EMIM 2019
To search for a specific ID please enter the hash sign followed by the ID number (e.g. #123).

POSTER SESSION II - Exhibition - Lunch Break (Hall II)

   
Shortcut: PO 02
Date: Thursday, 21 March, 2019, 12:45 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 the Heart and the Vascular System I

Session chair: Ulrich Flögel (Düsseldorf, Germany); Jereon Essers (Rotterdam, Netherlands)
 
Shortcut: PW09
Date: Thursday, 21 March, 2019, 12:45 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.

101

Manganese enhanced MRI can quantify myocardial infarct size earlier than gadolinium enhanced MRI (#81)

Nur H. Jasmin1, 2, May Zaw-Thin1, Mark F. Lythgoe1, Sean Davidson3, Daniel J. Stuckey1

1 University College London, Cantre for Advanced Biomedical Imaging, London, United Kingdom
2 Universiti Sultan Zainal Abidin, School of Medical Imaging, Terengganu Darul Iman, Malaysia
3 University College London, Hatter Institute, London, United Kingdom

Introduction

Late gadolinium enhanced MRI (LGE-MRI) can quantify of infarct size after myocardial infarction (MI) but is non-specific and reflects the increased membrane rupture and extracellular space that develop post MI (1). Mn is a potent T1-contrast agent that enters myocytes through active calcium channels, thus reducing T1 in viable myocardium (2). This active process rapid ceases under ischemia. Hence, we hypothesised that Mn-enhanced MRI (MEMRI) could quantify final infarct size earlier than LGE-MRI and tested this by applying both methods to mice at 1 & 24 hours after myocardial infarction

Methods

Myocardial infarction was induced in 7 mice which then underwent MEMRI (n=4, 0.1mmol/kg MnCl2) or LGE-MRI (n=3, 0.5mmol/kg Gd-DTPA) at 1 hour post MI. All animals then underwent both MEMRI and LGE-MRI at ~24 hours post MI with a contrast washout period of at least 5 hours between scans. Imaging was performed using a 9.4T Agilent MRI system and a multi-slice inversion recovery sequence in the short-axis orientation to covered the whole left ventricle (TE/TR =  3.04/1.11ms, TI = ~600ms for MEMRI and ~350ms for LGE-MRI, 90⁰ excitation pulse, slice thickness = 1.0mm, FOV = 25.6 x 25.6 mm, matrix size = 128 x 128) as described (3).

Results/Discussion

At 1-hour post MI, viable myocardium was enhanced in MEMRI, allowing early delineation of the occlusion zone as 41 ± 8% of the myocardium, whereas only subtle enhancement was seen on LGE-MRI, yielding a significantly lower value of 12 ± 2% (P=0.03. Fig1). At ~24 hours post MI, the MEMRI measure of infarct size remained constant (41 ± 5%) whilst LGE-MRI significantly increased to a level comparable with MEMRI (37 ± 3%). Fig2 shows a direct comparison of MEMRI and LGE-MRI acquired in the same animal at 22 and 27 hours after MRI, respectively, with matching histological TTC staining for infarct size.

Effected myocytes rapidly stop internalising Mn under ischemic conditions, allowing early delineation of the occlusion zone. The membrane rupture that underlies LGE-MRI occurs later, meaning LGE-MRI underestimates the occlusion zone during the first hours post MI

Conclusions

The present study shows MEMRI can quantify final infarct size earlier than LGE-MRI. This provides a sensitive approach which could be used as an early measure of cell death and myocardial viability when assessing the efficacy of new drugs which target acute MI.

References

(1) Doltra, A., Amundsen, B. H., Gebker, R., Fleck, E., & Kelle, S. (2013). Emerging concepts for myocardial late gadolinium enhancement MRI. Current Cardiology Reviews, 9. 185

(2) Waghorn, B., Schumacher, A., Liu, J., Jacobs, S., Baba, A., Matsuda, T,  Hu, T. C.-C. (2011). Indirectly probing Ca(2+) handling alterations following myocardial infarction in a murine model using T(1)-mapping manganese-enhanced magnetic resonance imaging. Magnetic Resonance in Medicine, 65. 239 .

(3) Chow A, Stuckey DJ, Kidher E, Rocco M, Jabbour RJ, Mansfield CA, Darzi A, Harding SE, Stevens MM, Athanasiou T (2017). Human induced pluripotent stem cell-derived cardiomyocyte encapsulating bioactive hydrogels improve rat heart function post myocardial infarction. Stem cell reports 9. 1415

Acknowledgement

Daniel J Stuckey would like to acknowledge the support of his BHF Fellowship FS/15/33/31608 and MRC Project Grant MR/R026416/1

Fig1: MEMRI and LGE-MRI acquired at 1 and 24 hours post MI

MEMRI or LGE-MRI acquired in the same mouse at 1 & 24h after myocardial infarction. At 1h post MI, Mn uptake in viable myocardium allowed the estimated final infarct area to be distinguished, whilst only subtle enhancement was seen in LGE-MRI resulting in a smaller measure of occlusion zone. At 24h post MI the infarct shows hypoenhancement in MEMRI and hyperenhancement in LGE of similar szes.

Fig2: Direct comparison of MEMRI and LGE-MRI

Fig2: Direct comparison of MEMRI and LGE-MRI acquired in the same animal at 22 and 27 hours after MI, respectively. Arrowhead shows hypoenhancement of the infarct in the MEMRI image and hyperenhancement of the infarct in LGE. In vivo data corresponded with TTC histological staining for infarct.

Keywords: MRI, cardiac, myocardial infarction, contrast, mouse
102

In vivo imaging of human monocytes reveals a distinct spatial infiltration pattern in the early and late inflammatory phase of mouse myocardial infarction (#254)

Janette Iking1, Lisa Honold2, Michael Kuhlmann2, Holger Reinecke1, Lars Stegger3, Johannes Waltenberger1, Michael Schäfers2, 3, Sven Hermann2, Evangelia Pardali1

1 University Hospital Münster, Dept. of Cardiology I – Coronary and Peripheral Vascular Disease, Heart Failure, Münster, North Rhine-Westphalia, Germany
2 University of Münster, European Institute for Molecular Imaging (EIMI), Münster, North Rhine-Westphalia, Germany
3 University Hospital Münster, Dept. of Nuclear Medicine, Münster, North Rhine-Westphalia, Germany

Introduction

Distinct infiltration patterns of human monocytes during the inflammatory and the proliferative phase of infarct healing have been shown post-mortem in human hearts. This could have an effect on the remodelling process and might be altered in patients with cardiovascular diseases, as monocytes isolated from these patients show defects in migration, extravasation, and adhesion. So far, no in vivo data is available concerning the accumulation of human monocytes in the infarcted heart. Here, we used an in vivo imaging approach to track human monocytes in mice with myocardial infarction (MI).

Methods

Human CD14++ CD16- monocytes were labelled with the 99mTc-HMPAO and the fluorescent dye DiD. Immunodeficient NOD/Scid mice underwent surgery for permanent ligation of the left anterior descending (LAD) coronary artery or a control sham surgery. The area at risk of infarction (AAR) was determined by myocardial perfusion SPECT/CT following surgery. On day 1 or 3 post-MI, 10x106 dual-labelled human monocytes were injected into the tail vein of operated NOD/Scid mice. In vivo SPECT/CT scans were acquired the next day approximately 18h post-injection (2 or 4 days post-MI). Subsequently, organs were explanted and further assessed for biodistribution of human monocytes by ex vivo SPECT/CT imaging, fluorescence reflectance imaging (FRI), autoradiography (AR), gamma counter analysis and histology.

Results/Discussion

Dual-labelled human monocytes were detected in vivo in the AAR by SPECT/CT imaging on day 2 and day 4 post-MI. In vivo and ex vivo SPECT/CT scans of the heart revealed that human monocytes mainly accumulated in the border zones of the AAR on day 2 post-MI. In contrast, on day 4 post-MI they accumulated more homogeneously within the infarct area. Similar results were obtained with ex vivo FRI of tissue sections. Quantification of the imaging data displayed significantly higher signals in the AAR of MI-operated hearts compared to remote myocardium and sham-operated hearts on both, day 2 and day 4 post-MI. In addition, linear regression analysis revealed that monocyte infiltration increases with infarct size indicating that infarct size is the main determinant for immune cell infiltration.  

Conclusions

By using a non-invasive in vivo imaging approach we have shown that human inflammatory monocytes accumulate in distinct regions of the infarcted myocardium in different phases of healing following murine MI. These results are in accordance with observations made in ischaemic human hearts post-mortem indicating that the presented imaging model can be a valuable tool to further characterize human immune cells in vivo in the context of MI.

References

Pardali, E., Schmitz, T., Borgscheiper, A., Iking, J., Stegger, L., & Waltenberger, J. (2016). Cryopreservation of primary human monocytes does not negatively affect their functionality or their ability to be labelled with radionuclides: basis for molecular imaging and cell therapy. EJNMMI Research, 6(1), 77.

Acknowledgement

This research was funded by DFG, CRC 656 Münster, project C12 and the DSHF, project F732/13.

SPECT/CT imaging of human CD14++ monocytes in the murine heart.
In vivo (A) & ex vivo (C) SPECT/CT images showing an accumulation of the cells in the border zones of the area at risk of infarction (AAR) on day 2 post-MI (d2). In contrast, on day 4 post-MI (d4), the cells localized homogeneously within the AAR. Quantification of the data (B+D) showing the %ID/ml of the AAR relative to the respective remote myocardium (n=5-9). *p<0.05, **p<0.01 (Kruskal-Wallis).
Keywords: Monocytes, myocardial infarction, in vivo, SPECT, human
103

Ultrasonographic cardiovascular phenotyping in ApoE-/-Fbn1C1039G +/- murine model of vulnerable atherosclerotic plaque (#265)

Sara Gargiulo1, Sandra Albanese1, Chetan Dhakan1, Matteo Gramanzini1, Adele Ferro1, Flavio Cristofani2, Sharmila Fagoonee1, Lorenzo Silengo2, Fiorella Altruda2, Marcello Mancini1

1 National Council of Research, Institute of Biostructure and Bioimaging , Naples, Italy
2 University of Turin, Department of Molecular Biotechnology and Health Sciences, Turin, Italy

Introduction

Noninvasive cardiovascular phenotyping using state-of-the-art ultrasound technology is an high-sensitivity method for evaluating genes of interest and novel theranostic approaches in murine models of atherosclerosis. The ApoE-/-Fbn1C1039G +/- mouse is an emerging model of vulnerable atherosclerotic plaques, but the in vivo alterations of heart and vessels have not yet been described. To the best of our knowledge, this proof-of-concept study provides for the first time a morphological and functional characterization of cardiovascular system in the ApoE-/-Fbn1C1039G +/- mouse by ultrasonography.

Methods

High frequency ultrasound (HFUS) was carried out in ApoE-/-Fbn1C1039G +/- mice (DKO) fed a western-type diet at 16 and 21 weeks of age, and in sex- and age-matched C57Bl/6J control mice (CTRL) fed a standard diet. Vevo 2100-Visualsonics system equipped with a 40 MHz probe and isoflurane anesthesia was used to perform left ventricle (LV) B-mode, M-mode and speckle tracking strain (STS) imaging, as well as B-mode and pulsed wave Doppler scans of aortic arch, abdominal aorta, brachiocephalic and carotid arteries. Cardiac structure, systolic and diastolic function, global and regional myocardial deformation, and arterial elasticity were assessed. Indipendent Student’s test was used to determine statistical significance between DKO and CTRL mice (P <0.05).

Results/Discussion

Aortic arch pulse wave velocity was significantly higher in DKO than CTRL mice at 16 and 21 weeks of age. The velocity time integral of ascending, descending and abdominal aorta, as of brachiocephalic and carotid arteries were significantly decreased in 21 weeks DKO mice. LV mass, cardiac output and stroke volume, normalized to body weight, were significantly increased in 16 weeks DKO mice. Afterwards, normalized LV diameter and volume in systole and Tei index were significantly decreased in 21 weeks DKO mice. Fractional shortening did not showed a significant difference between DKO and CTRL mice at both 16 and 21 weeks of age. However, LV wall global longitudinal strain decreasing trend was observed in DKO mice, according to age, while global radial strain and strain rate resulted significantly lower at 21 weeks. Regional radial strain and strain rate of LV anterior and posterior wall segments as well the circumferential strain rate were significantly decreased in 21 weeks DKO mice.

Conclusions

These results support that DKO mice develop LV hypertrophy, as a compensatory mechanism related to increased arterial stiffness and afterload. Afterwards, diastolic dysfunction and arteries dilation occurr. STS analysis showed more accuracy than conventional echocardiography to capture subtle changes in DKO mice LV function. Advanced HFUS technology is useful for multiparametric cardiovascular mouse characterization in early atherogenesis stages.

References

Van der Donckt C, Van Herck JL, Schrijvers DM, Vanhoutte G, Verhoye M, Blockx I, Van Der Linden A, Bauters D, Lijnen HR, Sluimer JC, Roth L, Van Hove CE, Fransen P, Knaapen MW, Hervent AS, De Keulenaer GW, Bult H, Martinet W, Herman AG, De Meyer GR. Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death. Eur Heart J. 2015;36(17):1049-58.

Van Herck JL, De Meyer GR, Martinet W, Van Hove CE, Foubert K, Theunis MH, Apers S, Bult H, Vrints CJ, Herman AG. Impaired fibrillin-1 function promotes features of plaque instability in apolipoprotein E-deficient mice. Circulation. 2009;120(24):2478-87.

Lee L, Cui JZ, Cua M, Esfandiarei M, Sheng X, Chui WA, Xu MH, Sarunic MV, Beg MF, van Breemen C, Sandor GG, Tibbits GF. Aortic and Cardiac Structure and Function Using High-Resolution Echocardiography and Optical Coherence Tomography in a Mouse Model of Marfan Syndrome. PLoS One. 2016;11(11):e0164778.

Schaefer A, Meyer GP, Hilfiker-Kleiner D, Brand B, Drexler H, Klein G. Evaluation of Tissue Doppler Tei index for global left ventricular function in mice after myocardial infarction: comparison with Pulsed Doppler Tei index. Eur J Echocardiogr. 2005;6(5):367-75.

Stypmann J, Engelen MA, Troatz C, Rothenburger M, Eckardt L, Tiemann K. Echocardiographic assessment of global left ventricular function in mice. Lab Anim. 2009;43(2):127-37.

Figure 1. Representative images on the Vevo 2100 system (Visualsonics, ON, Toronto, Canada)

Evaluation of left ventricular systolic and diastolic function

A) B-mode left ventricle short axis at the level of papillary miscles and tissue doppler imaging trace in corrispondence of posterior wall.

B)  B-mode left ventricle long axis and short axis scans with velocity vectrors and corresponding wall Colour M-mode map for speckle tracking-based strain analysis

Figure 2.High Frequency Ultrasound with Vevo 2100 system (Visualsonics, ON, Toronto, Canada)

Blood Flow Velocity Measurements.

B-mode long axis scan of vessels of interest and corresponding pulse wave doppler trace.

Keywords: mouse model of vulnerable plaque, cardiovascular phenotyping, High frequency ultrasound (HFUS)
104

Multimodal in vivo imaging highlights novel features of cardiovascular abnormalities in a new mouse model for Williams syndrome (#448)

Jeroen Essers1, Nicole van Vliet1, Yanto Ridwan1, Laurens Bosman2, Chris de Zeeuw2

1 Erasmus MC, Molecular Genetics, Rotterdam, Netherlands
2 Erasmus MC, Neuroscience, Rotterdam, Netherlands

Introduction

Williams-Beuren syndrome (WBS) is a developmental disorder that is characterized by distinct facial features, intellectual disability and cardiovascular abnormalities. The underlying etiopathogenesis of this rare disease is due to the de novo microdeletion of a region spanning up to 27 genes, including the elastin gene that is responsible for the elasticity of the arterial wall. Cardiovascular abnormalities encountered in WBS are supravalvular aortic stenosis as well as coronary artery stenosis. Here, we investigated cardiovascular abnormalities in a new WBS mouse model.

Methods

We used ultrasound imaging (vevo3100) to determine aortic dimensions, aortic distensibility and left ventricular ejection fraction (LVEF) in a new mouse model with a 18 gene deletion spanning the WBS region. In order to define biomarkers related to cardiac and vascular alterations, we tested the combined use of XCT-FMT using near infrared fluorescent (NIRF) probes. WBS mice and control mice were imaged in vivo using fast and low dose microCT scanning (QuantumFX, Perkin Elmer) and near infrared (NIRF) probes to monitor tissue remodeling activity (MMPsense680). Immediate contrast enhanced CT imaging using EXIA160 during its blood-pool phase allowed registration of changes in ventricular anatomy and of important global parameters, like end-diastolic volume (EDV) and end-systolic volume (ESV).

Results/Discussion

Extensive ultrasound and microCT analysis showed that aortas of WBS mice exhibited striking tortuosity, an increased length and a smaller internal diameter of the ascending aorta compared to control mice. Functional ultrasound analysis showed that WBS mice had a lower aortic distensibility, and an unexpected but significant increase in LVEF. These anatomical aberrations prompted us to analyze possible differences in remodeling activity in these WBS mice using smart optical probes that report on the activity of matrix metalloproteases; MMPs (MMPsense680, Perkin Elmer). While we detected less MMP activity in the aorta of WBS mice, an increase of MMP activity in the heart region was seen, which appeared to be due to specific activation of the MMPsense probe in the coronaries. Coronary insufficiency in WBS puts the patient at high risk of sudden death due to drop of coronary perfusion pressure which may occur during cardiac catheterization or after induction of anesthesia.

Conclusions

Arterial stenosis is a concern for the majority of patients with WBS. Affected patients require careful observation either with echocardiography or other noninvasive imaging. Molecular, non-invasive evaluation of complex cardiovascular abnormalities of WBS including coronary artery disease is feasible with novel probes and could provide accurate information for planning and noninvasive assessment of arterial changes in WBS patients.

Quantitative in and ex vivo imaging of cardiovascular failure in a WBS mouse model.
(A) In vivo microCT visualization of aorta anatomy in a control and WBS mouse highlighting severe tortuosity and lengthening of the aorta (B) Ex-vivo fluorescence analysis of the aorta and heart using a near-infrared MMP activity imaging (MMPsense680). Note the specific labeling of the coronary artery in the WBS mouse (C) Quantification of the MMPsense signal in heart and aorta
Keywords: Williams-Beuren, cardiovascular, microCT, ultrasound, optical imaging
105

Imaging of hydrogel biomaterials for myocardial regenerative medicine with Magnetization Transfer MRI (#349)

Vitaliy Khlebnikov1, Klaus Neef1, Annette van der Toorn1, Rick Dijkhuizen1, Caroline van Heijningen1, Patricia Dankers2, Carlijn Bouten2, Steven Chamuleau1, Dennis Klomp1, Jeanine J. Prompers1

1 University Medical Center Utrecht, Utrecht, Netherlands
2 Eindhoven University of Technology, Eindhoven, Netherlands

Introduction

The function of myocardial tissue post-infarction may be restored with the use of tissue engineering. An injectable hydrogel based on poly(ethylene glycol) modified with ureido-pyrimidinone (UPy) moieties (Fig.1a) was proposed as a new delivery system for therapeutic molecules and cells.1 UPy-gel is a pH-switchable hydrogel. At pH≥8.5, it is in a liquid state and can be injected through a catheter, whereas at physiological pH it forms a gel. In this study we developed a non-invasive MRI protocol not only capable of imaging hydrogel biomaterials, but also their pH-switchable sol-gel behavior.

Methods

Sample preparation: Liquid (pH≈9) and gelated (pH≈7) were prepared by dissolving UPy-gel in PBS at 3 concentrations (5%, 7.5% and 10% (w/w)).

Ex vivo hearts: UPy10liquid was injected into the myocardial tissue (ventricular wall) of adult rats after isoflurane overdose.

Magnetization transfer (MT) MRI experiments: Z-spectra were acquired by applying a saturation module composed of 100 Gaussian pulses (20ms each) at 137 frequency offsets with varying power levels.

