐dependent (TOLD) [3] can be evaluated combining different intrinsic MRI contrast mechanisms (T1, T2*). Our main goal is to assess both techniques to characterize the aggressiveness of different breast cancer models (MCF-7, SKBR-3, MDA-MB-231).

Methods

Human breast cancer cells of three different levels of aggressiveness were injected into the mammary gland of female nude mice. MRI measurements were performed using a 9.4T magnet system combined with a 1H volume coil (Bruker) when breast tumours reached a diameter of at least 10mm. The MR protocol includes a T1‐RARE sequence (TOLD) and T2* MGE/T2* EPI sequences (BOLD). A baseline measurement was acquired while the animal breathed air. The oxygenation challenge was performed in two different ways: A stepwise increase of the oxygen level (50%, 80%, 100%) and a direct increase to 100%. T1 and T2* maps were calculated using the ISA Tool fit routine. Data analysis of the calculated maps was performed on a voxel-by-voxel basis using Matlab. Imaging findings were compared with HIF-1alpha.

Results/Discussion

First results and a comparison of the two different oxygen challenges and the different aggressive breast cancer models will be presented. TOLD and BOLD provide different temporal and spatial information on breast cancer oxygenation. Typical results of TOLD/BOLD measurements for breast cancer of high aggressiveness are shown in figure 2, giving evidence of elevated hypoxia levels. For BOLD, the signal does not change heavily after the 3rd timepoint of the oxygenation challenge therefore we may shorten the measurement protocol accordingly. A stepwise oxygenation or starting the measurement after oxygen saturation could lead to different information. To make the technique even more efficient, T2* EPI might also be a potential sequence for fast BOLD measurements. Even if the scan protocol can still be optimized, the first measurements already provide impetus to use non-invasive BOLD/TOLD MRI for monitoring breast cancer oxygenation and drawing conclusions regarding aggressiveness.

Conclusions

Results give evidence of elevated hypoxia levels in breast cancer of high aggressiveness. First measurements provide further impetus to use non-invasive BOLD/TOLD MRI for monitoring tumor oxygenation and drawing conclusions regarding aggressiveness.

References

[1] O´Connor, J. P. B., et al. Int. J. Radiation Oncology Biol. Phys., 2009, 75(4):1209‐1215

[2] Christen, T., et al. AJNR Am J Neuroradiol., 2013, 34:1113‐1123

[3] Hallac, R. R., et al., Magnetic Resonance in Medicine, 201, 71:1863‐1873

[4] Zhao, D., et al., Magnetic Resonance in Medicine, 2009, 62:357‐364

[5] Baudelet, C., et al., Magnetic Resonance Imaging, 2004, 22:905‐912

[6] Jiang, L., et al., Magnetic Resonance in Medicine, 2004, 51:953‐960

Acknowledgement

This work is supported by the EU project Hallmarks of Cancer (667211‐HYPMED H2020‐PHC) and the Siemens project CA-ID C00220910 and covered by the ethics (66.009/0284‐WF/V/3b/2017). In part supported by a research grant from Bruker.

Figure 1: Scan protocol of the BOLD/TOLD MRI
Figure 2:

∆SI(%) for the TOLD measurement after oxygen challenge (left) and of the BOLD measurement (right) for five different time points during tumour oxygenation (B-F). Difference between the single time points is approximately 10 min. Tumour position in the BOLD images is shown in (A). The increases in ∆SI during oxygenation challenge are clearly visible.  

Keywords: MRI, BOLD, TOLD, breast cancer
733

Severity assessment – Influence of repeated MRI measurements on animal welfare (#170)

Jasmin Baier1, Anne Rix1, Milita Darguzyte1, Maike Baues1, Jan-Niklas May1, Diana Möckel1, Rupert Palme3, René H. Tolba2, Fabian Kiessling1

1 RWTH Aachen University, Institute for Experimental Molecular Imaging, Aachen, North Rhine-Westphalia, Germany
2 RWTH Aachen University, Institute for Laboratory Animal Science & Experimental Surgery and Central Laboratory for Laboratory Animal Science, Aachen, North Rhine-Westphalia, Germany
3 University of Veterinary Medicine Vienna, Department of Biomedical Sciences, Wien, Wien, Austria

Introduction

Non-invasive imaging is often used to observe physiological processes in small animals and most imaging devices are considered to be safe nowadays. However, the handling1 of animals, necessary anesthesia or the imaging procedures themselves can induce stress and therefore have an effect on animal welfare. Magnetic resonance imaging (MRI) measurements in particular can influence the behavior2 of animals due to the magnetic field. In the following study we investigate the influence of repeated MRI measurements on the welfare and different physiological parameters in mice.

Methods

For this purpose healthy, female BALB/C mice were exposed to 1T or 7T MRI field strength three times per week over a four weeks period. Imaging was performed under isoflurane anesthesia for 30 minutes using an established MRI protocol which is often applied in cancer research. A behavioral Rotarod test as well as blood pressure measurements were performed twice per week. Stress hormone levels in the feces and blood, the food intake and the nest-building behavior were examined. In addition, the general condition of the animals was evaluated daily using a score sheet. At the end of the experiment organs like brain, liver, spleen, lung, intestines, heart and kidneys were removed for histological examinations.

 

Results/Discussion

The investigations have shown that even after repeated MRI measurements there was no change in the behavior of the animals compared to different control groups (no imaging and no anesthesia or only anesthesia). Neither the Rotarod tests nor the blood pressure measurements showed any difference between the groups. Also, no differences were seen in the nest-building behavior, the food intake or the organ weights of the investigated groups. The evaluation of the stress hormone level (corticosterone) in feces and blood showed no increase in the animals exposed to MRI compared to the control groups.  Based on the score sheets, the burden of the animals measured with MRI is not significantly different from the burden of the control animals.

Conclusions

The results show that the burden of the animals during MRI imaging can be considered as mild. In conclusion, we evaluate single as well as repeated MRI measurements under isoflurane anesthesia as a safe tool for the non-invasive evaluation of physiological processes in laboratory mice.

References

1 Balcombe J P, Barnard N D, Sandusky C. Laboratory Routines Cause Animal Stress. Contemporary Topics. 2004, 43 (6): 42 - 51

2 Houpt T A, Pittman D W, Barranco J M, Brooks E H, Smith J C. Behavioral Effects of High-Strength Static Magnetic Fields on Rats. The Journal of Neuroscience. 2003, 23 (4): 1498 - 1505

Acknowledgement

This research was funded by the Deutsche Forschungsgesellschaft (DFG) within the framework of the research group FOR2591.

Influence of repeated MRI measurements on behavior and stress levels in mice

A control group, an anesthesia group (Isoflurane for 30 min), and two MRI groups (1T or 7T under Isoflurane anesthesia for 30 min) were examined.

a. Changes in Rotarod performance after MRI measurements compared to the baseline

b. Mean blood pressure after MRT measurements

c. Stress hormone (corticosterone) levels measured from feces throughout the experiment

Scores throughout the experiment after repeated 7T MRI measurements
Mice were scored daily. Parameters such as the appearance of the fur and eyes as well as the weight of the animals, the heart rate and the behavior were assessed. If an animal was evaluated with a total score of 1 - 9, it was exposed to a mild level of stress. 
Keywords: animal handling standardisation, stress level assessment, behavior evaluation, imaging influence
734

Strategies to avoid isoflurane chemical shift artefacts in high sensitivity in vivo19F MRI (#258)

Alexander H. Staal1, Andor Veltien2, N. Koen van Riessen1, Arend Heerschap2, Mangala Srinivas1

1 Radboud University Medical Centre, Tumor Immunology Lab, Nijmegen, Netherlands
2 Radboud University Medical Centre, Department of Radiology, Nijmegen, Netherlands

Introduction

19F MRI is an increasingly popular imaging technique exploiting the benefits of background free imaging with the stable 19F isotope in imaging agents mainly consisting of inert perfluorocarbons[1]. Isoflurane (ISO) is the anaesthetic of choice for preclinical imaging studies, however, as this compound contains 19F, it can be a complicating factor, and may result in chemical shift artefacts (CSA). Currently, ISO artefacts are avoided by short imaging times, injection anaesthesia or chemical shirt imaging[2]. Here, we show three distinct, easy to implement, strategies for avoiding ISO artefacts.

Methods

A phantom consisting of 50% PFCE and 50% ISO was imaged as a proof of concept. For in vivo imaging, C57Bl/6 mice were injected with 20mg PFCE containing nanoparticles[3] dissolved in 400µl 0,9% NaCl and imaged 2 days after injection. MRI was performed on an 11.7T BioSpec (Bruker, Ettlingen, Germany). 19F pulse-acquire with bandwidth=30ppm and points=8000. A RARE sequence was used with 4 different parameter sets. 1) standard: TE=15.2ms, TR=5000ms 2) out of plane shift with an extremely small bandwidth 3) suppression pulse 4) 3D selective excitation. In vitro imaging took <20 sec for all scans, in vivo imaging time was 12:48min. For in vivo1H reference scans a respiratory gated FLASH was used.

Results/Discussion

In vitro NMR spectra of the phantom show a sharp ISO and PFCE peak (Fig 1a), in vivoNMR spectra show multiple broad ISO peaks and a single sharp PFCE peak(Fig 1b). In vitro 19F MRI shows the CSA when using a standard sequence(Fig 1c), that can be shifted out of plane by using a very narrow acquisition bandwidth (Fig 2d). Figure 2e shows the two resonance frequencies of ISO and PFCE a fair distance apart, either signal can be supressed using a frequency selective 90°pulse before the excitation pulse. In vivo19F MRI with the standard sequence shows the isoflurane problem; ISO CSA creates ghosts that confound image interpretation and quantification (Fig 2a). Shifting these signals out of plane is feasible in vivo,but results in aggravated susceptibility artefacts(Fig 2b). Using our ISO suppression pulse, results in effective in vivosuppression of ISO signals(Fig 2c). Selective 3D slab excitation with a narrow excitation bandwidth results in artefact free images(Fig 2d).

Conclusions

Preclinical 19F MRI scan times are limed by isoflurane chemical shift artefacts, complicating imaging analysis and quantification. Here we show three strategies to avoid these chemical shift artefacts while maintaining a high signal to noise ratio. Having 3 different approaches allows for flexibility in sequence design.

References

1. M. Srinivas, A. Heerschap, E.T. Ahrens, C.G. Figdor, I.J.M. de Vries. 19F MRI for quantitative in vivo cell tracking. Trends in Biotechnol 2010; 28(7): 363-70.

2. S.M. Fox, J.M. Gaudet and P.J. Foster, Fluorine-19 Mri Contrast Agents for Cell Tracking and Lung Imaging’, Magnetic Resonance Insights. 2015; 8(1).

 

3. E. Swider, A. H. J. Staal, N. K. van Riessen, et al. Design of triphasic poly(lactic-co-glycolic acid) nanoparticles containing a perfluorocarbon phase for biomedical applications. RSC Adv. 2018; 8: 6460-6470

Figure 1
19F-spectra, (a) in vitro (b) in vivo. c-e In vitro ISO avoidance strategies, (c) standard imaging, (d) shift ISO out of plane (e) frequency specific suppression pulse. Note: in all images only one phantom is present
 
Figure 2: in vivo imaging

in vivo imaging 1H reference image in grey scale 19F image in red hot. (a) standard imaging with ISO ghosts overlapping the liver (b) ISO artefact is shifted out of plane, clear 19F susceptibility artefacts of the reference and liver projecting outside the animal, (c) ISO suppression pulse and (d) selective excitation 3D-RARE both resulting in an absence of ISO artefacts.

Keywords: 19F MRI, isoflurane, in vivo, chemical shift artifacts
735

In vivo Fast Field Cycling Relaxometry reports on the extra- and intracellular localization of iron oxide particles in tumour mice models. (#62)

Maria Rosaria Ruggiero1, Simona Baroni1, Valeria Bitonto1, Smeralda Rapisarda1, Silvio Aime1, Simonetta Geninatti Crich1

1 Università degli studi di Torino, torino, Italy

Introduction

The relationship“immune system/tumour”is considered an important hallmark of cancer1. Tumour associated macrophages (TAMs) adopt an anti-inflammatory phenotype and secrete factors to promote angiogenesis and tumor invasion.The use of Ultra Small Iron Oxides nanoparticles (USPIO) has been already proposed to the TAM detection generating contrast in T2-weighted images indipendently of extra and intracellular localization of the NPs. While, Tat different fields appear dependent on localization, especially at low field, of the NPs allowing an unambiguous TAM quantification.

Methods

In vitro studies have been carried out on a murine monocyte-derived macrophage cell line (J774) to evaluate the relaxivity changes due to the intracellular localization of ferumoxytol, clinical negative contrast agent. T1 were acquired on a FFC relaxometer able to switch over a large range of field stranghts (0.01-20MHz). In order to host a mouse, the commercially available relaxometer (Stelar, Mede, Italy) has been modified with the implementation of a 40 mm 0.5T Field Cycling magnet and a dedicated 11mm solenoid detection coil placed around the anatomical region of interest2. The tumour xenografts were prepared by injecting three tumour cell lines (B16 melanoma, 4T1 and 168FARN breast carcinoma) in the hindlimb muscle.

Results/Discussion

The relaxivity peak at ca. 8-10 MHz observed in water on ferumoxytol is shifted to lower magnetic field strengths (at 0.5-1 MHz) when the NPs were entrapped in macrophages (Fig.1). For in vivo model, the selected types of tumours (168FARN, 4T1 and B16) are characterized by different amount of necrotic zones and macrophages infiltrating the tumor stroma. Ferumoxytol was injected at a dose of 0.5 mmol/kg of Fe. The profile obtained 3h and 24h after the injection were significantly different. (Fig.2) The profile observed at 24h displays a bell-shaped profile with a maximum around 0.4-0.5 MHz similar to one found for ferumoxytol labelled macrophages. This finding clearly indicated the intracellular localization of ferumoxytol as confirmed by histological analysis by the Pearls assay.

Conclusions

The measured T1 at different field immediately reports on the intra- or extra-cellular localization of the investigated contrast agent. This information could be open new horizons for cell tracking applications. Despite the herein used prototype FFC-NMR, FFC has recently been applied to MRI, largely thanks to the work of the Lurie group at Aberdeen University where two prototype human whole-body sized FFC-MRI scanners have been built3.

References

[1] Morita Y, Zhang R, Leslie M, Adhikari S, Hasan N, Chervoneva I, Rui H, Tanaka T. Oncol Lett. 2017;14:2111-2118.

[2] Ruggiero MR, Baroni S, Pezzana S, Ferrante G, Geninatti Crich S, Aime S. Angew Chem Int Ed Engl. 2018, 57:7468-7472.

[3] Pine KJ, Davies GR, Lurie DJ. Magn Reson Med., 2010, 63, 1698-702.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 668119

Figure 1
NMRD profiles of J774 cells incubated for 24 h with different ferumoxytol concentrations or with ferumoxytol added to the external buffer. The indicated concentrations refer to the [Fe] in the measured cell pellets
Figure 2
A) NMRD profiles of B16 tumour bearing mouse leg 3 and 24h after (POST) the i.v. injection of ferumoxytol subtracted by the corresponding PRE profile (acquired before ferumoxytol injection). B) Different initial slopes of the NMRD profiles
Keywords: Iron oxide NPs, labelled macrophage, FFC-NMR
736

Hyperpolarisation of clinical agents using SABRE and SABRE-relay (#16)

Ben Tickner1, Alexandra Olaru1, Peter Rayner1, Soumya Roy1, Wissam Iali1, Aneurin Kennerley1, Simon Duckett1

1 Center for Hyperpolarisation in Magnetic Resonance, Department of Chemistry, University of York, York, United Kingdom

Introduction

Magnetic Resonance (MR) techniques are insensitive as signal intensities are derived from small population differences across nuclear spin energy levels. Hyperpolarisation techniques can be used to increase the number of spins that contribute to the MR signal. Here we use Signal Amplification By Reversible Exchange (SABRE) to make low concentration biomolecules visible to MR. This technique takes para-hydrogen (p-H2), a spin isomer of hydrogen, and catalytically transfers its latent hyperpolarisation into a target substrate using an iridium catalyst.

Methods

SABRE traditionally necessitates substrate hyperpolarization through coordination to an iridium catalyst, limiting substrate scope to N-heterocycles. A recent variation termed SABRE-Relay allows molecules to become hyperpolarised by exchange of hyperpolarised nuclei. Amines can act as hyperpolarization carriers by coordinating to the SABRE catalyst and relaying polarisation onto other functional groups including alcohols. This rapidly expands the substrate scope of SABRE by hyperpolarising molecules without the need for direct catalyst interaction. Here, we create active SABRE catalysts of the form [Ir(H)2(NHC)(Amine)3] to hyperpolarise molecules via SABRE-relay or metal complexes of the form [Ir(H)2(NHC)(α-carboxyimine)(amine)] which can be hyperpolarised by SABRE.

Results/Discussion

We use SABRE and SABRE-relay to produce 1H and 13C signals enhanced by 4 orders of magnitude for a range of different molecules including alcohols and pyruvate. The determining factor in achieving high alcohol enhancements is the level of carrier NH polarisation. The alcohol and carrier amounts should be equal for optimal alcohol enhancement; the best carrier NH3 has been identified. Such hyperpolarised molecules including pyruvate have been imaged in vitro. We can also create iridium α-carboxyimine complexes from the in situ reaction between pyruvate and amines. These complexes undergo rapid p-H2 exchange and isotopic labelling techniques were used to achieve 13C signal enhancements of 750-fold and a long-lived 13C2 state can be created in the first instance, the lifetimes of which approach 18 s. Such states may prove highly beneficial in the future when looking for intermediates in chemical reactions or as flow probes.

Conclusions

We present the hyperpolarisation of a wide range of molecules including organics (alcohols), biomolecules (pyruvate and ethyl lactate) and transition metal complexes (iridium α-carboxyimines). We show that SABRE can be used as a route to create hyperpolarised molecules in a fast, cheap, and refreshable manner. Current research is directed at improving biocompatibility and we expect SABRE will provide unique opportunities for molecular imaging.

References

  1. R. W. Adams, J. A. Aguilar, K. D. Atkinson, M. J. Cowley, P. I. Elliott, S. B. Duckett, G. G. Green, I. G. Khazal, J. López-Serrano and D. C. Williamson, Science, 2009, 323, 1708-171
  2. W. Iali, P. J. Rayner and S. B. Duckett, Sci. Adv., 2018, 4, eaao6250.
  3. B. J. Tickner, W. Iali, S. S. Roy, A. C. Whitwood and S. B. Duckett, ChemPhysChem, 2018.

Acknowledgement

The Wellcome Trust (092506 and 098335), MRC (R16249), and the ESPRC IAA G00251 (B.J.T. studentship) are thanked for supporting this work.

SABRE Mechanism
A substrate can become hyperpolarised by SABRE when it and para-hydrogen are in reversible exchange with an iridium catalyst. In a second step, polarisation can be relayed onto other molecules by exchange of nuclei
Keywords: SABRE, Hyperpolarisation, Para-hydrogen, Pyruvate
737

A spin echo pulse sequence with unpaired adiabatic refocusing pulse and a 3D cone readout for hyperpolarized 13C imaging (#181)

Vencel Somai1, Alan Wright1, Kevin M. Brindle1, 2

1 University of Cambridge, Cancer Research UK, Cambridge Institute, Cambridge, United Kingdom
2 University of Cambridge, Department of Biochemistry, Cambridge, United Kingdom

Introduction

Dynamic nuclear spin polarization has enabled dynamic measurements of tissue metabolism in vivo using 13C magnetic resonance spectroscopic imaging. However, this is demanding in terms of imaging speed due to the short lifetime of the hyperpolarization, hence the shortest possible sequence with the highest possible SNR is desirable for dynamic measurements. We have developed a single spin-echo sequence, which improves on that described in [1], with a shorter readout, better potential SNR efficiency and an isotropic PSF that does not degrade the nominal resolution.

Methods

The sequence uses a spatial spectral pulse set to the resonant frequency of the individual metabolites and a HS adiabatic refocusing pulse without slice selection. The gradient readout uses a 3D cone trajectory [2], which is built from 13 cones. Each of the cones is fully refocused with time optimal rewinders and the 7th cone is refocused at the time of the spin-echo. The total readout time is 18.576ms. The nominal isotropic resolution and FOV are 2 mm and 3.2 cm respectively. The total pulse sequence length is 43.73 ms. PSF of the sequence was simulated according to [3].

The pulse sequence was tested on two cylindrical phantoms filled and half-filled with thermally polarized 5M [1-13C]lactate and 3M [1-13C]acetate respectively, placed in the magnet isocentre and parallel with the z-axis.

Results/Discussion

The immunity of the proposed sequence to hardware imperfections was tested by measuring the resulting k-space, as described in [4]. Despite the slew-rate limited regime and high gradient amplitudes, no measurable delay was observable and the amplitude of the waveforms matched the design values within the detection noise. Simulations showed that the PSF of the sampling trajectory is isotropic and maintains a resolution of <4 mm in the x, y and z directions. The simulation results were validated with measurements on two phantoms containing 13C-labelled lactate and acetate using a 7T scanner (Agilent, Palo Alto, CA), with a 42 mm diameter bird‐cage volume coil for 1H transmission and reception, and a similar volume coil for 13C transmission and a 20 mm diameter surface coil for 13C detection (Rapid Biomedical, Rimpar, Germany). The sequence was designed with a maximum gradient strength of 0.4T/m and slew rate of 2000T/m/s.

Conclusions

The proposed sequence gave better resolution, potentially higher SNR efficiency and a shorter readout and TR when compared with the sequence described in [1]. The main advantage of the sequence is its isotropic sampling PSF.

References

  1. Wang J, Hesketh RL, Wright AJ, Brindle KM. Hyperpolarized 13C spectroscopic imaging using single‐shot 3D sequences with unpaired adiabatic refocusing pulses. NMR in Biomedicine. 2018;31:e4004. https://doi.org/10.1002/nbm.4004
  2. Gurney, P. T., Hargreaves, B. A. and Nishimura, D. G. (2006), Design and analysis of a practical 3D cones trajectory. Magn. Reson. Med., 55: 575-582. doi:10.1002/mrm.20796
  3. Wang, J. , Wright, A. J., Hu, D. , Hesketh, R. and Brindle, K. M. (2017), Single shot three‐dimensional pulse sequence for hyperpolarized 13C MRI. Magn. Reson. Med., 77: 740-752. doi:10.1002/mrm.26168
  4. Takahashi, A. and Peters, T. (1995), Compensation of multi‐dimensional selective excitation pulses using measured k‐space trajectories. Magn. Reson. Med., 34: 446-456. doi:10.1002/mrm.1910340323

Acknowledgement

The work was supported by a Cancer Research UK Programme grant (17242), by the CRUK-EPSRC Imaging Centre in Cambridge and Manchester (16465) and by the CRUK Cambridge Centre.

Pulse sequence
The pulse sequence uses a fly-back SpSp excitation (10.056ms), HS adiabatic refocusing pulse (10ms) and a 3D cones k-space trajectory. The excitation pulse has a bandwidth at half maximum of 350 Hz and a period of excitation bands of 1645 Hz. To reduce signal loss due to the quadratic phase imparted by the adiabatic pulse, the encoding gradients are turned off on all three axes during refocusing.
Reconstruction
Central slice of the images of the acetate (without 1H decoupling) and lactate phantoms. Due to 1H coupling and NOE the quartet spectrum of the acetate leads to inherently smaller signal compared to the singlet lactate. The reference 1H images were acquired using a 2D FSE sequence with the same isotropic 3.2 cm FOV, 2 mm slice thickness and 128x128 in-plane resolution. The echo train had 8 echoes.
Keywords: hyperpolarizaton, 13C, 3D single shot, spin-echo, 3D cones
738

Detection of T cell activation through metabolic changes using hyperpolarized 13C magnetic resonance (#268)

Emine Can1, Mor Mishkovsky1, Hikari Yoshihara1, Ulf Petrausch4, Marie-Agnès Doucey5, Arnaud Comment3, 2

1 EPFL, Lausanne, Switzerland
2 University of Cambridge, Cambridge, United Kingdom
3 GE Healthcare, Chalfont St Giles, United Kingdom
4 OnkoZentrum, Zurich, Switzerland
5 UNIL, Lausanne, Switzerland

Introduction

Upon activation, T cells rapidly increase their requirements in metabolic substrates to sustain their proliferation and function. While memory and naïve T cells mostly rely on the β-oxidation of fatty acids to produce their ATP, it has been shown that the higher need in energy and biosynthetic material required by activated T cells leads to an upregulation of the catabolism of glucose1. This has been observed through both an increase in glucose transporters as well as an upregulation in enzymes involved in glycolysis2. We aimed at detecting this metabolic shift by hyperpolarized 13C MRS.

Methods

CD4+T cells were isolated from healthy human peripheral mononuclear blood cells by immune magnetic selection. T cells were activated through a 5-day incubation in anti-TCR/CD3 (10µg/ml) coated tissue culture plates containing soluble anti-CD28 (1µg/ml) and 100U/ml of IL-2. At the end of the stimulation, ~5·106 T cells were transferred into a 5-mm NMR tube containing 30mM unlabeled lactate. Another 5-mm tube was prepared as control with non-activated T cells. The two tubes were placed in a dual 13C NMR probe and loaded in a 9.4T/31cm horizontal bore magnet at 37°C. Hyperpolarized [1-13C]pyruvate solution (300µl per tube) prepared in a 7T polarizer was injected in parallel using a flow splitter, and interleaved 13C MRS measurements were acquired on both samples (4s Trep, 5o flip angle).

Results/Discussion

In both non-activated and activated T cells, the formation of [1-13C]lactate was detected following the injection of hyperpolarized [1-13C]pyruvate. The lactate-to-pyruvate signal ratio recorded in activated T cells was about 3 times larger than in non-activated cells. This can be explained by the upregulation of glycolysis triggered by the activation, which leads to an increase in 13C labelling of the lactate pool through LDH activity. Although the observed lactate signal is the sum of both intra- and extracellular lactate since the line width was too large to differentiate the two pools3, most of the detected signal likely originated from extracellular lactate. It is in agreement with recent experiments showing that extracellular lactate increases upon T cell activation4. Despite the fairly high signal-to-noise ratio, no bicarbonate signal could be observed, which shows that the flux through PDH was low and that most of the injected pyruvate was not used to feed the TCA cycle.

Conclusions

We demonstrated that T cell activation can be detected by hyperpolarized 13C MRS. This method can complement the standard ECAR measurements (Seahorse) and could be used to analyze the behavior of T cells in presence of antigens. Since it is well known that cancer cells will also compete for pyruvate5, it is not the ideal substrate to detect T cell activation in the tumor microenvironment and alternative hyperpolarized 13C tracers shall be tested.