Data analysis: To distinguish two different states of the UPy10 biomaterial, i.e. liquid versus gelated, an artificial neural network (NN) was trained on the Z-spectra from in vitro (Z-spectra for UPy10liquid and UPy10gel) and ex vivo (Z-spectra for the myocardial tissue) experiments.

Results/Discussion

The UPy-gel generated a detectable MT effect in vitro (Fig.1b). The MT effect was higher in the gelated state when compared with the corresponding liquid biomaterial and scaled with the power of the saturation module. A power level as low as 15dB was sufficient to distinguish two different states of the UPy10 biomaterials. For comparison, we also performed imaging with more traditional MRI contrast, i.e. quantitative T1, T2-weighted, and diffusion-weighted MRI, but none of those was able to differentiate between the liquid and gelated states of the biomaterials. To test the pH-switchable behavior of the UPy-gel, two experiments were performed where UPy10liquid (100μL and 50μL, respectively) was injected in the myocardial tissue of a beating rat heart (Fig.2). A gelated rim and a liquid core (likely a result of hindered diffusion) were identified at the injection site of 100μL of UPy10liquid, whereas at the injection site of 50μL of UPy10liquid, only the gelated state was identified.

Conclusions

We developed and validated a MT-MRI protocol for imaging of hydrogel biomaterials and their pH-switchable behavior.

References

1. Bastings, M. M. C. et al. A fast pH-switchable and self-healing supramolecular hydrogel carrier for guided, local catheter injection in the infarcted myocardium. Adv Healthc Mater 3, 70–78 (2014).

Figure 1.
(a) A schematic of bifunctional UPy-PEG-UPy hydrogelator (Mn(PEG) = 10 kDa). (b) In vitro experiments. MT maps as a function of power (x-axis, in dB) of the MT prepulse for different concentrations of biomaterials: UPy5liquid (5% w/w, liquid), UPy5gel (5% w/w, gelated), UPy7.5liquid (7.5% w/w, liquid), UPy7.5gel (7.5% w/w, gelated), UPy10liquid (10% w/w, liquid), UPy10gel (10% w/w, gelated).
Figure 2. Ex vivo experiments.

MRI images showing the injection site of (a) 100µL and (d) 50µL UPy10liquid (10%, w/w) biomaterial in the rat myocardium. (d) is accompanied with a control-liquid biomaterial (Upy10liquid). (b) and (e) Z spectra at a B1 of 24dB averaged over the hydrogel voxels from the injection site for (a) and (d), respectively. (c) and (f) profiles of the predicted states for (a) and (d), respectively.

Keywords: Myocardial tissue engineering, Hydrogel, Magnetic resonance imaging, Magnetization transfer, Myocardial infarction
106

Evaluation of [68Ga]Ga-NODAGA-RGD for PET Imaging of Rat Autoimmune Myocarditis (#346)

Arghavan Jahandideh1, 3, Mia Ståhle1, Xiang-Guo Li1, 6, Heidi Liljenbäck1, 2, Juhani Knuuti1, 3, Anne Roivainen1, 2, 3, Antti Saraste1, 3, 4

1 University of Turku, Turku PET Centre, Turku, Finland
2 University of Turku, Turku Center for Disease Modeling, Turku, Finland
3 Turku University Hospital, Turku PET Centre, Turku, Finland
4 Turku University Hospital and University of Turku, Heart Center, Turku, Finland
5 Åbo Akademi University, Turku PET Centre, Turku, Finland

Introduction

Positron emission tomography (PET) imaging of myocarditis has been challenging due to high physiological uptake of 2-deoxy2-[18F]fluoroglucose tracer in the myocardium1. Therefore, new PET tracers are needed. The αvβ3 integrin receptor is expressed in angiogenic endothelial cells, macrophages and myofibroblasts during myocardial injury and inflammatory response 2,3. Arginyl-glycyl-aspartic acid (RGD) motif containing peptides can detect αvβ3 integrin expression. In this study, [68Ga]Ga-NODAGA-RGD PET for the detection of αvβ3 integrin expression in autoimmune myocarditis in rats was evaluated.

Methods

Rats (n=6) were immunized twice on day 0 and 7 with subcutaneous injection of porcine cardiac myosin in an equal volume of complete Freund’s adjuvant supplemented with mycobacterium tuberculosis and i.p. pertussis toxin injection. Control rats (n=8) were injected with complete Freund’s adjuvant alone. Twenty min static PET imaging at 60-80 min after i.v. [68Ga]Ga-NODAGA-RGD (50 ± 2.7 MBq) injection was performed on day 21 post-immunization followed by autoradiography and histological analysis of excised heart.

Results/Discussion

Focal myocardial inflammatory lesions containing high density of macrophages were detected in 5 of immunized rats. In the immunized rats, PET imaging revealed increased target-to-background ratio (maximum standardized uptake value in myocardium/mean standardized uptake value in blood) compared to control rats (2.3 ± 0.47 vs. 1.2 ± 0.15, respectively; P < 0.05).

Conclusions

[68Ga]Ga-NODAGA-RGD shows accumulation in cardiac inflammatory lesions in a rat model of autoimmune myocarditis. Our result indicates that, as a novel application, [68Ga]Ga-NODAGA-RGD is a potential PET tracer for detection and monitoring of active myocarditis.

References

  1. Grönman et al. J Transl Med (2017) 15:144.
  2. Tang et al. J Clin Nucl Med. (2016) 41(7):e327-39.
  3. Vancraeynest et al. EJNMMI Research (2016) 6:29.

Acknowledgement

The authors would like to thank Aake Honkaniemi and Erica Nyman (Histology Unit of Turku Center for Disease Modeling) for their assistance.

Autoimmune myocarditis in rat
Representative short axis histological sections of the left ventricle stained with hematoxylin and eosin (HE) or anti-CD68 antibody detecting macrophages in a rat with autoimmune myocarditis (A) and a control rat (B). In A, focal lesion close to the epicardium (red arrows) shows dense inflammatory cell infiltrate and myocyte damage at high magnification.
Keywords: Positron emission tomography, Myocarditis, Experimental autoimmune myocarditis
107

In vivo High Frequency Ultrasound detection and analysis of atherosclerotic lesions in ApoE-/-Fbn1C1039G +/- murine model of vulnerable plaque (#281)

Sandra Albanese1, Matteo Gramanzini1, Chetan Dhakan1, Juan C. Cutrin2, Valeria Bitonto2, Flavio Cristofani2, Sharmila Fagoonee1, Lorenzo Silengo2, Fiorella Altruda2, Sara Gargiulo1, Marcello Mancini1

1 National Council of Research, Institute of Biostructure and Bioimaging , Naples, Italy
2 University of Turin, Department of Molecular Biotechnology and Sciences for the Health, Turin, Italy

Introduction

Noninvasive imaging in mouse models of atherosclerosis has become increasingly important in vascular characterization at early disease stages. Recently, double knockout ApoE-/-Fbn1C1039G +/- mouse (DKO), fed a western type diet, was established as relevant model of human-like vulnerable atherosclerotic plaques, but almost all of the studies focus on ex vivo analysis. To the best of our knowledge, this pilot study is the first describing detection and monitoring of atherosclerotic plaques, and local hemodynamics, in living ApoE-/-Fbn1C1039G +/- mice by means of High Frequency Ultrasound (HFUS).

Methods

ApoE-/-Fbn1C1039G +/- mice, feeding 13 weeks of high-fat diet, were examined by HFUS at 16 and 21 weeks of age. Sex- and age-matched C57Bl/6J mice fed a standard diet were used as controls (CTRL). Vevo 2100-Visualsonics system (40 MHz probe) was used to perform aortic arch long-axis view, and lesion-prone sites were examined. Intima-media thickness (IMT) was measured as the distance between the leading edge of the lumen-intima echo and the one of the media-adventitia echo. Lesions were outlined and maximal plaque thickness (MPT) and area (PA) were calculated by leading-to-leading edge approach. Spatio-temporal changes of wall shear stress (WSS) were measured in the ascending and descending aorta. In vivo findings were compared with histology. T-test was used for comparisons (P <0.05).

Results/Discussion

IMT, an established surrogate marker for human atherosclerosis, was significantly increased for aortic arch lesser curvature in DKO from 16 to 21 weeks of age (p=0.04). Moreover, IMT was significantly increased in 21 weeks DKO at the level of aortic arch greater (p = 0.01) and lesser curvature (p = 0.005). Atherosclerotic plaques were visible in DKO from 16 weeks of age at the level of aortic arch lesser curvature and at the origin of the brachiocephalic artery, as more echolucent and elevated areas than surrounding normal wall and corresponding sites in matched CTRL. MPT and PA showed an increasing trend overtime in DKO from 16 to 21 weeks of age. WSS showed a decreasing trend overtime in DKO, probably related to disease progression, reaching a statistical significance at the level of ascending aorta (t-test p = 0.0002) proximally to detected plaques. Histological analysis confirmed the presence of aortic arch atherosclerotic lesions in DKO. No lesions were observed in CTRL mice.

Conclusions

This study supports the feasibility of noninvasively assessing atherosclerotic plaques and hemodynamic state in ApoE-/-Fbn1C1039G +/-  mice in the early atherogenesis using HFUS. By measuring IMT, PMT and PA in the aortic arch, and WSS in the region proximal and distal to lesion sites, HFUS allowed an exploratory monitoring of disease progression in the DKO murine model of vulnerable plaque under investigation.

References

Li RJ, Yang Y, Wang YH, Xie JJ, Song L, Wang Z, Zhang YZ, Qin YW, Li ZA, Zhang XS. Micro-Ultrasonographic Imaging of Atherosclerotic Progression and Correlation with Inflammatory Markers in Apolipoprotein-E Knockout Mice: Tex Heart Inst J 2011;38(4):364-70.

Gan LM, Grönros J, Hägg U, Wikström J, Theodoropoulos C, Friberg P, Fritsche-Danielson R. Non-invasive real-time imaging of atherosclerosis in mice using ultrasound biomicroscopy. Atherosclerosis. 2007 Feb;190(2):313-20. Epub 2006 May 4.

De Wilde D, Trachet B, Van der Donckt C, Vandeghinste B, Descamps B, Vanhove C, De Meyer GR, Segers P.Vulnerable plaque detection and quantification with gold particle-enhanced computed tomography in atherosclerotic mouse models. Mol Imaging. 2015;14.

Van der Veken B, De Meyer GRY, Martinet W. Axitinib attenuates intraplaque angiogenesis, haemorrhages and plaque destabilization in mice. Vascul Pharmacol. 2018 Jan;100:34-40. doi: 10.1016/j.vph.2017.10.004. Epub 2017 Oct 31.

Ding SF, Ni M, Liu XL, Qi LH, Zhang M, Liu CX, Wang Y, Xia LH, Zhang Y. A causal relationship between shear stress and atherosclerotic lesions in apolipoprotein E knockout mice assessed by ultrasound biomicroscopy; Am J Physiol Heart Circ Physiol 298: H2121–H2129, 2010.

Figure 1. Assessment of atherosclerotic plaques in ApoE-/-Fbn1C1039G+/- murine model
B-Mode longitudinal (A) and trasversal (B) scans (Vevo 2100 system, Visualsonics, ON, Toronto, Canada) of aortic arch in DKO mouse, showing an atherosclerotic plaque (arrow) at the level of the lesser curvature, confirmed by Hematoxylin and eosin (C) and Oil Red-O  staining (D) histological sections (x100). Corresponding ultrasonographic (E, F) and histological (G, H) evaluation in healthy CTRL.
Keywords: mouse model of vulnerable plaque, plaque characterization, High Frequency Ultrasound (HFUS)
109

Evaluation of 18F-FTHA as cardiac metabolism tracer for preclinical PET imaging (#574)

Mario González1, Juan Pellico1, Lorena Cussó1, 2, 3, Daniel Calle1, 2, Borja Ibáñez1, Manuel Desco1, 2, 3, Beatriz Salinas1, 2, 3

1 Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
2 Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
3 Departamento de Bioingeniería e Ingeniería Aeroespacial, Universidad Carlos III de Madrid, Leganés, Spain

Introduction

Free fatty acids (FFA) are the primary source of energy in the myocardium. In the development of new radiotracers based on the FFA, 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid (18F-FTHA) has been widely evaluated in pig and clinical studies [1-4]. However, its evaluation in small animals is almost nonexistent, with only a few studies about the use of the tracer for metabolic quantification[5] but without imaging evidences. In this work, we evaluate in vivo pharmacokinetic properties as well as optimal acquisition conditions for the use of this tracer as cardiac imaging agent on mice.

Methods

18F-FTHA was synthetized by nucleophilic substitution of the tosyl commercial precursor (90ºC, 8min) and further C-18 sep-pack purification [6]. In vitro stability in PBS (37ºC) of the tracer was measured by HPLC each hour during four hours. Pharmacokinetic evaluation of the radioactive fatty acid was carried out in healthy C57BL/6 mice by ex vivo biodistribution (BD) studies (5min, 20 min and 60 min post injection) and blood half-life analysis by blood extraction during 180 min. Plasma Protein Binding (PPB) was assessed ex vivo on mouse blood sample (0.77 MBq, 0.5 mL, 30 min incubation). Finally, in vivo PET/CT imaging was obtained on C57BL/6 mice (n=4), evaluating different acquisition conditions (uptake times: 5,10,15,20 and 60 min) and fasting/non-fasting conditions.

Results/Discussion

18F-FTHA was successfully synthetized with a 10% radio-chemical yield, as in previous references [6]. In vitro stability evaluation showed a 7.7% degradation of the tracer after 2h, increasing to 15.2% after 4h. Circulation profile confirmed fast clearance of the tracer, with blood half-life values lower than 5 min. PPB evaluation shown strong affinity between the radioactive fatty acid and plasma proteins, reaching values of 91.92% protein binding. Ex vivo BD studies showed uptake mainly in myocardium and liver, with highest values at 5 min (20.4±3.6% and ID/g31.6±4.8% respectively). Significant decrease in heart uptake is observed at longer time points (60 min= 12.61±7.20%). These results were confirmed by PET imaging, showing in vivo high and specific myocardium uptake as well as hepatic metabolism. Optimal uptake was observed 5 min post injection. In the preliminary evaluation of fasting effect, no significant differences were observed between fasting and non-fasting mice.

Conclusions

This is the first time the radiotracer 18F-FTHA is presented as cardiac metabolism imaging agent for PET on rodents. We successfully optimized acquisition parameter and performed a complete physical-chemical characterization of the radiotracer proving its promising use as PET tracer for cardiac imaging on mice.

References

[1] MÄKI, Maija T., et al. Free fatty acid uptake in the myocardium and skeletal muscle using fluorine-18-fluoro-6-thia-heptadecanoic acid. Journal of Nuclear Medicine, 1998, vol. 39, no 8, p. 1320-1327.

[2] KARMI, Anna, et al. Increased brain fatty acid uptake in metabolic syndrome. Diabetes, 2010.

[3] TAYLOR, Michael, et al. An evaluation of myocardial fatty acid and glucose uptake using PET with [18F] fluoro-6-thia-heptadecanoic acid and [18F] FDG in patients with congestive heart failure. Journal of Nuclear Medicine, 2001, vol. 42, no 1, p. 55-62.

[4] TAKALA, Teemu, et al. 14 (R, S)-[18 F] Fluoro-6-thia-heptadecanoic acid as a tracer of free fatty acid uptake and oxidation in myocardium and skeletal muscle. European journal of nuclear medicine and molecular imaging, 2002, vol. 29, no 12, p. 1617-1622.

[5] DEGRADO, Timothy R.; COENEN, Heinz H.; STOCKLIN, G. 14 (R, S)-[18F] fluoro-6-thia-heptadecanoic acid (FTHA): evaluation in mouse of a new probe of myocardial utilization of long chain fatty acids. Journal of nuclear medicine: official publication, Society of Nuclear Medicine, 1991, vol. 32, no 10, p. 1888-1896.

[6] DEGRADO, Timothy R. Synthesis of 14 (R, S)‐[18F] fluoro‐6‐thia‐heptadecanoic acid (FTHA). Journal of Labelled Compounds and Radiopharmaceuticals, 1991, vol. 29, no 9, p. 989-995.

Acknowledgement

This work has been supported by the Advanced Imaging Unit (UIA) of Centro Nacional de Investigaciones Cardiovasculares (CNIC), CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505) and Small Animal Image Core at Hospital General Gregorio Marañón (HGGM), Madrid, Spain. This work was partially supported by Comunidad de Madrid (S2017/BMD-3867 RENIM-CM, co-financed by European Structural and Investment Funds), and Ministry of Science, Innovation and Universities (ISCIII-FIS grants PI16/02037, co-financed by ERDF, FEDER, Funds from the European Commission, “A way of making Europe”)

Figure 1. Physicochemical characterization and pharmacokinetic evaluation of 18F-FTHA.

A) Synthesis of 18F-FTHA. B) HPLC chromatogram of purified 18F-FTHA. C) In vitro stability evaluation of the tracer in PBS by HPLC. D) Circulation blood half-life profile of 18F-FTHA in healthy mice. E) Ex vivo biodistribution studies at 5 min, 20 min and 60 min post injection.

Figure 2. In vivo PET/CT imaging of 18F-FTHA.

A) Axial PET/ CT imaging at 5 min, 10 min, 15 min and 20 min postinjection of 18F-FTHA (150-200 mCi) in healthy mice. B) Sagital PET/CT imaging of the tracer 5 mi postinjection.

Keywords: PET, Fatty acid, Tracer, Cardiac metabolism
110

Murine Atherosclerosis Characterization Using Lipid-Specific Photoacoustic Imaging and 4D Ultrasound Strain Mapping (#582)

Gurneet S. Sangha1, Katherine Leyba1, Craig J. Goergen1

1 Purdue University, Biomedical Engineering, West Lafayette, Indiana, United States of America

Introduction

Photoacoustic tomography (PAT) and 4D ultrasound (4DUS) imaging have recently been used to study cardiovascular disease in small animals [1-3]. PAT uses pulsed laser light induced acoustic waves to reconstruct lipid-specific compositional images. 4DUS captures dynamic volumetric information and can be used to estimate 3D Green-Lagrange strain using a direct deformation estimation method [4]. We aim to use a combined PAT/4DUS approach to correlate changes in arterial strain and hemodynamics with lipid localization in mice that have undergone partial carotid ligation induced-atherosclerosis.

Methods

A 40 MHz small animal transducer (Vevo2100, VisualSonics) and a Nd:YAG pulsed laser (Surelite EX, Continuum) were used to image five apolipoprotein-E deficient male mice that underwent partial carotid ligation of the left carotid artery while being fed a Western diet [5]. Animals were imaged using ultrasound at days 0, 1, 4, 7, 10, and 14 to obtain long-axis B-mode, M-mode, and pulsed-wave Doppler for morphological and hemodynamic characterization, as well as imaged via 4DUS for strain mapping. At day 14 all animals were euthanized and 3D in situ PAT images of the left carotid artery were acquired using 1210nm light.

Results/Discussion

Overall the results show that atherosclerotic lesions can be characterized via PAT/4DUS to localize both lipid accumulation and identify regions of low strain. We observed rapid decrease in left carotid artery peak velocity between baseline and day 1 imaging (64±3.8 cm/s to 18.6±7.2 cm/s). Additionally, baseline and day 5 imaging showed a steady decrease in the left carotid artery circumferential cyclic strain (31±6.2% to 11±2.5%) and lumen diameter (0.48±0.03 mm to 0.32±0.05 mm), as well as an increase in right carotid artery diameter (0.48±0.04 mm to 0.56±0.03 mm). Strain mapping from 4DUS images showed heterogenous maximum first principal strain values in both the left (Figure 1) and right carotid artery. The 3D in situ PAT imaging revealed lipid signal from atherosclerotic plaque accumulation in the left carotid artery with no signs of lipid accumulation in the contralateral control right carotid artery (Figure 2).

Conclusions

Dual-modality PAT/4DUS can be used to quantify changes in carotid artery hemodynamics and morphology, as well as lipid localization in a murine model of atherosclerosis. Future work will focus on utilizing this technique for cross-sectional studies to better understand how vessel hemodynamics and mechanics impact changes in atherosclerotic plaque composition.

References

[1] Sangha, Gurneet S., Evan H. Phillips, and Craig J. Goergen. "In vivo photoacoustic lipid imaging in mice using the second near-infrared window." Biomedical optics express 8.2 (2017): 736-742.

[2] Soepriatna, Arvin H., et al. "Cardiac and respiratory-gated volumetric murine ultrasound." The international journal of cardiovascular imaging (2018): 1-12.

[3] Damen, Frederick W., et al. "High-Frequency 4-Dimensional Ultrasound (4DUS): A Reliable Method for Assessing Murine Cardiac Function." Tomography: a journal for imaging research 3.4 (2017): 180-187.

[4] Cebull, Hannah., “Strain mapping from 4D ultrasound reveals complex remodeling in dissecting murine abdominal aortic aneurysms.” Journal of Biomechanical Engineering (2018). In Review.

[5] Nam, Douglas, et al. "Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis." American Journal of Physiology-Heart and Circulatory Physiology 297.4 (2009): H1535.

Acknowledgement

Funding was provided to Gurneet S. Sangha through the NSF Graduate Research Fellowship (DGE-1333468)

Figure 1:
3D maximum first principal strain of the left carotid artery (red dotted line) at day 14. Regions of high (red arrow) and low (green arrow) strain can be visualized near the aortic arch and carotid bifurcation, respectively.
Figure 2
Overlaid in situ PAT and ultrasound image of the left carotid artery (red dotted line). Lipid signal showed heterogenous plaque formation with regions of minimal (red arrow), mild (yellow arrow), and severe (green arrow) lipid accumulation.
Keywords: photoacoustic, atherosclerosis, murine, ultrasound, imaging
111

Single chain-antibody variable fragments for the molecular imaging of oxidized LDL in atherosclerosis   (#111)

Samata S. Pandey1, 2, Michael Mullin3, Laura Vazquez-Martinez1, Cleo Kontoravdi2, Dorian Haskard1, Ramzi Khamis1

1 National Heart and Lung Institute, Imperial College London, London, United Kingdom
2 Centre of Process Systems Engineering, Imperial College London, London, United Kingdom
3 Protein & Cell Sciences, GlaxoSmithKline , Stevenage, United Kingdom

Introduction

Atherosclerosis is the underlying pathophysiology of the world’s leading cause of death, ischemic heart disease. The high specificity of monoclonal antibodies (mAbs) makes them promising imaging agents for the molecular imaging of atherosclerosis. However, the high molecular weight of full mAbs hinders their ability to be effectively used as biological imaging agents, due to lower tissue penetrance and slower kinetics. Furthermore, many mAbs are hybridoma-derived and thus pose an immunological risk due to their murine origins.

Methods

LO1, a monoclonal autoantibody with specificity for malondialdehyde-modified low-density lipoprotein (MDA-LDL) was isolated from a non-immunized LDL receptor deficient mouse (LDLR-/-). Characterization by ELISA, immunohistochemistry on human and mouse atherosclerotic tissue and in vivo imaging using a Near-Infrared Fluorescence (NIRF) fluorophore demonstrated binding to atherosclerotic lesions. In a translational effort, antibody fragments of LO1 were generated. LO1 single-chain variable fragments (scFv) and LO1-humanized Fab (huFab) were expressed in mammalian cells. The humanized Fab was generated using CDR grafting and backmutations. Cys-tags were included on both constructs for labelling of the construct with a NIRF fluorophore using maleimide chemistry.

Results/Discussion

ScFv and Fabs are a fifth and third of the size of a mAb, respectively, whilst both retaining a full antigen binding site. In vitro characterization of LO1-scFv and LO1-huFab by ELISA and immunohistochemistry demonstrated retention of function and binding of LO1-scFv and LO1-huFab to atherosclerotic lesions and thrombus in mouse tissue sections (Figure 1). Control constructs demonstrated lysines in CDR-H3 to be crucial for binding to MDA-LDL. Humanization of the Fab allows for repeated dosing due to the lower threat of immunogenicity.

Conclusions

In this study, we demonstrated that antibody fragments of LO1 retain function to antigen and thereby have the potential to be used as molecular imaging tools and for future therapeutic targeting of oxidized LDL. Ongoing further work involves in vivo testing of constructs in atherosclerotic LDLR-/- mice for lesional staining.

Figure 1: LO1 antibody fragments staining LDLR-/- mice fed a high fat diet.
Keywords: single-chain variable fragment, atherosclerosis, humanization, molecular imaging

Data Processing & Quantification

Session chair: Adriana Tavares (Edinburgh, UK); Athanasios Zacharopolous (Heraklion, Greece)
 
Shortcut: PW10
Date: Thursday, 21 March, 2019, 12:45 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.

201

Fully Automated Radiomic Analysis for Tumor Detection and Classification in Ultrasound Images (#415)

Zuzanna A. Magnuska1, Tatjana Opacic1, Severine Iborra2, Elmar Stickeler2, Fabian Kiessling1, 3, Benjamin Theek1, 3

1 University Clinic Aachen, RWTH Aachen University, Institute for Experimental Molecular Imaging, Aachen, North Rhine-Westphalia, Germany
2 University Clinic Aachen, RWTH Aachen University, Department of Obstetrics and Gynecology, Aachen, North Rhine-Westphalia, Germany
3 Fraunhofer MEVIS, Bremen, Bremen, Germany

Introduction

The goal of radiomics is to extract quantitative features from radiological images and correlate them with clinical findings. This might improve precision medicine by developing algorithms supporting physicians in diagnosis and therapy. Thus far, only a few radiomic studies on ultrasound (US) images have been published [1,2]. Therefore, we created an user-independent workflow to conduct a radiomic analysis of US images and assessed its capability to automatically differentiate xenograft tumor models. Moreover, the developed algorithms were adapted to analyze clinical breast cancer US images.

Methods

US B-mode images of 3 different xenograft tumor models (lung cancer (n=3), ovarian cancer (n=3), SCC (n=3)) and clinical breast cancer types (fibroadenoma (n=6), HER2-positive (n=2), TNB (n=7)) were retrospectively analyzed. Cascade classifiers (CCs) were trained for an automated tumor detection (Fig. 1A) [3]. The tumor segmentation was based on an active contour model and morphological operations. From the segmented region, a total of 230 intensity-based, textural and wavelet-based features were mined (Fig. 1C). Feature stability, discriminative power and user-independence were evaluated to identify 3 non-correlating traits, composing the radiomic signature (RS). Using the RS as input, a k-NN based classification algorithm was evaluated with the leaving-one-out-cross-validation scheme.

Results/Discussion

The developed algorithm for the automated detection and segmentation of tumors achieved a high detection accuracy (89% of correct tumor detections), and automated segmentations overlapped with the manual segmentations (81% ± 24% overlap) (Fig. 1B). The developed imaging biomarker extraction and selection algorithm identified the following 3 non-correlating features as the RS: median (intensity-based feature), correlation (textural feature) and short run emphasis (wavelet-based feature). The tumor classification model assigned 80% of the analyzed images to the correct tumor type (p=0.8 [95% CI 0.6-0.9]) (Fig. 1C). The application of the established radiomic analysis on breast cancer US images resulted, thus far, in a similar detection accuracy (87% of correct tumor detections) (Fig. 2).

Conclusions

Our results show that a radiomic analysis of US images can be performed to classify tumors. Initial results on clinical data suggest that the established concept can be translated to the clinical setting as well. The developed automated workflow may assist clinicians in a more robust and reliable tumor recognition, characterisation and differentiation. More extensive evaluation of clinical data will be performed in future studies.

References

[1] Lee, S.E., et al., Scientific Reports 2018. 8(1): p. 13546.

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

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

Acknowledgement

This work has been supported by the German Research Foundation (KI1072/11-1).

 

The retrospective study of clinical breast cancer US images was approved by the ethics comity of RWTH Aachen University, Germany.

Fully Automated Radiomic Analysis of Experimental Tumor Models
Automated Tumor Detection in Clinical Breast Cancer US Images
Keywords: biomedical image processing, data analysis, texture analysis, automated segmentation, radiomics
202

Developing a customizable workflow engine for storing, sharing, processing and reusing medical images for preclinical imaging facilities (#140)

Sara Zullino1, Alessandro Paglialonga1, Walter Dastrù1, Alessandra Viale1, Silvio Aime1, Dario L. Longo1

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

Introduction

Medical imaging data is a highly valuable resource for research on diagnostics, epidemiology and drug development. However, a common limitation relies on the lack of common tools for sharing, processing and reusing medical imaging data acquired with different instrumentation among several imaging centers. Our aim is to overcome this urgent need through the integration of an open-source archiving platform with customizable tools for automated image processing. This workflow will allow the preclinical research community to store, process and reuse a large number of medical imaging data.

Methods

We have developed Python/Matlab-based tools for exporting, processing and archiving preclinical images exploiting the built-in Pydicom library [1]. These tools interface with the remotely accessible database XNAT [2], a widely used open source platform for managing, sharing and processing medical imaging data, via XNAT Python clients, xnatpy and Pyxnat [3, 4]. Since preclinical instrumentation adopts a proprietary format for their data, these tools can convert raw images to DICOM format from different vendors (MRI Bruker and Aspect Imaging) or can directly import DICOM images for several imaging modalities (PET/SPECT/CT/OI/OA). Dedicated tools have been implemented for automatically storing whole experiment datasets including annotations for experimental groups and timepoints.

Results/Discussion

The workflow is based on the following steps (Fig.1):

  1. a retrieving/archiving step to collect multiple imaging datasets acquired through several modalities;
  2. a Bruker/Aspect to DICOM format converter to import images to XNAT;
  3. an image processing step accepting as input either DICOM (3a) or raw data (3b) to produce parametric images related to biological aspects.

The workflow can decipher preclinical MRI raw data and convert proprietary images to DICOM, storing the parameters in both standard and private DICOM tags. To match our data organization, a XNAT feature to extend an existing data type using custom variables has been used. XNAT pipelines have been implemented to import large datasets arising from several studies/patients/sessions and to process MR images. XNAT can support the storage of several files at the resource level, such as binary masks, parametric images, report files and annotated information (Fig. 2). 

Conclusions

A user-friendly, customizable workflow has been developed to store and process image datasets from preclinical imaging centers using a XNAT-based archive system. This approach complies with the FAIR (Findable, Accessible, Interoperable and Reusable) guidelines. This engine will be available for the preclinical research community, allowing to access and share image data among preclinical imaging centers.         

References

[1]         D. Mason, “Pydicom: An Open Source DICOM Library,” in Medical Physics, 2011, vol. 38, no. 6, p. 3493.

[2]         D. S. Marcus, T. R. Olsen, M. Ramaratnam, and R. L. Buckner, “The extensible neuroimaging archive toolkit: An informatics platform for managing, exploring, and sharing neuroimaging data,” Neuroinformatics, vol. 5, no. 1, pp. 11–33, 2007.

[3]         Hakim Achterberg, “XNAT Python Client,” https://xnat.readthedocs.io/en/latest/

[4]         Y. Schwartz et al., “PyXNAT: XNAT in Python,” Front. Neuroinform., vol. 6, p. 12, May 2012.

Acknowledgement

European Union’s Horizon 2020 research and innovation programme under grant agreement No 654248 (CORBEL project), grant No 667510 (GLINT project) and from Compagnia San Paolo project (Regione Piemonte, grant #CSTO165925).

Figure 1
Schematic workflow for image archiving and processing.
Figure 2
Snapshots of the image session webpage on the XNAT platform: a) custom variables with subject information; b) pipeline launcher; c) resource folder containing parametric images, report files and annotated information.
Keywords: image processing, preclinical imaging, XNAT, MR imaging, DICOM
203

Standardization and traceability of FDG PET imaging may improve statistical analysis (#470)

Thulaciga Yoganathan1, 2, Amel Raboudi2, 3, 4, Lucile Offredo1, 2, Thomas Viel1, 2, Bertrand Tavitian1, 2

1 Université Paris Descartes, Sorbonne Paris Cité, PARIS , France
2 INSERM U970, Paris-Cardiovascular Research Center at HEGP, PARIS , France
3 FEALINX, Courbevoie, France
4 Université de Technologie de Compiègne (UTC), UMR 7337 Roberval, Compiègne, France

Introduction

Standardization of animal handling, data acquisition and image reconstruction is essential for the ethical use of animals in research and impacts quantification, accuracy and reproducibility of small animal 18F-FDG Positron Emission Tomography (FDG PET) (1,2,3). In theory, using standardized PET acquisitions and image analysis, FDG uptake in healthy animals could be reused for multiple studies that include a similar control group. The present study aimed at testing whether data traceability and standardization can indeed help to minimize the number of mice in PET imaging.

Methods

A group of n=7 (TTT group) sunitinib-treated (50 mg/kg p.d. during 2 weeks) C57BL/6 male mice were compared to (i) an untreated control group (CON, n=9) from the same study and (ii) an untreated reference group (REF, n=52) of mice from our in-house database. Non-fasted mice were anesthetized with isoflurane, injected intravenously with FDG and imaged on the NanoScan PET-CT (Mediso, Hungary). Body weight, glycaemia, isoflurane and respiration were monitored. PET scans were reconstructed using customized 3D-OSEM and quantified using PMOD (Switzerland) by the same examiner. Mean comparison tests were performed between all groups using the Student test for groups of equal variances according to the Fisher test or the Wilcoxon-Mann-Whitney non parametric test at a 5% significance level.

Results/Discussion

Two weeks of sunitinib treatment increased FDG uptake in heart and muscle by 8% and 19% respectively in the TTT vs. CON group. However, the difference did not differ significantly between the 2 groups in the heart (SUVmean 6.33±0.96 vs. 5.85±1.51; p=0.4609) nor in the muscle (0.41±0.07 vs. 0.34±0.09; p=0.1059). In contrast, comparing the TTT and the REF groups demonstrated a significant difference in FDG uptake in the heart (p=0.0469) as well as in the muscle (p=0.0032). Access to a standardized PET database allows to evidence statistically significant differences in cardiac and muscle uptake induced by sunitinib treatment that we had failed to evidence in the original study because of the small number of mice used. Based on these encouraging results, we have implemented our PET-FDG database and the research workflow of standardized PET acquisitions, reconstructions and analyses, in a data management technology using the BMI-LM data model (4) in order to continuously feed the database.

Conclusions

The 52 PET exams of our current database are continuously fed and will now be extended to other acquisition conditions (e.g. fasting, anesthesia, sex, strain…). In the long term, such databases are expected to improve the observance of the 3R principle and allow power calculations in order to estimate the sample size needed to obtain a statistically significant result.

References

  1. Mannheim, J. G., Kara, F., Doorduin, J., Fuchs, K., Reischl, G., Liang, S., ... & Huisman, M. C. (2017). Standardization of Small Animal Imaging—Current Status and Future Prospects. Molecular Imaging and Biology, 1-16.
  2. Vanhove, C., Bankstahl, J. P., Krämer, S. D., Visser, E., Belcari, N., & Vandenberghe, S. (2015). Accurate molecular imaging of small animals taking into account animal models, handling, anaesthesia, quality control and imaging system performance. EJNMMI physics, 2(1), 31.
  3. Nanni, C., Rubello, D., & Fanti, S. (2007). Role of small animal PET for molecular imaging in pre-clinical studies.
  4. Allanic, M., Hervé, P. Y., Pham, C. C., Lekkal, M., Durupt, A., Brial, T., ... & Joliot, M. (2017). BIoMIST: A platform for Biomedical Data lifecycle Management of neuroimaging cohorts. Frontiers in ICT, 3, 35.

Acknowledgement

In vivo imaging was performed at the Life Imaging Facility of Paris Descartes University (Plateforme Imageries du Vivant - PIV), supported by France Life Imaging (grant ANR-11-INBS-0006), Infrastructures Biologie-Santé (IBISA), CARPEM Siric grant and Plan Cancer (ASC16026HSA-C16026HS).

Improved statistical significance using a standardized reference database

(A) Glycaemia, Body weight and injected dose in REF, CON and TTT groups: no difference by one-way ANOVA test (glycaemia and injected dose) and nonparametric Kruskal-Wallis test (body weight).

(B) FDG uptake was significantly increased in TTT heart and muscle using the REF but not the CON group for comparison. Heart *: p=0.047; £: p=0.223; $: p=0.461 Muscle **: p=0.007; ££: p=0.311; $$: p=0.207

 

 

Keywords: FDG PET imaging, Standardization, Traceability, Statistical analysis
204

Integrating PET-CT imaging research workflow with end-to-end traceability for small animal research (#337)

Amel Raboudi1, 2, 3, Thulaciga Yoganathan1, Thomas Viel1, Marianne Allanic2, Daniel Balvay1, Bertrand Tavitian1

1 INSERM, UMR970, Paris-Cardiovascular Research Center at HEGP, Paris, France
2 FEALINX, Courbevoie, France
3 Université de Technologie de Compiègne (UTC), UMR 7337 Roberval, Compiègne, France

Introduction

Poor annotation of heterogeneous data is a barrier to its reuse. The data model of Biomedical Imaging-Lifecycle Management (BMI-LM) platform enables data reuse. It provides provenance and domain concepts, as shown previously in neuroimaging studies [1]. Here, we assume that BMI-LM data model could be applied to PET-CT imaging research lifecycle and we propose a BMI-LM model-driven integration of PET-CT research workflow. Our data of interest are PET images in DICOM standard format, acquired since 2015 on a nanoScan PET-CT (Mediso, Hungary) during different small animal imaging studies.

Methods

BMI-LM is applied to COS-TEP: a retrospective research workflow using preclinical PET images. COS-TEP uses data from several research studies acquired in many configurations by different operators. The proposed PET-CT research workflow integration method covers (i) study specification (ii) raw data acquisition (iii) derived data analysis and (iv) publication (fig.1). First, BMI-LM provenance concepts are used to predefine and describe the final data. Then, acquired and reconstructed DICOM data integration is performed on-demand and accordingly PMOD analysis results are integrated [2]. Finally, BMI-LM database export to Excel is used for statistical analysis. The resulting integrated research workflow is based on a systematic end-to-end traceability using BMI-LM concepts.

Results/Discussion

The BMI-LM data model originally proposed for neuroimaging studies can be applied to PET-CT imaging research workflow provided that the following adaptations are made: (i) three new concepts are required, besides the BMI-LM existent ones[1], in order to manage PET-CT imaging data heterogeneity: Agent, Intervention and Sample. Agent includes description radiotracer and products used during the acquisition. Intervention designates the pharmacological treatment, the animal model preparation,..etc. And Sample describes the use of animal derived materials for in vivo and ex-vivo studies. (ii) For fluent adoption of the proposed method by imaging researchers, it is necessary to adapt the DICOM data integration tool to the local vocabulary of a PET preclinical imaging laboratory. Therefore, alignments between BMI-LM concepts, local vocabulary terms and DICOM standard were established and a Matlab GUI tool was provided. This assures more flexibility while maintaining DICOM standard compliance.

Conclusions

The ongoing integration of preclinical PET-CT imaging research workflow allows its continuous use for newly acquired data. Here, we have pointed some specific requirements of this integration. In future work, we intend to simplify it for PET-CT researchers, especially the study specification step bottleneck, using a customized Data Management Plan (DMP). This will allow scientists to reuse their own research workflow once it has been established.

References

1. Allanic, M. et al.: BIOMIST: A Platform for Biomedical Data Lifecycle Management of Neuroimaging Cohorts. Frontiers in ICT. 3, (2017).

2. Raboudi, A. et al.: Traçabilité de l’intégration de données biomédicales hétérogènes dans le système SWOMed de gestion du cycle de vie des études biomédicales. In: actes du symposium SIIM 2017. , Toulouse (2017).