References

  1. Pearce E.L., Poffenberger M.C., Chang C.H., Jones R.G. (2013). Fueling immunity: insights into metabolism and lymphocyte function. Science 342: 1242454.
  2. Ghesquiere B., Wong B.W., Kuchnio A., Carmeliet P. (2014). Metabolism of stromal and immune cells in health and disease. Nature 511: 167-76.
  3. Breukels V. et al. (2015). Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with 13C hyperpolarised NMR. NMR Biomed. 28: 1040–1048.
  4. Grist J.T. et al. (2018). Extracellular lactate : a novel measure of T cell proliferation. J. Immun. 200:1220-1226.
  5. Comment A. and Merritt M.E. (2014). Hyperpolarized Magnetic Resonance as a Sensitive Detector of Metabolic Function. Biochem. 53: 7333-7357.

Acknowledgement

This work is part of a project that has received funding from the European Union’s Horizon 2020 European Research Council (ERC Consolidator Grant) under grant agreement no. 682574 (ASSIMILES).

Keywords: NMR, mri, hyperpolarization, immunology, metabolism
739

Active targeting of epicardium-derived progenitor cells (EPDC) by EPDC-specific peptides (#432)

Tamara Straub1, Julia Nave1, Pascal Bouvain1, Julia Kistner1, Zaoping Ding1, Aseel Marzoq1, Stefanie Stepanow2, Siva S. K. Dasa3, Brent A. French4, Julia Hesse1, Karl Köhrer2, Ulrich Flögel1, Jürgen Schrader1, Sebastian Temme1

1 Heinrich Heine University, Department of Molecular Cardiology, Düsseldorf, North Rhine-Westphalia, Germany
2 Heinrich Heine University, Genomics & Transcriptomics Laboratory, Düsseldorf, North Rhine-Westphalia, Germany
3 University of Virginia, Cardiovascular Research Center, Charlottesville, Virginia, United States of America
4 University of Virginia, Department of Biomedical Engineering, Charlottesville, Virginia, United States of America

Introduction

Epicardium-derived progenitor cells (EPDC) play an important role during heart development . After myocardial infarction (MI), EPDC are reactivated, proliferate and migrate into the damaged tissue where they can differentiate into fibroblasts, endothelial cells and possibly also to cardiomyocytes1. How EPDC precisely contribute to the healing process after MI is still unclear. To further study the role of EPDC after MI, the aim of this study was to identify EPDC-specific peptides to enable the in vivo visualization by 1H/19F magnetic resonance imaging (MRI).

Methods

About 1010 clones of a Ph7 phage-library (linear 7mer peptides) were incubated for 1.5 h at 37 °C on cultured rat EPDC, isolated 5 d after MI. Cells were washed, bound phages were eluted and subsequently incubated with rat whole blood to eliminate clones that bind to blood cells. Phage titer was determined by qPCR. Identification of individual peptide sequences was performed with deep sequencing (Illumina Platform) and subsequent bioinformatics analysis (PHASTPep). Peptides were commercially manufactured and equipped with a carboxyfluorescein (flow cytometry) and a cystein for coupling to maleimide-perfluorocarbons (PFC). MR-experiments were performed at a 9.4 T Bruker AVANCEIII Wide Bore NMR spectrometer. Data were acquired using a 25 mm birdcage resonator tuneable to 1H and 19F.

Results/Discussion

Sequential panning of the phage library on EPDC and blood followed by deep-sequencing and bioinformatics analysis (Fig.1) revealed a total of 78,300 ± 31,900 different peptide-insertion sequences. Five peptides (EP1-EP5) showed an increased abundance of more than 70 in all samples and flow cytometry revealed that EP1-EP5 strongly bind to EPDC (MFI range: 16,000–51,000; Fig.2A) but not to blood immune cells (MFI: 970±610; Fig.2A). EP5 displayed the best binding affinity followed by EP2, EP4, EP1 and EP3. EP-peptides also showed strong binding to non-cultured EPDC freshly isolated from infarct hearts. Coupling EP5 to maleimid-containing PFC nanoemulsions (EP5-PFCs), incubation with EPDC and subsequent FACS analysis revealed strong targeting of EP5-PFC to EPDC in comparison to control-PFCs without peptide. Furthermore, 1H/19F MRI showed a more than two-fold higher 19F-signal of EPDC treated with EP5-PFCs compared to controls (14.1±2.8 vs 6.8±2.9; Fig.2B).

Conclusions

In the present study, we identified linear peptides which specifically bind to rat EPDC but not to circulating immune cells. Coupling of these peptides to PFC nanoemulsions enabled the targeting of EP-PFCs to EPDC and the visualization by 19F MRI in vitro. Therefore, this approach holds the potential to specifically track EPDC also in vivo by 1H/19F MRI to further unravel their role in the healing phase after MI.

References

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants ST 1209/1-1, FL  303/6-1 and the Sonderforschungsbereich SFB 1116.

Acknowledgement

1. Smart N, Riley PR. The epicardium as a candidate for heart regeneration. Future Cardiology. 2012;8(1):53–69.

Fig. 1: Schematic structure of the phage-display with rat EPDC.
First, a Ph7 phage-library was incubated on rat EPDC. The cells were washed, bound phages were eluted and incubated on rat whole-blood. The blood was subsequently centrifuged to split plasma and serum. Plasma phages were subjected to a PCR amplification. The PCR products were used for deep-sequencing and subsequent bioinformatics revealed differend peptide sequences.
Fig. 2: Binding affinity of EPDC-peptides to rat EPDC

A) The peptides bind strongly to EPDC but not to immune cells. The MFI for blood immune cells is around 970±610. EP5 has the best binding affinity to EPDC with a MFI of 59246, whereas EP3 shows the lowest binding with a MFI of 16742. n = 6

B) 1H/19F MRI showed a more than two-fold higher 19F-signal of EPDCs treated with EP5-PFCs compared to controls (14.1±2.8 vs 6.8±2.9). n = 2

Keywords: myocardial infarction, magnetic resonance imaging, 19F MRI, Epicardium-derived progenitor cells, phage-display
740

Examination of photo-CIDNP-based 19F MR hyperpolarization in dependence of the temperature (#490)

Frederike Euchner1, Rainer Ringleb1, Christian Bruns1, Joachim Bargon2, Johannes Bernarding1, Markus Plaumann1

1 Otto-von-Guericke University Magdeburg, Institute for Biometrics and Medical Informatics, Magdeburg, Saxony-Anhalt, Germany
2 University of Bonn, Institute for Physical and Theoretical Chemistry, Bonn, North Rhine-Westphalia, Germany

Introduction

MR signal enhancements can be achieved by using the hyperpolarization technique photo-Chemical Induced Dynamic Nuclear Polarization (photo-CIDNP).[1,2] Previous studies have shown that 19F MR hyperpolarization in aqueous solutions is a challenge.[3,4] Metabolism examination of fluorinated drugs as well as their intermolecular interactions are of high interest.[4] To date, only photo-chemically induced dynamic nuclear polarization (photo-CIDNP) allows the 19F hyperpolarization in pure water.[4,5] Here, a LED-based set-up[6,7] was used and we investigate the effect of a temperature change.

Methods

The investigated sample contained 3-fluoro-DL-tyrosine (2mM) and riboflavin 5’-monophosphate sodium salt hydrate (0.21mM) dissolved in physiologic salt solution. NMR-spectroscopic measurements were performed in a 5mm NMR tube on a 7T MR system (Bruker WB-300 Ultrashield). In all examinations an optical fiber is connected to a Cree XP E high power LED (455nm) and was centrally positioned in the solution. Irradiation times between 0s and 15s were chosen and a 90° pulse was used for the detection of 19F (P1=32.5µs, PL1=17W) NMR spectra. For measuring the temperature effect on the photo-CIDNP, the temperature unit of the NMR spectrometer was used and spectra were acquired at defined temperature in the range of 300-318K. Temperatures up to 330K were set for chemical shift change determination.

Results/Discussion

Photo-CIDNP enables the hyperpolarization of fluorine nuclei in aqueous solution (Figure 1). The 19F MR spectra show that with increasing temperature the signal enhancement decreases slightly. Nevertheless, the increase at 318 K is still sufficient to allow use in the MRI. In addition, the measurable change in chemical shift of the 19F signal to lower fields is of high importance. It provides a very accurate determination of the temperature within an MRI system (within high magnetic fields) using the graphical plot and the determined trend line (Figure 2).

While e.g. the hyperpolarization method such as PHIP does not enable a 19F hyperpolarization in pure water, the hyperpolarization technique photo-CIDNP allows a repetitive hyperpolarization without adding new substrates.[8] In comparison to the laser-based CIDNP (standard method), the LED set-up allows irradiation without warming the sample.  

Conclusions

The photo-CIDNP measurements show significant enhancements of the 19F NMR signal of 3-fluoro-DL-tyrosine in a biocompatible system using flavins as photosensitizers. Here, the 19F MR hyperpolarization occurs directly at 7 T using a low-cost and easy-to-handle LED-based set-up. At higher temperatures, both the MR signal and the signal gain decrease slightly. However, the detected enhanced signal can still be used for imaging.

References

[1] J. Bargon, et al. Zeitschrift Naturforschung Teil A, 1967, 22, 1551-1555.

[2] M. Goez, Annual reports on NMR Spectroscopy, 2009, 66, 77-147.

[3] M. Plaumann, et al. EMIM, 2015, #127.

[4] U. Flögel, E. Ahrens, 1. Ed. , Fluorine Magnetic Resonance Imaging, Pan Stanford Publishing, Singapore, 2017.

[5] F. Khan, et al. JACS, 2006, 128, 10729–10737.

[6] C. Feldmeier, et al. J. Magn. Res., 2013, 232, 39-44.

[7] C. Feldmeier et al. Angew. Chem., 2015, 127, 1363-1367.

[8] J. Bernarding, et al. ChemPhysChem., 2018, 19, 2453–2456.

Acknowledgement

This work was supported by the Deutsche Forschungsgemeinschaft (DFG BE 1824/12-1).

Figure 1: 19F Hyperpolarization
The 19F NMR spectra of hyperpolarized 3-fluoro-DL-tyrosine dissolved in aqueous solution. The signal enhancement is dependent from the irradiation time.
Figure 2: Plot of the chemical shift vs. temperature
The plot shows the dependence of the chemical shift of the 19F signal (3-fluoro-D,L-tyrosine) when changing the temperature.
Keywords: hyperpolarization, 19F, photo-CIDNP, 3-fluoro-DL-tyrosine, riboflavin

Ultrasound and Opto-Acoustic | Technology

Session chair: Ferdinand Knieling (Erlangen, Germany); Chrit Moonen (Utrecht, Netherlands)
 
Shortcut: PW05
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

745

Spiral volumetric optoacoustic tomography visualizes 3D distribution of brown adipose tissue in diabetic mice (#23)

Avihai Ron1, 2, Xosé Luis Deán-Ben1, Josefine Reber1, Vasilis Ntziachristos1, 2, Daniel Razansky1, 2, 3

1 Helmholtz Center Munich, Institute for Biological and Medical Imaging,, Neuherberg, Germany
2 Technical University of Munich, Faculty of Medicine, Munich, Germany
3 University of Zurich, Faculty of Medicine and Institute of Pharmacology and Toxicology, Zurich, Switzerland

Introduction

Diabetes is linked to deterioration of vasculature in the brown adipose tissues and decrease in its metabolic activity [1]. Tracking metabolism-related changes and structural changes of microvasculature in brown adipose tissue (BAT) may thus contribute to a better understanding of metabolic alterations. Multispectral optoacoustic tomography (MSOT) has been recently proposed as a potent tool for differentiating healthy and diabetic BAT by observing hemoglobin gradients and microvasculature density [2].

Methods

Here we explore spiral volumetric optoacoustic tomography (SVOT) as a new imaging approach to provide 3D functional and molecular assessment of BAT at the whole-body level [3]. Streptozotocin (STZ)-induced diabetic mice were imaged with SVOT and compared with the corresponding results acquired from healthy controls. The system enabled rendering high-resolution 3D views of the distribution of BAT and its intricate microvasculature. Volumetric blood oxygen saturation (sO2) as well as total blood volume (TBV) in the subcutaneous interscapular BAT (iBAT) were quantified.  Segmentation further enabled separating feeding and draining vessels from the BAT anatomical structure.

Results/Discussion

The scans have clearly revealed both the distortion in the microvasculature patterns and angiopathy of the microvasculature in the diabetic animals when compared to healthy controls. Scanning further revealed a significant decrease in the sO2 of diabetic iBAT with respect to the healthy control.

Conclusions

The results suggest that SVOT may serve as an effective tool for longitudinal studies of the effects of diabetes on the brown adipose tissue metabolism. As compared to cross-sectional (2D) MSOT imaging approaches, the true volumetric imaging capacity approach of SVOT enables visualization of the iBAT with significantly better image quality and quantification abilities.

References

[1] Owens B. 2014. The changing colour of fat. Nature. 508(7496):S52.

[2] Reber J, Willershäuser M, Karlas A et al. 2018. Non-invasive measurement of brown fat metabolism based on optoacoustic imaging of hemoglobin gradients. Cell metabolism. 27(3):689-701. e684.

[3] Deán-Ben XL, Fehm TF, Ford SJ et al. 2017. Spiral volumetric optoacoustic tomography visualizes multi-scale dynamics in mice. Light: Science & Applications. 6(4):e16247.

Acknowledgement

The authors would like to acknowledge the Human Frontier Science Program (HFSP) Grant RGY0070/201 and the European Research Council Grant ERC-2015-CoG-682379. Support from the Deutsche Forschungsgemeinschaft (DFG), Germany [Gottfried Wilhelm Leibniz Prize 2013; NT 3/10-1] and from the European Research Council (ERC) under grant agreement No 694968 (PREMSOT) is further acknowledged.

Figure 1.
(Left) Schematic of the spiral volumetric optoacoustic tomography (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 spherical motion. (Right) The yielded 3D image, focusing at the region of the interscapular brown adipose tissue (iBAT).
Keywords: optoacoustic, brown fat, metabolism, hemoglobin, oxygen saturation
746

Multispectral optoacoustic tomography of the thyroid gland (#6)

Wolfgang Roll1, Niklas Markwardt2, 3, Max Masthoff4, Anne Helfen4, Jing Claussen5, Michel Eisenblätter4, Alexa Hasenbach6, Sven Hermann6, Angelos Karlas2, 3, Moritz Wildgruber4, 7, Vasilis Ntziachristos2, 3, Michael Schäfers1, 7

1 University Hospital Münster, Department of Nuclear Medicine, Münster, Germany
2 Helmholtz Zentrum München, Institute of Biological and Medical Imaging, München, Germany
3 Technische Universität München, Chair of Biological Imaging, TranslaTUM, München, Germany
4 University Hospital Münster, Institute of Clinical Radiology, Münster, Germany
5 iThera Medical, München, Germany
6 University of Münster, European Institute for Molecular Imaging, Münster, Germany
7 University of Münster, Cells in Motion (CiM) Cluster of Excellence, Münster, Germany

Introduction

Thyroid disorders like thyroid nodules and autoimmune diseases are common clinical issues. Standard diagnostic tools including ultrasound(US)/doppler imaging, laboratory testing and invasive procedures, such as fine needle aspiration (FNA) and thyroidectomy for thyroid nodules. Multispectral optoacoustic tomography (MSOT) as a functional, contrast-free and non-invasive imaging method might help to improve diagnostics of thyroid disease. We report initial results of hybrid MSOT/US of common thyroid disorders, including Graves’ disease and thyroid nodules.

Methods

18 subjects (median age: 52, range: 21-82 years) were imaged with a hybrid clinical MSOT/US imaging system (Fig. 1). Functional biomarkers, including oxygenated (HbO2), deoxygenated (HbR) and total (HbT) hemoglobin, saturation of hemoglobin (sO2), fat and water content, were analyzed in thyroid lobes affected by Graves’ disease (n=6), in benign (n=13) and malignant (n=3) thyroid nodules and in healthy tissue (n=8).

Results/Discussion

In Graves’ disease, we found significantly higher HbR and HbT values (p = 0.0055 and p = 0.0084, respectively) and significantly lower fat values (p = 0.0293) compared to healthy thyroid tissue (Fig. 2). Malignant thyroid nodules showed significantly lower sO2 (p = 0.0393) and lower fat values than benign nodules (Fig. 3).

Conclusions

This pilot study shows the potential of hybrid MSOT/US to resolve functional and morphological tissue parameters in thyroid disorders. Large prospective studies are needed to confirm our observations.

Acknowledgement

This study was supported in part by the IZKF Münster, project Z04 & Core Unit PIX. WR was funded by a rotational clinician scientist position of the Medical Faculty, University of Münster, Germany. NM has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 667933 (MIB) and AK by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich-824 (SFB-824), subproject A1.

Keywords: Multispectral optoacoustic tomography (MSOT), Graves’ disease, thyroid nodule, hemoglobin
747

High frequency ultrasound and clinical ultrasound to monitor embryos and fetus development in chinchilla (chinchilla lanigera) as a valuable models to study human pregnancy. (#431)

Adelaide Greco1, 3, 2, Leonardo Meomartino2, Gerardo Fatone4, Monica Ragucci3, Raffaele Liuzzi3, Marcello Mancini3

1 University of Naples Federico II, Department of Advanced Biomedical Science, Naples, Italy
2 University of Naples Federico II, Centro Interdipartimentale di Radiologia Veterinaria, Naples, Italy
3 CNR, Institute of Biostrutture and Bioimaging, Naples, Italy
4 University of Naples Federico II, Dipartimento di Medicina Veterinaria e Produzioni animali, Naples, Italy

Introduction

The laboratory mouse has emerged as a predictable model in placental and fetal development studies(1), however, a recent study, has demonstrated that the Chinchilla Lanigerais an animal model suitable for human obstetric research (2,3).Ultrasound has been proven to be a non invasive Imaging techniques for monitoring embryo development, congenital anomalies, and to determine embryos and foetuses gestational age. Aim of our study was to increase the knowledge on the chinchilla pregnancy using High Frequency Ultrasound (HFUS) and clinical ultrasound (US).

Methods

One to two embryos were imaged in 35 pregnant female. We divided the duration of pregnancy in 4 stage of observation.Very early stage (S1) from embryonic day (E) 14 to E30 days, early stage (S2) from E31 to E46 days, intermediate stage (S3) from E47 to E70 days and advanced stage (S4) from E71 to E115 days.All morphometric parameters were analyzed in each embyos in a longitudinal way, during the entire duration of the pregnancy.Spearman's rank correlation (Rs) was used to see the relationship between each measurement and the embryonic day. Continuous linear regression was adopted for multivariate analysis of significant parameters. All statistical tests were bilateral, and a p value of 0.05 was considered statistically significant (MedCalc 12, Ostend, Belgium).

Results/Discussion

The study describes the application of HFUS to assess changes in phenotypic parameters in chichilla embryo and fetus during pregnancy and to evaluate physiological fetal and placental growth. A total of 74 embryos were counted. All morphometric parameters, except for femoral length, were significantly correlated with gestational age. For the regression analysis we divided our variable in 3 groups, based on the fact that each variables considered was possible to be monitored at the same time: group 1 - included variables measurable between time S1 and S3; group 2 - included variables measurable between time S2 and S3; group 3 - included variables measurable between time S2 and S4. Using this analysis, three models useful to predict the gestational age were obtained.

Conclusions

This study describes the use of HFUS to assess changes in phenotypic parameters in the developing chichilla embryo and fetus during pregnancy. A database of normal structural and functional parameters of chinchilla pregnancy will provide a use­ful tool for the characterization and the study of human pregnancy diseases.

References

1.A. Greco, M. Ragucci, A.R.D. Coda, et al., “High frequency ultrasound for in vivo pregnancy diagnosis and staging of placental and fetal development in mice,” Plos One 8(10):e77205, 2013.

2.E. Mikkelsen, H. Lauridsen, P.R. Nielsen, et al., “The chinchilla as novel animal model of pregnancy,Royal Society Open Science,vol 4, no 4, Article ID 161098, 2016.

3.P.L. Grigsby, “Animal models to study placental development and function throughout normal and disfunctional human pregnancy,” Semin Reprod Me,dvol 34, no 1, pp. 11-6, 2016.

Keywords: High Frequency Ultrasound, Chinchilla Lanigerais, pregnancy, embryos
748

Fast Three‑Dimensional Weighted Optoacoustic Reconstruction using a Novel w-k algorithm (#422)

Qutaiba Mustafa1, 2, Murad Omar1, 2, Ludwig Prade1, 2, Pouyan Mohajerani1, 2, Vasilis Ntziachristos1, 2

1 Helmholtz Zentrum München, Institute of Biological and Medical Imaging, Munich, Bavaria, Germany
2 Technical University of Munich, Chair of Biological Imaging and TranslaTUM, Munich, Bavaria, Germany

Introduction

In this work, we aimed to accelerate RSOM reconstruction by taking advantage of the speed of FFT, without compromising reconstruction quality. Therefore we developed a fast weighted - (FWOK) algorithm that builds and improves upon the conventional - algorithm. FWOK weights the data according to the optical transfer function, then transforms them back into the time domain. This weighting allows us to account for the sensitivity field of the detector in a more computationally efficient way than conventional time-domain RSOM reconstruction.

Methods

The transducer response is modelled by its Optical Transfer Function OTF(kz,kx,ky) defined as the Fourier transform of its Point Spread Function PSF(z,x,y) 1. We build up on the methodology presented in the w-k algorithm2. Before the data are transformed back to the time domain, they are in the form S(kz,kx,ky), which allows us to easily account for the OTF. In the case of absence of noise, the data are simply multiplied by the conjugate of the OTF. But in presence of noise, the following equation is used: S = S*OTF/(OTF^2 + σ^2), where σ is the noise variance, which is usually found empirically. Finally, the results are subjected to inverse Fourier transform to produce the final image. Figure 1 depicts the steps in applying the FWOK algorithm to a scan involving two point sources.

Results/Discussion

Figure 2 shows the results comparing the ability of conventional time-domain backprojection, the w-k algorithm as well as our FWOK algorithm to reconstruct RSOM images of the suture phantom. Figure 2a shows the reconstruction with time-domain backprojection; Figure 2b, with the w-k algorithm; and Figure 2c, with the FWOK algorithm. All three reconstructions showed similar three-dimensional structures, but the time-domain backprojection showed some artefacts surrounding the sutures, as shown by the red arrows in the magnified views (Figure 2d-f). The FWOK algorithm generated fewer reconstruction artefacts. The whole-image CNR is evaluated and compared for the different reconstruction methods. Its value was 16 for conventional backprojection, 17 for the w-k algorithm, and 33.5 for the FWOK algorithm. For finer assessment of image quality, Figure 2i-j compares CNRlocal for the three reconstruction methods.

Conclusions

For raster scan optoacoustic imaging, we demonstrated that the FWOK algorithm provides faster computation and higher reconstruction quality than time-domain backprojection.  We showed that the transducer aberration can be corrected by taking into account its optical transfer function, and that the FWOK algorithm can enhance CNR across the entire reconstructed image

References

1           Omar, M. et al. in Photons Plus Ultrasound: Imaging and Sensing 2017.  100640Z (International Society for Optics and Photonics).

2          Köstli, K. P., Frenz, M., Bebie, H. & Weber, H. P. Temporal backward projection of optoacoustic pressure transients using Fourier transform methods.                  Physics in Medicine & Biology 46, 1863 (2001).

3        Yaroslavsky, L. P. & Caulfield, H. J. Deconvolution of multiple images of the same object. Applied optics 33, 2157-2162 (1994).

Figure 2a-c : Comparison of backprojection, w-k and FWOK algorithms using the phantom experiment.
(d-f) Magnified views of panels (a)-(c), respectively. Red arrows in panels (d)-(f) point to possible reconstruction artefacts around the suture. (g) Intensity profile across the red lines in panels (a)-(c). (h) 3D visualization of the reconstruction in panels (a) and (c), overlaid on top of each other. (j) CNRlocal comparison results for ROIs shown on (i).
Figure 1: A 2D demonstration of reconstruction steps in FWOK.

a) One B-Scan of raw data from two point sources. b) The positive part of the spectrum of the raw data within the transducer bandwidth. c) The spectrum after the change of variables (f to kz). d) Measurement of the optical transfer function and its application to the spectrum. e) The final reconstructed image.

Keywords: Fast Fourier Transform, Omega-k migration, Optical Transfer Function, Photoacoustics, Mesoscopy
749

Photoacoustic imaging for hemoglobin content and oxygenation detection, from preclinical to clinical applications. (#494)

Jonathan Lavaud1, Maxime Henry1, Jean-Luc Cracowski2, 3, Jean-Luc Coll1, Veronique Josserand1

1 University Grenoble Alpes, Institute fro Advanced Biosciences, Grenoble, France
2 University Grenoble Alpes, HP2, Grenoble, France
3 Centre Hospitalier Universitaire, Centre d'investigations Cliniques, Grenoble, France

Introduction

PAI provides unique opportunities to measure noninvasively some endogenous compounds such as oxy- and deoxy-hemoglobin and thus tissue oxygenation or hypoxia. These characteristics are of prime importance since they provide simultaneously physio-pathological and anatomical information. Photoacoustic imaging appears to be extremely useful in many preclinical and clinical applications. Faced with the growing interest in this new technology, it seems essential to accurately characterize the veracity of the information emanating from the photoacoustic imaging of endogenous contrast.

Methods

PAI was performed with the VevoLAZR system (FUJIFILM VisualSonics Inc.). The stability of either single-wavelength or spectroscopic measurements was evaluated on a phantom containing a stable absorber in a 30 min continuous acquisition and then at 10 different time points over two weeks. Both repeatability and reproducibility of hemoglobin content and tissue oxygenation measurements were evaluated in vivo on various organs of interest (liver, spleen, kidney, brain, muscle). For this, an oxy-hemo protocol was used at different time points over two weeks. Finally, an ischemic challenge was applied on the forearm of 13 healthy volunteers. In short, after a 2 minutes baseline recording, blood flow was partially occluded for 3 minutes. Tissue oxygenation was recorded during and after occlusion.

Results/Discussion

Results first showed an extremely high stability of measurement on phantoms (COV% < 2) both in single-wavelength and spectroscopic acquisitions.

In vivo in mice, hemoglobin content and tissue oxygenation measurements showed good repeatability (COV% between 4.9 and 12.4) and reproducibility (COV% between 1.9 and 14.6) and these were observed in all organs regardless of their depth or blood content.

Finally, photoacoustic imaging in healthy volunteers was able to evidence in real-time an experimentally induced ischemia in the forearm with a high reproducibility (COV% <20).

 

Conclusions

These results highlighted the high reliability of endogenous contrast based photoacoustic measurements in vivo and open a wide scope with high potential for preclinical applications as well as clinical investigations.