Acknowledgement

This work is supported by the DRIVE-SPC collaboration project between Fealinx Company and LRI-PARCC-Inserm with a grant from IDEX Université Paris Sorbonne Cité. Amel Raboudi has an ANRT PhD scholarship. Authors want to thank PIV platform, Dr. Pierre-Yves Hervé, Dr. Philippe Boutinaud, Ing.Thierry Brial, Ing. Arthur Grioche, Ing. Olivier Menuel and Ing. Jérôme Cornet for their time and help.

The BMI-LM platform showing in flows and out flows with corresponding interfaces and concepts
This figure presents the proposed model-driven workflow integration for COS-TEP research workflow. Data integration tool was previously presented in [2] (in french). Presenting each BMI-LM concept relevant to COS-TEP adds complexity to the figure, that's why, groups of concepts are present instead. Please refer to [1] for detailed information about BMI-LM data model.
Keywords: Data provenance, research workflow, small animal research, PET-CT imaging, model-based workflow
205

Dispersion Correction for Sampled Blood Input function in Rats (#589)

Marie-Claude Asselin1, Daniela Bochicchio1, Rainer Hinz1, Hervé Boutin2

1 University of Manchester, Division of Informatics, Imaging & Data Sciences, Manchester, United Kingdom
2 University of Manchester, Division of Neuroscience & Experimental Psychology, Manchester, United Kingdom

Introduction

Tracer kinetic modeling of dynamic PET images requires an input function. Preclinically, blood-derived input functions suffer from severe dispersion in the narrow bore tubing connecting the animal to the blood sampler. Dispersion depends on the sampling rate, tubing material, inner diameter and length to and within the detector, and the dispersion correction must be derived for each experimental set-up and radiotracer. The aim of the study was to assess the accuracy of the dispersion correction proposed by Munk et al [1] as implemented in PMOD for use with the Twilite detector in rodents [2].

Methods

Solutions of approx. 1.2 MBq/mL were prepared by adding [F-18]FDG to water or blood. The dispersion function of PE tubing of 0.58-mm inner diameter and varying lengths (30, 45 or 60 cm) was measured by transferring the tubing from a beaker containing the non-radioactive solution to another beaker with the radioactive solution and back, creating a step function. Another 15 or 30 cm of tubing was winded in the F-18 or C-11 guide inserted into the Twilite detector. At the recommended sampling rate of 0.350 mL/min, the transit time in the detector is 6.8 s in the F-18 or 13.6 s in the C-11 guide. The sampling rate was also halved and doubled. The transmission-dispersion model [1], which does not account for the transit time in the detector, was used to fit the rising part of the step function

Results/Discussion

As shown in Fig. 1A, doubling and halving the sampling rate increased and decreased the slope of the rising step function. At the recommended sampling rate, the slope became shallower by lengthening the tubing to the detector (Fig. 1B) or inside the detector by replacing the F-18 guide with the C-11 guide (Fig. 1C). Substituting water with blood also led to shallower slope, to a similar extent an extra 15-cm of tubing (Fig. 1D). The Munk model did not fit the Twilite data because it was unable to follow the shape of the rising step function. The fitted parameters (alpha and kappa) were highly dependent on the initial values, particularly the transit time to the detector. For comparison, Munk et al [1] validated their model in pigs using the Allogg blood sampler operated at 7 mL/min and connected to PVC tubing of 1.65-mm inner diameter and 38-cm in length plus 5-cm inside the detector, corresponding to a transit time of only 0.9 sec.

Conclusions

By winding the tubing into the guide inserted in the Twilite detector in order to increase detection sensitivity, the Twilite detector acts as an integrator of the radioactive solution slowly transiting through it. The Munk model, which assumes instantaneous measurement of radioactivity, fails to fit the data and its parameters cannot be used to accurately correct the sampled blood input function for dispersion.

References

[1] Munk O.L. et al (2008) Med Phys 35(8): 3471-81.

[2] Alf M.F. et al (2013) J Nucl Med 54(1): 132-38.

Acknowledgement

To the EPRSC for contributing to the purchase of the Twilite blood sampler and to GSK for funding the PhD studentship of D. Bochicchio.

Figure 1
Effects of changing A) sampling rate, B) tubing length to the detector, C) guide and D) fluid (from water to blood) on the step function characterising the dispersion in the PE tubing connected to the Twilite detector.
Keywords: Blood input function, dispersion correction, tracer kinetic modeling
206

Low rank approximation as an alternative to accumulation for noise reduction in hyperpolarized 13C spectroscopy:  preliminary results for synthetic, phantom and in vivo spectra. (#560)

Roberto Francischello1, 2, Alessandra Flori3, 4, Luca Menichetti2

1 University of Pisa, Department of chemistry and industrial chemistry, Pisa, Italy
2 CNR, Clinical Physiology Institute, Pisa, Italy
3 Fondazione CNR/Toscana Gabriele Monasterio, Bioengineering and clinical engineering, Pisa, Italy
4 Scuola Superiore Sant'Anna, Institute of Life Sciences, Pisa, Italy

Introduction

Dissolution dynamic nuclear polarization allows in-vivo studies of metabolic flux using hyperpolarized 13C tracer by enhancing signal intensity.

To reduce the noise intensity and further enhance the signal-to-noise ratio Brender, et all. used low rank approximation on the real part of phase corrected or magnitude spectra for hyperpolarized 1-13C-pyruvate (13C-Pyr) [1,2].

Since the effect of the phase manipulation could alter the quantification of metabolites in these studies, we adapted the method of low rank approximation to be applied to complex value signal.

Methods

Let A be the NxM signal matrix which rows are made by the N acquired signal of n metabolites with n<N. Since the presence of additive noise makes A full rank it is possible to reduce the noise by searching for a low rank approximation of A.

The truncation of the last N-k singular value, obtained using singular value decomposition, gives the best approximation in Frobenius norm with rank k for A. The choice of k is related to the a-priori knowledge on the number of metabolites and to the variation of singular value.

We tested this method on simulated data and applied to panthom and in-vivo dataset. The phantom was made after dissolution of hyperpolarized 13C-Pyr in a standard volume. The animal model of rodent was injected with hyperpolarized 13C butyrate as previously reported [3].

Results/Discussion

To estimate the noise intensity, we take the standard deviation of the last points of the time-domain signal.

As shown in fig.1 the presented method is able to reduce the noise intensity in all the dataset, allowing the retrieval of signal even in extremely noisy data without the needing of phase pre-processing.

For the in-vivo sample the presence of a peak at ≈2 ppm was known from previews analysis, Gaussian apodization and sum of magnitude spectra [3]. Using low rank reduction was possible to observe a peak in the same spectral region, while retaining temporal resolution and spectral resolution thanks to a lower level of Gaussian apoditazion fig.2.

Conclusions

Our preliminary result shows that low rank approximation by SVD on complex signal matrix could be considered a valid alternative to accumulation of spectra for enhancing signal to noise ratio preserving temporal resolution in metabolic flux spectroscopy without the need of previous phase manipulation.

References

[1] KISHIMOTO, Shun, et al. Distinguishing Closely Related Pancreatic Cancer Subtypes In Vivo by 13C Glucose MRI without Hyperpolarization. BioRxiv, 2019, 511543

[2] BRENDER, Jeffrey R., et al. PET by MRI: Glucose Imaging by 13C-MRS without Dynamic Nuclear Polarization by Noise Suppression through Tensor Decomposition Rank Reduction. bioRxiv, 2018, 265793.

[3] FLORI, Alessandra, et al. Biomolecular imaging of 13C-butyrate with dissolution-DNP: Polarization enhancement and formulation for in vivo studies. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2018, 199: 153-160.

Acknowledgement

The author would like to thank the Fondazione CNR/Regione Toscana ‘G. Monasterio’ (Pisa, Italy) for funding this study.

Fig.1: Effect of denoise alghorithem on different dataset

Panel A): comparison between the spectra before and after the noise reduction for the 3 dataset. Panel B): comparison between the noise intensity before and after the noise reduction.

Fig.2: Comparison between magnitude spectra of butyrate
Comparison between the results of different post-processing technique on 13C butyrate. The ‘Sum’ spectrum was obtained by summing 20 Gaussian apodized (10Hz) magnitude spectra. The ‘SVD’ is the magnitude of the SVD low rank approximation of a single Gaussian apodized(1.5Hz) spectra.
Keywords: Hyperpolarization, Nuclear magnetic spectroscopy, Noise reduction, Low rank approximation
207

SPILL-IN EFFECTS IN POSITRON EMISSION TOMOGRAPHY (PET) IMAGING OF ABDOMINAL AORTA ANEURYSM (AAA) (#557)

Mercy I. Akerele1, Daniel Deidda1, 2, Jacobo Cal-Gonzalez3, Rachael O. Forsythe4, Marc R. Dweck4, Nicolas A. Karakatsanis5, 6, Robert G. Aykroyd2, Steven Sourbron1, Charalampos Tsoumpas1, 6

1 University of Leeds, Biomedical Imaging Science Department, Leeds, United Kingdom
2 University of Leeds, Department of Statistics, Leeds, United Kingdom
3 Medical University of Vienna, Centre for Medical Physics and Biomedical Engineering, Vienna, Wien, Austria
4 University of Edinburgh, Centre for Cardiovascular Science, Edinburgh, United Kingdom
5 Weil Cornell Medical University, Department of Radiology, New York, New York, United States of America
6 Icahn School of Medicine, Translational and Molecular Imaging Institute, New York, New York, United States of America

Introduction

Positron emission tomography (PET) is an imaging tool with primary applications in oncology, neurology and cardiology. However, accurate clinical assessment is often affected by the partial volume effect (PVE), leading to overestimation (spill-in) or underestimation (spill-out) of activity in various small regions. Many techniques are used to correct for PVE, but past studies have shown that most of these techniques cannot effectively correct for the spill-in effects when the target region is within 1-5 cm to a highly radioactive region such as the urinary bladder, myocardium or bone.

Methods

This study evaluated the spill-in effects using 3 patients [18F]-Sodium Fluoride PET data of abdominal aortic aneurysms (AAA) adjacent to active bone, acquired with the Siemens Biograph mCTTM scanner. We also compared the performance of various recently developed spill-in correction techniques, namely: (1) background correction (BC), (2) local projection (LP), and (3) hybrid kernel method (HKEM) methods. Iterative reconstructions, including point spread function (PSF) modelling, were performed with the Software for Tomographic Image Reconstruction (STIR) library. Region of Interest (ROI) analysis was performed by comparing the SUVmax and target-to-background ratio (TBR) of two delineating ROIs: one covering the entire aneurysm and the other excluding the parts very close to the bone.

Results/Discussion

The results demonstrated large differences in corrected SUVmax and TBRmax scores between the ROIs drawn over the entire aneurysm (AAA) and ROIs excluding some regions close to the bone (AAA-exc) for both cSUVmax and TBRmax especially for patient 3. This discrepancy in SUV values between the two ROIs is likely due to the spill-in effect emanating from the adjacent active bone. LP and HKM performed well in reducing this spill-in activity for patients 1 and 2, but not for patient 3. PSF+BC had the least difference for all 3 patients under review, suggesting the best spill-in correction performance. These results show that the uptake measurement in the abdominal aneurysm is highly dependent on how the ROI is drawn, and most importantly on clinician expertise. In addition, excluding some regions close to the bone in a bid to avoid the spill-in effect from the bone, could result in a certain degree of potentially important physiological information being lost from the excluded regions.

Conclusions

Overall, the BC technique yielded the best performance in spill-in correction for the patient data. This implies that BC can be used to effectively correct the spill-in effect from the bone into the aneurysm. Additionally, BC method is also robust to ROI-selection variability and could thereby enhance accurate quantification in regions of interest.

References

[1] Y. Liu. “Invalidity of SUV measurements of lesions in close proximity to hot sources due to shine-through effect on FDG PET-CT interpretation,” Radiol. Res. Pract. 868218, 2012.

[2] M. I. Akerele, P. Wadhwa, J. Silva-Rodriguez, W. Hallett and C. Tsoumpas, “Validation of the physiological background correction method for the suppression of the spill-in effect near highly radioactive regions in positron emission tomography,” EJNMMI Phys., 5:34, 2018.

[3] R. O. Forsythe, M. R. Dweck, O. M. B. McBride, A. T. Vesey, S. I. Semple, A. S. V. Shah, et al., “18F-Sodium Fluoride Uptake in Abdominal Aortic Aneurysms: The SoFIA3 Study,” J. Am. Coll. Cardiol., vol. 71, no. 5, pp. 513-523, 2018.

[4] J. Cal-Gonzalez, X. Li, D. Heber, I. Rausch, S. C. Moore, K. Schafers, et al., “Partial volume correction for improved PET quantification in 18F-NaF imaging of atherosclerotic plaques,” J. Nucl. Cardiol., vol. 25, no. 5, pp. 1742-1756, 2017.

[5] D. Deidda, N. A. Karakatsanis, P. M. Robson, N. Efthimiou, Z. A. Fayad, R. G. Aykroyd, and C. Tsoumpas, “Effect of PET-MR inconsistency in the kernel image reconstruction method,” IEEE Trans. Radiat. Plasma Med. Sci, 2018 (in press).

Acknowledgement

This work was undertaken on MARC1, part of the High-Performance Computing and Leeds Institute for Data Analytics (LIDA) facilities at the University of Leeds, UK.

Corrected SUVmax (cSUVmax) and maximum target-to-backgriound ratio (TBRmax)

Fig. 1. The cSUVmax and the TBRmax analysis from patients 1 - 3 (left-to-right columns), as estimated for all evaluated reconstruction algorithms at 3 iterations and with 3 mm Gaussian post-filter.

Reconstructed Images from all correction techniques

Fig. 2. Sagittal views of the PET reconstructed images at 3 full iterations with 3 mm Gaussian post-filter, shown for all 3 patients (top-to-bottom rows). The ROIs used to extract the SUVs for AAA (outer sphere) and AAA-exc (inner sphere) are shown on the CTAC image.

Keywords: PET, PVE, spill-in effect, quantification
208

Biological diversity or numeric artifact? MSI data clustering robustness. (#587)

Grzegorz Mrukwa1, Katarzyna Fratczak1, Marta Gawin2, Mykola Chekan2, Monika Pietrowska2, Piotr Widlak2, Joanna Polanska1

1 Silesian University of Technology, Data Mining Group, Gliwice, Poland
2 The Centre of Oncology -Maria Skłodowska-Curie Institute, branch in Gliwice, Gliwice, Poland

Introduction

Mass Spectrometry Imaging is a recent technique providing vast amounts of data about the distribution of hundreds of compounds within a tissue, revealing implicit structure crucial for investigating tumor or neurodegenerative diseases. Despite dedicated preprocessing, data is heavily duplicated (thus overly multidimensional) and noisy. Moreover, in molecularly diverse regions, different descriptors play the key role in heterogeneity analysis. A number of clustering algorithms were presented, as well as dimensionality reduction techniques. But which combination provides justified results?

Methods

Data from two Head and Neck Cancer cases (8005 and 11 869 spectra, 109 568 mass channels each) was subject to the standard pipeline of Savitzky-Golay smoothing, Peak Alignment via Fast Fourier Transform, baseline removal, and TIC normalization. Instead of overly simplistic procedures for peak selection, peak modeling via a Gaussian Mixture Model (GMM) was applied and 3 714 new descriptors were obtained.

Three clustering algorithms were included in considerations: standard K-Means algorithm, graph-cuts based clustering, and Divisive intelligent K-Means. Each of them ran independently on GMM-modelled data, PCA-reduced dataset (most relevant components w.r.t. knee-method or EXIMS score) and dataset reduced by nonlinear manifold embedding. The number of clusters was indicated by GAP index.

Results/Discussion

From each scenario, clusters were selected to compose regions of tumor and epithelium (figure). Clusters from the graph-cuts deformed region of tumor and epithelium marked by pathologist and exhibit fragmentation over homogeneous regions of the epithelium.

K-means provides the most spatially consistent clusters (except EXIMS), but only with manifold embedding is capable to reveal major diversity within the healthy epithelium.

DiviK for GMM components reveals both tumor and epithelium molecular diversity. Results are stable for PCA transformation, despite small clusters appear (knee) or vanish (EXIMS). With manifold embedding provides a similar result to k-means (manifold), and, similarly to the DiviK (GMM), diversity within the cancer region is captured.

The effect size of features was computed and features with at least large effect size between clusters were measured. Top 3 counts correspond to DiviK algorithm with PCA (knee), GMM and manifold. Most of the counted features are the same.

Conclusions

Inspection of clusters indicates three candidates for biological diversity analyses: K-means and DiviK after manifold embedding, as well as DiviK in GMM space. PCA (dependent on the component selection method) tends to lose or exaggerate nuances in the data. Spectral clustering introduces fragmentation that is hardly corresponding to tissue diversity. Finally, DiviK finds regions which could be differentiated with the greatest number of features.

References

  1. Dexter, Alex, et al. "Two-phase and graph-based clustering methods for accurate and efficient segmentation of large mass spectrometry images." Analytical chemistry 89.21 (2017): 11293-11300.
  2. Pietrowska, Monika, et al. "Molecular profiles of thyroid cancer subtypes: Classification based on features of tissue revealed by mass spectrometry imaging." Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1865.7 (2017): 837-845.
  3. Tibshirani, Robert, Guenther Walther, and Trevor Hastie. "Estimating the number of clusters in a data set via the gap statistic." Journal of the Royal Statistical Society: Series B (Statistical Methodology) 63.2 (2001): 411-423.
  4. Wijetunge, Chalini D., et al. "EXIMS: an improved data analysis pipeline based on a new peak picking method for EXploring Imaging Mass Spectrometry data." Bioinformatics 31.19 (2015): 3198-3206.
  5. Polanski, Andrzej, et al. "Signal partitioning algorithm for highly efficient Gaussian mixture modeling in mass spectrometry." PloS one 10.7 (2015): e0134256.

Acknowledgement

This project was financially supported by the European Union through the European Social Fund (grant POWR.03.02.00-00-I029) (GM) and NCN grant BITIMS no. UMO-2015/19/B/ST6/01736 (GM, JP, KF).

Optical scan of analyzed tissue
Tissue regions are marked over the preparation area. Red color corresponds to tumor tissue and inflammatory response. Cyan color corresponds to the healthy epithelium.
Visualization of clustering results
Clustering results are limited to the area corresponding to the tumor and healthy epithelium. Different colors indicate separate clusters. Colors were selected in a way that matches original H&E coloring the most.
Keywords: mass spectrometry imaging, tissue heterogeneity, clustering, dimensionality reduction
209

PSMA-PET/CT texture analysis to characterise unaffected bone and bone metastases of patients with prostate cancer (#408)

Robert Seifert1, 2, Aaron Scherzinger3, Bastian Zinnhardt1, 2, Florian Büther1, Matthias Weckesser1, Kambiz Rahbar1, Michael Schäfers1, 2

1 University of Münster, Department of Nuclear Medicine, Münster, North Rhine-Westphalia, Germany
2 University of Münster, European Institute for Molecular Imaging, Münster, North Rhine-Westphalia, Germany
3 University of Münster, Department of Computer Science, Münster, North Rhine-Westphalia, Germany

Introduction

Bone metastases of prostate cancer can be detected by various modalities like bone scintigraphy, CT or PSMA PET acquisitions. Yet the characterization of Disseminated Tumour Cells (DTCs) in the bone marrow by imaging means is an unmet clinical challenge. Texture analysis (TA) is an established tool to reveal image features of clinical significance and has recently been applied to PSMA PET acquisitions [1]. TA is often utilised for the analysis of tumours or metastases. Here, TA is used to characterise bone metastases and apparently unaffected bone parts, i.e. with no visible bone metastases.

Methods

98 [68Ga]-PSMA-11 PET/CT acquisitions were automatically analysed (25 without, 73 with bone metastases). CT acquisitions were used to segment a bone mask for each PET/CT, which was then applied to the PET acquisitions to exclude non-bone structures. Further, a global SUV threshold was defined to segment bone metastases in each PET acquisition. To cope for potential spill-out artefacts, each binary bone metastases mask was expanded by a Gaussian smoothing of 8.6 mm FWHM (thus minimising effects of positron ranges and system resolution) and thresholded to include 99.9 % of the Gauss distribution. Fifteen texture analysis features were calculated for bone metastases and unaffected bone of each PET acquisition, including Grey Level Cooccurrence Matrix (GLCM) features [2].

Results/Discussion

The global threshold to segment visible bone metastases was set to 1.6 SUV. In an exploratory approach, Receiver Operating Characteristics (ROC) were calculated for each textural feature to assess the performance of unaffected bone TA to classify acquisitions as either bone metastases free or osseous metastasized. Highest Area Under Curve (AUC) value was obtained by the GLCM feature ‘angular second moment’ (GLCMASM; AUC = 0.711), whereas the best statistical image feature was ‘image entropy’ (AUC = 0.334; for comparison AUC of SUVmean: 0.352) [2]. Analysing unaffected bone, there was a significant difference comparing GLCMASM of patients with and without bone metastases (0.013 vs. 0.002, respectively; p < 0.005). However, TA of bone metastases seemed of little clinical relevance: PSA levels correlated with SUVmean but not with GLCMASM of bone metastases (Spearman: p < 0.001 vs. p > 0.05).

Conclusions

Automated TA of visually unaffected bone is feasible and reveals image features that were significantly different between PET acquisitions with and without bone metastases. TA of bone metastases seems inferior to SUVmean. Future studies have to show if image features of unaffected bone have a predictive value for the outcome and if they correlate with the presence of DTCs.

References

  1. Khurshid Z, Ahmadzadehfar H, Gaertner FC, et al (2018) Role of textural heterogeneity parameters in patient selection for 177Lu-PSMA therapy via response prediction. Oncotarget 9:33312–33321. https://doi.org/10.18632/oncotarget.26051
  2. Haralick RM, Shanmugam K, Dinstein I (1973) Textural Features for Image Classification. IEEE Trans Syst Man Cybern SMC-3:610–621

Acknowledgement

none.

Keywords: PSMA PET/CT, texture-analysis, bone metastases, prostate cancer.
210

Simplified estimation of binding potential for TSPO tracer 18F-DPA714 without a reference region (#236)

Claire Leroy2, Sonia Lavisse2, 4, Martin Schain3, Michel Bottlaender2, 5, Irène Buvat2, Catriona Wimberley1, 2

1 University of Edinburgh, Edinburgh Imaging Facility QMRI, Edinburgh, United Kingdom
2 CEA, Inserm, Université Paris-Sud, Université Paris Saclay, IMIV, Orsay, France
3 Copenhagen University Hospital, Neurobiology Research Unit, Copenhagen, Denmark
4 CEA and CNRS-UMR9199, Université Paris-Sud, MIRCen, Fontenay-aux-Roses, France
5 CEA, Neurospin, Gif-sur-Yvette, France

Introduction

Quantification of TSPO tracers such as 18F-DPA714 faces several challenges1 including lack of a reference region. Arterial blood sampling is gold standard, but not always possible so alternative quantification methods are valuable. There are BPND estimation methods for 18F-DPA714 such as supervised cluster analysis to extract a reference curve (SVCA)2,3 however, these methods are susceptible to bias from whole brain TSPO. The aim of this study is to assess a previously developed method of estimating BPND (SIME) 4 on 18F-DPA714 scans using an individual AIF and a template input function (tIF).

Methods

Ten healthy subjects (3 mixed (MAB), 7 high affinity binders (HAB)) underwent 18F-DPA-714 scans with metabolite corrected arterial input functions (AIF). For each subject, SIME analysis used the 2TCM with a fixed VND across 15 brain regions, fitting all regions simultaneously4. A grid of 100 VND values was tested (0.1 to 10) and the VND that produced the lowest cost function was selected. The regional BPNDs were calculated using the VTs and VND. This was done with two input functions and compared:

  1. Individual AIF
  2. A tIF was created for each subject by averaging the AIFs: each AIF was normalised (area under the curve=1) and the peaks temporally shifted so they were lined up with each other. For each subject, a tIF was generated from the 9 other subjects, to emulate when there is no AIF

Results/Discussion

The SIME method successfully identified VND values for each subject using its own AIF. Mean VND values were HABs: 2.7±1.30 ml/cm3 and MABs: 2.0±0.06 ml/cm3 which were used to estimate regional BPND as shown in Fig 1. The BPND estimates using the tIF were strongly correlated with those using individual AIFs in HAB and MAB groups: r2 = 0.98 and 0.95 respectively (Fig 2).

To assess the value of the method, it could be tested in a cohort of pathological subjects and compared against SUVr or BPNDs calculated using reference tissue models with the cerebellum or extracted SVCA curve. This would allow us to see if SIME is more sensitive to regional changes in TSPO than the other methods, which could be susceptible to bias from TSPO expression across the whole brain or unexpected regional increases in TSPO expression. On top of that, the SIME method could be tested with a model including a component for vascular TSPO.

Conclusions

We have applied the previously developed SIME method of estimating BPND to 18F-DPA714, which identified BPND values using individual AIFs and also the population based tIF. The BPND values between the two methods were highly correlated. The SIME method combined with a tIF will be useful for obtaining regional BPND estimates without individually sampled AIFs or a reference region.

References

  1. Turkheimer et al.; The methodology of TSPO imaging with positron emission tomography; Biochem. Soc. Trans., 2015
  2. Garcia Lorenzo et al.; Validation of an automatic reference region extraction for the quantification of [18F]DPA-714 in dynamic brain PET studies; JCBFM, 2017
  3. Turkheimer et al.; Reference and target region modelling of 11C-R-PK11195 brain studies; JNM, 2007
  4. Schain et al.; Non-invasive estimation of 11C-PBR28 binding potential; Neuroimage, 2018

Acknowledgement

Dr Catriona Wimberley's position as Research Fellow in PET-MRI physics at the University of Edinburgh is funded by Siemens Healthcare Limited, and her previous post doctoral position was supported by a CEA-Enhanced Eurotalents cofund with FP7 Marie Sklodowska-Curie COFUND Program (600382).

Regional BPND estimates

Average regional BPND estimates for HABs and MABs using the SIME 2TCM with individual AIFs

Correlation between AIFs and tIFs.

Correlations between regional BPND estimates calculated using the individual AIFs and the tIF for HABs and MABs

Keywords: TSPO, quantification, kinetic modelling, 18F-DPA714, simplified
211

Segmentation of amyloid-β plaques in three mouse models of Alzheimer’s disease using X-ray phase contrast-computed tomography (#362)

Coralie Gislard1, Carlie Boisvert1, Cécile Olivier2, Françoise Peyrin2, Marlène Wiart3, Hervé Boutin4, Hugo Rositi5, Fabien Chauveau1

1 Univ. Lyon, Lyon Neuroscience Research Center; CNRS UMR5292; INSERM U1028, Univ. Lyon 1, Lyon, France
2 Univ.Lyon, CREATIS; CNRS UMR5220; INSERM U1044; INSA-Lyon; Univ. Lyon 1, Lyon, France
3 Univ. Lyon, CarMeN laboratory; INSERM U1060; INRA U1397; Hospices Civils de Lyon, Lyon, France
4 Univ. Manchester, Faculty of Biology Medicine and Health, Wolfson Molecular Imaging Centre, Manchester, United Kingdom
5 Univ. Clermont Auvergne; CNRS; SIGMA Clermont; Institut Pascal, , Clermont-Ferrand, France

Introduction

X-ray Phase Contrast Tomography (XPCT) uses highly coherent synchrotron radiation to image soft tissues [1]. Ex vivo brain XPCT enables a virtual histology of cerebral structures [2], myelinated tracts [3], but also amyloid plaques (Aβ) [4]. Previous reports on Aβ detection have been mostly restricted to qualitative observations [5], owing to the difficulty to process the large amount of data arising from whole-brain imaging at a µm scale. The present work aims to develop a segmentation pipeline to extract relevant measurements on Aβ plaques across several Alzheimer’s transgenic lines.

Methods

Three transgenic lines were used, for a total of eight brains: i) 3xTg (n=3, 1 y.o.), ii) APP-PS1 (n=3, 1 y.o.), iii) PDAPP-J20 (n=2, 2 y.o.) [6].

Fixed brains were dehydrated in ethanol and placed in test tubes for imaging at ESRF beamline ID-19 (Table 1). Paraffin embedding and standard immunofluorescence using 4G8 antibody and thioflavin S (ThS) were performed afterwards.

Semi-automated detection of Aβ plaques in the hippocampus used Fiji software and the following plugins: segmentation editor (to isolate hippocampus), trainable WEKA segmentation (to identify plaques) and 3D objects counter (to extract relevant parameters). Accuracy of segmentation was visually evaluated.

Results/Discussion

The semi-automated detection pipeline identified, in the hippocampus of a single hemisphere, 25±5 plaques in 3xTg, 1350±377 plaques in APP-PS1 and 2577±457 plaques in PDAPP-J20, which presence was confirmed by immunofluorescence (Fig. 1). No false-positive structures, such as hippocampal neurons, were segmented. The mean volume size of individual plaques in 3xTg was about twice the ones of APP-PS1 and PDAPP-J20 (25470±4360 vs 14150±4360 vs 13650±1720 µm3 respectively), and the fraction volume of hippocampus occupied by Aβ was 0.01% in 3xTg, 0.22% for APP-PS1, and 0.48% for PDAPP-J20. In all cases, the majority of plaques were ellipsoidal, with 15 to 40% being detected as circular.

Conclusions

A pipeline using freely available tools was successfully tested on three transgenic lines, and accurately detected visible Aβ plaques. The present work leveraged 3D-analysis to extract quantitative parameters hardly available with standard histology. The validation of such quantification pipelines is a necessary step to realize the full potential of XPCT in neuroscience research.

References

[1] Albers et al. Mol Imaging Biol. 2018.

[2] Marinescu et al. Mol Imaging Biol. 2013; Barbone et al. IJROBP 2018.

[3] Rositi et al. In: 12h European Molecular Imaging Meeting (EMIM), Cologne, Germany. 2017.

[4] Chauveau et al. In: 13th European Molecular Imaging Meeting (EMIM), San Sebastian, Spain. 2018.

[5] Noda-Saita  et al. Neuroscience 2006 ; Connor et al. NeuroImage 2009 ; Pinzer et al. NeuroImage 2012 ; Astolfo et al. J Synchrotron Radiat. 2016 ; Massimi et al. NeuroImage 2019.

[6] Oddo et al. Neuron. 2003 ; Jankowsky et al. Hum Mol Genet. 2001 ; Mucke et al. J Neurosci. 2000.

Acknowledgement

MITACS grant (Carlie Boisvert); LABEX  PRIMES  (ANR-11-LABX-0063).

Table 1.
Acquisition parameters used for whole-brain imaging of mouse brains at beamline ID-19 of the European Synchrotron Radiation Facility (ESRF)
Figure 1.
Corresponding (A) XPCT image, (B) amyloid staining with 4G8 antibody, and (C) Thioflavin S staining, for the three transgenic mouse strains (I- 3xTg; II- PDAPP-J20; III- APP-PS1).
Keywords: X-ray Phase Contrast Tomography, amyloid, Alzheimer's disease, virtual histology
212

Effect of image-derived input function extraction method on quantification of 18F-FDG uptake in mice with different dietary conditions (#192)

Celia De La Calle3, Natalia Magro1, Marta Ibañez1, Marta Oteo1, Guillermo Garaulet2, Francisca Mulero2, Alejo Efeyan3, Miguel Angel Morcillo1

1 CIEMAT, Biomedical Applications of Radioisotopes and Pharmacokinetics Unit, Madrid, Spain
2 CNIO, Molecular Imaging Core Unit, Madrid, Spain
3 CNIO, Metabolism and Cell Signalling Lab, Madrid, Spain

Introduction

Measurement of an accurate plasma input function is required for quantitative 18F-FDG PET studies in mice. We aimed to compare the effect of method of generating image-derived input function (IDIF) on the quantification of 18F-FDG uptake in mice PET studies under different dietary states.

Methods

Mice (C57BL6x129S2/SvPasCrl) were studied under different conditions: nonfasted, fasted and insulin administration. 60-min dynamic PET was performed under isoflurane anesthesia after 18F-FDG injection. Regions of interest were manually drawn over: myocardium, liver, cerebral cortex, brown adipose tissue, muscle and white fat. IDIFs were obtained from the left ventricle cavity (LV) using the COMKAT software1 or vena cava (VC) fitted to the 3-compartment FDG model using nonlinear regression2. The 18F-FDG uptake constant (Ki) was estimated by Patlak graphical analysis3 using input function and time-activity data from 15-60 min. When the resulting plot became horizontal, a Logan plot was applied to calculate the distribution volume4. Tracer uptake was also quantified as SUVs.

Results/Discussion

Arterial time-activity curves (TAC) normalized by injected dose (SUV) are illustrated in Figure 1. No significant differences in the area under the arterial curve (AUC; expressed as SUV x min) for LV and VC were observed between nonfasted and fasted or insulin mice (333±189 vs. 301±126 and 413±26 for LV and 108±13 vs. 114±33 and 79±30 for VC). The AUCs were significantly higher in TAC obtained from LV cavity than from VC due the spillover from the myocardial wall and the partial-volume non-correction of the VC.  Result of the graphical analysis of PET data is shown in Table 1. No significant difference in the Ki estimates from myocardium and cerebral cortex was obtained with LV and VC IDIF, although the values were higher in those estimated from LV IDIF. The myocardium Ki values were significant higher in insulin than in nonfasted mice. There were no significant differences in distribution volume values obtained with both IDIFs in the rest of organs, irrespective of dietary state.

Conclusions

The arterial left ventricle and vena cava time-activity curves can be used both as an easy accessible input function for kinetic modeling of 18F-FDG uptake. Although a slight underestimation of Patlak/Logan slope from the vena cava as IDIF compared to the obtained from left ventricle was found, the choice of IDIF had not significant effect on Patlak/Logan kinetics and calculated 18F-FDG uptake under different dietary conditions.

References

1Muzic RF, Jr, Cornelius S. COMKAT: compartment model kinetic analysis tool. J Nucl Med. 2001;42:636–45. (http//comkat.case.edu)

2Feng D, Wong KP, Wu CM, Siu WC. A technique for extracting physiological parameters and the required input function simultaneously from PET image measurements: theory and simulation study. IEEE Trans Inf Technol Biomed. 1997;1:243–54.

3 Patlak CS, Blasberg RG. Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. Generalizations. J Cereb Blood Flow Metab. 1985;5:584-590.

4 Logan J. Graphical analysis of PET data applied to reversible and irreversible tracers. Nucl Med Biol. 2000;27:661-670.

Figure 1. Time-activity curves
Time-activity curves averaged for nonfasted (n=4, black), fasted (n=4, red) and insulin-treated (n=3, blue). Given are blood activities divided by injected dose per Kg of body weight (SUV) for left ventricle cavity and vena cava.
Table 1. Graphical analysis

Ki (mL tissue/mL plasma*min) and DV (mL tissue/mL plasma) values estimated by graphical analysis using IDIFs obtained from left ventricle and vena cava. Mice: nonfasted (n=4), overnight fasted (n=4) and insulin-treated (n=3). (a) Significant differences were found between nonfasted and insulin-treated mice. (b) Significant difference was found between LV and VC IDIF for nonfasted mice.

Keywords: PET, quantitative image analysis, IDIF
213

Radiographic quantitative values measured from 18F-NaF PET/CT bone scan for Alkaptonuria patients at Lumbar spine and femoral region. (#158)

Eman H. Alawadhi1, Sobhan Vinjamuri2, James Gallagher1, Ranganath Lakshminarayn3

1 The University of Liverpool, Musculoskeletal Biology I, Institute of Ageing & Chronic Disease, William Henry Duncan Building, Liverpool, United Kingdom
2 Royal Liverpool University Hospital, Department Of Nuclear Medicine, Liverpool, United Kingdom
3 Royal Liverpool & Broad green University Hospitals , Liverpool Clinical Laboratories, Liverpool, United Kingdom

Introduction

Alkaptonuria is a genetioarthritic disorder caused by a deficiency of HGA enzyme leading to increasing homogentisic acid forming cartilage pigmentation and arthritis called ochronosis. Radiographic quantitative assessment from PET/CT using bone radiotracer can be used to assess the progression of skeletal involvement in AKU.  

The study aim is to assess bone involvement in lumbar and hip regions for AKU by measuring Hounsfield units from CT and standardised uptake value from PET. We aimed also to determin the correlation between HU, SUV and bone density value and tseting age and gender affect.

Methods

A total of 39 AKU patients (males 24, female 15) who involved in the SONIA2 trial and underwent the whole body 18F-NaF PET/CT and DEXA scans were enrolled in our study. The mean age of the patients was 50 ± 10, and each gender was grouped into four groups stratified by decade of life. A HERMES software system was used to measure an average HU from CT image and SUVmax from PET image for lumbar vertebrae L1-L5 and head of the femur. For each measurement, the largest possible elliptical region covering the ROI was drawn in the axial slice, excluding the cortical margins to avoid volume averaging (fig1).The effect of age in each gender for HU and SUVmax was tested. The measured quantitative values were compared between each other age and correlated with bone mineral density from a DEXA scan.

Results/Discussion

In our study, although that males have higher T-score values compared with females, there was no a significant difference in HU and SUVmax between genders. The mean HU across L1-L5 was 145 for males and 152 for female.HU values showed a significant correlation with increased age for both gender in average lumbar vertebral bodies (male, p= 0.014, r = 0.48; female p= 0.0005, r =0.79) but only for female in head of the femur with increasing age (r = 0.83 and p < 0.0005). SUV values were not changed significantly between the groups of age except in average lumbar vertebrae for males (r= 0.47, p= 0.021). The range of lumbar vertebrae SUVmax was between 5.2 to 15.91 while femoral SUVmax was between 2.2 to 21. There was no correlation coefficient of SUVmax with T score (p > 0.05) in both lesions, but moderate correlations with HU in lumbar lesion only. There was a significant correlation coefficient of HU and T score in lumbar and femur region (p < 0.005).

 

Conclusions

The present study proposes a novel radiographic quantitative method by measuring morphological and functional quantitative data from a single scan which is readily available but rarely used. SUV can be used as a quantitative method to quantify bone PET/CT studies which convey bone metabolism and structural information. HU values from PET/CT could be useful in measuring bone density based on the strong correlation between HU and bone density valu.

References

[1] W. J. Introne and W. A. Gahl, “Alkaptonuria,” 2016.

[2] P. Omoumi, G. A. Mercier, F. Lecouvet, P. Simoni, and B. C. Vande Berg, “CT Arthrography, MR Arthrography, PET, and Scintigraphy in Osteoarthritis,” Radiol. Clin. North Am., vol. 47, no. 4, pp. 595–615, 2009.

[3] N. Kobayashi et al., “New application of 18F-fluoride PET for the detection of bone remodeling in early-stage osteoarthritis of the hip.,” Clin. Nucl. Med., vol. 38, no. 10, pp. e379-83, 2013.

[4] N. Kobayashi et al., “Use of 18F-fluoride positron emission tomography as a predictor of the hip osteoarthritis progression.,” Mod. Rheumatol., vol. 25, no. 6, pp. 925–930, 2015.

Acknowledgement

The author specially thanks to Professor James Gallagher, Professor Sobhan Vinjamuri and Professor Ranganath Lakshminarayn for supporting and supervise me in my PhD research

PET/CT scan

Upper images, illustrating the methods of determining the HU values and SUV using an elliptical ROI. a. Shows the vertebral bodies in a sagittal slice of PET/CT of the lumbar SPINE. In the panel, three axial locations are selected b. immediately superior to inferior endplate c. middle vertebra and d. inferior to the superior endplate.