References

 

  1. Wang, X., et al., Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain.Nat Biotechnol, 2003. 21(7): p. 803-6.
  2. Wang, X., et al., Noninvasive imaging of hemoglobin concentration and oxygenation in the rat brain using high-resolution photoacoustic tomography.J Biomed Opt, 2006. 11(2): p. 024015.
  3. Lavaud, J., et al., Exploration of melanoma metastases in mice brains using endogenous contrast photoacoustic imaging.Int J Pharm, 2017. 532(2): p. 704-709.
Keywords: photoacoustic, endogenous contrast, oxygenation, in vivo, human
750

Longitudinal studies of mouse mammary gland tumour models using photoacoustics (#323)

Jakub L. Czuchnowski1, 4, Robert Prevedel1, 2, 3

1 EMBL Heidelberg, Cell Biology and Biophysics Unit, Heidelberg, Baden-Württemberg, Germany
2 EMBL Heidelberg, Developmental Biology Unit, Heidelberg, Baden-Württemberg, Germany
3 EMBL Rome, Epigenetics and Neurobiology Unit, Monterotondo , Italy
4 Heidelberg University, Faculty of Biosciences, Heidelberg, Baden-Württemberg, Germany

Introduction

Targeted therapies against cancer often prove to be only partially successful due to relapses occurring during long-term treatment, raising the need for better understanding of cancer drug resistance and relapse. Transgenic mouse models with inducible oncogene expression provide highly tractable experimental systems for such purposes1. Unfortunately most of those systems rely on ex vivo imaging of excised tumours which prohibit real longitudinal studies. Therefore we present a photoacoustic tomography (PAT) based approach to monitor tumour progression and therapy in vivo.

Methods

Our method is based on an optical acoustic wave detection scheme employing an Fabry-Perot interferometer (FPI) acting as the active element of the detector2. This system has proven successful in proof of principle studies of tumour progression. Here, we want to apply it for longitudinal in vivo monitoring of mammary gland tumour progression and therapy in a clinically relevant mouse model system. We characterise the setup by ex vivo imaging of tyrosine expressing cells in hydrogels before tracking tumour progression in vivo in mammary gland tumours based on stereo-tactical injection of cells into the fat pad of adenectomised mice.

Results/Discussion

We present the details of our PAT system design, including a robust optical interrogation path and low-noise amplified photodiode detectors as well as their functional characterisation. The functionality of the setup was validated and cross-checked with acoustic signals generated by an ultrasound transducer. We further performed computer simulations  taking into account all known physical parameters, including properties of the imaged tissue and tyrosinase reporter system and compared them to the actual system’s performance. Furthermore, we present our results towards in-vivo experiments and discuss the limitations of our approach in terms of achievable tissue depths and clonal population sizes.

Conclusions

We believe that our optical PAT approach can be a reliable method for fast assessment of tumour size and morphology, enabling studies of dynamics of cancer growth, regression and relapse in vivo. The technical advantage provided by a high performance photoacoustic setup in combination with clinically relevant mouse cancer models will lead to better understanding of mechanisms leading to drug resistance and relapse of breast tumours.

References

 1. Jechlinger, M., Podsypanina, K., & Varmus, H. (2009). Regulation of transgenes in three-dimensional cultures of primary mouse mammary cells demonstrates oncogene dependence and identifies cells that survive deinduction. Genes & development, 23(14), 1677-1688.

2.  Jathoul, A. P., Laufer, J., Ogunlade, O., Treeby, B., Cox, B., Zhang, E., ... & Lythgoe, M. F. (2015). Deep in vivo photoacoustic imaging of mammalian tissues using a tyrosinase-based genetic reporter. Nature Photonics, 9(4), 239.

Acknowledgement

We want to acknowledge Florian Mathies, Johannes Zimmermann and Gerardo Hernandez Sosa from Innovation Lab Heidelberg for their assistance in manufacturing the Fabry-Perot interferometer sensors. We want to acknowledge Lucas Chaible and Martin Jechlinger from EMBL Heidelberg for their help in preparing cell line and mouse experiments.

Keywords: photoacoustics, photoacoustic tomography, cancer
751

Multispectral Optoacoustic Tomography: A Novel Label-Free Imaging Technique for the Assessment of Graves’ disease and autonomous adenomas (#319)

Markus Kroenke1, Angelos Karlas2, 3, Nikolina Fasoula3, Niklas Markwardt3, Klemens Scheidhauer1, Wolfgang Weber1, Vasilis Ntziachristos3

1 Klinikum rechts der Isar, TUM, Department of Nuclear Medicine, München, Germany
2 Klinikum rechts der Isar, TUM, Department of Vascular Surgery, München, Germany
3 Klinikum rechts der Isar, TUM, Department of Medical Imaging, München, Germany

Introduction

MSOT is a novel imaging technique that can provide morphological and functional tissue characterization without the need for contrast agents. Tissue is illuminated by laser pulses and ultrasound signals are produced and collected to form high-resolution tomographic images. Oxy- and deoxy-hemoglobin and other natural chromophores’ (such as lipids and water) concentrations can be measured. In the current study, we explore the capabilities of MSOT to characterize functional changes of the thyroid parenchyma in patients with Graves’ disease and autonomous adenomas.

Methods

In this ongoing prospective study, we have so far enrolled 8 healthy volunteers and 19 patients (17 women, 2 men, median age: 53.5 years, range: 20-78). Nine patients suffered from Graves’ disease and ten from autonomous adenoma. All patients received axial MSOT, ultrasound (b-mode and Doppler-mode) and scintigraphy of the thyroid. The levels of oxy- and deoxy-hemoglobin, the local oxygen saturation (sO2) as well as the lipid and water content of the thyroid gland were extracted by means of spectral unmixing from the corresponding MSOT readouts. For data analysis, different techniques were applied (backprojection and model-based reconstruction, deconvolution with electrical and spatial impulse response, use of ultrasound priors, linear unmixing and spectral tissue characterization).

Results/Discussion

So far, model-based reconstruction without deconvolution seems to offer highest imaging accuracy and contrast. The use of ultrasound priors might help improve image quality by noise reduction. The normal thyroid gland demonstrated significantly higher sO2 than skeletal muscle (58% vs 46% uncorrected) while lipid content was low and similar in both tissues. Patients with Graves’ disease had normal fT3 and fT4 values, while patients with autonomous adenoma had a suppressed TSH. Graves’ disease was confirmed by antibody titers. MSOT was capable of detecting the autonomous adenoma and Graves’ disease by showing in both increased perfusion (as confirmed by Doppler-ultrasonography), increased deoxyHb, 0.55 au and 0.79 au compared to 0.49 au, p<0.05, increased oxyHb, 0,86 au, 1,2 au compared 0,77 au p<0.05, and decreased levels of sO2, 0.56 and 0.54 compared to 0.58, p<0.05. There were no significant differences in fat and water content found.

Conclusions

In contrast to the intensity-based signal of traditional ultrasonography MSOT of the thyroid can provide a variety of functional parameters including oxygenation, lipid and water content in one imaging session. Since MSOT is non-invasive and does not require ionizing radiation, this new technology shows significant potential for studies of thyroid pathophysiology in various diseases.

Keywords: Multispectral Optoacoustic Tomography, Graves’ disease, autonomous adenomas, Optoacoustic
752

Power doppler ultrasound quantification in infrapatellar fat pad vasculature during patellar tendinopathy (#8)

Juan Antonio Cámara Serrano1, Anna Pujol Esclusa1, Paola Contreras Muñoz2, 3, David Dominguez4, Mario Marotta2, 3

1 Vall d´Hebron Campus, Preclinical Imaging Platform, Barcelona, Spain
2 Vall d´Hebron Institue of Research, Bioengineering, Cell therapy and Surgery in Congenital Malformations Lab, Barcelona, Spain
3 Leitat Technological Center, Barcelona, Spain
4 Futbol club Barcelona, Medical Services, Barcelona, Spain

Introduction

Ultrasound exams have always been classified as “semi-quantitative” technique. Recent advances in imaging technologies and processing software have opened the door to B-mode studies quantification. Power Doppler ultrasound is the next step for quantification protocols.

Tendinosis is a recurrent pathology affecting tendon. Owing to its nature, this structures are perfect targets for ultrasonography, as well as other soft tissues affected in tendon pathologies.

We present a protocol for Power Doppler ultrasound quantification of infrapatellar fat vasculature in a tendinosis model in rats.

Methods

7 rats where scanned two weeks after an unilateral tendinopathy induction. Joint was positioned in forced flexion to expose the patellar tendon. B-Mode scans were firstly performed to localize the patellar tendon and infrapatellar fat pad. The probe was fixed to a bracket for immobilizing and reduce the Doppler artifacts due to involuntary movements.

Scanning parameters were: B-Mode - 18 Mhz frequency, 1cm depth and 25 frames per second. Power Doppler mode - 12.5 Mhz and PRF 750 Hz.

8-10 seconds (200-250 frames) videos were analyzed with Image J. Color channel was made binary and particles counted. Erratic frames originated from artifacts were discarded. Analyzed parameters were: Doppler pulse, number of Doppler signals, signal intensity, Doppler signal size and Total Doppler signal area.

Results/Discussion

In healthy tendons, longitudinal view offers more Doppler signals and higher total Doppler area. In pathologic samples, longitudinal views have more total area and mean signal size. There are no differences in other parameters. Mean Doppler signal size and Total Doppler area are bigger in pathologic tendons and  Doppler signals increase in transversal view of pathologic samples.

Longitudinal ultrasound scan of patellar tendon as well as infrapatellar fat pad exams are more feasible than transversal exams. In this view, the number of Doppler signals are higher than in transversal view. Pathologic process of tendon increases Doppler signals in the infrapatellar fat pad. This could be due to it is supposed to bring blood supply to patellar tendon during recovery and healing process. This tendon has been described as a poorly vascularized tissue.

Power Doppler quantification can be used for evaluating infrapatellar fat pad vascularization during patellar tendon inflammatory process.

Conclusions

Power Doppler quantification can be used for evaluating tendon inflammatory processes in patellar tendon. During the healing process of the patellar tendon, infrapatellar fat pad increase its vascularization. This could be because the fat pad is supposed to bring blood supply to patellar tendon during recovery and healing process. This tendon has been described as a poorly vascularized tissue.

References

Bouta EM, Banik PD, Wood RW, Rahimi H, Ritchlin CT, Thiele RG, Schwarz EM. Validation of power Doppler versus contrast-enhanced magnetic resonance imaging quantification of joint inflammation in murine inflammatory arthritis. J Bone Miner Res. 2015 Apr;30(4):690-4

Xu H, Bouta EM, Wood RW, Schwarz EM, Wang Y, Xing L. Utilization of longitudinal ultrasound to quantify joint soft-tissue changes in a mouse model of posttraumatic osteoarthritis. Bone Res. 2017 Jun 13;5:17012

Acknowledgement

Authors would like to thank the animal facility staff at Vall d´Hebron Research Institute for their help during this experiment.

Ultrasound analysis
Power Doppler ultrasound in a healthy (A) and pathologic (B) tendon. Note the increase of Doppler signals. Total Doppler signal area (square pixels) of both tendons is displayed in C.
Statistics
Resume of results and graphic representation of analyzed parameters.
Keywords: ultrasound, power doppler, quantification, tendon
753

Quantitative photoacoustic hyperspectral imaging using a non-iterative approach (#142)

Hisham Assi1, Andrew Heinmiller2, Joseph C. Kumaradas1

1 Ryerson University , Department of Physics, Toronto, Ontario, Canada
2 FUJIFILM, VisualSonics Inc., Toronto, Ontario, Canada

Introduction

Photoacoustic (PA) imaging is an emerging hybrid modality that has the contrast of the optical imaging and the high resolution of the ultrasound imaging [1, 2]. Quantitative photoacoustic hyperspectral imaging is a modality that uses a range of optical wavelengths to quantify the concentration of an optical absorber which is needed for molecular and functional imaging [3]. The photoacoustic signal depends on both the absorber concentration in the imaged media and the optical fluence in that media. Hence, information about the fluence distribution is needed to obtain a quantitative PA image.

Methods

Fluence distribution in tissue can be described by the radiative transfer equation or by its diffusion-approximation. These have been used as forward models in iterative inverse solvers to find tissues absorption in tomographic systems[4,5]. In this work, we propose a simple non-interactive approach to correct PA images obtained by linear array transducers. In the equation of light diffusion in a medium, one of the terms is the fluence multiplied by the absorption coefficient. This product is, in fact, proportional to the uncorrected PA image. We used finite element simulations to calculate the fluence distribution using the uncorrected PA image as an input. The corrected PA image can, therefore, be obtained by dividing the uncorrected PA image by the calculated fluence distribution.

Results/Discussion

Figure 1A shows the uncorrected PA image obtained in-vivo and figure 1B shows the estimated fluence distribution based on the uncorrected PA image. The uncorrected PA image (in C) can then be corrected (as shown in D) for the non-uniform fluence distribution by dividing it by the estimated fluence (in B). The in-vivo experiment consisted of an abdominal section of a mouse that had two tubes with the same absorbing dye. One was placed above the abdomen and one below the abdomen, as indicated by the circles in figure 1A. The result for three wavelengths, 685 nm, 750 nm, and 830 nm, is shown in figure 5. The two sets of values which should ideally be alike are substantially different as shown in Figure 2A. The correction not only increases the penetration depth of the PA image at all wavelengths (making the further tube visible as shown by the arrow in Figure 1D) it also corrects the spectrum of the lower tube, as shown in Figure 2B.

Conclusions

We have introduced a fluence correction technique for photoacoustic imaging to aid in quantification of absorber concentration. It is a non-iterative method that uses both the uncorrected PA image and the physics of light diffusion in tissue to calculate the fluence distribution needed to quantify the absorption coefficient. It also corrects the absorption spectrum, which is not possible using other methods such as time-gain compensation (TGC).

References

[1] L. V. Wang, “Tutorial on Photoacoustic Microscopy and Computed Tomography,” IEEE J. Sel. Top. Quantum Electron., vol. 14, no. 1, pp. 171–179, 2008.

[2] P. Beard, “Biomedical photoacoustic imaging,” Interface Focus, vol. 1, no. 4, pp. 602–631, 2011.

[3] B. Cox, J. G. Laufer, S. R. Arridge, and P. C. Beard, “Quantitative spectroscopic photoacoustic imaging: a review,” J. Biomed. Opt., vol. 17, no. 6, p. 061202, Jun. 2012.

[4] G. Bal and K. Ren, “On multi-spectral quantitative photoacoustic tomography in diffusive regime,” Inverse Probl., vol. 28, no. 2, p. 025010, 2012.

[5] F. M. Brochu, J. Joseph, M. Tomaszewski, and S. E. Bohndiek, “Light fluence correction for quantitative determination of tissue absorption coefficient using multi-spectral optoacoustic tomography,” in Opto-Acoustic Methods and Applications in Biophotonics II, 2015.

Acknowledgement

The authors would like to thank Ontario Centres of Excellence and FUJIFILM VisualSonics, Inc. for supporting this research.

Figure 1

A mouse experiment with two tubes above (tube 1) and below (tube 2) the mouse, circled in (A), that contain the same dye. Both ultrasound image (grayscale) and the PA image (red scale) are shown in (A). The uncorrected PA image in the selected region in (A) was used to calculate the fluence distribution in (B). (C) and (D) shown the uncorrected and corrected (using C) PA images, respectively.

Figure 2
(A) shows the normalized values of the uncorrected PA signals at tube 1 (blue line) and tube 2 (red line) at three different wavelengths. The two sets of values are different; even if the tube 2 values were scaled up (dotted-line), the spectrum of the two tubes would not be similar. (B) shows the values of the corrected PA signal, which are close to each other, and have the same spectral shape.
Keywords: Quantitative photoacoustics, fluence correction, diffusion approximation, hyperspectral imaging.
754

Photoacoustic blood oxygenation measurements combined with online pO2 detection and flow spectrometry (#135)

Marcel Gehrung1, Sarah E. Bohndiek1, 2, Joanna Brunker1, 2

1 University of Cambridge, CRUK Cambridge Institute, Cambridge, United Kingdom
2 University of Cambridge, Department of Physics, Cambridge, United Kingdom

Introduction

Optoacoustic (OA) imaging is intrinsically sensitive to blood oxygen saturation (sO2), which is calculated from measured oxy- (HbO2) and deoxy- (Hb) haemoglobin concentrations. These measurements are however affected by unknown tissue and instrumentation-related correction factors. We have developed a flow system for calibrating OA images by monitoring sO2 using a flow spectrometer and probes sensitive to oxygen partial pressure (pO2). This ex vivo dynamic assessment of sO2 enables biological validation for in vivo studies relevant to conditions such as sickle-cell anaemia and tumour hypoxia.

Methods

The flow system (Fig. 1) enabled fluids to be circulated within a vessel-mimicking tube and characterised using an online spectrometer and pO2 probes, while acquiring OA images of the tube embedded within an agar phantom. First, dyes (methylene blue, MB, and indocyanine green, ICG) were injected into the system and the known concentrations compared with measured values calculated using linear spectral unmixing on the OA images1. Subsequently, blood (oxygenated using hydrogen peroxide) was circulated within the system and slowly deoxygenated by injecting sodium hydrosulfite. The accuracy of spectral unmixing was investigated for a range of sO2 values. Finally, the effect of spectral colouring was explored using agar impregnated with Nigrosin dye providing tissue-like background absorption.

Results/Discussion

Spectra measured using the online spectrometer had different spectral shapes compared to the literature spectra (used by default). Using default spectra resulted in inaccurate estimations for dye concentrations (MB and ICG) as well as for blood sO2. Blood sO2 remained stable while circulating within the flow system for at least three minutes. During chemically induced deoxygenation from sO2 = 100% to 0% (Fig. 2), unmixing with the online spectrometer yielded an accurately calculated upper limit of 100%, but overestimated the lower limit to be 18%; using the default unmixing spectra resulted in a highly supressed range of calculated sO2 (85% to 60%). The sO2 values calculated from pO2 measurements showed different dynamics due to the pO2-sO2 conversion. Spectral colouring within the tube was responsible for overestimation of the sO2 for deoxygenated blood. A simple exponential decay fluence correction mitigated the effects of spectral colouring produced by the tissue-mimicking phantom.

Conclusions

Optoacoustic imaging allows estimation of blood sO2 but unmixing with literature spectra of Hb and HbO2 compromises measurement accuracy. In addition, fluence correction methods are essential for mitigating the adverse effects of spectral colouring on sO2 quantification. In general, to ensure biological validity of functional measurements in vivo, it would be necessary to define system specific unmixing spectra for endogenous chromophores.

References

  1. S. Tzoumas, N. Deliolanis, S. Morscher, and V. Ntziachristos, “Unmixing molecular agents from absorbing tissue in multispectral optoacoustic tomography,” IEEE Transactions on Med. Imaging 33, 48–60 (2014).

Acknowledgement

The authors would like to thank Ayaka Shinozaki for her assistance with experiments, James Joseph for his contributions to the experimental design and Michael Schneider for his helpful input regarding data interpretation.

Overview of the flow system.
1 Injection site; 2 light source and spectrometer record spectra of flowing fluids (e.g. blood); 3a/b needle probes measure temperature and pO2 before/after blood flows through an agar phantom in the optoacoustic imaging system; 4 MSOT (iThera); 5 touch-screen display for temperature and pO2 data, which are downloaded 6 via an Arduino UNO onto a laptop; 7 peristaltic pump provides circulation.
Dynamic blood deoxygenation.
(a) Change in sO2 measured using the MSOT, the online spectrometer and the pO2 probes while injecting 3% w/v sodium hydrosulfite in PBS over nine minutes. (b, top) MSOT default spectra for oxy- and deoxy- haemoglobin used for MSOT “Vendor” unmixing. (b, bottom) Spectra obtained from the online spectrometer at the start and end of the dynamic deoxygenation, and used for “Online" unmixing.
Keywords: oxygenation, optoacoustic, unmixing, blood, spectrometer
755

An Ultrasound-Guided Injection method for a refinement and efficient Breast Tumor Induction into mice. (#540)

Francesca La Cava1, Alberto Fringuello Mingo2, Sonia Colombo Serra2, Aldo Di Vito2, Claudia Cabella2, Paolo Oliva2, Alessia Cordaro2, Chiara Brioschi2, Luisa Poggi2, Enzo Terreno1, Luigi Miragoli2

1 University of Turin, Department of Molecular Biotechnology and Health, Turin , Italy
2 Bracco Imaging SpA, Laboratory of Preclinical Imaging, BioIndustry park of Canavese, Colleretto Giacosa, Italy

Introduction

Breast cancer is the most common cancer for US women.1MRI with the use of Contrast Agents (CAs) is a recommended screening, especially for high-risk women.2

The use of animal models is essential to test contrast efficacy of CAs under development. Breast cancer orthotopic models can be achieved by implantation under direct vision of the mammary fat pad or orthotopic percutaneous blind injection (OP) in the nipple area.3-4 To overcome the invasiveness of surgery and the limited accuracy of OP, ultrasound was introduced as a guide for precise localization. The model was then characterized by MRI.

Methods

BT20 cells (106 to 107 in 100 µL) were injected into the fourth mammary fat pad (mfp) of female nu/nu mice via classic surgery (n=6), and ultrasound (US) guided injection (n=16) by using a Vevo Lazr 2100 US System with a MS700 Transducer (55 MHz). 4T1 (5x105 to 106 in 50 µL) and TS/A (5x104 to 5x105 in 50 µL) cells were injected into the mfp of 26 Balb/C female mice by using US guided injection.

All the animals survived the tumor induction procedure and did not show suffering signs. Tumor growth was monitored by MRI/US imaging weekly or closer. Mice developing noticeable masses were recruited for Contrast Enhanced (CE) MRI studies, performed by a Bruker Biospec 3 T equipped with a 20 mm diameter surface coil. At the end of the study, tumor identity was confirmed by histology.

Results/Discussion

Both surgery and US-guided injection were successful in locating the BT20 tumor mass into the mfp, as confirmed by MRI and histology. Despite no intraoperative death occurred, the invasiveness of surgery was not negligible. US-guided injection, reducing the animal post-operative suffering, resulted an optimized refinement procedure.

The growth of BT20 tumor was limited, reaching a maximum volume of 10 mm3 in 6 weeks. When detectable the diseased mass showed a 50-80% signal enhancement after CA administration at 0.1 mmol/kg.

4T1 and TS/A cells were injected into the mfp to confirm the validity of the US-guided injection. 4T1 tumor growth was noticeably fast, leading to observable masses (10 mm3) already 4 days after inoculation and progressively increasing to the endpoint (300 mm3) in 2 weeks or earlier. After CA administration, a 100% enhancement was observed with spin and gradient echo sequences.

TS/A tumor growth curve is under monitoring and the characterization by CE MRI is ongoing.

Conclusions

Breast cancer drug development is ineffective without a reliable preclinical model, which is hard to identify. In this study, a novel and refinement method to induce breast cancer was developed and three different cell lines were tested. The use of CE MRI served as a way to fully characterize the tumor and study its vascularization, rendering this model useful for efficacy studies, thus bridging the gap between clinical and preclinical research.

References

1. Centers for Disease Control and Prevention. Breast Cancer Statistics. Available at: https://www.cdc.gov/cancer/breast/statistics/. Accessed January 16, 2019

2. Mainiero MB, Lourenco A, Mahoney MC, et al. ACR Appropriateness Criteria breast cancer screening. JAmCollRadiol 2016; 13(11S): R45–R49.

3. Talmadge JE, Singh RK, Fidler IJ, et al. Murine models to evaluate novel and conventional therapeutic strategies for cancer. Am J Pathol. 2007; 170(3):793–804. [PubMed: 17322365]

4. Rashid OM. Et al. Breast Cancer Res Treat. 2014 Oct; 147(3):501-12. doi: 10.1007/s10549-014-3118-0

 

 

 

Ultrasound Guided Injection of breast tumor cells into the mammary fat pad
4T1 Orthotopic Tumor after GBCA Injection
4T1 Tumor (red arrow) MRI characterization before, 1 min and 10 min after administration of a GBCA. Mammary fat pad (green arrow) demonstrates the correct localization of the tumor.
Keywords: Breast cancer, mri, ultrasound, gbca, tumor models

Imaging Cancer Therapy I

Session chair: Christine Goze (Bourgogne, France); Jan Grimm (New York, US)
 
Shortcut: PW06
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

826

Stress induced by iron-overload as a novel treatment for breast cancer. (#374)

Valeria Bitonto1, Diego Alberti1, Simonetta Geninatti Crich1, Silvio Aime1, Juan C. Cutrin1

1 University of Turin, Department of Molecular Biotechnology and Health Sciences, Torino, Italy

Introduction

New insights are linking the presence of iron excess and altered iron metabolism to cancer1. Since iron is crucial to many fundamental cellular processes, many cancer cells reprogram iron metabolism in ways that result in net iron influx2. However, it has been reported that tumor cells appear unable to properly manage iron excess removal. In this perspective, iron overload by means of horse spleen ferritin (HoS-Ferritin) appears to be an attractive therapeutic idea for cancer treatment due to its potential to activate mechanisms of redox cell injury.

Methods

In order to evaluate if the amount of HoS-Ferritin taken-up by tumors is sufficient to visualize tumor lesions, a group of female BALB/c mice were inoculated subcutaneously with 3x105 TS/A cells. When tumors reached a dimension of approximately 10 mm3, mice were intravenous injected with HoS-Ferritin (0,2 mmol kg-1 of iron). MRI images were acquired before HoS-Ferritin administration and 3h, 6h and 24h after using a T2-Weighted RARE sequence protocol.  For tumor treatment a group of female BALB/C mice, inoculated with 3x105 TS/A cells, were treated with HoS-Ferritin (0,1 mmol kg-1 of iron) every two days; tumor volume growth was assessed by MRI. After 5 administrations mice were sacrified and ICP-MS analysis of iron content was performed ex vivo in tumor, liver and spleen.

Results/Discussion

In all TS/A tumor-bearing mice, contrast enhancements upon a single intravenous injection of HoS-Ferritin were successfully observed. Contrast enhancement in tumor, spleen and liver was already visible 3h after the injection, while the maximum signal was recorded after 6h.

After HoS-Ferritin treatment a significant decrease in tumor growth (*p<0,05) and a significant difference in MR signal intensity was observed in the tumor mass of treated mice. Histological analysis showed that the signal intensity changes in MR correlated with the presence of necrotic tissue.

ICP-MS analysis and Perls staining showed a significant increase in iron content in the liver (p<0.01) and tumor (p<0.05) after HoS-Ferritin treatment. AST enzymatic activity and liver histology showed no difference between the control and treated group, indicating that iron accumulation doesn’t have toxic effects on liver structure and function.  

Conclusions

HoS-Ferritin, as a natural theranostic agent, was exploited for MRI visualization and treatment of breast cancer. After HoS-Ferritin administration an intense signal intensity change, due to the protein accumulation in mice bearing TS/A breast tumor was detected. To our knowledge iron-uploading dependent mechanism of cell damage and death, caused by HoS-ferritin internalization, could be used as a novel strategy to treat breast cancer.