Lower images; axial slice of head of the femur.
Keywords: semiquantitative assessment, Alkaptonuria, standarised uptake value, Hounsfield Units, 18F-NaF PET/CT
214

An Open-Source Pipeline for Correlation and Visualization of Multi-Modal Multi-Scale Imaging Data in Tumor Vascularization (#114)

Verena Stanzl1, Lydia M. Zopf2, Stefan H. Geyer3, Nicole Swiadek4, Jelena Zinnanti2, Paul Slezak4, Wolfgang J. Weninger3, Andreas Walter5, Katja Bühler1

1 VRVis Zentrum für Virtual Reality und Visualisierung Forschungs-GmbH, Vienna, Austria
2 Vienna Biocenter Core Facilities GmbH, Preclinical Imaging Facility, Vienna, Austria
3 Medical University of Vienna, Division of Anatomy, Center for Anatomy and Cell Biology & MIC, Vienna, Austria
4 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
5 BioImaging Austria - Correlated Multimodal Imaging Node Austria (CMI), Vienna, Austria

Introduction

Activating angiogenesis is one of the main indicators of cancer supporting rapid tumor growth and metastasis [1]. Current treatment strategies aim at inhibiting tumor neo-angiogenesis, but tumor vessels are disorganized, leaky and dysfunctional [2]. To understand how tumors instruct the formation and maintenance of blood vessels, we apply ultra-high field MRI, µCT and HREM to a xenograft mouse model and correlate the tumor vessels. The proposed open source visualization pipeline supports the analysis of angiogenesis by depicting the different vessels and anatomical context in a single model.

Methods

Vessel segmentation is performed using semi-automatic vessel tracking using [3] and Amira. The visualization pipeline provides an open source approach to integrate the multi-modal multi-scale imaging data. µCT is used as reference space for data integration. Its resolution is high enough to show enough detail from HREM data while providing a data size manageable by most software tools. Thus, all data is rescaled to µCT resolution using 3D Slicer [4]. We use CustusX [5] and its 3D visualization to perform pairwise affine landmark-based registration as spatial orientation is crucial to mark corresponding landmarks at branching points. The generated transformation matrices are used to resample all data to the µCT reference in 3D Slicer. Multi-volume rendering is performed with tomviz [6].

Results/Discussion

The visualization in our example (see Figure 1) shows the tumor's blood supply provided by surrounding vessels and reveals the finer vessels subserving the tumor itself. Due to different resolutions, the main branch visible in MRI is also detected in the other modalities, whereas smaller vessels can only be seen in µCT and HREM.

Currently affine image correlation is performed to establish correspondence of matching branching points. However, non-rigid registration would be necessary to correlate the vessels more accurate compensating for deformations the vessels undergo during preparation processes for image acquisition. This is an open problem even for state-of-the-art registration software, as corresponding landmarks along incomplete vessel segments are unknown and image context cannot easily be correlated.

Conclusions

We presented a processing pipeline for visualizing the tumor vascularization in a xenograft mouse model gained by MRI, µCT and HREM to support the analysis of angiogenesis.

The pipeline is still fragmented and the development of a tool providing an integrated correlation and visualization workflow remains an open issue and future work.

References

[1] Folkman, J. (2002). Role of angiogenesis in tumor growth and metastasis. In Seminars in oncology (Vol. 29, No. 6, pp. 15-18). WB Saunders.

[2] Cao, Y. (2008). Molecular mechanisms and therapeutic development of angiogenesis inhibitors. Advances in cancer research, 100, 113-131.

[3] Novikov, A. A., Major, D., Wimmer, M., Sluiter, G., & Bühler, K. (2017). Automated Anatomy-Based Tracking of Systemic Arteries in Arbitrary Field-of-View CTA Scans. IEEE transactions on medical imaging, 36(6), 1359-1371.

[4] Fedorov, A., Beichel, R., Kalpathy-Cramer, J., Finet, J., Fillion-Robin, J. C., Pujol, S., ... & Buatti, J. (2012). 3D Slicer as an image computing platform for the Quantitative Imaging Network. Magnetic resonance imaging, 30(9), 1323-1341.

[5] Askeland, C., Solberg, O. V., Bakeng, J. B. L., Reinertsen, I., Tangen, G. A., Hofstad, E. F., ... & Hernes, T. A. N. (2016). CustusX: an open-source research platform for image-guided therapy. International journal of computer assisted radiology and surgery, 11(4), 505-519. https://www.custusx.org

[6] Tomviz. https//www.tomviz.org. [Online; accessed 28-November-2018]

Acknowledgement

This work was funded by Talente (FEMtech Praktika, project no. 869267) via the Austrian Research Promotion Agency (FFG) and supported by the Correlated Multi Modal Imaging Node Austria. VRVis is funded by BMVIT, BMDW, Styria, SFG and Vienna Business Agency in the scope of COMET - Competence Centers for Excellent Technologies (854174) which is managed by FFG.

Visualization of Tumor Vascularization
Figure 1: Visualization result showing anatomical context from µCT, tumor hull, tumor blood vessels from ultra-high field MRI (orange), µCT (red) and HREM (yellow). The additional vessel parts are gained from µCt by manual labeling (green) and thresholding (blue).
Flowchart of the proposed Pipeline
Figure 2: Overview of the processing pipeline consisting of Image Acquisition, Vessel Segmentation, Correlation and Visualization. The orange rectangles depict the imaging modalites, the light blue rectangles show the used software tools and the dark blue circles illustrate the performed processings.
Keywords: tumor vascularization, visualization, image correlation, multi-modal multi-scale imaging, image processing
215

Effect of different ROI definitions on the quantification of SUV (#197)

Bashair Alhummiany1, Richa Gandhi1, Steve J. Archibald2, Christopher Cawthorne3, Marc A. Bailey1, Charalampos Tsoumpas1

1 University of Leeds, Department of Biomedical Imaging Science, Leeds Institute of Cardiovascular and Metabolic Medicine, Leeds, United Kingdom
2 University of Hull, PET Research Centre, School of Life Sciences, Hull, United Kingdom
3 KU Leuven, Leuven, Belgium

Introduction

Although the standardised uptake value (SUV) has been widely used to quantify tracer uptake in positron emission tomography-computed tomography (PET-CT) images, it can be influenced by the method of defining regions of interest (ROIs)1. Since the maximum SUV can be affected by noise, another method was introduced to improve the accuracy of SUV by applying a threshold to estimate the average SUV of a group of voxels2. Our aim was thus to identify a reproducible threshold value using two segmentation methods. We also assessed the applicability of using these methods in preclinical PET-CT.

Methods

Fourteen mice (10 with abdominal aortic aneurysms, 4 controls) underwent 90-min dynamic PET-CT acquisitions using the Sedecal Super Argus PET-CT scanner. Images were reconstructed with the 3D ordered subset expectation maximisation algorithm (2 iterations, 16 subsets). Using Amide software, all data were segmented using manual and fixed-size ROIs at 80–90 min to compute the mean uptake (SUVmean); maximum uptake (SUVmax); and mean uptake for voxels higher than 40%, 50%, 70%, and 90% of the maximum (SUV40, SUV50, SUV70, and SUV90, respectively) (Fig 1). A fixed 4-mm sphere was used for delineation; the size was chosen based on the maximum aortic aneurysmal diameter (3 mm). Volumetric differences at each threshold and the delineation times were analysed to assess the segmentation methods.

Results/Discussion

By comparing threshold values for each pair of SUVs calculated using the manual and fixed-size methods, significant differences were found at the 40%, 50%, and 90% thresholds (all P < 0.05). However, SUV70showed no statistically significant difference between the two segmentation techniques (P=0.72), indicating that applying a 70% threshold on ROIs can minimise operator bias and result in a more stable SUV measurement. The average volumes of fixed-size ROIs were relatively larger than those of the manual ROIs, since the size of the fixed-size ROIs was chosen to segment different aneurysm diameters. The result also demonstrated that the volumes of ROIs using both methods decreased when a higher threshold value was applied,indicating that fewer voxels were accounted for with a higher threshold.Finally, we found that the average time required for delineation using manual segmentation was up to three times more than that needed to draw the fixed-size ROIs.

Conclusions

In this study, we assessed the reproducibility of four different threshold values for voxels greater than the maximum when two different segmentation methods were used. We concluded that the implementation of fixed-size ROIs with a 70% threshold appears to provide the most stable SUV measurement in the analysis of aortae in preclinical PET-CT images of mice.

References

  1. Boellaard R. "Standards for PET image acquisition and quantitative data analysis." J Nucl Med 50(S1), 2009
  2. Lee JR, et al. A threshold method to improve standardized uptake value reproducibility. Nucl Med Comm 21(7) 2000

 

Placement of ROIs

Figure1 The placement of manual (A and C), and fixed-size (B and D) ROIs on transverse, coronal, and sagittal views in PET-CT images of two different mice with AAA. The bottom panels show AAA that is close to the urinary bladder (UB).

Keywords: PET-CT, Standardised uptake value (SUV), Image segmentation

Imaging Metabolism

Session chair: David Lewis (Glasgow, UK); Bernard Lanz (Lausanne, Switzerland)
 
Shortcut: PW11
Date: Thursday, 21 March, 2019, 12:45 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.

301

In vivo metabolic profile characterization in a mouse model of vulnerable atherosclerotic plaque. (#272)

Chetan Dhakan1, Sandra Albanese1, Matteo Gramanzini1, Juan C. Cutrin2, Valeria Bitonto2, Flavio Cristofani2, Sharmila Fagoonee1, Lorenzo Silengo2, Fiorella Altruda2, Marcello Mancini1, Sara Gargiulo1

1 National Council of Research, Institute of Biostructure and Bioimaging , Naples, Italy
2 University of Turin, Department of Molecular Biotechnology and Health Sciences, Turin, Italy

Introduction

Atherosclerosis is often associated with altered metabolism in humans and in mouse model. Therefore, it would be of great interest to investigate in murine model the effect of diet in combination with genetic background. The double knockout ApoE-/-Fbn1C1039G+/− (DKO) mice fed an high-fat diet is a valuable model of vulnerable plaques, but information on their metabolic profile are still scanty. To the best of our knowledge, this exploratory study is the first to provide in vivo, a morphofunctional evaluation of renal, hepatic and body composition phenotypes in DKO through noninvasive imaging.

Methods

Body weight, food intake, plasmatic glucose, triglycerides and cholesterol were assessed in DKO mice on 13 weeks of high-fat diet, as in sex-/age-matched C57Bl/6J control (CTRL) mice fed standard diet. Data were supplemented by lean and fat mass, bone mineral concentration (BMC) and density (BMD) using DEXA (Lunar Piximus-GE). High Frequency Ultrasound (Vevo 2100-Visualsonics) was used to characterize kidney and liver. B-mode scans were used to evaluate hepatic echostructure, and a mean-gray-level ratio between caudate lobe and renal cortex was calculated as index of echogenicity (HR). Renal segmental artery flow velocity was measured using Pulse Wave Doppler, to derive Pulsatility and Resistivity indexes. Data were analyzed by T-test and P<0.05 was considered statistically significant.

Results/Discussion

Body weight in female DKO mice was higher than CTRL from 8 to 19 weeks of age. Moreover, global and abdominal fat were higher in 16 weeks female DKO than CTRL, similarly to BMC and BMD at 21 weeks of age. In DKO, liver echostructure appeared more heterogeneous and echogenic and HR showed increasing trend from 8 to 21 weeks of age. Basal plasma lipid levels were higher in DKO, and hypercholesterolemia persisted at 21 weeks of age. Female DKO basal glycaemia resulted higher, while showed a significant lowering at 21 weeks of age compared to CTRL. No significant differences in food intake and renal indexes were observed. DKO mice disclosed lipoprotein profile significantly different compared with CTRL with double levels of total cholesterol and develop a pathological liver phenotype after 13 weeks of high-fat diet. Metabolic analysis integrated by imaging highlights the effects of genes and diet manipulations on diseases development.

Conclusions

ApoE-/-Fbn1+/- mice under high-fat diet develop a significant sex- and age- dependent alteration of body composition and plasma biochemical parameters, together with changes of liver echostructure at early stage of atherogenesis. Multimodal imaging can provide relevant complementary information to conventional metabolic analysis for phenotyping of mouse model in a non-invasive and longitudinal way.

References

Neuhofer A, Wernly B, Leitner L, Sarabi A, Sommer NG, Staffler G, Zeyda M, Stulnig TM. An accelerated mouse model for atherosclerosis and adipose tissue inflammation. Cardiovasc Diabetol. 2014;13-23.

Van Herck JL, De Meyer GR, Martinet W, Van Hove CE, Foubert K, Theunis MH, Apers S, Bult H, Vrints CJ, Herman AG. Impaired fibrillin-1 function promotes features of plaque instability in apolipoprotein E-deficient mice. Circulation. 2009;120(24):2478-87.

Faita F, Di Lascio N, Rossi C, Kusmic C, Solini A. Ultrasonographic Characterization of the db/db Mouse: An Animal Model of Metabolic Abnormalities. J Diabetes Res. 2018;2018:4561309.

Figure 1 In vivo characterization of metabolic profile in ApoE-/-Fbn1C1039G+/- mouse model
significant differences (p<0.05) in plasma cholesterol, triglycerides and glucose levels with respect to healthy controls were found.
Figure 2. In vivo imaging characterization of metabolic profile in ApoE-/-Fbn1C1039G +/- mouse

A) DEXA scan and body composition analysis (Lunar Piximus, GE Medical Systems Madison, WI).

B) Kidney Color Doppler- and Pulsed Wave-mode images

C) Hepatic and renal B-mode  (Vevo 2100 system, Visualsonics, ON, Toronto, Canada) for echostructure and echogenicity analysis, and hystological section  of liver in DKO (Oil Red-O staining, x100)

D) Corresponding evaluation in healthy CTRL

        

Keywords: mouse model of vulnerable plaque, metabolic characterization, DEXA, High frequency ultrasound (HFUS)
302

Assessment of intrahepatic islet of Langerhans grafts with dynamic PET imaging using 68Ga-labeled exendin-4 (#266)

Tom Jansen1, Mijke Buitinga1, Marti Boss1, Eelco de Koning2, Marten Engelse2, Michiel Nijhoff2, Olle Korsgren3, Olof Eriksson4, Maarten Brom1, Martin Gotthardt1

1 Radboud university medical center, Radiology and Nuclear Medicine, Nijmegen, Netherlands
2 Leiden University Medical Center, Internal Medicine, Leiden, Netherlands
3 Uppsala University, Immunology, Genetics and Pathology, Uppsala, Sweden
4 Uppsala University, Medicinal Chemistry, Uppsala, Sweden

Introduction

Patients with complicated type 1 diabetes (T1D) and unstable glycemic control can receive intrahepatic islet transplantation as treatment. This procedure leads to an improved glycemic control and quality of life. Various factors can affect the function of the transplanted grafts and therefore graft function deteriorates over time. A clinical tool to assess transplantation success and monitor islet survival and functionality would be of great value. We applied PET imaging with the beta cell-specific tracer [68Ga]Ga-NODAGA-exendin-4, to study the presence of transplanted islets in T1D patients.

Methods

Dynamic PET scans of 60 minutes were acquired after intravenous injection of 1.2 MBq/kg [68Ga]Ga-NODAGA-exendin-4 in 5 T1D patients that previously received intrahepatic islet grafts (Tx-group: 2 men, 3 women). Graft function in these patients was biochemically proven prior to imaging. In addition, 3 control patients were imaged that awaited islet transplantation (2 men, 1 woman). Regions with high hepatic uptake were obtained applying thresholding. The accumulation of the exendin-based tracer in the liver was measured by kinetic modeling using the Logan model. In each patient a mixed-meal tolerance test (MMTT) was performed. These results were expressed as the area under the curve (AUC) of the measured c-peptide. Function was compared to the PET signal obtained from the dynamic PET scans.

Results/Discussion

The controls and Tx-group did not differ in age (58.7±5.5 vs. 54±9.5 years, p=0.57), BMI (24.5±4.5 vs. 21.8±1.4 kg/m2, p=0.57) and HbA1c (62.3±6.1 vs. 45.4±12.8 mmol/mol, p=0.14), though the results of the MMTT, expressed as the AUC for c-peptide (22.6 vs. 151.8 nmol.min/L, p<0.05)), significantly differed. The amount of transplanted islet equivalents (IEQ: measure for islet volume) in the Tx-group was 9.6*105±3.5*105. The distribution volume (Vt) of the PET tracer was significantly higher in the Tx-group, indicating an increased retention of 68Ga-exendin in the liver i.e. the presence of intrahepatic islets (0.53±0.02 vs. 0.77±0.06, p=0.036). No significant correlation was found in the Tx-group between Vt and IEQ, neither between Vt and c-peptide production. Larger datasets will provide the ability to study possible correlations more extensively and could further strengthen our observation that hepatic uptake significantly differs between both groups.

Conclusions

The possibility to monitor islet grafts after transplantation would be of great use to further optimize islet transplantation and prolong islet survival. Our preliminary data of this explorative study indicate that dynamic PET imaging using 68Ga-labeled exendin is a highly promising tool to monitor pancreatic islet grafts in T1D patients. The relation between Vt and c-peptide production should be further investigated using larger datasets.

Distribution volume of gallium-68-labeled exendin in the liver of T1D patients
Distribution volume (Vt) of 68Ga-labeled exendin in the liver of T1D patients who are on the waiting list for islet transplantation (“Controls”, n=3) and who received intrahepatic islet grafts (“Transplanted”, n=5) determined on dynamic PET scans using the Logan model. Data was analyzed with the Mann-Whitney U test.
Keywords: Exendin-4, Dynamic PET imaging, islets of Langerhans transplantation, beta cell, Type 1 diabetes (T1D)
303

Investigating radiation-free MRI-CEST technique for the assessment of cancer metabolism in two tumor murine models (#356)

Annasofia Anemone1, Martina Capozza1, Chetan Dhakan2, 1, Sara Zullino1, Bhavna Rani1, Pietro Irrera3, 1, Paola Bardini1, Francesca Arena1, Giada Marini1, Enzo Terreno1, Silivo Aime1, Dario Longo1

1 University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy
2 Institute of Biostructure and Bioimaging (IBB), National Research Council of Italy (CNR), Napoli, Italy
3 University of Campania “Luigi Vanvitelli” , Italian National Research Counsil – Biostructure and Bioimaging Institute, Caserta, Italy

Introduction

Cancer metabolism is routinely assessed by the quantification of the FDG uptake by PET imaging, despite radiation exposure issues and reduced spatial resolution that limits heterogeneity assessment. The aim of this study was to exploit radiation-free MRI-CEST (Chemical Exchange Saturation Transfer) methods for investigating different aspect of tumour metabolism, including dysregulated glucose uptake following the injection of natural D-glucoseor of 3-O-Methyl-D-glucose2 and tumor acidosis following iopamidol administration3 in two murine models exhibiting distinct metabolic rate.

Methods

4T1 (mouse mammary carcinoma) and PC3 (human prostate cancer) cells were subcutaneously implanted in both flanks of female BALB/c and male Athymic Nude-Foxn1nu mice, respectively. CEST MRI protocol was performed on a Bruker 7T MRI scanner. Each mouse underwent i.v. injection of glucose (dose 5g/kg) or of 3OMG (dose 3g/kg) followed by Iopamidol injection (dose 4gI/Kg) 30 min later. Z-spectra before and after contrast media injections were acquired and CEST contrast was calculated between POST and PRE images. Contrast enhanced maps were calculated for glucose or 3OMG and tumor pHe maps for iopamidol. Two days after the MRI acquisition mice were kept fasted overnight and injected with 18F-FDG for PET imaging. GLUT1 and NHE1 expression were quantified by Western Blot.

Results/Discussion

The two tumour models showed a marked different FDG uptake: significantly higher uptake in 4T1 tumours than in PC3 tumours (SUV: 0.62 and 0.32; %ID/cc: 3.23 and1.15 for 4T1 and PC3 respectively, P<0.0001, Figure 1e,f and 2d,e). Similarly, CEST analysis revealed that the PC3 prostate tumour model displayed a lower glucose contrast in comparison to the 4T1 breast tumour model (ΔST% = 3.0 and 2.5 for 4T1 and PC3, respectively) and a significantly lower 3OMG uptake (ΔST% 2.0 and 1.5 for 4T1 and PC3, respectively, P=0.03, Figure 1b,g and 2a,b). Consistently with the higher glucose uptake, 4T1 tumour model presented a more acidic extracellular pH than the PC3 model, reflecting an increased tumour acidosis (mean pHe 6.7 and 6.8 for 4T1 and PC3, P=0.02, Figure 1c,d,h,i and 2c). Western blot analysis showed an increased expression of GLUT1 in 4T1 cell line compared to PC3, reflecting the higher glucose metabolism and tumor acidosis for the 4T1 breast tumor model (Fig. 2f) displayed in vivo.