References

  1. Richardson DR, Ponka P. The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta. 1997; 1331:1–40.
  2. Lui GY, Kovacevic Z, Richardson V, Merlot AM, Kalinowski DS, Richardson DR. Targeting cancer by binding iron: Dissecting cellular signaling pathways. Oncotarget. 2015; 6:18748–79
In vivo MR and ex vivo histological (H&E) images of TS/A tumors

T2-Weighted MR and H&E images of veichle and HoS-Ferritin treated TS/A tumors. After the last treatment a significant difference in MR signal intensity was observed in the tumor mass of treated mice. Histological analysis showed that the signal intensity change in MR correlated with the presence of necrotic tissue (asterisks).

Evaluation of TS/A tumor volume
TS/A tumor volume was calculated by means of MRI: a significant decrease in tumor growth (*p<0,05) was observed in the HoS-Ferritin treated group.
Keywords: Iron, cancer treatment, ferritin, breast cancer
827

Intratumoral activity distribution of 177Lu-PSMA-617 compared with diagnostic tracers in preclinical prostate cancer models (#405)

Anders Örbom1, Sven-Erik Strand2, Oskar Vilhelmsson Timmermand1

1 Lund University, Department of Clinical Sicences Lund / Section Oncology and Pathology, Lund, Sweden
2 Lund University, Department of Clinical Sicences Lund / Section Medical Radiation Physics, Lund, Sweden

Introduction

This is a continuation of our project to examine the intratumoral distribution of PSMA-targeted peptide for radionuclide therapy of disseminated prostate cancer, which we presented the first results from at EMIM 2018. We have continued these studies in order to try to fill the lack of published preclinical data on PSMA radionuclide therapy. Following the feedback from the former meeting, we have used additional cell-lines, as well as compared the distribution of 177Lu-PSMA-617 with the diagnostic tracer 68Ga-PSMA-11.

Methods

BALB/c nude male mice were given subcutaneous tumor xenografts of human PSMA-expressing LNCaP (L) (n=24) or 22Rv1 (R) (n=9) cells. PSMA-617 (ABX, Radeberg, Germany) was labeled with 177Lu and the mice were given 5-20 MBq by i.v. injection. Some animals were co-injected with 18FDG (L n=7, R n=3), 18Fluorocholine (L n=5) or 68Ga-PSMA-11 (L n=1, R n=1). All co-injected animals were sacrificed 1 h p.i., and the rest at 20 min (L n=3), 70 min (L n=2), 90 min (L n=1), 2 h (L n=2, R n=5), 2 weeks (L n=1) and 3 weeks (L n=2) p.i.. Blood, tumor, and kidneys activity was measured by gamma counter. Part of the tumor was frozen on dry ice and cryosectioned at 10 µm. Autoradiography was performed using a detector at 50 µm intrinsic spatial resolution [1] after which sections were stained for histology.

Results/Discussion

The tumor activity distribution of 177Lu-PSMA-617 appears to become more homogeneous over time from 20-120 min p.i., however there are still areas with viable tumor cells with comparably low uptake at that time (Figure 1). For tumors from co-injected animals, the difference in half-life between isotopes was exploited to separate the signal from each radionuclide in the images. Comparing 177Lu-PSMA-617 with diagnostic tracers (Figure 2) show an almost identical intratumoral distribution of 68Ga-PSMA-11. Whereas both 18F-FDG and 18F-Fluorocholine seem to penetrate better into dense viable tumor tissue, especially for LNCaP tumors, but also have some non-specific uptake in tumor capsule or binding tissue.

Conclusions

The small-scale activity distribution influences the absorbed dose delivered to the tumor and is relevant to the choice of fractionation schedule etc. Our results so far indicate that 177Lu-PSMA-617 takes some time to fully penetrate the tumor, while some diagnostic tracers may penetrate better but less specifically. We are working on and hope to present adjacent sections stained for PSMA expression at the meeting.

References

1. Örbom, Anders, et al. "Characterization of a double‐sided silicon strip detector autoradiography system." Medical physics 42.2 (2015): 575-584.

Acknowledgement

The authors wish to thank Pontus Kjellman and Wahed Zedan for their assistance during this study. The study was made feasible with Grants from Mrs berta Kamprad Foundation, Swedish Cancer Society and the Swedish Prostate Cancer Association.

Figure 1

Histology and activity distribution at different times for 177Lu-PSMA-617.

Figure 2
Histology and activity distribution of 177Lu-PSMA-617 compared with diagnostic tracers at 1 h p.i.
Keywords: PSMA, Lu-177, Autoradiography, Small-scale, Prostate Cancer
828

Imaging the effect of local drug delivery on the tumor drug distribution (#350)

Helena Besse1, Angelique Barten-van Rijbroek1, Kim van der Wurff-Jacobs1, Clemens Bos1, Chrit Moonen1, Roel Deckers1

1 University Medical Center Utrecht, Center of Imaging Sciences, Utrecht, Netherlands

Introduction

Drug resistance is a multifactorial phenomenon, however many studies only focus on the cellular level1. Though, several studies have shown that drugs can be very heterogeneously distributed through the tumor and as such cause reduced drug efficacy2. Nanomedicine is used to improve tumor drug concentration, however it is unclear to what extend it improves the drug distribution. We measured tumor growth in vivo and drug distribution by fluorescence after treatment of free doxorubicin (DOX) and nanomedicine containing DOX (stealth liposomes (DOXIL) and thermosensitive liposomes (ThermoDox)).

Methods

Immune deficient mice bearing a subcutaneous HT1080 (human fibroscarcoma) xenograft were treated with a single iv injection of DOX, DOXIL or ThermoDox at a concentration of 2.5, 5 or 10 mg/kg. Tumors of animals treated with ThermoDox were additionally treated with hyperthermia (42°C, 1 hour, tumor submerged in water bath) immediately after drug treatment. Tumors were either used for tumor follow up (n=5) or harvested 24 hours after drug treatment for histology (n=1). Fluorescent images of whole tumor slices were acquired from doxorubicin autofluorescence, dividing cells (BrdU), perfused areas (Hoechst), vessels (CD31) and hypoxia (pimonidazole). Survival was compared with drug distribution. In addition, drug distribution was correlated with dividing cells, perfused vessels and hypoxia.

Results/Discussion

Animals treated with increasing DOX, DOXIL and ThermoDox concentrations showed increased survival. In addition, at equal doxorubicin concentrations treatment with DOXIL and ThermoDox resulted in increased survival compared to DOX, Fig 1a-c. At tumor level, tumors treated with increasing DOX, DOXIL or ThermoDox concentrations resulted in a more homogeneously drug distribution. In addition, the mean doxorubicin concentration was increased in tumors after treatment with DOXIL or ThermoDox compared to DOX at equal concentrations, Fig 1d-f. For all treatments the distribution of dividing cells was negatively correlated with the drug distribution. In contrast, the distribution of hypoxic areas and perfused vessels was comparable for all tumors indicating that observed difference in drug distribution were not caused by different tumor microenviroments.

Conclusions

Drug delivery by nanomedicine (both DOXIL and ThermoDox) increased the survival compared to free drug as well as the delivered drug concentration. Local drug concentration strongly determines arresting cell proliferation. Homogeneity of drug concentration was more strongly associated with injected drug concentration than with the mode of delivery, presence of functional vessels or hypoxia.

References

1Alfarouk, Cancer Cell Int. 2015; 2Baker, C&EM, 2018

Acknowledgement

ERC Sound Pharma – 268906 (CM)

Survival and drug distribution of doxorubicin after treatment with DOX, DOXIL or ThermoDox

Survival curves of animals treated with DOX (A), DOXIL (B) or ThermoDox (C) at 2.5, 5 and 10 mg/kg. Tumor slices of animals treated with 5 mg/kg DOX (D), DOXIL (E) or ThermoDox (F), doxorubicin (red), hypoxic areas (green) and vessels (white). Scale bar 1 mm

Keywords: Drug distribution, nanomedicine, tumor microenvironment
829

Preclinical evaluation of Magnesium-phthalocyanine/albumin adduct as theranostic agents for photodynamic therapy in a glioma murine model (#368)

Martina Capozza1, Francesca Arena1, Bhavna Rani2, Silvio Aime2, Enzo Terreno1, 2, Dario L. Longo2

1 University of Torino, Center for Preclinical Imaging, Dept. of Molecular Biotechnologies and Health Sciences, Colleretto Giacosa, Italy
2 University of Torino, Molecular Imaging Center, Dept. of Molecular Biotechnologies and Health Sciences, Torino, Italy

Introduction

Several molecules have been proposed as photosensitizers for cancer photodynamic therapy (PDT)  but their low solubility usually require chemical modifications (solfunation1) or nanoparticles encapsulation2, reducing both their PDT efficacy and fluorescence imaging detectability. The aim of this study was to develop Metal-Phthalocyanine (M-Pc) uncovalenty bound to bovine serum albumin (BSA)3 as soluble theranostic agents for photoacoustic imaging application4, testing both their PDT efficacy and multi-modality imaging detectability in a glioma tumor murine model. 

Methods

MgPc are dissolved in DMSO and added to stirring BSA solution (1h, RT). To evaluate the photodynamic activity, U-87MG cells are pre-incubated with MgPc-BSA (0.5µM, 3h, 37°C) and irradiated (4 min, 500 W, λ = 635nm). After 1 and 24h, cell viability is tested via MTT assay. MgPc-BSA (50nmol, 0.2mL) tumor uptake is imaged in U-87MG tumor bearing mice with Photoacoustic and Optical Imaging. The in vivo PDT efficacy of MgPc-BSA (100nmol, 0.2mL) is assessed by comparing 4 groups: MgPc-BSA+laser (group 1), MgPc (group 2), laser (group 3) and control (group 4) by measuring tumor growth and vascularization via 3D US and CEUS (microbubbles: Micromarker, Visualsonics). H&E staining is performed on paraffin sections of tumors of group1 and group 4 for necrotic scoring with Olympus BX41 microscope.

Results/Discussion

In tumor bearing mice, MgPc-BSA complex shows a strong tumor enhancement 10 min after injection. At later time point, the signal reached a plateau at 50% up to 4h. OI shows a marked hepatic clearance (Fig1). MgPc-BSA and laser irradiation induces a decrease (70%) in cell number after 1h of dark incubation; this difference becomes larger 24h after irradiation (40%). The U-87MG cells treated with only BSA or laser didn’t show any phototoxicity (Fig2A). To test the PDT efficacy, 4h post MgPc-BSA injection, the tumor region is irradiated for 15min (500W, λ = 635nm). Tumor volume of group1 measured 12 days post treatment was significantly lower compared to the volume of all the others groups (p>0.001, Fig 2B). Contrast enhanced-ultrasound shows a necrotic tumor center with lower peak enhancement in group1 than in the other groups (Fig2C). In agreement with the US data, H&E staining revealed a higher number of necrotic patches in MgPc-BSA + Laser group as compared with the control (Fig2D).

Conclusions

These results demonstrate the advantages of this theranostic agent, where the albumin binding with Mg-phthalocyanine improves  tumor uptake, photoacoustic enhancement/detection and efficacy as photosensitizer for photodynamic therapy both in vitro and in vivo application. These favorable properties could be further investigated with different central metal ion and cancer murine models. 

References

[1] A.B. Attia, G. Balasundaram, W. Driessen, V. Ntziachristos, M. Olivo, Phthalocyanine photosensitizers as contrast agents for in vivo photoacoustic tumor imaging, Biomed Opt Express 6(2) (2015) 591-8.

[2] W. Yu, M. Ye, J. Zhu, Y. Wang, C. Liang, J. Tang, H. Tao, Y. Shen, Zinc phthalocyanine encapsulated in polymer micelles as a potent photosensitizer for the photodynamic therapy of osteosarcoma, Nanomedicine 14(4) (2018) 1099-1110.

[3] A. M Garcia, H. de Alwis Weerasekera, S.P. Pitre, B. McNeill, E. Lissi, A.M. Edwards, E.I. Alarcon, Photodynamic performance of zinc phthalocyanine in HeLa cells: A comparison between DPCC liposomes and BSA as delivery systems, J Photochem Photobiol B 163 (2016) 385-90.

[4] S. Zackrisson, S.M. van de Ven, S.S. Gambhir, Light in and sound out: emerging translational strategies for photoacoustic imaging, Cancer Res 74(4) (2014) 979-1004.

Photoacoustic and Optical imaging of U-87MG bearing mice after MgPc-BSA administration

A) Photoacoustic spectrally unmixed representative images. B) graph representing PA enhancement percentage calculated as (PASIpost-PASIpre)/PASIpre*100 , data reported as mean ± SD, n=5. C) Optical imaging acquired 4 and 24 hours post injection 

Phototoxic efficacy of MgPc-BSA adduct tested in vitro and in vivo.

A) %of viable cells; data reported as mean±SEM, ANOVA 1-way Bonferroni.**p=0.0012. B) tumor volume measured via 3D-US. data reported as mean±SEM, ANOVA 2-way Bonferroni.* p<0.002, **p<0.001. C) Peak enhancement with CE-US. Data reported as mean±SD, ANOVA 1-way Bonferroni. *p<0.01. D) H&E staining for necrotic scoring, data reported as mean±SD, student t-test ** p<0.005

Keywords: photodynamic therapy, photoacoustic imaging, phthalocyanine, albumin complex, Magnesium-phthalocyanine
830

Suitability of PET Metrics to Detect Specific Drug Effects in Hypoxia Targeted Therapies (#504)

David M. Gorman1, Victoria Tessyman2, Duncan Forster1, James P. O'Connor3, 4, Kaye J. Williams2, Marie-Claude Asselin1

1 University of Manchester, Division of Informatics, Imaging and Data Sciences, Manchester, United Kingdom
2 University of Manchester, Research, Division of Pharmacy and Optometry, Manchester, United Kingdom
3 University of Manchester, Division of Cancer Sciences, Manchester, United Kingdom
4 The Christie Hospital NHS Trust, Department of Radiology, Manchester, United Kingdom

Introduction

Hypoxia occurs within solid tumours when oxygen delivery is insufficient to meet local metabolic demand [1]. Numerous hypoxia-modifying drugs are in development [2] with PET being a likely candidate for their assessment [3]. However, the most appropriate PET metric to quantify hypoxia is undetermined [3]. In this study, immunohistochemistry (IHC) quantified the effects of Atovaquone, a drug that reduces cellular oxygen consumption, on tissue morphology at sub PET voxel resolution to inform the suitability of various PET hypoxia metrics to detect the expected drug effect in Calu-6 xenografts.

Methods

The three PET metrics under investigation were SUVmean, tumour to muscle ratio (T:M), and the hypoxic fraction (HF). In PET, the HF is typically defined by applying a threshold relative to a reference tissue or blood. Here, we used a confidence interval (c.i.)-based threshold derived using pooled muscle data to define an HF from [18F]FAZA PET images of 17 mice with subcutaneous Calu-6 xenografts; this approach has previously been applied in clinical studies [4] and has been adapted by us for preclinical application. Each mouse was scanned twice using PET, at baseline and 7 days after treatment or vehicle (controls), at the end of which the tumour was excised for IHC analysis.

Results/Discussion

Figure 1a shows that PET derived HFs are 4 times higher than, and uncorrelated with, the IHC derived HFs despite in-vivo accumulation of pimonidazole and [18F]FAZA occurring via a similar mechanism. Furthermore, other hypoxia related metrics, SUVmean and T:M ratio, did not correlate with IHC derived HF. Busk et al. demonstrated that spatial resolution effects inhibit cross-validation between PET and IHC [5,6], however, in this instance, both methods independently demonstrate the drug effect on hypoxia (Figure 1b). 

Figure 2 shows an example of histological staining of Calu-6. Large internal regions of liquefactive necrosis reticulated with anuclear and pyknotic cells are observed using H&E with viable tissue at the edges of the tumour and as islands. Fluorescence microscopy shows the antibody to pimonidazole, a hypoxia marker, to accumulate along the edges of viable tissue and throughout the reticulated cells. CD-31, a microvascular marker, is observed within the viable regions.

Conclusions

The histologically observed diffusely distributed hypoxic tissue saturates the HF suggesting that metrics derived from or including intensity information would be better suited to this tumour model. The lack of correlation between PET and IHC HFs suggests that [18F]FAZA contrast is sufficient for even low subvoxel HFs to elevate voxel intensity above the threshold. Work is ongoing to characterise the effect of Atovaquone on tissue morphology.

References

  1. Hockel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. Journal of the National Cancer Institute. 2001:934:266–76.
  2. Manoochehri Khoshinani H, Afshar S, Najafi R. Hypoxia: a double-edged sword in cancer therapy. Cancer investigation. 2016:3410:536–45.
  3. Challapalli A, Carroll L, Aboagye EO. Molecular mechanisms of hypoxia in cancer. Clinical and translational imaging. 2017:53:225–53.
  4. Mortensen LS, Johansen J, Kallehauge J, et al. Radiotherapy and Oncol, 2012,105(1): 14–20.
  5. Busk M., Munk O.L., Jakobsen S.S., et al., Acta Oncol, 2017, 56(11): 1583–90.
  6. Busk M., Munk O.L., Jakobsen S.S., et al., Acta Oncol, 2010, 49(7): 922–33.
Figure 1
a) Correlation between the HF, SUVmean, or T:M ratio of subcutaneous Calu-6 xerographs measured using PET and IHC derived HF, b) HF measured using PET and IHC at baseline and/or after Atovaquone treatment.
Figure 2

Histological staining of Calu-6: H&E and Pimonidazole immunohistochemical fluorescence microscopy segmented into normoxic, hypoxic and necrotic regions using artificial neural network.

Keywords: Calu-6, hypoxia, Hypoxic Fraction, Atovaquone, PET
831

Design of a simple multifunctionalizable BODIPY platform and application to the facile elaboration of a large series of gold(I)-based optical theranostics (#257)

Jacques Pliquett1, Franck Denat1, Ali Bettaieb2, Souheila Amor1, Catherine Paul2, Christine Goze1, Ewen Bodio1

1 Université de Bourgogne - Franche Comté, ICMUB - UMR CNRS 6302, DIJON, France
2 Université de Bourgogne - Franche Comté, LIIC - (EPHE PSL), DIJON, France

Introduction

Multimodality is one of the researchers’ main interest of these last years, especially in medicinal chemistry. Indeed, the number of studies dealing with bimodal imaging, theranostic, or bitherapeutic agent have literally exploded. These elaborated structures often require the use of a multifunctionalizable platform, which enables to introduce step by step the different functionalities. A much less explored strategy consists in using one of the component of the multimodal object as a platform itself, which can enable, when chosen correctly, to win an additional functionality on the structures.

Methods

The BODIPY platform was synthesized is 4 steps. The introduction of 1 to 3 different nucleophiles (gold(I) complex, thioglucose derivative, or phosphonium) onto the platform enabled to rapidly synthesized 19 derivatives, 12 of which contain gold(I) therapeutic moiety. Their cytotoxic potential were evaluated on different types of cancer cells – human breast (MDA-MB-231), murine mammary (EMT6), and human melanoma (B16F10) cancer cell lines – after 48 h of incubation (MTS assay). Concerning optical imaging, MDA-MB-231 was treated with 10 μM of the compounds for 1, 4 or 24 h, and their cellular location was evaluated by confocal microscopy. Gold uptake has been determined by ICP-MS measurement, after the incubation of 40 μM solution of the different gold(I) complexes for 4 h on MDA-MB-231.

Results/Discussion

In this context, and working on BODIPY-based multimodal and theranostic compounds, we were interested in developing simple and versatile platforms based on this fluorescent probe. During the last decades the potential of BODIPY (boron dipyrromethene) dyes has emerged in many different fields, from material sciences to molecular imaging. This high interest is mainly due to the very good photophysical properties of the BODIPY dyes and their chemical and photochemical stability. However, their main advantage over other classic fluorophores is their chemical structure, which can be easily functionalized, making BODIPYs a perfect candidate for developing a multifunctionalizable platform.

Thus, drawing our inspiration from the pioneering work of Dehaen and coll,[1, 2] who investigated the difunctionalization of dichloro BODIPY, we designed a very simple trifunctional BODIPY platform. The potential of this platform was validated via the rapid synthesis of 12 optical theranostics (Figure 1).[3]

Conclusions

We reported on the synthesis, the characterization, the photophysical properties, and the evaluation of the water-soluble properties of 19 different BODIPY derivatives, as well as a theoretical rationalization of some results. Moreover, we performed antiproliferative evaluation and confocal imaging of the different compounds in three human and murine cancer cell lines, along with a measure of gold(I) uptake in one cancer cell line via ICP-MS.

References

1             T. Rohand, M. Baruah, W. Qin, N. Boens and W. Dehaen, Chem. Commun., 2006, 266–268.

2             V. Leen, T. Leemans, N. Boens and W. Dehaen, Eur. J. Org. Chem., 2011, 2011, 4386–4396.

3             J. Pliquett, S. Amor, M. Ponce-Vargas, M. Laly, C. Racoeur, Y. Rousselin, F. Denat, A. Bettaeib, P. Fleurat-Lessard, C. Paul, C. Goze and E. Bodio, Dalton Trans., 2018, DOI:10.1039/C8DT02364F.

Acknowledgement

Support was provided by the Conseil Régional de Bourgogne (PhD JCE grant # 2015-9205AAO033S04139/BG0003226), the Conseil Régional de Bourgogne Franche-Comté, the Ministère de l’Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique (CNRS), and the French Research National Agency (ANR) via the project JCJC “SPID” ANR-16-CE07-0020, the Université de Bourgogne, and the École Pratique des Hautes Études (EPHE). This work is part of the project PHARMACOIMAGERIE ET AGENTS THERANOSTIQUES and of the project CHIMIE DURABLE, ENVIRONNEMENT ET AGROALIMENTAIRE, supported by the Université de Bourgogne, Conseil Régional de Bourgogne through the plan d’actions régional pour l’innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs. FrenchBIC and GDR CNRS AIM are acknowledged for fruitful discussion. Mr Soustelle and Ms M.-J. Penouilh are gratefully acknowledged for HR-MS, and NMR analyses, and Dr M. Laly for ICP-MS measurements. Plateforme DimaCell, U. Bourgogne Franche- Q11 Comté, F21000 Dijon, France. The society Oncodesign® is acknowledged for fruitful discussion and for their support. Mr Jacques Pliquett gratefully acknowledges Ms Lucile Dondaine for her valuable advice on the biological tests. The calculations were performed using HPC resources from DNUM CCUB (Centre de Calcul de l’Université de Bourgogne).

Figure 1
(left) general structure of the compounds / (right) real time tracking of one of the theranostic in MDA-MB-231 cell line for 24 h.
Keywords: theranostics, gold complex, fluorescence, optical imaging, BODIPY
832

Detection of lentiviral suicide gene therapy in C6 Glioma using hyperpolarised [1-13C]pyruvate (#389)

Riikka Nivajärvi1, Venla Olsson2, Jan Henrik Ardenkjaer-Larsen3, Olli Gröhn1, Seppo Ylä-Herttuala2, Mikko Kettunen1

1 University of Eastern Finland, Biomedical Imaging unit, A.I. Virtanen institute, Kuopio, Finland
2 University of Eastern Finland, Molecular Sciences, A.I. Virtanen institute, Kuopio, Finland
3 Technical University of Denmark, Center for Magnetic Resonance, Department of Electrical Engineering, Center for Hyperpolarization in Magnetic Resonance, Center, Lyngby, Denmark

Introduction

Malignant glioma is one of the most aggressive and lethal human cancers 1, 2. Early diagnosis of successful therapy is therefore crucial. There has been lack of robust tool to study repeatedly cancer metabolism in vivo. Hyperpolarised (dDNP) MRI, which allows amplification of 13C signal over 10 000-fold 3 , has shown promise in therapy detection in a number of cancers including glioma 4. In this study, we assessed the response to the lentiviral suicide gene therapy of Herpex Simplex virus thymidine kinase with the prodrug ganciclovir (HSV-TK/GCV).

Methods

Female Wistar rats (n=33, 190-230g) were inoculated with 106 C6 glioma cells. Intratumoral lentiviral HSV-TK (FinVector Vision Therapies, Finland) gene transfers were made on post-implantation (p.i.) days 7 and 8 to therapy group. Ganciclovir (GCV) (Cymevene, Roche, Finland, i.p. 25 mg/kg twice a day, 14 days) therapy was started on p.i. day 10. Volumetric MRI data (n=33, T2-weighted multi-slice RARE, echo time 72ms) were collected at 9.4 T. 13C chemical shift imaging (n=16, repetition time 50 ms, echo time 0.7 ms, voxel 2x2x4-6 mm3, center-out spiral, 16x16 matrix, collected 30 seconds after i.v. injection of hyperpolarised (HYPERMAG 5, Denmark) [1-13C]pyruvate (Sigma, 80 mM, 5 ml/kg). CSI was repeated on both weeks of therapy. Survival was assessed up to day 30 post therapy.

Results/Discussion

HSV-TK/GCV Gene therapy lengthened the median survival from 13 days to 25 days (p=0.02) and the same was seen in relative tumor volumes, where an apparent growth arrest was seen in therapy group after day 6 (Fig. 1). Interestingly, a fraction of animals survived also in control groups. In DNP, therapy animals showed a decrease in lactate/pyruvate between therapy weeks (-26±15%, n=6) whereas an opposite trend was observed in control animals (+44±18%, n=6, p<0.05, Fig. 2). The two controls animals showing either a decrease or no change in ratio survived to the end of the study. Gene therapy, which caused an apparent growth arrest rather than cure in the majority of therapy animals, led to lowered lactate labeling which is consistent with other, more immediate, therapy models 4. Interestingly, some control animals also showed an altered metabolic pattern associated with improved survival possibly due to inherent immunoresponse 6.

Conclusions

The hyperpolarisation MRI, which offers non-invasive metabolic information on tissue, can be used to follow-up gene therapy. It can therefore offer complementary information to traditional MR methods to give a fuller picture of the therapy response.

References

1. Surawicz TS, Davis F, et al. Brain tumor survival: results from the national cancer data base. J Neurooncol. 1998;40(2):151–160.

2. Deangelis LM. Brain tumors. N Engl J Med. 2001;344(2):114–123.

3. Ardenkjær-Larsen J, Fridlund B, et al. Increase in signal-to-noise ratio of < 10,000 times in liquid-state NMR. PNAS 2003;100(18):10158-10163

4. Day SE, Kettunen MI, Cherukuri MK, et al. Detecting responce of rat C6 glioma tumors to radiotherapy using hyperpolarized [1- 13C]pyruvate and 13C magnetic resonance spectroscopic imaging. Magn Reson Med 2011;65(2):557-563

5. Ardenkjaer-Larsen JH, Bowen S, Petersen JR, Rybalko O, Vinding MS, Ullisch M, Nielsen NC. Cryogen-free dissolution dynamic nuclear polarization polarizer operating at 3.35 T, 6.70 T, and 10.1 T. Magn Reson Med. 2018 Oct 25. doi: 10.1002/mrm.27537.