Conclusions

These findings highlight a good characterization of different aspects of tumour metabolism by exploiting complimentary MRI-CEST approaches in comparison to PET imaging. Considering the relationship between increased glucose uptake and tumour acidosis, the metabolic MRI-CEST profiling of tumours may be a new radiation-free approach to distinguish differences in cancer aggressiveness and heterogeneity.

References

  1. van Zijl PC, et al. Proc Natl Acad Sci U S A. 2007,4359
  2. Navon G, et al. Magn Res Med 2018 79,1061
  3. Longo DL, et al.; Cancer Research 2016, 76, 6463

Acknowledgement

This work was supported by grants from European Union‘s Horizon 2020 research and innovation programme (GLINT project #667510), the Associazione Italiana Ricerca Cancro (AIRC MFAG #20153) and from Compagnia San Paolo project (Regione Piemonte, grant #CSTO165925).

Figure 1.
T2w images of representative 4T1 and PC3 tumour bearing mice (a,f). GlucoCEST map obtained after D-glucose (b) or 3OMG injection(g). Contrast enhanced maps upon injection of iopamidol as MRI-CEST ΔST% maps (c and h). pH map of each tumour model (d and i). Fused PET/CT axial view images of representative 4T1 and PC3 tumour bearing mice injected with 18F-FDG (e and j); data are expressed as % ID/cc.
Figure 2.

Average values calculated for each tumor model of a, Glucose ΔST% contrast b, 3OMG ΔST% contrast, c Tumor extracellular pH, d 18F-FDG PET uptake reported as standardized uptake values (SUV) and e 18F-FDG PET uptake reported as percentage of injected dose per cubic centimeter (%ID/cc). Bar graphs show mean ± SD. *: P <0.05, ***: P<0.0001. f, Western blot analysis in 4T1 and PC3 cell lines.

Keywords: D-Glucose, 3-O-Methyl-D-glucose, MRI-CEST, extracellular pH, PET
304

Towards Fast Deuterium Metabolic Imaging (#79)

Felix Kreis1, Alan J. Wright1, De-En Hu1, 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

Deuterium Metabolic Imaging (DMI), has great potential for quantitative imaging of tumour metabolism1,2, although there is a need to accelerate image acquisition for kinetic studies. We are working towards a pulse sequence that can image the dynamics of glucose metabolism following a bolus injection of d-[6,6’-2H2]glucose.

Methods

EL4 tumour-bearing mice were imaged in a 9.4 T MRI scanner. A 2D CSI sequence using Hamming window weighting for the number of averages per k-space point was employed. Total acquisition (TA) time was 54 min. For dynamic studies sequential 2H spectra were acquired. Both acquisitions were performed following injection of 2 g/kg d-[6,6’-2H2]glucose.

Experiments with an accelerated 3D CSI sequence were performed on a spherical phantom with three compartments containing 20 mM of [3,3-2H2]lactate,d-[6,6’-2H2]glucose and D2O respectively. The sequence used a 3D Hamming weighting (Fig. 2A). TA was 10 min. For each voxel the peaks were fitted individually using an AMARES implementation in MATLAB3.

Results/Discussion

2D chemical shift images show labelled glucose, lactate and water metabolite maps in the sensitive region of the surface coil, which covers the tumour(Fig. 1D-F). The time course of metabolite peaks extracted from spectra acquired serially is shown in Fig. 1B. First the glucose and then the lactate signals increased and then both of the labelled metabolites were flushed out of the tumour. The HOD peak increased throughout the experiment.

These data show that in order to capture the kinetics of glucose utilisation a time resolution of around 10 to 15 minutes is necessary. With that in mind we developed a fast 3D CSI pulse sequence (Fig 2). An image of a phantom using this sequence gave maps of deuterated metabolites at concentrations of 20 mM in 10 min (Fig 2D-F).

Conclusions

Serial acquisition of 2H spectra gave us the dynamics of glucose utilization in the tumour but not its precise localization. 2D CSI showed the labelled metabolite distribution but was to slow for dynamic measurements. We used this information to define the temporal and spatial demands of a dynamic imaging sequence. We demonstrated a fast 2H 3D CSI imaging sequence on a phantom and plan to use this soon for in vivo experiments.

References

  1. Lu, M., Zhu, X.-H., Zhang, Y., Mateescu, G. & Chen, W. Quantitative assessment of brain glucose metabolic rates using in vivo deuterium magnetic resonance spectroscopy. J. Cereb. Blood Flow Metab.37,3518–3530 (2017).
  2. De Feyter, H. M. et al.Deuterium metabolic imaging (DMI) for MRI-based 3D mapping of metabolism in vivo. Sci. Adv.4,eaat7314 (2018).
  3. Vanhamme, L., Van Den Boogaart, A. & Van Huffel, S. Improved Method for Accurate and Efficient Quantification of MRS Data with Use of Prior Knowledge. J. Magn. Reson.129,35–43 (1997).
in vivo DMI
A) Sum of the 80 spectra recorded in 88 min. B) Time course of the peak amplitudes. C) 1H reference image. D-F) Metabolite maps (36x36x10 mm3, matrix size of 12x12, TR=0.26 s, 12388 excitations, TA=54 min) acquired following injection of 2g/kg [6,6’-2H2]glucose. The colour scale represents the amplitudes of the fitted peaks. For all experiments a home-built 18 mm diameter surface coil was used.
Accelerated 3D 2H Chemical Shift Imaging
A) Averages per k-space position (10 to 128). B) Excitation and C) phase profile of the 50° BIR4 pulse. D-E): 3D CS images(FOV=27x27x27 mm3, matrix size=9x9x3, TR=0.14 s,256 points per spectrum, BW=2000 Hz), overlaid on a 1H image of the phantom. The colour scale represents the amplitudes of the fitted peaks.To acquire one CSI, the 4D k-space was sampled over 4328 transientsin a TA of 10 min.
Keywords: Deuterium Metabolic Imaging, DMI, Tumour Metabolism, MRI
305

MR spectroscopy to assess decreased tumor choline as a marker of response to choline kinase inhibitors (#416)

Claire L. Kelly1, Clementine Lesbats1, Sofya Osharovich2, Arthur Taylor1, Violaine See3, Edward J. Delikatny2, Harish Poptani1

1 University of Liverpool, Centre for Preclinical Imaging, Liverpool, United Kingdom
2 University of Pennsylvania, Department of Radiology, Philadelphia, Pennsylvania, United States of America
3 University of Liverpool, Centre for Cell Imaging, Liverpool, United Kingdom

Introduction

Increased total choline (tCho) has been observed in tumors1,2, manifested by an increase in phosphocholine (PC)1,4. A mediator of PC is an over-expression of Choline Kinase alpha (ChoKα)4 and Inhibition of ChoKα has shown promising results in vivo1,2,3, particularly the development of JAS239, a novel ChoKα inhibitor with inherent fluorescence1,5. JAS239 has shown efficacy in a breast cancer xenograft model1, however its efficacy in brain tumor models has not been reported. Our study investigated the effect of JAS239 on tumor growth and tCho using MRS and T2-weighted MRI.

Methods

Five Fischer F344 (120-130 g) female rats were injected with 5x104 F98 glioblastoma cells in the right cortex. Animals were injected intraperitoneally with 4 mg/kg JAS239 (n=3) or saline (n=2) for 5 consecutive days. Anatomical T2-weighted images and spectroscopy data were acquired at day 0 and day 5 after treatment. A single voxel of 2x2x2 mm3 spectrum was acquired using a PRESS sequence: TR= 2500 ms, TE1= 8.79 ms and TE2= 7.71 ms, number of averages= 256, complex points= 2048 and spectral width=4401 Hz. Effect of JAS239 on tumor growth was determined from tumour ROIs on T2-weighted images. Final volume was calculated by sum of all pixels and multiplied by section thickness. Metabolite concentrations were obtained using LC-Model software.

Results/Discussion

Control rats showed a 3.5 increase in tumor volume (Fig 1), compared to JAS239 treated animals which only demonstrated a 2.5-fold increase. In vivo MR spectra from the tumor and contralateral brain of a representative rat treated with JAS239 and from a control animal treated with saline are shown in Figure 2. A reduction in tCho peak was observed with JAS239 treatment (Fig 2B) compared to control (Fig 2C) and baseline values. Contralateral regions (Fig 2 E&F) show minimal change in tCho in control and treated animals before and after treatment. JAS239 selectively inhibits proliferating cells within the tumour and not in the normal brain. Percentage changes in tCho from baseline to post treatment are shown in Fig 2G. JAS239 treated animals exhibited a reduction (-24%) in tCho compared to control tumors which showed an increase (+22%).

Conclusions

The changes in tCho seem to be more pronounced than changes in tumor volume after JAS239, indicating not only the higher sensitivity of MRS in assessing treatment response, but that decreases in tCho may also be used as a pharmacodynamic marker of ChoKα inhibition.

References

  1. Arlauckas, S. P., Kumar, M., Popov, A. V, Poptani, H., & Delikatny, E. J. (2017). Near infrared fluorescent imaging of choline kinase alpha expression and inhibition in breast tumors. Oncotarget, 8(10), 16518–16530.
  2. Kumar, M., Arlauckas, S. P., Saksena, S., Verma, G., Ittyerah, R., Pickup, S., … Poptani, H. (2015). Magnetic resonance spectroscopy for detection of choline kinase inhibition in the treatment of brain tumors. Molecular Cancer Therapeutics, 14(4), 899–908.
  3. Mazarico, J. M., Sanchez-Arevalo Lobo, V. J., Favicchio, R., Greenhalf, W., Costello, E., Carrillo-de Santa Pau, E.,Real, F. X. (2016). Choline kinase alpha (CHK ) as a therapeutic target in pancreatic ductal adenocarcinoma: expression, predictive value, and sensitivity to inhibitors. Molecular Cancer Therapeutics, 15(2), 323–33
  4. Arlauckas, S. P., Popov, A. V., & Delikatny, E. J. (2016). Choline kinase alpha - Putting the ChoK-hold on tumor metabolism. Progress in Lipid Research, 63, 28–40.
  5. Arlauckas, S. P., Popov, A. V., & Delikatny, E. J. (2014). Direct inhibition of choline kinase by a near-infrared fluorescent carbocyanine. Molecular Cancer Therapeutics, 13(9), 2149–2158.
  6. Al-Saffar, N. M. S., Troy, H., De Molina, A. R., Jackson, L. E., Madhu, B., Griffiths, J. R., … Chung, Y. L. (2006). Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of the choline kinase inhibitor MN58b in human carcinoma models. Cancer Research, 66(1), 427–434.

 

Acknowledgement

All in vivo imaging was carried out in the Centre for Preclinical Imaging, University of Liverpool.

Figure 1.

Fold change in tumour volume from baseline.JAS239 treated tumours show a 2.5 fold increase compared to saline treated tumors, which showed a 3.5 fold increase in tumor volume.

Figure 2.

T₂-weighted images showing voxel placement in tumour (A) and contralateral regions(B). ¹H MR spectra showing JAS239 reduces tCho (B) compared to saline (C) after 5 days of treatment.¹H MR spectra of contralateral regions from treated (E) and untreated (F). % Change in tCho of JAS239 treated animals from baseline (G) show a 24% reduction in tCho.

Keywords: MRS, animal model, glioma
306

Visualizing the metabolism of glioblastoma patient-derived orthotopic xenografts by mass spectrometry imaging (#329)

Jyotsna Upendra Rao1, Katherine Gibson2, Gregory Hamm2, Alan Wright1, Maria Fala1, Richard Mair1, 3, Richard Goodwin2, Kevin M. Brindle1, 4

1 University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom
2 AstraZeneca, Pathology, Drug Safety and Metabolism, Cambridge, United Kingdom
3 University of Cambridge, Department of Clinical Neurosciences, Cambridge, United Kingdom
4 University of Cambridge, Department of Biochemistry, Cambridge, United Kingdom

Introduction

Glioblastoma multiforme (GBM) has a dismal prognosis with a mean survival of 15 months post-diagnosis. This poor prognosis has been attributed to extensive inter- and intra-tumoural heterogeneity at molecular and microenvironmental levels and tumour cell invasiveness. To understand the relationship between regional heterogeneity and metabolism, we employed Mass Spectrometry Imaging (MSI) on rapidly excised and freeze-clamped samples of GBM patient-derived orthotopic xenografts (PDOX) in rat brain following infusion of the animals with [U-13C]glucose.

Methods

Athymic nude rats (n=3) were infused with [U-13C]glucose (0.4 mg/g body weight bolus, 0.012 mg/g/min at 300 µL/h infusion1) for 40, 80 and 120 min followed by cardiac puncture to measure blood [U-13C]glucose levels using 13C NMR spectroscopy. A second set of rats (n=2) were implanted intracranially with patient-derived GBM cells (GB4). At 105 days post implantation, the presence of tumour was confirmed by T2-weighted MRI.  One animal was infused with [U-13C]glucose for 2 h and another with [12C]glucose, which was used as control for natural abundance 13C. The brains were then fixed by rapid freezing in liquid nitrogen followed by desorption electrospray ionization (DESI) MSI of coronal cryosections in negative ion mode (100 µm resolution).

Results/Discussion

After a 2 h infusion, 43% of the circulating glucose was 13C labelled. Metabolites of interest detected by DESI-MSI are reported in Table 1. Unsupervised spatial clustering of the MSI data resulted in segmentation of normal brain from tumour. Distribution of 12C and 13C labelled glucose, lactate and glutamate are shown in Figure 1.  The peritumoural area had high levels of [12C and 13C]glucose, with low intratumoural levels (Fig.1a,b) while [12C and 13C]lactate showed the opposite distribution, indicating that the rate of glucose utilization far exceeded the rate of delivery (Fig.1c,d). The peri-necrotic and peri-tumoural areas had higher levels of glutamate compared to the rest of the tumour (Fig.1e,f).  Based on these data, GB4 is predominantly glycolytic, as was shown by hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging in vivo2, but glutamate formation showed that in some tumour regions there is oxidative metabolism.

Conclusions

These results have demonstrated that a glioma PDOX is metabolically heterogeneous and different from surrounding brain tissue. Comparison of MSI data with histological staining of brain sections should yield additional information about the relationship between the tumour and its microenvironment.

References

1. Marin-Valencia, I., et al., Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse human glioblastomas in the mouse brain in vivo. Cell Metab, 2012. 15(6): p. 827-37.

2. Mair, R., et al., Metabolic Imaging Detects Low Levels of Glycolytic Activity That Vary with Levels of c-Myc Expression in Patient-Derived Xenograft Models of Glioblastoma. Cancer Res, 2018. 78(18): p. 5408-5418.

Acknowledgement

CRUK Grand Challenge Award 2015 (C197/A25040).

Table 1
List of m/z theoretical and measured values corresponding to metabolites of interest
Figure 1
Images of 12C and 13C labelled glucose (a,b), lactate (c,d) and glutamate (e,f) showing the distribution of the metabolites in GB4 GBM PDOXs.  The line represents 3 mm.
Keywords: glioblastoma, tumour heterogeneity, mass spectrometry imaging
307

Investigating metabolism and invasion of hepatocellular carcinoma cells during p53 re-activation induced senescence using a MR compatible cell perfusion system (#218)

Philipp Knopf1, 2, Jesus Pacheco-Torres2, Flonne Wildes2, Christoph Trautwein1, Benyuan Zhou1, Balaji Krishnamachary2, Lars Zender3, Bernd Pichler1, Zaver Bhujwalla2, 4, 5

1 Eberhard Karls University Tübingen, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Baden-Württemberg, Germany
2 The Johns Hopkins University, School of Medicine, Division of Cancer Imaging Research, The Russell H Morgan Department of Radiology and Radiological Science, Baltimore, Maryland, United States of America
3 Eberhard Karls University Tübingen, Department of Internal Medicine VIII, Tübingen, Baden-Württemberg, Germany
4 The Johns Hopkins University, School of Medicine, Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, United States of America
5 The Johns Hopkins University, School of Medicine, Department of Radiation Oncology and Molecular Radiation Sciences, Baltimore, Maryland, United States of America

Introduction

Cellular senescence is characterized by a stable cell-cycle arrest, whereas replicative, DNA damage-induced and oncogene-induced senescence can be distinguished being either beneficial or detrimental in tumors. The senescence associated secretory phenotype is a characteristic feature of senescent cells and can alter the metabolic and extracellular matrix degradative characteristics of p53 re-activation induced senescence in murine liver carcinoma cells.

We found alterations in creatine, phosphocreatine and glycerophosphocholine indicating differences in energy and phospholipid metabolism.

Methods

A murine liver carcinoma cell model, where p53 is silenced in the presence of doxycycline hyclate and senescence is induced within three days after doxycycline hyclate withdrawal and subsequent p53 re-activation was used.

Ex-vivo analysis of control and senescent H-Ras cells were performed on a high-resolution 1H NMR spectrometer (600 MHz) equipped with a triple resonance 5 mm TXI probe. Cell extracts were prepared using a 2-phase MeOH:MTBE solvent system.

A MR compatible cell perfusion was used for metabolic and degradative investigations over 48 h of H-Ras cells cultured on micro-carriers using a 9.4 T MR spectrometer [1]. T1-weighted 1H MR imaging was performed to visualize extracellular matrix degradation. Intracellular metabolite levels were derived from unlocalized DW 1H MR spectra.

Results/Discussion

Control H-Ras cell extracts were high in lactate, glycine and choline derivate peaks, whereas p53 re-activation induced senescent cells were high in alanine and creatine peaks. A principal component analysis showed a clear separation between the two groups.  

Using our MR compatible cell perfusion system, we found that p53-reactivation induced senescent hepatocellular carcinoma cells show a higher degradation index compared to control cells indicating an increase in ECM degradation.  Our data revealed alterations of metabolites in p53-reactivation induced senescent H-Ras cells compared to control cells, including creatine, phosphocreatine and glycerophosphocholine indicating differences in energy and phospholipid metabolism. 

Conclusions

Metabolite profiles of senescent and non-senescent H-Ras cells can be characterized using ex-vivo high-resolution 1H NMR spectroscopy of cell extracts. The use of a MR compatible cell perfusion system furthermore allows investigations of metabolic alterations together with dynamic quantification of ECM degradation during senescence of intact perfused cells. The study provides insights into the metabolic processes underlying senescence in cancer.

References

1. Ackerstaff E, Artemov D, Gillies RJ, Bhujwalla ZM. Hypoxia and the presence of human vascular endothelial cells affect prostate cancer cell invasion and metabolism. Neoplasia. 2007;9(12):1138-1151.

Acknowledgement

PK was supported by a grant from the German Academic Exchange Service (DAAD PPP USA 2018, Project-ID 57387312). Studies supported by NIH R35 CA209960.

Keywords: Senescence, MRI/MRS, 1H NMR, Cell Perfusion System, Oncogene-induced Senescence
308

[18F]Fluoride uptake in various bone types and soft tissues in rat (#243)

Nina Savisto1, Tove J. Grönroos1, 3, 5, Vesa Oikonen1, Johan Rajander2, Jörgen Bergman1, Sarita Forsback1, 4, Olof Solin1, 4, Merja Haaparanta-Solin1, 3

1 University of Turku, Turku PET Centre, Turku, Finland
2 Åbo Akademi University, Turku PET Centre Accelerator lab., Turku, Finland
3 University of Turku, MediCity Research Laboratory, Turku, Finland
4 University of Turku, Department of Chemistry, Turku, Finland
5 Turku University Hospital, Department of Oncology and Radiotherapy, Turku, Finland

Introduction

Fluorine-18 labeled sodium fluoride PET ([18F]NaF PET) produces high quality images due to high extraction of [18F]fluoride by bone. Importantly, all 18F-labeled PET-tracers are prone, to lesser or higher degree, to undergo defluorination, with subsequent release of [18F]fluoride. When developing new 18F-labeled tracers, it is important to assess the amount of released [18F]fluoride taken up in the bones of experimental animals. However, the pharmacokinetics of [18F]fluoride in bones and other organs of healthy experimental animals have not been well documented in a comprehensive manner.