6. Yang L, Zhao J, et al. The 9L LUC/Wistar rat glioma model is not suitable for immunotherapy. Neural Regen Res. 2012;7(18):1406-1411

Acknowledgement

This work was supported by The Academy of Finland (project grant #286895 and FIRI2014 funding), TEKES/EU Regional Development Fund (4298/31/2014) and Biocenter Finland.

Figure 1. Survival and tumor volume.
The survival improved (p=0.023) due to therapy. Median survival was 12 days longer in therapy group than in control group. In tumor volumes the growth arrest was statistical significant on days 6 (p=0.0025) and 11 (p=0.0048), when the therapy was efficient. Therapy ended at day 14 (dashed line). MRI was used to detect tumor volumes.
Figure 2. dDNP.
The therapy animals have decrease in lactate/pyruvate between therapy weeks (-26±15%, n=6), the opposite trend is observed in control animals (+44±18%, n=6, p<0.05). Blue arrows are repeated measurements with the same animal. Black lines are means of lactate/pyruvate in the control (beginning 0.86, end 1.10) and therapy (beginning 0.82, end 0.68) group.
Keywords: MRI, hyperpolarisation, Glioma, suicide gene therapy
833

Theranostic innovative nano-compounds for fluorescent imaging and boron neutron capture therapy (BNCT) (#153)

Ghadir Kalot1, Vanina Cosenza2, Benoit Busser1, 3, Robin Poirot2, Ulli Köster4, Jean-Luc Coll1, Christine Goze5, Rachel Auzely-Velty2, Lucie Sancey1

1 Institute for Advanced Biosciences, Grenoble Alpes University/INSERM U1209/ CNRS UMR5309, Team CTET, La Tronche, France
2 Grenoble Alpes University, Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRSGrenoble Alpes University, Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS, Grenoble, France
3 Grenoble Alpes University Hospital, Grenoble, France
4 Institut Laue Langevin, Grenoble, France
5 ICMUB U6302, Dijon, France

This work was partly funded by France Life Imaging (grant ANR-11-INBS-0006) from the French Investissements d’Avenir” program, and CNRS interdisciplinary projet ISOTOP.

Introduction

Boron Neutron Capture Therapy (BNCT) is an innovative form of radiotherapy based on neutron exposure. BNCT, considered as a promising treatment for malignant brain tumors, relies on the high propensity of boron-10 atoms to absorb slow thermal neutrons, which results in a nuclear fission reaction, releasing locally very energetic particles. BNCT-based treatments will induce important tissue damages in the Boron-10 rich tissues. There is an urgent need for designing innovative theranostic BNCT agents with efficient drug carriers, for preliminary small animal evaluation using optical imaging.

Methods

Biocompatible polymer-based nanogels were produced with functionalized hyaluronic acid (HA) moieties and selective core-crosslinking method. For BNCT applications, sodium borocaptate (BSH) was grafted on the HA chain. As a fluorescent probe for optical imaging in the NIR, aza-BODIPYs which contain one boron atom were encapsulated in the nanogel.

In vitro, the nanogels were evaluated in U87-MG glioma and A375 melanoma human cells. Internalization studies were conducted using confocal imaging, and flow cytometry. Distribution was observed using optical imaging in the NIR on tumor engrafted on chicken embryo chorioallantoic membrane- (CAM-) and mice-bearing tumors.

BNCT assay was performed at the Institut Laue Langevin (Grenoble, France) on cells and CAM-bearing tumors.

Results/Discussion

In this multidisciplinary project, we designed, synthetized, and characterized innovative boron-rich nanocompounds based on hyaluronic acid, boron-rich molecules, and aza-BODIPYs.

We studied their distribution and kinetics in vitro in A375 melanoma and U87-MG glioma cells. The nanogels were efficiently and highly internalized in these cell lines, without any evidence of toxicity.

In vivo, the distributions were observed using non-invasive optical imaging. The nanogels (HD size 150nm) were mainly eliminated through the liver and the spleen. The tumor accumulation was time- and EPR-dependent. Optical imaging, performed on CAM-bearing A375 tumors, was used to determine the optimal treatment plan for BNCT experiment. In ovo, the tumors treated with boron-rich nanogels and neutrons showed a reduced tumor development as compared to other conditions.

Conclusions

Altogether, this approach confirms that BNCT can be very efficient against boron-rich tumor areas, while sparing the surrounding healthy tissues. The use of theranostic nanogel is crucial for optimization of the treatment plan. Our results are promising and should be confirmed using boron-10 enriched compounds.

Acknowledgement

This work was partly funded by France Life Imaging (grant ANR-11-INBS-0006) from the French Investissements d’Avenir” program and CNRS interdisciplinary project ISOTOP.

Keywords: theranostic nanoparticles, BNCT, optical imaging

Onco-Immunology Imaging

Session chair: Nicolas Beziere (Tübingen, Germany); Filippo Galli (Terni, Italy)
 
Shortcut: PW07
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

850

Imaging of therapy-induced immune activation in glioma by [18F]DPA-714 (#393)

Claudia Foray1, 4, 11, Silvia Valtorta7, 8, 9, Cristina Barca1, 4, 11, Oliver Grauer10, 11, Michael Schäfers1, 5, 6, Rosa M. Moresco7, 8, 9, Andreas Jacobs1, 3, 4, Bastian Zinnhardt1, 2, 4

1 European Institute for Molecular Imaging - EIMI, Münster , Germany
2 Imaging Neuroinflammation in Neurodegenerative Diseases (INMIND) EU FP7 consortium, Münster , Germany
3 Department of Geriatrics, Johanniter Hospital, Evangelische Kliniken, Bonn, Germany
4 PET Imaging in Drug Design and Development (PET3D), Münster , Germany
5 Cells in Motion (CiM) Cluster of Excellence, Münster , Germany
6 University Hospital Muenster, Department of Nuclear Medicine, Münster , Germany
7 Tecnomed Foundation and Medicine and Surgery Department, University of Milano-Bicocca, Milan, Italy
8 Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Germany
9 SYSBIO.IT, Centre of Systems Biology, Milan, Germany
10 University Hospital Muenster, Departmen of Neurology, Münster, Germany
11 Westfälische Wilhelms-Universität Münster, Münster, Germany

Introduction

Glioblastoma is the primary malignant brain tumor in adults with a limited overall survival of only 14-15 months. The heterogeneous nature of gliomas and the dynamic interplay of tumor cells and immune cells in the disease course make the development and application of novel treatments challenging. To improve the understanding of the complex interaction of the tumor and the immune component of gliomas, we employed non-invasive PET/MRI using a combination of [18F]FET (aa-metabolism) and [18F]DPA-714 (TSPO) in order to monitor temozolomide (TMZ) treatment response.

Methods

N=12 female NMRI nude mice underwent MRI and PET/CT scans after intra-striatal implantation of 2x10^5 Gli36ΔEGFR-LITG glioma cells. 10 days p.i. mice were treated with 50 mg/kg of TMZ or DMSO vehicle intraperitoneally for 5 days. Mice were subjected to PET imaging at d0 (before) and at d6 after treatment with [18F]DPA-714 (TSPO; 20 MBq; 60-80 min p.i.) and [18F]FET (amino acid transport; 10 MBq, 20-30 min p.i.). Images were co-registered. Tumor-to-background (striatum) uptake ratios (T/B) and unique area of tracer uptake were calculated using an atlas-based volumetric approach. ROIs defining the area of interest were placed using the brain atlas as reference and a threshold based on 3.5xSD of the contralateral striatum area was applied. Brains were harvested for histological analysis.

Results/Discussion

TMZ treatment significantly reduced [18F]FET-derived tumor volume (0.009±0.008; p<0.05), while in DMSO treated animal it was significantly increased at day 6 (0.022±0.007; p<0.05). The tumor volume was also significantly reduced (p<0.0001) comparing treatment outcomes at day 6 between TMZ- and DMSO-treated groups (Fig 1A). TMZ treatment significantly reduced T/B ratio [18F]FET signal overtime (0.58±0.16; p<0.05) as well as [18F]DPA-714 signal (0.74±0.3; p<0.05). Comparison between DMSO- and TMZ-treated animals at day 6 showed also a significant decrease of [18F]FET signal (p<0.0001) (Fig 1B). The volumetric analysis of unique tracer areas indicated a reduction of [18F]FET uptake, between day 0 and day 6 in TMZ treated mice, as well as in DMSO control group, but the results did not reach significance (Fig. 2A). The analysis of unique [18F]DPA-714 tracer areas has shown a significant increase in TMZ treated mice (30.6±24.9; p<0.05) while in DMSO group the uptake remained stable(Fig 2B).

Conclusions

[18F]FET- and [18F]DPA-714-PET with MRI allow the non-invasive assessment of glioma growth and its associated inflammation. TMZ therapy effects can be detected by [18F]FET, while [18F]DPA-714 does not seem to be suitable for this purpose. However, [18F]DPA-714 seems to shift in response to therapy to areas which are not detected by [18F]FET. Additional analysis are currently ongoing to understand the therapy effects of the inflammatory component.

References

 

 

Acknowledgement

This work was supported by the EU 7th Framework Programme (FP7/2007-2013) under grant agreement n° 278850 (INMiND) and Horizon2020 Programme under grant agreement n° 675417 (PET3D).

Quantitative analysis of [18F]FET and [18F]DPA-714 T/B uptake ratios and tumor volume

Fig.1 A) Quantitative analysis of the changes in tumor volume (cm3/ml) after 6 days of therapy with TMZ. B) Quantitative analysis of changes in [18F]FET and [18F]DPA-714 T/B uptake ratio after 6 days from the beginning of the therapy with DMSO (vehicle) and 50 mg/kg TMZ. Differences between the treated (TMZ) and control (DMSO) groups were tested for significance using t-Test.

Volumetric analysis of unique tracers uptake areas
Fig.2 Quantitative analysis of changes in [18F]FET and [18F]DPA-714 tracers uptake volumes in DMSO and TMZ treated groups. Differences were tested for significance using t-Test. Data are represented as mean±SD.
Keywords: glioma, imaging, therapy-response, animal model, PET
851

In vitro assessment of radiolabelling effects on two types of cellular immunotherapy (#497)

Cameron Lang1, Francis Man1, Alessia Volpe1, Candice Ashmore-Harris1, 3, Lindsay Lim1, Ewelina Kurtys1, Rafael Torres Martin de Rosales1, Gilbert Fruhwirth1, 2

1 King's College London, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom
2 KCL & UCL Comprehensive Cancer Imaging Centre, London, United Kingdom
3 King's College London, Centre for Stem Cells & Regenerative Medicine, London, United Kingdom

Introduction

Chimeric antigen receptor (CAR) T cells and γδ-T cells are emerging anti-cancer cellular immunotherapies, but their fate after administration is unclear. Non-invasive tracking of T cell therapies by nuclear imaging, either through direct or reporter gene-based (indirect) radiolabelling, can shed light over the in vivo fate of adoptively transferred cells and potentially explain therapeutic outcomes. However, the effect of radiolabelling on T cell function and survival must be evaluated. Here we assessed the effect of radiolabelling with imaging radionuclides on CAR-T and γδ-T cells.

Methods

Primary human T cells were engineered using a lentiviral construct1 to co-express the anti-ErbB family CAR T1E28z2 and the radionuclide-fluorescence reporter human sodium iodide symporter (NIS)-TagRFP3 (T4:NT). T4:NT CAR‑T were selectively expanded via the co-expressed IL-4:IL-2/15 chimera4 and characterized in vitro for viability, CAR (tumour cell killing, IFNγ release) and reporter function after radiolabelling with the NIS radiotracers [18F]BF4- and 99mTcO4- (40 kBq/106 cells/mL). Human Vγ9Vδ2 T cells were isolated from peripheral blood, expanded in vitro, and directly radiolabelled with 89Zr(oxine)4.5 In addition, for both therapeutic cell types we assessed radiation-induced DNA damage by quantifying γH2AX expression by immunofluorescence staining.

Results/Discussion

T4:NT CAR-T were generated and confirmed fully functional. Radiolabelling with either 99mTcO4- or [18F]BF4- resulted in T4:NT containing 5.0±2.0 mBq 99mTcO4- or 3.6±1.7 mBq [18F]BF4- per cell, similar to what NIS reporter expressing cells experience upon in vivo imaging with these radiotracers. Neither CAR-T viability (5d post radiolabelling) nor tumour cell killing nor IFNγ were impaired by radiolabelling (Fig.1A-D). γH2AX foci (DNA double-strand breaks) were elevated upon external beam radiation (positive control, Fig.2A) and radiolabelling with 99mTcO4- and 89Zr(oxine)4 (Fig. 2B). γδ-T cells were directly radiolabelled to reach cellular 89Zr levels of [6-20], [50-90] and [150-450] mBq/cells5. Cell viability was impaired by radiolabelling higher than [6-20] mBq/cell, while tumour cell killing capacity did not change significantly5 (Fig. 1E-F). γH2AX foci increased in a radiation-dependent manner5 (Fig. 2C-D).

Conclusions

Therapeutic T-cells were radiolabelled and characterised. Radiolabelling of reporter gene-expressing CAR-T was confirmed to be non-detrimental to cells. Direct radiolabelling conditions were identified that enabled γδ-T cells to remain functional5, and are therefore suitable for in vivo tracking. We identified conditions allowing safe in vivo tracking of both cell types, which can serve as guidelines for other cellular T-cell immunotherapies.

References

1 Volpe et al., J Vis Exp. 2018; 133; doi: 10.3791/57088.

2 Davies et al., Mol Med 2012; 18:565-76; doi: 10.2119/molmed.2011.00493.

3 Diocou et al., Sci Rep 2017; 7:946; doi: 10.1038/s41598-017-01044-4.

4 Wilkie et al., J Biol Chem 2010; 285:25538-44; doi: 10.1074/jbc.M110.127951.

5 Man et al., Mol Ther 2018 (in press); doi: 10.1016/j.ymthe.2018.10.006.

Acknowledgement

We thank Dr John Maher for the T1E28z CAR construct and Cancer Research UK, Worldwide Cancer Research, and King’s Health Partners for financial support. We declare no conflicts of interest.

In vitro characterisation of NIS-RFP:CAR-T cells and radiolabelled γδ-T cells

A Viability of 18F- and 99mTc-labelled CAR-T cells. B Radiotracer uptake of CAR-T cells with (T4:NT) or without (T4) NIS expression. C Viability of cancer cells incubated with labelled CAR-T cells. D IFNγ production by labelled CAR-T cells. E Proliferation of 89Zr-labelled γδ-T cells. F Viability of MDA-MB-231 cancer cells after incubation with 89Zr-labelled γδ-T cells. Panels E,F from ref.5.

DNA damage in radiolabelled NIS-RFP:CAR-T and γδ-T cells

A γH2AX foci quantification in CAR-T cells after external beam irradiation. B Confocal microscopy images of γH2AX foci (green) and nuclei (blue) in CAR-T cells radiolabelled with 89Zr(oxine)4 or 99mTcO4-. C γH2AX foci quantification in 89Zr-labelled γδ-T cells. D Confocal images of γH2AX foci in 89Zr-labelled γδ-T cells. Scale bars = 10 μm. Panels C,D from Man et al. (ref. 5).

Keywords: CAR-T, Immunotherapy, Cell tracking, Reporter gene, Radiobiology
852

Investigating the Effect of Tumour Size and Fibrosis on Trafficking of 89Zr-labelled Gamma-delta T-cells and 111In-labelled PEGylated Liposomes to Xenograft Breast Tumours (#144)

Francis Man1, Alessia Volpe1, Alberto Gabizon2, Philip Blower1, Gilbert Fruhwirth1, Rafael TM de Rosales1

1 King's College London, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom
2 Hebrew University, School of Medicine, Oncology Institute, Shaare Zedek Medical Center, Jerusalem, Israel

Introduction

In vivo cell tracking by nuclear imaging could improve the development and clinical efficacy of anti-cancer T-cell therapies. Gamma-delta (γδ) T cells are highly cytotoxic immune cells, used successfully in several clinical trials in cancer immunotherapy (1,2). Using the tracers 89Zr(oxine)4 for PET (3,4) and 111In(oxine)3 for SPECT, we tracked human γδ-T cells and the tumour-sensitizing liposomal alendronate (4) in a xenograft breast cancer model to investigate the effect of tumour size on cell and liposome uptake. The effect of the antifibrotic drug tranilast was also investigated (5).

Methods

Human γδ-T cells were isolated from peripheral blood and expanded in vitro using zoledronate and IL-2. MBA-MB-231.hNIS-GFP cells were injected subcutaneously in the mammary fat pad of female SCID/beige mice. For tumour sensitisation, animals were treated with liposomal alendronate (5 mg/kg) 3 days before γδ-T cell injection. Liposomes were radiolabelled with 111In(oxine)3 (7 MBq/mouse). γδ-T cells were radiolabelled with 89Zr(oxine)4 (40 kBq/106 cells) and injected in the tail vein (11x106 cells/animal). To reduce tumour fibrosis, animals were administered tranilast (200 mg/kg, oral) daily from day 7 post-implantation. Animals were imaged by SPECT or PET/CT 48 h after administration of liposomes or γδ-T cells. Biodistribution studies were performed on day 7 after γδ-T cell administration.

Results/Discussion

Tumour uptake of 89Zr-labelled γδ-T cells and 111In-labelled liposomal alendronate was visible by PET and SPECT respectively after 48 h (Fig. 1) and measured by image-based quantification. Uptake after 7 days was measured by gamma-counting (Fig. 2). Tumour uptake of 111In-labelled liposomes was 22.8±3.6 %ID/mL (n=9) after 48 h and did not appear to correlate with tumour size. After 7 days, uptake was 26.1+11.1 %ID/g and was higher in smaller tumours, with the exception of very small, poorly vascularized tumours. Uptake of 89Zr-γδ-T cells was 1.5±1.1 %ID/mL (n=20) after 48 h and correlated with tumour size (Spearman r=0.5263, p=0.0171). After 7 days, uptake increased to 3.3±1.8 %ID/g and was independent of tumour size. No significant effect of tranilast was observed on the uptake of liposomes or γδ-T cells (p≥0.05, Mann-Whitney test).

Conclusions

We tracked γδ-T cells and liposomes in a xenograft cancer model to investigate parameters governing their uptake. Fibrosis appeared unaffected by tranilast, possibly explaining the absence of effect on cell/liposome uptake. Unlike liposomes, γδ-T cell uptake depended on tumour size at 48 h only, suggesting T-cell uptake is not solely governed by vascular permeability. Future studies will help elucidate the in vivo dynamics of γδ-T cell therapies.

References

(1) Silva-Santos B et al. Nat Rev Immunol (2015) 15:683-91

(2) Fisher JPH et al. Oncoimmunology (2014) 3(1):e27572

(3) Charoenphun P et al. Eur J Nucl Med Mol Imaging (2015) 42:278-87

(4) Man F et al. Mol Ther (2018) (in press). DOI: 10.1016/j.ymthe.2018.10.006

(5) Papageorgis P et al. Sci Rep (2017) 7:46140

Acknowledgement

This work was supported by Cancer Research UK (CRUK), the KCL/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 KCL, the Medical Research Council Confidence in Concepts scheme, the Experimental Cancer Medicine Centre at KCL, the KHP/KCL CRUK Cancer Centre and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and KCL. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. The authors declare no conflict of interest.

PET and SPECT tracking of gamma-delta T cells and liposomes
(A) 111In in tumour-bearing mice 48 h post-injection of 111In-labelled liposomal alendronate. (B) 89Zr in tumour-bearing mice 48 h post-injection of 89Zr-labelled γδ-T cells. MIP: maximum intensity projection; Sagitt: sagittal view; Liv: liver; Sp: spleen; Tum: tumour. (C) Histology: CD3+ (γδ-T) cells in formalin-fixed, paraffin-embedded excised tumours. Arrows indicate representative cells.
Quantification of gammadelta-T cell and liposome uptake

A-D: Effect of tumour size on cell and liposome uptake. Image-based quantification (A,B) and gamma-counting (C,D,E,F) measurements of 111In-labelled liposomal alendronate (A,C,E) and 89Zr-labelled γδ-T cells (B,C,F) in MDA-MB-231.hNIS-GFP tumour-bearing mice at 48 h and 7 days post-injection. E,F: effect of tranilast on cell and liposomal uptake after 7 days. ns: p≥0.05 (Mann-Whitney test).

Keywords: T-cell therapy, Cell tracking, Zr-89, Gamma-delta T cells
853

Pharmacokinetics of 111In-anti-mPD-L1 in immune challenged tumor-bearing mice (#74)

Gerwin Sandker1, Peter Wierstra1, Janneke Molkenboer-Kuenen1, Martin Gotthardt1, Gosse Adema2, Johan Bussink2, Erik H. J. G. Aarntzen1, Sandra Heskamp1

1 Radboudumc, Department of Radiology and Nuclear Medicine, Nijmegen, Netherlands
2 Radboudumc, Department of Radiation Oncology, Nijmegen, Netherlands

Introduction

Immune checkpoint inhibitors show impressive anti-tumor efficacy in cancer patients. However, mixed treatment responses and serious side effects call for predictive biomarkers. Preclinical studies show that microSPECT/CT using radiolabeled anti-programmed death-ligand 1 (PD-L1) antibodies (Abs) can be used to quantify PD-L1 expression in vivo. However, the activation status of PD-L1+ immune cells may influence anti-PD-L1 Ab pharmacokinetics (PK). Therefore, we investigated the effects of lipopolysaccharide-mediated (LPS) immune activation on the PK and tumor-targeting of 111In-anti-PD-L1.

Methods

The effect of immune activation on anti-PD-L1 in vivo biodistributions was evaluated in three conditions; healthy BALB/c mice, Renca tumor-bearing BALB/c mice, and LPS-challenged (0.6 mg/kg body weight) Renca tumor-bearing BALB/c mice. Mice were intravenously injected with 30 or 100 µg 111In-labeled anti-mouse PD-L1. PK was assessed by taking blood samples and biodistribution was quantified by microSPECT/CT and ex vivo biodistribution studies 72 h after tracer injection. PD-L1 expression in organs of interest was evaluated immunohistochemically.

Results/Discussion

There were no statistically significant differences in the in vivo biodistribution of 111In-anti-PD-L1 between tumor-bearing and non-tumor-bearing mice. However, in immune-challenged mice, splenic tracer uptake significantly increased compared with non-LPS challenged tumor-bearing mice (65.0±10.3 %ID/g vs. 35.2±4.9 %ID/g; p<0.001), resulting in accelerated blood clearance and reduced tumor targeting (Blood 24 hours pi: 4.2±0.3 %ID/g vs. 9.3±3.1 %ID/g; p<0.05, Tumor: 7.9±5.3 %ID/g vs. 25.9±11.6 %ID/g; p<0.05). Increasing the tracer dose to 100 µg resulted in reduced splenic uptake (27.3±4.9 %ID/g vs. 12.1±3.5 %ID/g; p<0.05) and slower blood clearance (Blood 24 hours pi:12.2±2.5 %ID/g vs. 14.3±2.6 %ID/g; ns), and restored tumor targeting (18.1±1.7 %ID/g vs. 15.3±4.3 %ID/g; ns).

Conclusions

This study shows that systemic inflammatory responses can significantly alter PK and tumor targeting of anti-PD-L1 Abs. Increasing the anti-PD-L1 Ab dose saturates splenic uptake and restores efficient tumor targeting. This information is essential to better understand alterations in in vivo anti-PD-L1 Ab biodistribution and to avoid suboptimal Ab-dosing.

SPECT/CT images of 111In-anti-mPD-L1 in vehicle and LPS treated mice
Figure 1. MIP thresholded microSPECT/CT images at 72 hours post injection of 111In-anti-mPD-L1 in vehicle treated or LPS challenged tumor-bearing BALB/c mice. Increased splenic uptake and decreased tumor targeting can be observed in the LPS treated mice. Increasing anti-PD-L1 dose restores tumor targeting.
Keywords: Immune checkpoint, 111In-anti-mPD-L1 Ab, Immune challenge, Pharmacokinetics, Tumor targeting
854

In vivo live imaging of human T/B cell lymphoma cross-linking mediated by bispecific CD20-TCB antibody (#183)

Elena Menietti1, Dario Speziale1, Johannes Sam1, Stefano Sammicheli1, Stenford Chen1, Marine Richard1, Klein Christian1, 2, Pablo Umana1, 2, Marina Bacac1, 2, Mario Perro1, Sara Colombetti1

1 Roche , Pharmacology Department, pRED, Roche Innovation Center Zurich, , Schlieren, Switzerland
2 Roche , Cancer Immunotherapy Discovery, pRED, Roche Innovation Center Zurich, , Schlieren, Switzerland

Introduction

Cancer Immune Therapies have shown unprecedented results in improving tumor control [1-3]. However, many patients are still refractory to treatment. A deeper understanding of the mode of action of the different CITs sub-classes may help improving therapeutic approaches to reach better anti-tumor response. For this reason, we developed a multi-photon intra-vital microscopy (MP-IVM) approach to study in vivo, at single cell level, the tumor microenvironment upon treatment with CD20-targeting T-cell bispecific antibodies (TCB) [4] in a preclinical model of diffuse large B cell lymphomas (DLBCL)

Methods

To selectively monitor clinical lead molecules in the context of human T cell responses, we developed a skinfold chamber model [5] in last generation humanized mice [6] that allows visualization, by MP-IVM, of labelled human T cells co-injected intra-dermally with WSU-DLCL2, a human DLBCL. We have used this model to investigate T cells recruitment to tumors upon CD20-TCB therapy: by intra-venously injecting labeled T cells in mice treated with selected blocking antibodies, we were able to identify dedicated pathways induced by CD20-TCB and regulating T cell influx into the tumor bed. Furthermore, we developed a user-independent quantification platform to assess changes in the dynamics of T cell motility and time of interaction with tumor cells

Results/Discussion

We have developed an experimental preclinical model that aims to reduce xenoreaction (human T cell reaction against mouse tissue) by utilizing T cells derived from humanized mice, educated within murine thymus. We demonstrate that such model is optimal to quantify human T cell dynamics in vivo. We show that CD20-TCB localizes in the tumor and acts on tumor-resident T cell motility within 1 hour post i.v. injection (defined as functional PK), causing a sharp reduction in their speed (from 4 to 2 µm/min) and an increase in tumor/T cell interaction time; those changes last up to 72h post-treatment. In addition, we prove how the initial tumor/T cell interaction mediated by CD20-TCB lead to peripheral T cells recruitment into the tumor. This mechanism is dependent on the presence of tumor-resident T cells and on IFNg-CXCL10 pathway. Inhibiting any of these two parameters resulted in reduced T cells infiltration from the periphery and reduced anti-tumor efficacy

Conclusions

We developed a reliable imaging and analysis pipeline to investigate in vivo T cell dynamics and recruitment and applied it to the study of CD20-TCB treatment of DLBCL model. Our approach has shed new lights into the MoA of this new class of immune-therapeutics, demonstrating that the IFNɣ-CXCL10 pathway is involved in T cell recruitment upon CD20-TCB treatment

References

  1. Yousefi, H., et al., Expert Rev Clin Immunol, 2017. 13(10): p. 1001-1015.
  2. Pishko, A. and S.D. Nasta, T Transl Cancer Res, 2017. 6(1): p. 93-103.
  3. Emens, L.A., et al., Eur J Cancer, 2017. 81: p. 116-129.
  4. Bacac, M., et al., Clin Cancer Res, 2018. 24(19): p. 4785-4797.
  5. Koehl, G.E., A. Gaumann, and E.K. Clin Exp Metastasis, 2009. 26(4): p. 329-44.
  6. Shultz, L.D., F. Ishikawa, and D.L. Greiner, Nat Rev Immunol, 2007. 7(2): p. 118-30.

Acknowledgement

We would like to acknowledge the oDTA of Roche pRED for the support of the project. We would like to thanks our colleagues of Large Molecule Research for providing the therapeutics, their  fluorescent conjugation as well as the genetically modified fluorescent cells. We would like to thanks the informatic teams for the help with analysis of the data and maintenance of the informatic infrastructure

Keywords: In vivo Imaging immunotherapy, T cell bi-specific, cancer immunotherapy, humanize mouse model, multi-photon
855

Comparison between PET and MRI techniques for tracking T-cells in an anti-PD1 immunotherapy animal model (#341)

Solenne Vaillant1, 2, Erwan Selingue2, Sébastien d’Heilly1, Françoise Geffroy2, Erwan Jouannot1, Sébastien Mériaux2

1 Sanofi, Bioimaging, Vitry-Sur-Seine, France
2 CEA, NeuroSpin, Gif-sur-Yvette, France

Introduction

With the breakthrough of new innovative cell-based therapies, molecular imaging could be increasingly used in clinical practice to non-invasively predict the efficiency of treatments and in preclinical studies to better understand the mechanisms involved. For this purpose, the present study aims to implement specific methodologies to label immune cells for their detection by either nuclear imaging or MRI. The more appropriate labeling technique will be used to characterize an anti-PD1 immunotherapy animal model.

Methods

Human T cell line was labeled with 89Zr and USPIO. To assess radiosensitivity and early tissue distribution, 12 SCID mice were randomized in 4 groups and injected via the tail vein (IV) or peritoneal (IP) route with labeled cells. Images were acquired from 20 minutes to 11 days after injection on preclinical PET/CT (Inveon, Siemens) and 11.7 T MRI (BioSpec, Bruker) scanners. MRI sequences (T2-wieghted RARE and MGE) were acquired with a volume radiofrequency coil dedicated to mouse body.

To obtain an anti-PD1 animal model, 6 C57BL/6 mice were implanted with MC38 murine tumor and after 10 days, injected (IP) with murine T cells labeled with 89Zr and randomized in 2 groups (treated with anti-PD1 antibody or with control isotype). Mice were imaged during 3 days with PET/CT scanner.

Results/Discussion

For both labelings, in vitro studies showed viability and cell proliferation similar to the control group without labeling (Fig. 1A and 1B). However, different results have been obtained for the stability study: the MRI labeling was found stable (decrease of fluorescence signal inside cells correlated with cell proliferation), whereas an efflux of 50% in three days was observed for the nuclear labeling (Fig. 1C and 1D).

For in vivo experiments, as expected, both labelings revealed migration of cells first in the lungs, then in the liver after IV injections and in the spleen for IP injections (Fig. 1 C/D/E/F).

Finally, for the anti-PD1 animal model, radioactivity was found in the spleen for isotype group, but close to the noise in the tumor for both groups (Fig. 2). However, after quantification, radioactivity seems to increase in the tumor for anti-PD1 group (Fig. 2 A). Furthermore, tumor of anti-PD1 group presents more radioactivity after ex vivo counting than control group (Fig. 2 B).

Conclusions

This study presents two complementary methods to track immune cells in vivo: nuclear imaging enables to track cells with higher sensitivity, whereas MRI may enable to track cells more precisely. Finally, 89Zr labeling was applied to an anti-PD1 model and, if the images didn’t show significant increase of radioactivity in the tumor for treated mice, quantification and ex vivo analysis confirm that T cells are recruited by the tumor with treatment.

References

1. Bansal, A. et al. Novel 89Zr cell labeling approach for PET-based cell trafficking studies. EJNMMI Research 5, 19 (2015).

2. Krüger, K. & Mooren, F. C. T cell homing and exercise. Exerc Immunol Rev 13, 37–54 (2007).

3. Fischer, U. M. et al. Pulmonary Passage is a Major Obstacle for Intravenous Stem Cell Delivery: The Pulmonary First-Pass Effect. Stem Cells Dev 18, 683–691 (2009).

Acknowledgement

France Life Imaging is acknowledged for funding the 11.7 T preclinical MRI scanner of NeuroSpin.

Figure 1. Comparison between PET and MRI techniques for tracking T-cells

In vitro studies. Efflux for PET labeling (A) and MFI per cell for MRI labeling (B, red) compared with cell proliferation (B, blue).

In vivo studies. MRI: C (resp. D) shows RARE image of mouse abdomen 24h after IV (resp. IP) injection of labeled cells. Blue (resp. red) arrows show the spleen (resp. liver). PET: E (resp. F) shows image of mouse 24h after IP (resp. IV) injection of labeled cells.

Figure 2. Tracking T-cells with PET imaging in an anti-PD1 immunotherapy animal model
PET/CT images of mice bearing MC38 tumor (red arrows), injected with murine T cells labeled with 89Zr and treated with an anti-PD1 antibody or control isotype. Graphics represent the evolution of volume radioactivity in tumor (A) for the anti-PD1 (blue) and isotype (red) groups and the residual radioactivity (B) in organs after sacrifices.
Keywords: Immunotherapy, Cell tracking, 89Zr labeling, USPIO labeling, anti-PD1 treatment
856

Nanobody-based imaging of Macrophage Mannose Receptor expressing Tumor-Associated Macrophages by Fluorescence Molecular Tomography (#136)

Marco Erreni1, Evangelia Bolli4, 5, Roberta Avigni2, Francesca D'Autilia1, Andrea Doni1, Paola Allavena2, 3, Cecilia Garlanda2, 3, Alberto Mantovani2, 3, 7, Sophie Hernot6, Jo Van Ginderachter4, 5

1 Humanitas Clinical and Research Center, Unit of Advanced Optical Microscopy, Rozzano, Italy
2 Humanitas Clinical and Research Center, Immunology and Inflammation, Rozzano, Italy
3 Humanitas University, Rozzano, Italy
4 Vrije Universiteit Brussel, Lab of Cellular and Molecular Immunology, Brussels, Belgium
5 VIB Center for Inflammation Research, Lab of Myeloid Cell Immunology, Brussels, Belgium
6 Vrije Universiteit Brussel, Lab for in vivo cellular and molecular imaging, ICMI-BEFY/MIMA, Brussels, Belgium
7 The William Harvey Research Institute, Queen Mary University of London, London, United Kingdom

Introduction

Tumor-associated macrophages (TAMs) have been shown to impact tumor progression by promoting angiogenesis, metastasis and tumor-immune suppression. In this context, targeting TAMs represents an effective prognostic and therapeutic anti-tumor approach.

Nanobodies (Nbs), the smallest available antigen-binding fragments derived from Camelid heavy-chain-only antibodies, are suitable for tumor targeting, due to their fast kinetics, low background and deep tissue penetration. 99mTc-labeled anti-mannose receptor (MMR)-Nbs have been successfully used to image pro-angiogenic MMR+-TAMs in vivo.

Methods

IRDye680RD-conjugated anti-MMR Nbs (IRDye-MMR-Nb) were used to image MMR+-TAMs in a mouse model of transplanted fibrosarcoma by Fluorescence Molecular Tomography (FMT). MN/MCA cells were intramuscularly injected in the mouse hind leg and MMRTAMs visualized every week, for 4 weeks. Hereto, IRDye-MMR-Nb was injected intravenously and animals imaged 1h post-injection. Intraperitoneal pre-injection with 20x molar excess of unconjugated-bivalent anti-MMR Nb (biMMR-Nb) was used to minimize the binding of IRDye-MMR-Nb in specific off-tumor sites, such as liver and spleen. Every week, a cohort of mice was sacrificed, organs were collected and imaged to analyze IRDye-MMR-Nb biodistribution. Localization of MMRTAMs by IRDye-MMR-Nb targeting in liver, tumor and lung was visualized by microscopy.

Results/Discussion

IRDye-MMR-Nb accumulation in the tumor-bearing mouse hind leg compared to healthy counterpart was already visible 1 week after cancer cell injection and the signal increased along with tumor progression. Importantly, pre-injection of unlabeled biMMR-Nb reduced the uptake of IRDye-MMR-Nb in extratumoral sites (mainly liver), without affecting tumor-specific uptake. Ex vivo IRDye-MMR-Nb biodistribution analysis confirmed the in vivo results, with tumor tissue and liver displaying the highest accumulation. MN/MCA-derived tumors develop lung metastasis at week 4 after inoculation. Accordingly, a weak IRDye-MMR-Nb accumulation could also be observed in vivo in the lungs of tumor-bearing mice. This was confirmed ex vivo, where signal in the lungs strongly increased at week 4 after tumor cell injection, compatibly with the occurrence of small metastatic foci. A colocalization between IRDye-MMR-Nb and a subpopulation of F4/80tumor-infiltrating macrophages was observed by confocal microscopy

Conclusions

TAMs are key components of the tumor microenvironment, orchestrating different aspect of cancer progression. We showed that IRDye-MMR-Nb can be used for efficient follow-up of disease progression through the in vivo imaging of TAMs by FMT. Since MMRTAMs are a major stromal component in many cancer types, IRDye-MMR-Nb can represent a tool to monitor TAM infiltration by optical in vivo imaging in a variety of unrelated preclinical tumor models.

References

Tumour-associated macrophages as treatment targets in oncology.

Mantovani A, Marchesi F, Malesci A, Laghi L, Allavena P.

Nat Rev Clin Oncol. 2017 Jul;14(7):399-416. doi: 10.1038/nrclinonc.2016.217

 

Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages.

Movahedi K, Schoonooghe S, Laoui D, Houbracken I, Waelput W, Breckpot K, Bouwens L, Lahoutte T, De Baetselier P, Raes G, Devoogdt N, Van Ginderachter JA.

Cancer Res. 2012 Aug 15;72(16):4165-77. doi: 10.1158/0008-5472.CAN-11-2994

 

Macrophage polarization in pathology.

Sica A, Erreni M, Allavena P, Porta C.

Cell Mol Life Sci. 2015 Nov;72(21):4111-26. doi: 10.1007/s00018-015-1995-y.

 

Immuno-imaging using nanobodies.

Vaneycken I, D'huyvetter M, Hernot S, De Vos J, Xavier C, Devoogdt N, Caveliers V, Lahoutte T.

Curr Opin Biotechnol. 2011 Dec;22(6):877-81. doi: 10.1016/j.copbio.2011.06.009

Acknowledgement

This work was supported by the Italian Association for Cancer Research (AIRC), Kom op tegen Kanker, Stichting tegen Kanker, FWO and EU-COST action Mye-EUNITER.

Keywords: Nanobodies, Macrophage Mannose Receptor, Tumor-associated Macrophages, Fluorescence Molecular Tomography, Optical Imaging
857

In vivo imaging of CD8+ T cell tumor infiltration following radiotherapy (#78)

Peter Wierstra1, René Raavé1, Gerwin Sandker1, Janneke Molkenboer-Kuenen1, Milou Boswinkel1, Gosse Adema2, Johan Bussink2, Martin Gotthardt1, Erik H. J. G. Aarntzen1, Sandra Heskamp1

1 Radboud University Medical Centre, Radiology and Nuclear Medicine, Nijmegen, Netherlands
2 Radboud University Medical Centre, Radiation Oncology, Nijmegen, Netherlands

Introduction

In recent years, cancer immunotherapy has proven shown clinical efficacy, but responses are heterogeneous across patients. Tumor characterization prior to treatment and early response monitoring are imperative for treatment personalization. One facet strongly associated with treatment response is increased infiltration of the tumor by CD8+ cytotoxic T cells. Quantitative imaging could allow response monitoring in a non-invasive manner. Presently, we investigated an 111In-labeled CD8 antibody for imaging influx of CD8+ T cells in irradiated tumors in vivo in mice bearing murine B16-F1 tumors.

Methods

C57Bl/6 mice of 12 weeks old (n=20) were injected with subcutaneous melanoma B16-F1 tumor cells on both hind legs. In half of the mice, right side tumors were irradiated with a single dose of 10 Gy. One day later, mice were intravenously injected with either: 8.1 MBq [111In]In-DTPA-anti-CD8 antibody (8.5 µg, n=10) or 8.7 MBq [111In]In-DTPA-Isotype rat IgG2A (8.5 µg, n=10), followed by SPECT/CT at day 2. After image acquisition, mice were dissected for ex vivo biodistribution, immunohistochemical analysis of CD8 in tumors, draining and distant lymph nodes, SPECT quantification and autoradiography.

Results/Discussion

Biodistribution data showed significant increased uptake of 111In-CD8 antibody in irradiated tumors compared to non irradiated tumors in different mice (10.19 ± 3.37 vs. 6.19 ± 1.36 %ID/g ± SD, p = 0.02). Uptake in left side non irradiated tumors of irradiated mice was slightly increased compared to tumors of non irradiated mice (7.76 ± 1.73 vs. 6.49 ± 1.28 %ID/g ± SD, p = 0.12 ). No statistically significantly differences in uptake were observed for the aspecific control IgG. Uptake of control IgG was significantly lower in all groups and tumors irradiated. Notably, irradiated right side tumor (10.19 ± 3.37 vs. 4.7 ± 1.04 %ID/g ± SD, p = 0.005). Tumor draining lymph nodes showed no significant difference between irradiated or non irradiated mice. Preliminary SPECT quantification shows a trend confirming the above findings but these analyses are ongoing.

Conclusions

In this study we demonstrate that 111In-CD8 antibody specifically targets tissues containing CD8+ cells and irradiation induces a significant increase in tracer uptake in B16-F1 tumors. This increase was not observed for the non specific IgG, suggesting a CD8 specific process instead of radiation induced changes in tumor perfusion. In the future, non invasive imaging of CD8+ T cells could assist in immunotherapy response monitoring.

Acknowledgement

This project is funded by the Netherlands Organization for Scientific Research (NWO, project number 91617039) and Dutch Cancer Society (KWF, project number 10099)

Biodistributions of CD8 antibody and control IgG in C57Bl/6 mice bearing two B16-F1 tumors
Top figure: Biodistribution of CD8 antibody. Bottom figure: Biodistribution of control IgG. All mice bearing two B16-F1 tumors on hind legs. In red: irradiated mice, in blue: non irradiated mice. Relevant organs shown.
Comparison of tumor uptake differences between CD8 antibody and control IgG
Significant difference in uptake between CD8 and its irrelevant control IgG in irradiated but not in non irradiated tumors.
Keywords: CD8, Radiotherapy, SPECT, Quantification, Monitoring
858

Development of a CD8 tracer for in vivo evaluation of CD8 T cell tumor infiltration during immunotherapy (#345)

René Raavé1, Milou Boswinkel1, Gerwin Sandker1, Peter Wierstra1, Erik H. J. G. Aarntzen1, Sandra Heskamp1

1 Radboud university medical center, Radboud Institute for Molecular Life Sciences, dept. of Radiology and Nuclear Medicine, Nijmegen, Netherlands

Introduction

Immunotherapy is considered a hallmark in cancer treatment by its profound and durable clinical responses in patients with various types of cancer. However, only a subgroup of patients responds to immunotherapy and methods for accurate early response monitoring are lacking. Noninvasive quantitative imaging of CD8+ cytotoxic T cells can provide dynamic and spatial information of anti-tumor response. In the present study we characterized an 111In-labeled anti-mouse CD8 antibody for imaging of tumor infiltrating CD8+ cytotoxic T cells in vitro and in vivo in mice bearing murine CT26 colon tumors.

Methods

An anti-mouse CD8 antibody (clone: YTS 169.4) was randomly conjugated with a 30 times molar excess of NCS-DTPA and radiolabeled with 111In. Using CD8+ TK1 mice lymphoma cells, the immunoreactivity, IC50, internalization and affinity characteristics were determined.

CT26 tumor bearing BALB/c mice (10-12 weeks old) were intravenously injected with 8.5 µg (8.5 MBq) [111In]In-DTPA-anti-CD8. One group received an excess of non-radiolabeled CD8 antibody (250 µg). SPECT/CT imaging was performed and organs were collected to quantify tracer uptake at 4h, 24h, 48h and 72h after injection. Autoradiography and immunohistochemistry were performed on paraffin embedded  tissue sections of tumor, spleen, lymph nodes and duodenum.

Results/Discussion

In vitro assays demonstrated that the immunoreactive fraction was 44%, IC50 was 1.77 nM, Kd was 3.83 nM, and 6.5% internalization of the total membrane bound activity after 4.5 h of incubation. CD8+ T cell containing organs (lymph nodes, spleen and duodenum) were clearly visible on SPECT scans of mice injected with [111In]In-DTPA-CD8-antibody at all time points (Fig. 1A). Mice that received an excess of non-radiolabeled CD8-antibody showed most uptake in the spleen (Fig . 1B). Low to moderate tumor uptake was visible in all groups. Ex vivo biodistribution data confirmed results from SPECT imaging. In the lymph nodes, spleen, duodenum and tumor, an uptake of 38.6 ± 12.3% ID/g (±SD), 87.1 ± 18.0% ID/g, 31.7 ± 16.9% ID/g, and 12.9 ±2.9% ID/g at 24h after injection, respectively. The tumor-to-blood ratio increased from 0.48 at 4h after injection to 2.23 at 72h after injection. Autoradiography and immunohistochemistry confirmed these findings.

Conclusions

The CD8 antibody showed specific uptake in CD8+ T cell containing tissues in vivo, but uptake in the tumor was limited because of presence low number of CD8+ T cells. In the future, this tracer has potential for in vivo evaluation of CD8+ T cell infiltration in tumors and lymphoid tissues before and during immunotherapy.

Acknowledgement

This project receives funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 116106. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA.

Figure 1. SPECT/CT images of [111In]In-DTPA-antimouse-CD8 antibody in BALB/c mice with CT26 tumors
CT26 tumor bearing mice were injected with 111In labeled CD8 antibody and imaged with SPECT/CT at 4, 24, 48, and 72 h after injection (A). One group was co-injected with 250 µg unlabeled antibody (B). Tumors are indicated with white arrows. Images (ventral view) are manually scaled.
Keywords: CD8 T-lympocytes, Tracer development, immunotherapy, Monitoring, in vivo
859

T-cell tracking using Cerenkov and radioluminescence imaging (#131)

Federico Boschi1, Francesco De Sanctis2, Stefano Ugel2, Antonello Spinelli3

1 University of Verona, Department of Computer Science, Verona, Italy
2 University of Verona, Department of Medicine, Verona, Italy
3 San Raffaele Scientific Institute, Experimental Imaging Centre, Milan, Italy

Introduction

Cancer immunotherapy includes a wide group of treatments focused to restrict tumor growth and possibly induce cancer remission by manipulating and restoring a functional immune response in the host. The availability of imaging technologies that follow in vivo T-cell trafficking in tissues and functionality after infusion in the host are mandatory to predict the outcome of an adoptive cell transfer (ACT) based immunotherapy approach.

Here, we report the potentialities of optical detection of radiolabeled T-cells via Cerenkov luminescence imaging (CLI) and radio-luminescence imaging (RLI).

Methods

Mixed leukocyte peptide cultures (MLPCs): spleens from OT-I CD45.1 mice were collected and mashed to isolate splenocytes. Then, OT-I splenocytes were plated at a concentration of 5 × 10^6/well in 24-well plates in complete media in the presence of 1 μg/mL OVA257–264 peptide and 20 IU/mL interleukin-2 to stimulate the proliferation of OVA-specific CD8+ CD45.1+T lymphocytes. The culture was split every other day andreplenished with fresh media containing 20 IU/mL interleukin-2. After 5 days of culture, MLPC was washed twice and dead cell were removed by Ficoll under standard procedures. T-cells were labeled with 32P-ATP for 1 hour at 37C and then washed in phosphate buffered saline.

CLI and RLI acquisitions were performed by using the in vivo imaging systems (IVIS) Spectrum optical imager.

Results/Discussion

We found that T-cell biodistribution can be followed during in vivo acquisitions for hours after cells injection, in particular we described a new method to in vivo follow T lymphocyte trafficking without sacrificing the animals based on CLI and RLI (see figure 1).The light signal is well detectable directly with CLI without the use of any other device allowing to track the biodistribution of T-cells. Instead, by using scintillator materials, the light emission coming from the conversion of radioactivity in light increases.

Conclusions

We conclude that 32P-ATP T-cell labeling can provide several benefits: it allows the imaging of T-cell trafficking and supports the cytotoxic activity on tumor cells by the “cross fire” exerted by the high-penetrating beta particles of 32P-ATP.

Figure 1

Cerenkov luminescence images of mice treated with 32P-ATP labeled T lymphocytes at different time points (from 30 minutes to 18 hours) after i.v. (left) and i.p. (right) injection.

Keywords: optical imaging, Cerenkov luminescence imaging, adoptive cell transfer immunotherapy, cancer imaging
860

Fixed and live imaging of neutrophils in the lung pre-metastatic niche (#565)

Amanda McFarlane1, Judith Secklehner1, Ximena Raffo1, John Mackey1, Grant McGregor1, David Novo1, Louise Mitchell1, Jen Morton1, Jim Norman1, Leo Carlin1

1 The Beatson Institute for Cancer Research, Glasgow, United Kingdom

Introduction

The establishment of the premetastatic niche (PMN) during cancer is highly complicated, involving many factors including the immune system and the extracellular matrix (ECM). The lung is a complex and highly vascularized organ prone to metastasis. Previous data has revealed changes in ECM deposition in the lung during early pancreatic cancer prior to metastasis (1). We investigated if such changes in ECM organisation might affect immune cell composition, localisation and trafficking in the lung prior to and during the formation of the PMN.

Methods

KPC (Pdx1-Cre, KrasG12D/+, p53R172H/+), KFC (Pdx1-Cre, KrasG12D/+, p53fl/+), KC (Pdx1-Cre, KrasG12D/+) and WT (Pdx1-Cre) mice as previously described (2) were used. When a small palpable pancreatic tumour was detected in experimental mice, they were humanely killed and the lungs inflated with agarose, before producing precision cut lung slices (PCLS) cut at 300μm thickness using a vibratome. For live PCLS, slices were stained and imaged directly using the Zeiss 880 Airyscan Fast confocal microscope to produce time-lapse images which were segmented and tracked. For fixed PCLS imaging a Perkin Elmer Opera Phenix was used to evaluate whole lung slices by high-speed image tiling and high content segmentation and analysis.

Results/Discussion

Increased leukocyte numbers were observed in the lungs of mutant p53 bearing mice, with an increase in the ratio of myeloid to lymphoid cells. Fixed PCLS from KPC (highly invasive & metastatic), KFC (non-metastatic), KC and WT (controls) were stained to localize neutrophils within the tissue in the context of the intact stroma. Imaging of live PCLS revealed many more neutrophils present in KPC lungs compared with controls as expected. Analysis of their movement and trafficking through the vasculature has revealed differences which may be as a result of the changes in the ECM of these mice. High content analysis of the neutrophil positions across whole lobe PCLS allowed us to observe and quantify heterogeneity in the localisation of leukocyte subsets.

Conclusions

Investigating the formation of the PMN aids our understanding of metastasis and potential targets for therapy. We have imaged fixed and live mouse PCLS to investigate both localisation and trafficking of neutrophils in the lung vasculature of pre-metastatic pancreatic cancer. Identifying the localisation and motility of different leukocyte subsets in the lung in the context of intact stroma may shed light on their function in metastasis.

References

(1) Hingorani, S. R. et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7, 469–483 (2005).

(2) Novo, D. et al. Mutant p53s generate pro-invasive niches by influencing exosome podocalyxin levels. Nature Communications 9, 5069 (2018).

Keywords: pre-metastatic niche, lung, leukocytes, high content imaging
861

Imaging the tumor microenvironment in vivo – origin and regulatory mechanisms of tumor promoting S100A9 (#550)

Anne Helfen1, Jan Rieß1, Olesja Fehler2, Annika Schnepel1, Miriam Stölting1, Mirjam Gerwing1, Max Masthoff1, Walter Heindel1, Moritz Wildgruber1, 3, Michel Eisenblätter1, 4

1 University of Muenster, Institute of Clinical Radiology, Muenster, Germany
2 University of Muenster, Institute of Immunology, Muenster, Germany
3 University of Muenster, DFG EXC 1003 Cluster of Excellence `Cells in Motion`, Muenster, Germany
4 King´s College London, Division of Imaging Sciences & Biomedical Engineering, London, United Kingdom

Introduction

Tumor progression and metastasis depend on tumor-infiltrating immune cells, which form a characteristic inflammatory tumor microenvironment (TME). Within this microenvironment but also in premetastatic niches, the protein heterodimer S100A8/A9 is released by activated infiltrating monocytes [1]. As a promoter of tumor invasion and TME formation, it has been associated with poor prognosis [2]. Our aim was to develop an in vivo imaging tool serving as a biomarker for TME influence on tumor behavior.

Methods

From syngeneic murine breast cancer tumors 4T1 (highly malignant) and 67NR (low malignancy), wildtype (wt) and S100A9 knock out cells (CRISPR/cas9-method, ko) were created and implanted into either female BALB/c wildtype or S100A9-/--mice (n=10 each). At 4 mm tumor diameter, anti-S100A9-Cy5.5-driven flurorescence reflectance imaging has been performed 0 and 24h after injection (contrast-to-noise ratios of fluorescence intensities in arbitrary units, AU). An isotype IgG-Cy5.5 served as a control (n=5) for unspecific label distribution in 4T1 tumors. In vivo imaging was correlated with immunohistology, Western Blot and FACS analyses. Statistical analysis was performed using unpaired t test and one-way ANOVA with Bonferroni post test.

Results/Discussion

24h after injection, anti-S100A9-Cy5.5 showed significantly higher fluorescence signals as compared to IgG-Cy5.5.

In S100A9-/--mice, fluorescence signals were significantly reduced for wildtype (4T1: 62,9 vs. 43,02AU;p=0,048, 67NR: 52,73 vs. 27,26;p=0,033) and S100A9-/--tumors (4T1ko: 65,33 vs. 42,51AU;p=0,005; 67NRko: 54,61 vs. 34,19AU;p=0,006, Figure). However, no significant differences were detected for 4T1ko and 67NRko cells as compared with wildtype cells (4T1:p=0.543, 67NR:p=0.85). In all subgroups, the fluorescence signal was significantly higher in 4T1 as compared to 67NR tumors, reflecting their higher malignant potential.Imaging results were confirmed by ex-vivo analyses.

Furthermore, FACS of 4T1 tumors revealed a significantly lower amount of CD115+/F4/80+ cells (15,8 vs. 30,9%;p=0,001) and a higher of Ly6C+ cells (82,2 vs. 47,53%;p=0,001) in wildtype mice as compared to S100A9-/--mice, indicating an immune cell shift to immature monocytes within the S100A9-positive TME.

Conclusions

Our results in the 4T1/67NR breast cancer model system confirm a secretion of S100A8/A9 by components of the TME, while tumor cells do apparently not release S100A8/A9. S100A9-specific in vivo imaging reflects tumor malignancy and may serve as a biomarker for TME formation and activity.

References

1) Arai, K., et al. (2008) S100A8 and S100A9 overexpression is associated with poor pathological parameters in invasive ductal carcinoma of the breast. Curr Cancer Drug Targets.

2) Roth, J., et al. (2003) Phagocyte-specific S100 proteins: a novel group of proinflammatory molecules. Trends Immunol.

Acknowledgement

This work has been funded by DFG EXC 1003 (PP-2017-01).

S100A9-specific fluorescence signals in 4T1 and 67NR tumors

Calculated contrast-to-noise ratios of fluorescence intensities for 4T1 (highly malignant) and 67NR (low malignancy) wildtype as well as S100A9 knock out tumors in female BALB/c wildtype and S100A9-/--mice (n=10 each).

Keywords: tumor microenvironment, tumor immunology, S100A9, fluorescence reflectance imaging
862

Visualising pharmacodynamic changes in the tumour microenvironment with in vivo imaging (#523)

Stefanie R. Mullins1, Aimee Ruffle1, 2, Thomas Murray2, Judit Espana-Agusti3, Shannon Burke1, Nadia Luheshi1, Simon Dovedi1, Robert Wilkinson1

1 MedImmune, Research Oncology, Cambridge, United Kingdom
2 MedImmune, ADPE, Cambridge, United Kingdom
3 MedImmune, IVS, Cambridge, United Kingdom

Introduction

Efficacy of cancer immunotherapy is associated with pharmacodynamic (PD) changes in the tumour microenvironment (TME). The presence of proinflammatory cytokines, including interferon-γ (IFNγ), are associated with positive therapeutic responses to immuno-oncology (IO) drugs (Higgs et al.). We have developed an imaging model that detects changes in IFNγ in the TME to measure the proinflammatory response to IO therapeutics, which will allow for measurement of real-time PD changes in the same animal and provide further insight into the differences between IO responsive and resistant tumours.

Methods

CT26 and B16-F10 AP3 cells lines responsive to IFNγ were generated using a pCDH-IFNγ-luc2P-PGK(-)-mKATE2 lentiviral vector containing a constitutive mKATE2 fluorescent reporter and IFNγ-inducible luc2P bioluminescent reporter. FACS sorted clones were characterised in vitro by flow cytometry (mKATE2) and IFNγ-induced luciferase expression (luc2P). IFNγ reporter tumour (CT26 or B16-F10 AP3) bearing mice were injected with an IFNγ neutralizing antibody. Fluorescence and bioluminescence images were taken using the IVIS Spectrum. Intratumoural IFNγ was calculated as (total flux [p/s]) divided by (total radiant efficiency [RFU]; CT26) or tumour volume [mm3] (B16-F10 AP3). IFNγ-responsiveness was confirmed ex vivo by incubating tumour tissue slice cultures with IFNγ (0-10ng/ml).

Results/Discussion

Luc2P bioluminescence was observed in all mice injected with IFNγ-reporter cells and B16-F10 AP3 transduced cells had lower baseline IFNγ response compared with CT26. Tissue slices from tumours grown in vivo, then treated with recombinant IFNγ, also demonstrated that both CT26 and B16-F10 AP3 transduced tumours were responsive to IFNγ in a dose dependent manner. Anti-IFNγ-mAb treatment in mice was associated with a significant decrease in intratumoural IFNγ in CT26 IFNγ-reporter 7 and B16-F10 AP3 IFNγ-reporter 22 tumours respectively from 3 days and 6 days post first dose.

Conclusions

Changes in IFNγ signalling were observed in two models using in vivo imaging. Selectivity for IFNγ was validated using in vivo imaging and ex vivo tissue slice culture. It also revealed differences in IFNγ signalling between IO-responsive (CT26) and IO-resistant (B16-F10 AP3) tumours. This TME in vivo imaging method has several merits, compared with standard PD approaches, as it is less invasive and has the potential to reduce animal numbers.

References

Higgs, B.W., et al., ESMO presentation, 2015

Keywords: IFNgamma, immuno-oncology, PD

Disease Models, Translational Approaches I | Neuroimaging

Session chair: Andreas Hess (Erlangen, Germany); Cornelius Faber (Münster, Germany)
 
Shortcut: PW08
Date: Wednesday, 20 March, 2019, 4:00 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

909

Serial BLI of Hydrogel-Scaffolded Glial Progenitors Transplanted into Mouse Brain (#196)

Shreyas Kuddannaya1, Wei Zhu1, Chengyan Chu1, Anirudha Singh2, Piotr Walczak1, Jeff Bulte1

1 Johns Hopkins University School of Medicine, Dept. of Radiology, Baltimore, Maryland, United States of America
2 Johns Hopkins University School of Medicine, Dept. of Urology, BALTIMORE, Maryland, United States of America

Introduction

Neurorestorative cell therapies are prone to substantial cell loss (up to 90 %) owing to mechanical stress, hypoxia and immunorejection [1]. We assessed whether the fate and function of glial-restricted progenitor cells (GRPs), myelinating cells that support neuronal survival, can be improved following their scaffolding in hyaluronic acid (HA)-based composite hydrogels. Here, we determined the optimal composition of hydrogel formulations to facilitate injection, enhance survival, and aid with proper tissue integration of scaffolded GRPs.

Methods

Mouse GRPs (mGRPs) expressing GFP and transduced with firefly luciferase (pLenti4-CMV-Luc) were encapsulated within composite hydrogels containing thiolated hyaluronic acid (HA) and gelatin (Gelin-S) with polyethylene (glycol) diacrylate (PEGDA) as crosslinker. Using a 2:2:1 ratio of HA, GelinS and PEGDA, various composite formulations ranging from 5 to 20 mg/ml (all components) were synthesized. Rheological properties were determined using an ARES-G2 rheometer and the in vitro proliferation was assessed with a CCK-8 assay. The in vivo survival of (scaffolded) mGRPs (2x106 cells/100 µl hydrogel or saline) transplanted in immunocompetent BALB/c mice was assessed with bioluminescent imaging (BLI) using an IVIS-Spectrum CT imaging system and validated with histological fluorescence staining.

Results/Discussion

Small variations in the individual components of hydrogel formulations critically affect hydrogel stiffness (Fig. 1a). Scaffolded mGRPs showed enhanced proliferation compared to naked cells (Fig. 1b). In vitro BLI indicated differential cell survival in different hydrogel compositions (Fig. 1c) and with syringe injection a slight cell death was noted (Fig. 1d). On day 10, GFP-positive mGRPs showed distinct cell migration patterns away from the hydrogel scaffolding boundary (Fig. 1c). In vivo BLI readouts from sub-cutaneous and intrastriatal transplantation demonstrated a higher survival of scaffolded mGRPs (Fig. 2a,b) compared to the unscaffolded (naked) mGRPs. T2 weighted MRI and histology revealed a restricted localization of the mGRP-hydrogel graft at the injection/target site (Fig. 2c,d).

Conclusions

Composite hydrogel formulations can be customized to facilitate cell injection and enhance post-transplantation survival of scaffolded GRPs for use in neurorestorative therapies.

References

[1] Marquardt L.M., et al, Design of Injectable Materials to Improve Stem Cell Transplantation. Curr Stem Cell Rep., 2016. 2(3): p. 207–220.

Figure 1
(a) Storage modulus. (b) In vitro mGRP proliferation counts. (c) In vitro BLI of mGRPs scaffolded in different hydrogel formulations including PBS as control. (d) In vitro BLI for injection and normal pipetting conditions. (e) Cell migration from hydrogel boundary (red dotted line) to the surrounding regions. Images were obtained 10 days after plating. Scale bar=100 µm.
Figure 2

(a) Serial in vivo BLI of transplanted mGRPs scaffolded in various hydrogels or PBS. (b) Serial in vivo BLI of 3x105 mGRPs transplanted in mouse brain following scaffolding with optimized (10 mg/ml) hydrogel formulation or PBS as control. (c, d) T2-weighted MRI at day 1 and histological staining at day 16 post-intrastriatal transplantation of hydrogel-scaffolded GFP+ mGRPs.

Keywords: BLI, Cell therapy, Hydrogel
910

Erythropoietin receptors can be targeted in human and rat stroke tissue using [89Zr] ‑Deferoxamine-EPO (#496)

Kristin J. Patzwaldt1, Ramona Stumm1, Francesca Russo2, Laura Kuebler1, Dominik Seyfried1, Andreas Maurer1, Manuela Neumann4, Sven Poli2, Bernd Pichler1, Salvador Castaneda Vega1, 3

1 Eberhard Karls University of Tuebingen, Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Tübingen, Baden-Württemberg, Germany
2 Eberhard Karls University of Tuebingen, Department of Neurology & Stroke, and Hertie Institute for Clinical Brain Research, Tübingen, Baden-Württemberg, Germany
3 Eberhard Karls University of Tuebingen, Department of Nuclear Medicine and Clinical Molecular Imaging, Tübingen, Baden-Württemberg, Germany
4 Eberhard Karls University of Tuebingen, Division of Neuropathology, Tübingen, Baden-Württemberg, Germany

Introduction

Erythropoietin (EPO) and EPO receptor (EPOr) expression are directly linked to hypoxia in tissues including brain, heart, liver and kidney. EPO is a cytoprotective agent showing neuroprotective effects by inducing angiogenesis, neurogenesis and inhibiting apoptosis [1]. In this study, we prepared [89Zr]-Deferoxamine (Dfo)-EPO and evaluated EPOr expression in the stroke region of human and rat brains. The measurement of EPOr presence through [89Zr]-Dfo-EPO might be a potential tool to measure a tissue’s response to hypoxia.

Methods

Rats underwent surgery using the middle cerebral artery occlusion stroke model (n=9) or sham surgery (n=7). Animals were injected i.v. with [89Zr]-Dfo-EPO 3 h after stroke induction and scanned using PET and MRI at 24, 48 and 72 h after injection. Images were co-registered to MRI and evaluated using Pmod software.

Autoradiography (AR) was performed on human and rat ischemic brain tissues in the acute stroke phase. To determine total binding, tissue sections were incubated with 4.28 nM of [89Zr]-Dfo-EPO for 60 min. Blocking was performed on separate sections using 1320 nM of the nonradioactive EPO. After washing, the dried sections were exposed to AR plates for 24 h and read out with a phosphorimager at 50µm resolution. Image analysis was performed using ImageJ software.

Results/Discussion

Our in vivo results show significant accumulation of [89Zr]-Dfo-EPO starting at 24 h after stroke induction in the stroke area of rats in comparison to healthy controls (p = 0.05) and contralateral hemispheres (p = 0.02). A significant increment continued at 48 and 72 h in comparison to sham (p < 0.01, at 48 & 72 h) and contralateral hemispheres (p < 0.01, at 48 & 72 h).

A specific binding to the stroke area in human and rat samples was observed. Human tissue sections showed binding in stroke tissue of 64.47 ± 18.9 fmol of [89Zr]-Dfo-EPO per mg of brain tissue. Blocked tissue sections showed a significantly lower binding (14.1 ± 4.0 fmol per mg of tissue, p < 0.01).  The stroke area presented significantly increased binding of [89Zr]-Dfo-EPO in contrast to the unspecific background and healthy non-stroke brain tissue (p < 0.01).

Conclusions

In this study we show for the first time that human stroke tissues can be targeted using [89Zr]-Dfo-EPO. The amount of [89Zr]-Dfo-EPO binding in human and rat stroke tissues was consistent between both species. These results demonstrate the potential of [89Zr]-Dfo-EPO to evaluate tissue response to hypoxia [2].

References

[1] M. Buemi et al., “The pleiotropic effects of erythropoietin in the central nervous system,” J. Neuropathol. Exp. Neurol., vol. 62, no. 3, pp. 228–36, Mar. 2003.

[2] M. Brines and A. Cerami, “The receptor that tames the innate immune response,” Mol. Med., vol. 18, no. 3, p. 1, Jan. 2012.

Keywords: Erythropoietin, Stroke, [89Zr]-Desferoxamine-EPO, Hypoxia
911

Lasting effects of adolescence cannabinoid exposure on the brain glucose metabolism and endogenous cannabinoid system after adult morphine consumption (#377)

Nicolás Lamanna1, Marta Casquero-Veiga1, 2, Alejandro Higuera-Matas4, Karina MacDowell5, 6, Emilio Ambrosio4, Manuel Desco1, 2, 3, Maria Luisa Soto-Montenegro1, 2

1 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
2 CIBER de Salud Mental (CIBERSAM), Madrid, Spain
3 Universidad Carlos III de Madrid, Departamento de Bioingeniería e Ingeniería Aeroespacial, Leganés, Spain
4 Universidad Nacional de Educación a Distancia (UNED), Departament of Psychobiology, School of Psychology, Madrid, Spain
5 Complutense University, Department of Pharmacology, Faculty of Medicine, Madrid, Spain
6 Instituto de Investigación Sanitaria Hospital 12 de Octubre (I+12), Madrid, Spain

Introduction

The endocannabinoid system (ECS) is the target of the psychoactive component of cannabis and modulates the rewarding effects of addictive drugs. This study examines the influence of a chronic peripuberal treatment with the synthetic cannabinoid CP-55,940 (CP) on morphine (M) self-administration at adulthood in rats.  Our work benefits from the detection of the brain glucose metabolism through positron emission tomography (PET) studies, as well as from the evaluation of EC receptors and enzymes by means of western blot (WB) analyses.

Methods

37 male and 35 female Wistar rats were daily injected with CP or its vehicle (VH) from postnatal day (PND) 28 to 38. At PND100, rats were trained in an operant task and then in a self-administration  (SA) procedure,  for 15 d. After MSA they underwent extinction sessions for 15 d with saline (sal) instead of M. 4 study groups were evaluated according to 2 factors: pretreatment (CP, VH) and treatment (M, sal). PET (Argus PET/CT, SEDECAL) and MR (Bruker, 7T) images were acquired after 16 weeks of withdrawal. [18F]FDG was injected and, after 45 min of uptake, animals were scanned for 45 min. PET data were analyzed by SPM12 analyses. ROI analyses were performed in amygdala and Caudate-Putamen (CPu). CB1R, CB2R, DAGL, MAGL and FAAH protein expression was measured with WB assays in brain tissue.

Results/Discussion

PET data: Global analysis of brain metabolism revealed an interaction between the pre-treatment and M in both males and females, with a reduction of brain metabolism after MSA in CP-treated animals. M-treatment reduced [18F]FDG uptake in nucleus accumbens (nacc), amygdala, entorhinal cortex (Ent) and CPu in males; and reduced metabolism in the cerebellum and Ent in females as compared to sal-treated rats.  On the other hand, M-treatment in CP pre-exposed animals induced an opposite metabolic pattern. WB data: We found an effect of the sex factor in CB1 and CB2 receptors and the enzymes studied. An effect of the phenotype was found in CB2-CPu and MAGL-nacc in males, CB2-st and CB1-nacc in females. An interaction between pre-treatment and treatment was found in CB1-nacc and DAGL-nacc in males and CB2-st and CB2-nacc in females

Conclusions

M altered the metabolism in limbic and reward areas in a sex- dependent manner. This pattern turned opposite when CP was present; hence suggesting that CP may cause permanent metabolic changes. Also, CP modulated the ECS mainly through receptors and enzymes in the nacc. Together, our results suggest that CP pre-exposure contributes to modulate reward and EC systems, influencing the different vulnerability to abuse drugs such as the opiates.

 

Acknowledgement

This work was partially supported by the Ministry of Economy and Competitiveness ISCIII-FIS grants (PI14/00860, CPII/00005, PI17/01766), co-financed by ERDF (FEDER) Funds from the European Commission, “A way of making Europe”, Fundación Alicia Koplowitz, Fundación Tatiana Pérez de Guzmán el Bueno and Delegación del Gobierno para el Plan Nacional sobre Drogas.

Brain metabolic changes
[A] SPM results in T-maps overlaid on a T2 MR image, showing the brain metabolic changes in morphine and CP-morphine-treated animals in male (left side) and females (right side). Color bars show the T values (lower and higher FDG uptake). Right (R) and Left (L). [B] ROI analysis. Values expressed as mean ± EEM.  ***p<0.001, **p<0.01, *p<0.05 vs saline; &&p<0.01, &p<0.05 vs VH-saline, 2-way ANOVA .
Changes in the endocannabinoid system
Effects of morphine on the expression of receptors [A] and enzymes [B] in males and females pre-exposed to CP55,940 during adolescence. [A] CB1 and CB2 receptor expression in the caudate-putamen (CPu) and nucleus accumbens (nacc). [B] Synthesis enzyme (DAGL) and degradation enzymes (MAGL, FAAH) in the nacc. Data are expressed as the mean ± SEM. ***p<0.001, **p<0.01, *p<0.05 2-way ANOVA.
Keywords: drug addiction, opiates, THC, endocannabionid system, FDG-PET
912

Detection of accumulated iron and microglia in the striatum of Huntington’s disease patients: evidence from post-mortem MRI and histology (#155)

Marjolein Bulk1, Ernst Suidgeest1, Ingrid Hegeman-Kleinn3, Sjoerd van Duinen3, Jan Lewerenz2, Bernhard Landwehrmeyer2, Itamar Ronen1, Louise van der Weerd1

1 Leiden University Medical Center, Radiology, Leiden, Netherlands
2 Ulm University Hospital, Neurology, Ulm, Germany
3 Leiden University Medical Center, Pathology, Leiden, Netherlands

Introduction

T2*-weighted MRI and quantitative susceptibility mapping (QSM) in Huntington’s Disease (HD) patients showed iron accumulation in the striatum correlated with disease state1,2. Although previous studies showed the usefulness of iron-sensitive MRI scans in detecting iron accumulation in HD patients and its potential as a biomarker for disease progression, the underlying pathological substrates have never been investigated. Here we investigated the correlation between 7T MRI and histopathology in the striatum of HD patients and their association with disease severity.

Methods

Formalin-fixed brain material, one whole HD brain and coronal brain slabs including the striatum of HD patients (N=6) and controls (N=3), were obtained respectively from Ulm University Hospital, the local neuropathology tissue collection LUMC, and the Normal Ageing Brain Collection Amsterdam. The whole HD brain and individual brain slabs were scanned on a whole body human 7T MR system. Smaller tissue blocks including the caudate nucleus and putamen were resected and scanned at ultra-high resolution (100μm isotropic) on a 7T Bruker system (Bruker Biospin, Germany). R2*-maps were calculated using an in-house written Matlab pipeline. The same tissue blocks as used for MRI were used for iron histochemistry (Meguro staining).

Results/Discussion

On MRI, lowest signal amplitude was found in the myelin-rich areas of the white matter, followed by the striatum. Additionally, large focal hypointensities were observed in the striatum in HD patients but not in controls (Fig. 1). Striatal R2*-values were significantly increased in HD patients compared to controls and were correlated with neuropathological disease severity as defined by the Vonsattel grade. Currently, we are quantifying the susceptibility changes using QSM. Striatal hypointense regions on MRI colocalized macroscopically with increased iron staining intensity (Fig.1). Microscopic analysis showed an increase of iron in regions which most likely represented the matrix component of the striatum and in cells morphologically resembling glial cells (Fig. 2). Specifically in controls, the microglial-like positive stainings had the appearance of densely packed cells with long, thin processes. In contrast, in HD patients these cells had fewer, thicker, more twisted processes.

Conclusions

Using high-field MRI, we clearly distinguished HD patients from controls, which is mirrored in the histology, mostly in differences in iron. Clinically, the role of iron accumulation is increasingly recognized and shown to be a potential imaging biomarker for disease progression, possibly reflecting neuroinflammation. Susceptibility-based MRI methods could therefore play an important role for further clinical and mechanistic studies.

References

1.            van Bergen JM, Hua J, Unschuld PG, Lim IA, Jones CK, Margolis RL, et al. Quantitative susceptibility mapping suggests altered brain iron in premanifest huntington disease. AJNR Am J Neuroradiol. 2016;37:789-796

2.            Dominguez JF, Ng AC, Poudel G, Stout JC, Churchyard A, Chua P, et al. Iron accumulation in the basal ganglia in huntington's disease: Cross-sectional data from the image-hd study. J Neurol Neurosurg Psychiatry. 2016;87:545-549

Figure 1. Post-mortem 7T MRI of brain slabs of an HD patient and a control.
Lowest signal amplitude was found in the white matter followed by the striatum. In HD patients large focal hypo intensities were found in the striatum. High-resolution 7T MRI ((a,d) magnitude and (b,e) R2*-maps) of smaller tissue blocks of the striatum showed spatial colocalization with histological iron (c,f) in both HD patients and controls. Color bars in R2*-maps range from 0-0.2. CN = caudate nucleus; P = putamen.
Figure 2. Microscopic images of striatal regions with high staining intensity.
Microscopic images of striatal regions with high staining intensity showed a general increase of iron in the matrix and in cells which morphologically closely resemble glial cells (arrows). In HD patients (a) these cells have fewer, thicker, more twisted processes than controls (b). Scale bars: 50 μm.
Keywords: Huntington's disease, iron, microglia, MRI, neuroinflammation
913

N-acetylcysteine and physical exercise as preventive therapies in the schizophrenia onset: A behavioral and imaging study in an animal model (#384)

Diego Romero-Miguel1, Marta Casquero-Veiga1, 2, Vanessa Gómez-Rangel1, Alejandro Higuera-Matas4, Emilio Ambrosio4, Manuel Desco3, 2, 1, María Luisa Soto-Montenegro1, 2

1 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
2 CIBER de Salud Mental (CIBERSAM), Madrid, Spain
3 Universidad Carlos III de Madrid, Departamento de Bioingeniería e Ingeniería Aeroespacial, Madrid, Spain
4 Universidad Nacional de Educación a Distancia, School of Psychology, Department of Psychobiology, Madrid, Spain

Introduction

Oxidative stress and inflammation play an important role in the biochemical mechanisms underlying schizophrenia and other mental disorders. In this sense, N-acetylcysteine (NAC) and physical exercise (Ex) have been reported to induce anti-inflammatory and antioxidant effects. Therefore, the aim of this study is to evaluate whether the combined treatment with NAC and exercise during adolescence in the maternal immune stimulation (MIS) rodent model of schizophrenia could prevent 1) the behavioral deficits and 2) the metabolic and volumetric brain changes described at adulthood.

Methods

At gestational day 15, Poly I:C  or saline were injected to pregnant Wistar rats. 6 study groups were evaluated attending to the factors: phenotype (VH, MIS) & treatment (saline, NAC, NAC+Ex). Treatments started at postnatal day (PND) 35. NAC was orally administered (500mg/kg) for 15 days, and then in the drinking water until PND60. Exercise lasted 12 weeks1. At PND60, sensorimotor gating was tested by prepulse inhibition (PPI) test2. Animals were imaged at PND120. After 45 min of 18FDG uptake, rats were scanned for 45 min (Argus PET/CT, SEDECAL). 2D-OSEM reconstruction was applied and PET images were analyzed by SPM12. T2W-MRI images were acquired with a 7T MRI scanner (Bruker), and ROIs were studied. MRI and PPI data were analyzed with 2 way-ANOVAs and Bonferroni post-hoc test.

Results/Discussion

PET: In VH-offspring, NAC reduced the metabolism in the brain stem (BS), entorhinal cortex and piriform cortex (Pir), while increased it in caudate-putamen and motor and primary somatosensory cortex. In MIS-offspring, NAC reduced the metabolism in the Pir and septum and increase it in the BS. Comparing to VH-saline, VH-NAC+Ex group revealed lower FDG uptake in substantia nigra-hypothalamus-Pir area and BS-cerebellum, and cortical FDG increase. Also, higher cortical FDG uptake was shown in MIS-NAC+Ex animals compared to MIS-saline rats.

MRI: MIS-offspring showed significant volumetric reductions in whole brain, cortex and hippocampus, and volumetric enlargement in ventricles (V), compared to VH. Both hippocampus and V differences were reverted after NAC or NAC+Ex in MIS animals.

PPI: MIS-offspring showed significant PPI deficit (120 ms PP) comparing with VH-offspring. NAC and NAC+Ex treatments induced PPI improvements in both phenotypes, although they were no significant.

Conclusions

NAC and NAC+Ex were able to slightly improve some of the attentional, metabolic and volumetric alterations present in the MIS model3. Thus, both approaches have shown a moderate capability as preventive strategies of the functional and structural schizophrenia deficits, making necessary further research to uncover their real potential in this field.

References

  1. Sastre E, Caracuel L, Balfagón G, & Blanco-Rivero J (2015). Aerobic exercise training increases nitrergic innervation function and decreases sympathetic innervation function in mesenteric artery from rats fed a high-fat diet. Journal of Hypertension, 33(9): 1819–1830.
  2. Santos-Toscano R, Borcel É, Ucha M, et al (2016). Unaltered cocaine self-administration in the prenatal LPS rat model of schizophrenia. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 69: 38-48.
  3. Hadar R, Soto-Montenegro ML, Götz T, et al. (2015). Using a maternal immune stimulation model of schizophrenia to study behavioral and neurobiological alterations over the developmental course. Schizophrenia Research, 166(1-3), 238–247.

Acknowledgement

This work was partially supported by the Ministry of Economy and Competitiveness ISCIII-FIS grants (PI14/00860, CPII/00005, PI17/01766), co-financed by ERDF (FEDER) Funds from the European Commission, “A way of making Europe”, Fundación Alicia Koplowitz, Fundación Tatiana Pérez de Guzmán el Bueno and Delegación del Gobierno para el Plan Nacional sobre Drogas.

Prepulse inhibition protocol
Figure 1 - Behavioural results. Prepulse inhibition (PPI) percentage in two sets of prepulses (12 db 30 ms; 12 db 120 ms) for the 6 groups: VH-saline (n=15), VH-NAC (n=11), VH-NAC+Ex (n=10), MIS-saline (n=13), MIS-NAC (n=13), MIS-NAC+Ex (n=12). Data are expressed as mean ± SEM, p<0.05.
Metabolic and structural imaging results
A) Voxel-based SPM results in T-maps overlaid on a T2 MR image. Color bars in the right represent T values corresponding to lower and higher FDG uptake. Statistical threshold: k>50; p<0.01 (unc.). B) Region of interest (ROI) analysis in the whole brain (WB), cortex (Cx), hippocampus (Hi) and ventricles (V). Values are expressed as mean ± SEM. *p<0.05; ***p<0.001 (Bonferroni post-hoc test).
Keywords: positron emission tomography (PET), magnetic resonance (MRI), N-acetylcysteine, physical exercise, MIS model
914

Multimodal combination of in vivo bioluminescence and magnetic resonance imaging reveales enhanced elements of repair after ischemic brain lesion in Tlr2-deficient mouse (#480)

Srecko Gajovic1, Dunja Gorup1, Siniša Škokić1, Jasna Križ2

1 University of Zagreb School of Medicine, Croatian Institute for Brain Research, Zagreb, Croatia
2 Laval University Faculty of Medicine, Department of Psychiatry and Neuroscience, Quebec, Québec, Canada

Introduction

Previous studies of brain ischemia using Tlr2-deficient mice, including our own, have shown that altering neuroinflammatory response did not result in either beneficial or harmful consequences with regards to the lesion size, but in a combination of both, depending on the time or phase following ischemia. In the acute phase, Tlr2-deficiency reduces the volume of the ischemic lesion, however in the later phase, the resulting modified inflammation leads to delayed apoptosis and a larger sized ischemic lesion at later time points compared to the wild type (WT) animals (1,2).

Methods

To clarify the consequences of the modulated neuroinflammation due to Tlr2-deficiency, this study monitored evolution of ischemic brain lesion through time by multimodal in vivo imaging combining magnetic resonance imaging (MRI), bioluminescence imaging (BLI) and caged-luciferin-BLI (cagedBLI). Ischemic lesion was obtained by temporary middle cerebral artery occlusion. MRI was done using T2 modality, BLI used Gap43-luc/GFP transgenic animals crossbred to Tlr2-deficient mice, and cagedBLI on the same animals but using sensitive to caspases 3 and 7 DEVD-luciferin (VivoGlo, Promega) as a substrate (3). The animals were functionally assessed by neruological scoring, accelerating rotarod, Y-maze, Schallert’s cylinder, and bilateral tactile stimulation.

Results/Discussion

When used as a single modality Gap43 BLI and cagedBLI in 4 different time points (3, 7, 14 and 28 days after ischemia) were comparable between Tlr2-deficient and wild type animals. As the same animals were imaged in the same time points by MRI, we could show the positive correlation of the bioluminescence signal with the MRI-obtained volume of the ischemic lesion. Subsequently, this allowed to normalise the BLI to the lesion size, which revealed statistically significantly higher Gap43 and Casp3 activity in the brains of Tlr-2 deficient animals. The imaging data were confirmed by Western blot, which as well showed the higher levels of synaptic markers DLG and synaptophysin.

Conclusions

Altered inflammation in Tlr2-deficient mice was accompanied by enhanced elements of poststroke repair, in particular during the chronic phase of recovery, but also with delayed apoptosis and final consolidation of the brain lesion. Multimodal imaging allowed to compare the animal groups in a more informative way than single modalities only.

References

  1. Bohacek I, et al. J Neuroinflammation. 2012 Aug 8;9:191.
  2. Winters L,  et al. Neuroscience. 2013;238:87-96.
  3. Gorup D et al. Neurosci Lett. 2015;597:176-82.

Acknowledgement

This study was supported by EU European Regional Development Fund, Operational Programme Competitiveness and Cohesion, grant agreement No.KK.01.1.1.01.0007, CoRE – Neuro, and by the Croatian Science Foundation under the project IP-06-2016-1892 (RepairStroke). Multimodal imaging was done at Laboratory for Regenerative Neuroscience - GlowLab, University of Zagreb School of Medicine.

Keywords: MRI, BLI, stroke, ischemia
915

[11C]leucine PET imaging to measure cerebral protein synthesis rate in rats. (#478)

Daniela Bochicchio2, 3, Matthias Vandesquille1, 3, Karl Herholz1, 3, Christine Parker4, Rainer Hinz2, 3, Hervé Boutin1, 3

1 University of Manchester, Faculty of Biology, Medicine and Health, School of Biological Sciences, Division of Neuroscience and Experimental Psychology, Manchester, United Kingdom
2 University of Manchester, Faculty of Biology, Medicine and Health, School of Health Sciences, Division of Informatics, Imaging and Data Sciences, Manchester, United Kingdom
3 University of Manchester, Faculty of Biology, Medicine and Health, Wolfson Molecular Imaging Centre, Manchester, United Kingdom
4 GlaxoSmithKline Research and Development Limited, Experimental Medicine Imaging, Stevenage, United Kingdom

Introduction

Alzheimer’s disease (AD) is characterised by severe alterations of cognitive function and memory which depend on protein synthesis1. Protein synthesis is essential to neuronal plasticity and new connections. The various protein synthesis pathways have been shown to be altered in animal models of AD2 and patients3-5. Here we set up a measurement of protein synthesis rate (PSR) by [11C]leucine PET in rats to characterise the arterial input function (AIF) and determine an image-derived input function (IDIF). These methods will be applied to a longitudinal study in the TgF344-AD rat model of AD.

Methods

Wistar rats (n=6) were anaesthetised with isoflurane (2-2.5% in O2/NO2 30%/70%). A CT scan was first performed; [11C]leucine was injected in the tail vein at the start of the PET scan. Arterial blood activity was monitored continuously over the 60min scans using a Twilite Swisstrace. Discrete blood samples were collected at 2, 5, 10, 20, 30, 40, and 60min post-injection. Whole-blood and plasma samples were then counted in a γ-counter. Free leucine in plasma was measured by γ-counting after precipitation of plasma proteins using 5% perchloric acid. the images. ROIs for the heart left ventricle and vena-cava were segmented automatically using local means analysis (LMA) in the BrainVisa/Anatomist framework6. The [11C]leucine uptake in the brain was quantified with an MRI rat brain atlas7.

Results/Discussion

The PET [11C]leucine time-activity curve (TAC) in the heart ventricle peaked at 22s post-injection (SUV=7.7±1.61), followed by a rapid wash-out, a plateau between 7 and 20min (0.67±0.11) and then a slight increase from 20 to 60min (0.82±0.06) post-injection (Fig. 1a). Whole-blood and plasma γ-counting confirmed these observations. The ratio of free/protein-bound [11C]leucine decreased with time (Fig. 1b). These data are in agreement with previous studies in human1,8 and in rat9.
[11C]leucine uptake in various brain regions showed similar pharmacokinetics with a rapid uptake within 5 minutes post-injection, reaching a plateau from 10 minutes onwards (Fig. 2). PET analysis and γ-counting of different brain regions were in good agreement:  highest [11C]-leucine uptake in cerebellum and cortical regions (0.62±0.07), intermediate in the hippocampus region (0.59±0.06), thalamus (0.54±0.06) and lowest in the striatum (0.51±0.03). These data are also in agreement with previously results1,10,11.

Conclusions

The biodistribution and pharmacokinetics of [11C]-leucine in blood, plasma and brain observed here are similar to those previously reported. γ-Counting and heart-ventricular values as measured by PET are also in good agreement, highlighting the accuracy of PET measurements, and support the feasibility of modelling an IDIF for [11C]-leucine, which we are currently performing. A longitudinal study in the TgF344-AD rat model of AD is also ongoing.

References

1. Bishu S. et al. (2008). J Cereb Blood Flow Metab.28:1502-1513.

2. Pasini S. et al. (2015). Cell Rep.11:183-191.

3. Hoozemans J. J. et al. (2005). Acta Neuropathol.110:165-172.

4. Ma T. et al. (2013). Nat Neurosci.16:1299-1305.

5. Garcia-Esparcia P. et al. (2017). Am J Neurodegener Dis.6:15-25.

6. Maroy R. et al. (2008). IEEE Trans Med Imaging.27:342-354.

7. Schwarz A. J. et al. (2006). Neuroimage.32:538-550.

8. Sundaram S. K. et al. (2006). J Nucl Med.47:1787-1795.

9. Lauenstein L. et al. (1987). Neurosurg Rev.10:147-150.

10. Smith C. B. et al. (2005). J Cereb Blood Flow Metab.25:629-640.

11. Hawkins R. A. et al. (1989). J Cereb Blood Flow Metab.9:446-460.

Acknowledgement

CB and this study are funded by a GSK-University of Manchester PhD studentship.

MV was funded by the EPSRC project EP/M005909/1.

The authors wish to thank all the personnel of the WMIC, especially Ms. Lidan Christie, Ms. Carol Brough, Mr Michael Green and Mr Hamza Al-qasmi for facilitating the study.

Conflict of interest:

Christine A. Parker is an employee of GSK. GSK was not involved in the study design or data analysis.

Figure 1

[11C]leucine in whole-blood (WB) and plasma (P). (A) Whole blood (PET in blue and γ-counting in purple) and plasma (γ-counting in red) time-activity curves. Data shown as standard uptake values (mean±SD, n=6). (B) Percentage of free and protein-bound leucine in plasma at different time point (n=1).

Figure 2

Time-activity curves of [11C]leucine in different brain regions: somatosensory cortex, posterior hippocampus and thalamus (A), frontal cortex and anterior hippocampus (B) and caudate-putamen and cerebellum (C) in Wistar rats. Data shown as SUV (mean±SD, n=6). (D) Coronal, axial and sagittal view of a representative PET scan (20-60min sum image in SUV).

Keywords: brain metabolism, protein synthesis, PET, Input function, Alzheimer's disease
916

Identifying the cerebrovascular changes associated with lipopolysaccharide treatment in a mouse model of Alzheimer’s disease (#461)

Sebastien Serres1, Nerissa Culi1, Faiza Bukenya3, Malcom Prior2, Alessandra Agostini1, Bai Li3, Marie-Christine Pardon1

1 University of Nottingham, School of Life Sciences, Nottingham, United Kingdom
2 University of Nottingham, School of Medicine, Nottingham, United Kingdom
3 University of Nottingham, School of Computer Sciences, Nottingham, United Kingdom

Introduction

Systemic inflammation is regarded as a key contributor to Alzheimer’s disease (AD) with proinflammatory markers associated with cognitive decline and amyloid plaque load. This is in part mediated by vascular inflammation, and can be mimicked in mouse models of AD using lipopolysaccharide (LPS). We therefore tested the hypothesis that LPS, administered systemically to model peripheral inflammation, or intranasally to overcome the blood-brain barrier (BBB) will damage cerebral blood vessels in a mouse model of AD using in vivo magnetic resonance imaging and ex vivo histological analysis. 

Methods

APP/PS1 mice and their wildtype littermates were challenged with LPS (100μg/kg, i.v.) or its vehicle PBS as follows: (i) one injection was given (LPS or PBS) to 16 APP/PS1 and 16 WT littermates and two injections were given. (LPS/LPS, LPS/PBS, PBS/PBS, 7 days apart) to 24 APP/PS1 and 24 WT littermates, all aged 4.5-months-old at the start of the experiment. Histological analysis of cerebral vessel diameter in the cortex and hippocampus was performed using anti-collagen IV. (ii) To assess blood-brain barrier permeability, one injection was given i.n. (LPS, on one side, PBS on the other side) to 4 APP/PS1 and 4 WT females, aged 4 months and 5-month-old APP/PS1 females. Mice underwent T2 map and contrast enhanced T1-weighted MRI to assess oedema and BBB permeability, respectively.

Results/Discussion

Transverse assessment of blood vessels (Fig1A) showed in the single challenge, that females exhibiting significantly larger vessels than males (p<0.05) regardless of genotype and treatment. Longitudinal measurement of blood vessels (Fig1B) showed that a single systemic challenge with LPS significant increased vessel diameters (p<0.001), but this was only significant in WT males. This was also the case in the double challenge experiment (p<0.01). MRI analysis after intranasal administration of PBS and LPS, showed a significant decrease in intensity between hippocampus and cortex on T1 scans in 4-month-old APP/PS1 vs WT mice (p<0.001, Fig2A) and 5-month-old APP/PS1 mice (p<0.05, Fig2C), but a significant increase on T2 map in these mice, regardless of age, treatment and genotype (p<0.05 in all cases, Fig2B-C).

Conclusions

Our findings suggest that vessel diameter respond to systemic inflammation in a sex-dependent manner, with males showing smaller inner lumen diameters than females whilst responding to systemic infection by increasing vessel diameters. MRI scans suggest that BBB is more permeable in the cortex than in the hippocampus, whilst oedema is more likely to occur in the hippocampus rather than the cortex but not affected by intranasal LPS administration.

Acknowledgement

This work was funded by a British Association of Psychopharmacology in vivo training award NRS and MCP and an University of Nottingham Vice-Chancellor scholarship to AA.

Fig1: The effect of systemic injection of LPS on cerebral blood vessels.
(A) Representative image of transverse measurement using collagen IV staining and graphs showing vessel diameter for one or two challenges in APP and WT mice. (B) Representative image of longitudinal measurement using collagen IV staining and graphs showing vessel diameter for one or two challenges in APP and WT mice. N=16-24, mean ± SEM, ANOVA and post-hoc t test; *p<0.05, **p<0.01 and ***p<0.001
Fig2: The effect of systemic injection of LPS on blood-brain barrier and tissue oedema

(A) Representative contrast enhanced T1-weighted MR images of WT (top), APP (4 months; middle), and APP (5 months, bottom) mice. Graphs showing signal intensity for T1-weighted Gadolinium (Gd) enhanced MRI and T2 map in WT and APP (4 months) mice (B) and in APP (4 months) and APP (5 months) mice (C) after LPS or PBS challenge (male or female; N=3-4; mean ± SEM, ANOVA and post-hoc t test;*p <0.05).

Keywords: Alzheimer's disease, MRI, cerebral blood vessels, systemic inflammation, amyloid-beta
917

Plasma pharmacokinetic and metabolism of (S)-[18F]THK5117 and [18F]THK5351 are dependent on sex (#120)

Severin Mairinger1, Thomas Filip1, Michael Sauberer1, Stefanie Flunkert2, Thomas Wanek1, Johann Stanek1, Nobuyuki Okamura3, Claudia Kuntner1

1 AIT Austrian Institute of Technology, Center for Health & Bioresources, Seibersdorf, Austria
2 QPS Austria, Neuropharmacology, Grambach, Austria
3 Tohoku Medical and Pharmaceutical University, Division of Pharmacology, Sendai, Japan

Introduction

Tau deposition is one of the hallmarks of Alzheimer’s disease (AD). The 2-arylquinoline derivatives (S)-[18F]THK5117 and [18F]THK5351 show high affinity for neurofibrillary tangles [1, 2]. Kinetic modelling has been proposed to determine tau binding. However, kinetic modelling is precluded in mouse AD models by their small blood volume, thus rat models with tau pathology may offer the possibility to perform arterial blood sampling and kinetic modelling. To determine the feasibility of this approach, we measured blood pharmacokinetics and radiotracer metabolism in female and male rats.

Methods

Female and male rats (n=10-12) were cannulated via the femoral artery for continuous blood sampling. Blood sampling was performed every 5-6 sec for the first 3 min and then at 5, 10, 20, 30, 40, 50 and 60 min after intravenous injection of (S)-[18F]THK5117 or [18F]THK5351. After collection of the 60 min blood sample, animals were sacrificed and organs were excised. Blood from minute 5, 20 and 60 was centrifuged to obtain plasma. Radiolabelled metabolites in plasma, brain, liver and urine were analyzed by radio-thin-layer chromatography (radio-TLC).

Results/Discussion

Plasma pharmacokinetics and metabolism were significantly different between female and male rats for both radiotracers. (S)-[18F]THK5117 plasma clearance was faster in female (0.66±0.08 mL/h/kg BW) than in male (0.52±0.11 mL/h/kg BW) rats (p=0.005). For (S)-[18F]THK5117, the percentage of unmetabolized parent in plasma was different between both sexes (5 min: 63% (f) vs 69% (m), ns; 20 min: 49% (f) vs 28% (m), p<0.001; 60 min: 32% (f) vs 13% (m), p<0.001) see Fig 1. [18F]THK5351 plasma clearance was faster in female compared to male rats (1.07±0.23 mL/h/kg BW vs 0.67±0.04 mL/h/kg BW) and percentage of unmetabolized parent was also different (5 min: 83% (f) vs 76% (m), ns; 20 min: 50% (f) vs 19% (m), p=0.01; 60 min: 37% (f) vs 4% (m), p<0.001). In the liver, 7% (f) vs 6% (m) unchanged parent was measured for (S)-[18F]THK5117 and 16% (f) vs 3% (m) for [18F]THK5351 (Fig 2). In the brain, 90-98% and 84-90% of total radioactivity consisted of (S)-[18F]THK5117 and [18F]THK5351, respectively.

Conclusions

Our results show pronounced sex differences in blood pharmacokinetics and metabolism of (S)-[18F]THK5117 and [18F]THK5351 in rats. Female animals showed a faster plasma clearance of both radiotracers. These results underline the importance of investigating both sexes and also support the notion that individual input functions are needed for kinetic modelling analyses.

References

[1] Okamura N, Furumoto S, Harada R, Tago T, Yoshikawa T, Fodero-Tavoletti M, et al. Novel 18F-labeled arylquinoline derivatives for noninvasive imaging of tau pathology in Alzheimer disease. J Nucl Med 2013;54:1420-7.

[2] Harada R, Okamura N, Furumoto S, Furukawa K, Ishiki A, Tomita N, et al. 18F-THK5351: A Novel PET Radiotracer for Imaging Neurofibrillary Pathology in Alzheimer Disease. J Nucl Med 2016;57:208-14.

Acknowledgement

The research leading to these results has been funded by the Austrian Research Promotion Agency (FFG) through project 853256.

Figure 1.
Unchanged parent fraction determined in plasma at 5, 20 and 60 min after injection.
Figure 2
Unchanged parent fraction determined in brain, liver and urine at 60 min after injection.
Keywords: tau tracer, plasma PK, metabolism
918

MRI investigation of neuro-anatomical and neuro-functional differences in models of Rett Syndrome (#575)

Sara Carli1, Clarissa Butti2, Angelisa Frasca2, Nicoletta Landsberger2, 1, Linda Chaabane1, 3

1 IRCCS San Raffaele Scientific Institute, Dept. of Neuroscience, Milano, Italy
2 University of Milan, Dept. of Medical Biotechnology and Translational Medicine, Milano, Italy
3 San Raffaele Hospital, INSPE-CIS, Milano, Italy

Introduction

Rett syndrome (RTT) is a devastating neurological disorder caused by mutations in the Methyl-CpG-binding protein-2 (MECP2) gene. Interestingly, metabolic dysregulations have been found in RTT patients and mice models using 1H-MR-Spectroscopy (MRS)1,2. Thus, in order to identify the key brain areas to study by MRS, we initially used manganese contrast (Mn) to highlight the neuronal dysfunctions in two models of RTT, Mecp2-null and knock-in Mecp2-Y120D mice that have similar behavioural phenotypes, but different molecular expressions3.

Methods

Ex-vivo MRI experiments were conducted on a 7-Tesla MRI scanner (Biospec, Bruker-biospin). Brains were taken and fixed 24h after the administration of manganese in all transgenic mice (P150 females and P39 males) and their respective littermate controls. Brains from mice untreated with manganese were also used as negative controls. To quantify Mn uptake, T1 maps were acquired by varying the repetition time (294-4000 ms). To evaluate the anatomical differences, serial transversal sections covering the entire brain (except olfactive bulbs) were acquired with T2-weighted fast spin-echo sequence with a relative high resolution (0.083x0.083x0.7 mm).

Results/Discussion

For both genders, the volume of brains were clearly smaller for both Mecp2-null and Mecp2-Y120D mice (Fig. 1). Interestingly, brain's atrophy is more caudal in the Mecp2-null mice while Mecp2-Y120D animals showed a diffused atrophy throughout the whole brain (Fig. 1). From T1 maps, significant manganese contrast uptake compared to negative controls were measured in several brain's area as the striatum and hippocampus, as expected. Different responses to Mn contrast were observed between genotypes and genders. A diffuse hypo-neuronal activity was found in Mecp2-Y120D males while significant hyperactivity was measured in the pons and regions of the medulla in Mecp2-null males. In females, hypo and hyperactive areas were measured in the mid-brain with a more significant activity of the somatosensorial cortex in Mecp2-Y120D females.

Conclusions

Differences in brain size and manganese uptakes were observed between mice genotypes and gender that are supporting the molecular differences previously found in these models of RTT. According to these results, we will further investigate the metabolic profile of these different brain areas with major impairments with 1H-MR spectroscopy to monitor the cerebral dysregulation at different age.

References

1. Orska A, et al. Quantitative 1H MR spectroscopic imaging in early Rett syndrome. Neurology 2000, 54, 715–722. doi:10.1212/WNL.54.3.715

2. De Filippis B, et al. Modulation of RhoGTPases improves the behavioral phenotype and reverses astrocytic deficits in a mouse model of Rett syndrome. Neuropsychopharmacology. 2012 Apr;37(5):1152-63. doi: 10.1038/npp.2011.301.

3. Gandaglia A, et al. Novel Mecp2(Y120D) Knock-in Model Displays Similar Behavioral Traits But Distinct Molecular Features Compared to the Mecp2-Null Mouse Implying Precision Medicine for the Treatment of Rett Syndrome. Mol Neurobiol. 2018 Nov 6. doi: 10.1007/s12035-018-1412-2.

Figure 1
Example of T2 weighted section of brains from a wild type, Mecp2 null and Y120D mice showing the reduced size of transgenic mice. The brain size measured along the different MRI sections (slice 1 to 10: rostral to caudal) differs between both genotypes (Left graph: Mecp2 null female; Right graph: Mecp2 Y120D female). Statistical analysis were done using Two-way ANOVA, Bonferroni post-hoc test.
Keywords: Rett syndrome, Mecp2 models, MRI anatomy, Manganese Enhanced MRI, Brain atrophy
919

In vivo longitudinal tracking of anti-inflammatory, cerebrovascular and functional effects of a hydroxytyrosol enriched diet after stroke (#559)

Cristina Barca1, 2, Bastian Zinnhardt1, 2, 3, Maximilian Wiesmann4, Claudia Foray1, 2, Michael Schäfers1, 3, Amanda J. Kiliaan4, Andreas H. Jacobs1, 2, 5

1 Westfälische Wilhelms University , European Institute for Molecular Imaging (EIMI) , Münster , North Rhine-Westphalia, Germany
2 PET imaging in Drug Design and Development , PET3D, Münster, Germany
3 Universitätklinikum Münster, Department of Nuclear Medicine, Münster, North Rhine-Westphalia, Germany
4 Radboud University medical center , Department of Anatomy, Nijmegen, Netherlands
5 Johanniter Hospital , Department of Geriatrics, Bonn, Germany

Introduction

Novel dietary therapies aim to facilitate stroke recovery by e.g. counteracting immune cells activation/neuroinflammation1,2,3,4. Hydroxytyrosol (HT) shows broad anti-inflammatory and anti-oxidant properties in vitro5, 6.  The aim of this project is therefore to investigate the effects of a long-term HT diet in a murine ischemic stroke on neuroinflammation, cerebrovascular and functional parameters using non-invasive multimodal imaging. We hypothetized HT may positively alter neuroinflammation and so help tissue recovery and functional outcomes. 

Methods

A total of N=10 C57BL/6 mice were provided either a HT-enriched (n=6) or control diet (n=4) for 35 days after a 30 min tMCAo. They underwent combined [18F]DPA-714 (TSPO) PET-CT and MR imaging (T2w imaging, perfusion-/diffusion weighted imaging) to assess immune cell activity, cerebral blood flow and water diffusion respectively at 3, 7, 14, 21 and 30/34 days after tMCAo, as well as behavioural parameters. All imaging datasets were co-registered and analysed using an atlas-based approach. The percentage of injected tracer (%ID/mL) and mean lesion-to-contralateral ratios were calculated (mean±sd).

Results/Discussion

Peak [18F]DPA-714 uptake was observed between day 14 and 21 post ischemia with no significant difference between control and HT fed mice over time, indicating that HT has no effects on TSPO levels.

HT fed mice exhibited enhanced water diffusion (HT: 1.10 ± 0.08, control: 0.97 ± 0.098, ANOVA on ranks, p = 0.038), a sign of altered tissue integrity. However, PW imaging data indicated improved reperfusion of the lesioned compared to control mice at day 34 post-stroke (HT: 0.98 ± 0.13, control: 0.81 ± 0.080, ANOVA on ranks, p = 0.049).

Ex vivo validation and analysis of behavioural data are currently ongoing.

Conclusions

This study indicates that a HT diet may modulate tissue recovery: we didn't observe any change in inflammation in vivo but mice fed with HT showed improved perfusion of the lesioned tissue along with higher water diffusivity, indicative of altered tissue. Still, behavioural and ex vivo data must be added to better appreciate therapeutic effects of HT.

References

1 Lourbopoulos A, Ertürk A, Hellal F. Microglia in action: how aging and injury can change the brain's guardians. Front Cell Neurosci. 2015;9:54

2 Wiesmann M, Zinnhardt B, Reinhardt D, Eligehausen S, Wachsmuth L, Hermann S, Dederen PJ, Hellwich M, Kuhlmann MT, Broersen LM, Heerschap A, Jacobs AH, Kiliaan AJ. A specific dietary intervention to restore brain structure and function after ischemic stroke. Theranostics 2017; 7(2):493-512.

3 Aquilani R, Scocchi M, Boschi F, Viglio S, Iadarola P, Pastoris O. et al. Effect of calorie-protein supplementation on the cognitive recovery of patients with subacute stroke. Nutr Neurosci. 2008;11:235-40.

4 Gardener H, Wright CB, Gu Y, Demmer RT, Boden-Albala B, Elkind MS. et al. Mediterranean-style diet and risk of ischemic stroke, myocardial infarction, and vascular death: the Northern Manhattan Study. Am J Clin Nutr. 2011;94:1458-64

5 Fuccelli R, Fabiani R, Rosignoli P. Hydroxytyrosol Exerts Anti-Inflammatory and Anti-Oxidant Activities in a Mouse Model of Systemic Inflammation. Molecules. 2018 Dec 5;23(12).

6 Illesca P, Valenzuela R, Espinosa A, Echeverría F, Soto-Alarcon S, Ortiz M, Videla LA. Hydroxytyrosol supplementation ameliorates the metabolic disturbances in white adipose tissue from mice fed a high-fat diet through recovery of transcription factors Nrf2, SREBP-1c, PPAR-γ and NF-κB. Biomed Pharmacother. 2019 Jan;109:2472-2481

Acknowledgement

This research was funded by the Horizon2020 Programme under grant agreement n°675417 (PET3D).

Fig 1.
Similar temporal [18F]DPA-714 uptake (%ID/mL) in mice fed with the control diet (green) versus mice fed with the HT-enriched diet (red) after a 30 min tMCAo.
Fig. 2
Water diffusion and cerebral blood flow (CBF) determined by diffusion weighted (DWI)- and perfusion weighted (PWI)- MR imaging indicate hyperdiffusion and higher CBF in HT fed mice compared to control mice at day 34 post ischemia. (*p< 0.05, **p<0.01)
Keywords: Stroke, hydroxytyrosol, neuroinflammation, multimodal imaging