Methods

We studied [18F]fluoride uptake in Sprague Dawley rat bones, including the epiphyseal parts of the tibia and radius, the mandible, ilium, lumbar vertebrae, costochondral joints, tibia, radius, and ribs, with 60-min in vivo PET/CT imaging. Kinetic parameters, K1, Ki, Ki/K1, and k3 were calculated with a three-compartment model. In addition, separate groups of male and female rats (n=6 for each of 6 time points) were studied with ex vivo tissue harvesting and gamma counting over a 6-h period.]

Results/Discussion

High 18F-radioactivity uptake was detected in the epiphyseal regions of long bones, the mandible, lumbar vertebrae, and parts of the pelvis. The mean bone perfusion rate (K1) was significantly higher (p = 0.035) at the tibia head and mandible compared to other bones. Bone osteoblastic activity (Ki) was significantly higher (p = 0.005) in the tibia head, mandible, and lumbar vertebrae, than in the tibia, radius, parietal bone, and ribs. The costochondral joints showed higher uptake than other parts of the ribs. The tibia, radius, parietal bone, and ribs showed the lowest uptake. Increased organ-to-blood ratios were detected in the eyes, testes, lung, brain, and ovaries. [18F]fluoride was mainly excreted via the kidneys, and at later time points, also via the gastrointestinal tract.

Conclusions

We showed that 18F-uptake was higher in trabecular bones, due to high perfusion and osteoblastic activity, compared to cortical bones. Understanding the pharmacokinetics of [18F] in various bones and tissues is highly useful for assessing 18F-labeled radiotracers that release [18F]fluoride. The same applies when [18F]NaF is used in human bone or calcification studies.

Acknowledgement

This study was supported by the Academy of Finland (project no. 266891), the State Funding for University Level Health Research for the Turku University Hospital (13250), and the University of Turku Foundation.

Keywords: PET, biodistribution, metabolism, bone perfusion, [18F]NaF
309

Metabolomics using NMR spectroscopy to detect IDH1/2 and ATRX in glioma  (#151)

Thomas W. Leather1, Marie Phelan1, Michael Jenkinson2, 1, Khaja Syed2, Nitika Rathi2, Eva C. Gutierrez1, Kumar Das2, Harish Poptani1

1 University of Liverpool, Cellular and Molecular Physiology, Institute of Translational Medicine , Liverpool, United Kingdom
2 The Walton Centre NHS Foundation Trust, Liverpool, United Kingdom

Introduction

The World Health Organization has revised the  guidelines for diagnosis of gliomas incorporating several molecular markers with diagnostic, prognostic and predictive value1, which rely on the availability of tissue specimen. 2-hydroxyglutarate (2-HG), has been proposed as a NMR detectable oncometabolite for mutations in the IDH1/2 gene, however no such markers have been proposed for other molecular alterations2-5. We have performed high resolution NMR of brain tumour samples and, using a metabolomics approach, aim to identify metabolic aberrations correlating with tumour molecular phenotype.

Methods

Tumour samples from 17 patients were collected during surgical resection and snap frozen in liquid nitrogen. Metabolites were extracted using a solution of 50% acetonitrile and 50% H2O and sonication, followed by centrifugation and the resulting supernatant was lyophilised. 1D 1H-NMR spectra were acquired using a CPMG pulse sequence on a Bruker 700MHz spectrometer. Metabolomic analysis was carried out using in-house scripts in RStudio. Spectral data were bucketed using AMIX and then samples grouped according to the molecular diagnosis. The groups consisted of IDH1/2 mutation, ATRX loss, 1p/19q co-deletion or MGMT methylation. Two-sided independent Welch test (p-values adjusted for multiple tests) were performed for each grouping parameter to identify metabolites of significant difference.

Results/Discussion

From the 17 tumours 1H NMR spectra significant differences were observed for multiple metabolite resonance peaks between IDH mutated and wild type tumours. In addition, significant differences were observed in tumours with ATRX loss or retention. No differences were noted in samples with 1p/19q codeletion and MGMT methylation versus the one’s without. Similar to previous findings, our results suggest that metabolomics may not only be useful for detecting IDH mutations, but also ATRX loss. The presence of the mutation has prognostic implications whereby those patients presenting low grade glioma with ATRX retention and IDH mutation have lower progression-free survival and overall survival than tumours with 1p/19q co-deletion and IDH mutation, as well as longer time to treatment failure than those patients with IDH mutation and ATRX retention10,11.

Conclusions

In addition to confirming 2HG as a marker for IDH mutations, our studies suggest that metabolomics may be able to identify tumours with ATRX loss and the elucidation of the role of this mutation in tumorigenesis and malignant progression. These results may lead to a deeper understanding of glioma progression in the presence of both IDH and ATRX mutation, as well as providing a potential biomarker for this molecular signature.

References

1.        Louis, D. N. et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 131, 803–820 (2016).

2.        Gliomas, I. L. et al. Magnetic Resonance of 2-Hydroxyglutarate in. Sci. Transl. Med. 4, 1–10 (2012).

3.        Losman, J. A. & Kaelin, W. G. What a difference a hydroxyl makes: Mutant IDH, (R)-2-hydroxyglutarate, and cancer. Genes Dev. 27, 836–852 (2013).

4.        Berrington, A. et al. Improved localisation for 2-hydroxyglutarate detection at 3T using long-TE semi-LASER. Tomogr.  a J. imaging Res. 2, 94–105 (2016).

5.        Juratli, T. A. et al. a Biomarker for Malignant Progression. 15, 682–690 (2013).

6.        Fuente, M. I. De et al. Neuro-Oncology. 18, 283–290 (2016).

7.        Yang, H. et al. IDH1 and IDH2 mutations in tumorigenesis: Mechanistic insights and clinical perspectives. Genes Dev. 27, 5562–5571 (2013).

8.        Xu, W. et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of ??-ketoglutarate-dependent dioxygenases. Cancer Cell 19, 17–30 (2011).

9.        Nandakumar, P., Mansouri, A. & Das, S. The Role of ATRX in Glioma Biology. Front. Oncol. 7, 1–8 (2017).

10.      Leeper, H. E. et al. IDH mutation, 1p19q codeletion and ATRX loss in WHO grade II gliomas. Oncotarget6, 30295–30305 (2015).

11.      Wiestler, B. et al. ATRX loss refines the classification of anaplastic gliomas and identifies a subgroup of IDH mutant astrocytic tumors with better prognosis. Acta Neuropathol. 126, 443–451 (2013).

Acknowledgement

Tissues samples were collected and kindly donated for this study by the Walton Centre NHS Foundation Trust.

All spectra were acquired at the University of Liverpool NMR Centre.

Figure 1.
Spectra obtained from IDH positive (mutated) and negative (wild type) glioma samples. The 2-HG peak is clearly identifiable at 2.25ppm, and assesment of further metabolite differences can aid in elucidating the progression of malignancy in glioma.
Keywords: Glioma, Metabolomics, IDH, ATRX, NMR
310

Optimising timing and animal handling for [18F]FDG-PET imaging in mice (#189)

Richard Hesketh1, 3, David Lewis1, 2, Kevin M. Brindle1, 4

1 University of Cambridge, CRUK Cambridge Institute, Cambridge, United Kingdom
2 Beatson Institute of Cancer Research, Glasgow, United Kingdom
3 University College London Hospitals NHS Foundation Trust, Department of Radiology, London, United Kingdom
4 University of Cambridge, Department of Biochemsitry, Cambridge, United Kingdom

Introduction

 [18F]FDG biodistribution is affected by many controllable variables including blood glucose concentration and body temperature(1). Dynamic PET imaging is often impractical, therefore static imaging is typically performed at a convenient, but essentially arbitrary, timepoint of 60 min post-injection. Here, by performing long dynamic scans of three different mouse tumour models following different animal handling conditions we provide a framework for an evidence based, preclinical static [18F]FDG-PET tumour imaging protocol.

Methods

C57BL6/J mice bearing subcutaneous EL4 tumours underwent 4 h dynamic [18F]FDG-PET imaging under four different animal handling conditions prior to anaesthesia: (i) fasted for 6 – 10 h and warmed in a 31 ºC chamber for 1 h; (ii)  fasted and not warmed (at room temperature); (iii) fed and warmed and (iv) fed and not warmed. Eµ-Myc mice that develop autochthonous lymphomas (fed and warmed) and BALB/c nude mice bearing subcutaneous Colo205 tumours (fasted and warmed) underwent 3 h dynamic scans. Animals were anaesthetised with isoflurane and respiratory rate and temperature monitored as 12.75 ± 4.2 MBq [18F]FDG was injected intravenously and images acquired using a Mediso NanoScan PET/CT scanner. Images were analysed using VivoQuant 3.0.

Results/Discussion

Twenty eight mice underwent dynamic PET imaging. In EL4 tumour-bearing mice, fasting caused a reduction in blood glucose from 8.3 ± 1.8 to 5.2 ± 1.4 (P = 0.003, n = 8 in each group). Warming prior to anaesthesia had no significant effect on myocardial or tumour [18F]FDG uptake. However, fasting resulted in a decrease in myocardial SUVmean from 13.5 ± 2.9 to 4.90 ± 1.67 (P = 0.0015) and an increase in tumour SUVmean from 4.09 ± 0.39 to 5.13 ± 0.4 (P = 0.01) (Figure 1).

            SUVmean and the tumour/blood ratio peaked at approximately 60 min post-injection in autochthonous Eµ-Myc tumours (Figure 2). However, subcutaneous EL4 and Colo205 tumours demonstrated slower uptake of [18F]FDG with peak SUVmean occurring at ≥ 90 min and tumour/blood ratios continued to increase throughout scans (≥ 170 min) for subcutaneous tumours under most animal handling conditions.

Conclusions

Mice should be fasted prior to imaging but warming prior to anaesthesia is unnecessary. Static PET imaging of autochthonous tumours can be performed >60 min post-injection. However, imaging of subcutaneous tumours should be delayed until at least 90 min post-injection of [18F]FDG to allow time for SUV to plateau and maximise tumour/background signal.

References

Fueger BJ, Czernin J, Hildebrandt I, Tran C, Halpern BS, Stout D, et al. Impact of animal handling on the results of 18F-FDG PET studies in mice. J Nucl Med 2006;47:999-1006

Acknowledgement

We would like to thank Matt Clayton, Mike Mitchell and Ryan Asby for their help with animal experiments. We would also like to thank the members of the PET-CT department at Addenbrookes’ Hospital for their generous provision of [18F]FDG.

Dynamic [18F]FDG uptake over 4 h in a fasted, warmed EL4 tumour-bearing mouse.
(Rows a & d) [18F]FDG maximum intensity projections. Solid blue arrow – inferior vena cava (at 10 s); outline white arrow – right kidney (at 55 s); solid white arrow – heart (240 min); solid yellow arrow – tumour (at 240 min); outline yellow arrow – bladder (240 min). Axial [18F]FDG-PET/CT slice through heart (rows b & e) and tumour (rows c & f).
4 h [18F]FDG-PET time activity curves for tumour bearing mice.
(a) SUVmean for different organs of EL4 tumour-bearing mice (n = 3) that were warmed and fasted prior to anaesthesia; (b) SUVmean for different tumour types under different animal handling conditions and (c) tumour/blood ratios over time for the different tumours and animal handling conditions.
Keywords: Animal handling, FDG-PET, Standardisation
311

1-[11C] Acetate Imaging of Membrane Biosynthesis in Glioma Patients (#586)

Mark Muzi1, Tao Lan2, Fengyun Gu2, Finbarr O’Sullivan2

1 University of Washington, Department of Radiology, Seattle, Washington, United States of America
2 University College Cork, Department of Statistics, Cork, Ireland

Introduction

1-[11C]-Acetate (Ac) has been used in prostate cancer, where it enters the TCA cycle as Acetyl CoA and offers the ability to estimate membrane biosynthesis. For Ac PET imaging, radioactivity in blood includes a significant amount of [11C]-CO2 (CO2) from body Ac metabolism contaminating the imaging signal. Separate injections of CO2 and Ac with compartmental modeling can account for CO2 contamination and estimation of Ac biosynthetic Flux [Muzi 2011]. A non-parametric modelling approach mapping Ac transport and metabolism was developed and evaluated relative to conventional ROI analysis.

Methods

Glioma patients (6 low, 6 high grade) underwent two-injection serial dynamic PET imaging of CO2 (60 min) followed by Ac (60 min) with arterial blood sampling. The metabolic imaging procedure involves representation of the complete voxel-level time-course as a positive linear combination/mixture of underlying time-courses (sub-TACs). Kinetic analysis of sub-TACs is used to construct a voxel-level tissue residue from which metabolic parameters are extracted. The sub-TAC kinetic modelling uses a dual tracer procedure with arterial input functions for CO2 & Ac. This allows the tissue impulse response (tissue residue function) for CO2 and Acetate to be determined. The Ac flux constant for membrane biosynthesis (KiAc) and other quantities were computed from these residues.

Results/Discussion

CO2 and Ac parameters in brain and glioma were comparable to prior reports. KiAc in gliomas (0.035 ± 0.010 mL/min/g) was higher than brain (0.017 ± 0.016 mL/min/g)(p<0.005). A sample case is shown in Fig 1(a). Close agreement is found between values of kinetic parameters recovered from tumor regions of interest (ROIs) versus values obtained in the conventional manner - by applying kinetic analyses to time-course recovered from those same ROIs. Correlations for KiAC are in excess of 0.95. Similar results are found for parameters describing the Ac volume of distribution and flow - see Fig 1(b). Further analysis was undertaken to evaluate the extent to which data from Ac PET imaging only (without the CO2 PET) might provide a flux variable that might be predictive of membrane biosynthesis. Our data also show a strong correlation (0.97) between the true KiAC and a value of flux recovered from analysis of raw Acetate tissue signal using the (CO2-corrected) Acetate blood input.

Conclusions

Uncorrected glioma Ac imaging shows high uptake in tumor, yet the image is contaminated with CO2. Dual CO2/Ac injections with methods that account for labeled CO2 metabolites can effectively estimate Ac membrane biosynthesis. Mixture modeling facilitates this metabolic mapping. There are significant computational advantages, not least the ability to map the complex membrane biosynthesis without reliance on uncertain kinetics modeling assumptions.

References

O’Sullivan F, Muzi M, Mankoff DM, O’Sullivan JN, Spence AM, Krohn KA.   Voxel-level mapping of tracer kinetics in PET  studies: a statistical approach emphasizing tissue life tables. Annals of Applied Statistics. 2014 Nov.

Muzi M, Link J, Shoner S, McLaughlin L, Mankoff D, O'Sullivan F, Krohn K. 1-[11C] Acetate model for membrane biosynthesis in glioma patients. Journal of Nuclear Medicine. 52:2026. 2011 May.

Acknowledgement

This work is supported by NIH grants R50-CA211270, P01 CA042045, R33-CA225310; and by Science Foundation Ireland Grant SFI-PI-11/1027.

Patient example of 11C-Acetate Imaging.
Patient example: Low-grade brain tumor with no contrast enhancement on MR T1+Gd. Dynamic imaging protocol is 11C-CO2 for 60 min, 11C-Acetate for 60 min then 18F-FDG for 90 min. Residue analysis generated parametric images of acetate metabolism accounting for 11C-CO2 contamination with similar values as those from compartmental model analysis of dynamic time-series data extracted from tissue ROIs.
Keywords: Membrane Biosynthesis, Glioma, PET, 1-[11C] Acetate

MRI and Multimodal I | New Probes

Session chair: Ali Barandov (Cambridge, US); Kristina Djanashvili (Delft, Netherlands)
 
Shortcut: PW12
Date: Thursday, 21 March, 2019, 12:45 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.

615

Monitoring cell proliferation by exploiting CEST-MRI methods (#228)

Giuseppe Ferrauto1, Enza Di Gregorio1, Daniela Delli Castelli1, Silvio Aime1

1 University of Torino, Molecular Biotechnologies and Health Sciences, Torino, Italy

Introduction

The monitoring of amministrated cells has a central role in regenerative medicine, with the aim of visualizing their localization and their fate (e.g. proliferation rate). Magnetic resonance Imaging(MRI) has been extensively used for this purpose, by using relaxation enhancers which allow detecting only one cell population. Conversely, Chemical Exchange Saturation Transfer(CEST) agents allows visualizing multiple cells populations. Herein, it is reported that the loading of Lanthanide Shift Reagents(SR) inside cells allows their MRI visualization and the monitoring of proliferation rate1,2.

Methods

Two different cell lines (i.e. murine macrophages J774A.1 and  murine breast cancer cells TS/A) have been labelled with paramagnetci shift reagents (Eu, Dy, Gd and Tm-HPDO3A) by hypotonic swelling and pinocytosis. CEST-MR images have been acquired at 7T and Saturation Transfer (ST%) effect has been measured. The effect of the chosen metal complex, number of cells and intracellular compartmentalization (cytoplasm or endosomes) on ST% has been exploited. A method for the assessment of cell proliferation rate by using CEST-MRI has been developped and applied both in vitr and in viivo, in subcutaouse murine tumors

Results/Discussion

The entrapment of the paramagnetic SRs into cells leads to the onset of a CEST effect in the 3-8 ppm range, depending on the metal. The intracellular water resonance is proportional to the the effective magnetic moment of the metal. Among the tested complexes, the best one is Dy-HPDO3A (highest magnetic moment) and the worse one is Eu-HPDO3A (lowest magnetic moment)(Fig.1).

The shift is higher when the complexes are inside endoeomes (e.g. upon macropinocytosis loading). When they are loaded in the cytoplasm (by using hypotonic swelling3), the shift is much lower. Dy-labelled cells are detectable when they are less than 10% of the total numer of cells in the analysed sample.

Cells proliferation index has been assessed (in vitro and in vivo) by evaluating the reduction of intracellualr water chemical shift in the days after the labeling. In fact, with mitosis, cells diluite their content of SRs, thus reducing the shift. An example of in vivo proliferation rate has been reported in Fig.2.

Conclusions

SR- labelled cells can be visualized by CEST-MRI, because of the occurrence of shift of intracellular water NMR signal. The shift is proportional to the intracellular concentration of the metal, to the metal magnetic susceptibility and it is affected by the probe localization. Cells proliferation processes can be monitored through the measurement of intracellular water chemical shift

References

1) Ferrauto G, et al. J. Am. Chem. Soc. 2014;136:638-41

2)Ferrauto G. et al. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2016; 8 :602-18

3) Di Gregorio E. et al. Contrast Media Mol Imaging. 2013;8:475-86

 

Figure 1

A) Comparison among ST profiles of cells labelled by pinocytosis with Dy- (blue), Eu- (red) or Tm-(green) HPDO3A at the same intracellular concentration. B) Relationship between the induced chemical shift and the effective magnetic moment of the Lanthanide. The number of Ln-HPDO3A/Cell is maintained constant for all the cellular samples to ca. 1.5x1010 Ln/cell.

Figure 2

A) In vivo visualization of Dy-labelled TSA cells (yellow arrow) 24h post injection by irradiating at 4.8 ppm. Unlabelled TSA cells have been injected as control (white arrow) ; B) Decreasing of chemical shift inside tumor region during the days post injection of Dy-labelled TSA cells.

 

Keywords: MRi, Chemical excahnge saturation trasnfer, cell labeling, paramagnetic complexes
616

Enzyme-driven multimodal detection of atherosclerosis with iron oxide nanoparticles (#277)

María Muñoz-Hernando1, 2, Paula Nogales1, Ana Victoria1, Juan Pellico5, Jesús Ruiz-Cabello6, 7, 8, Jacob F. Benzton1, 3, 4, Fernando Herranz2, 6

1 Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
2 Instituto de Química Médica. Consejo Superior de Investigaciones Científicas (IQM-CSIC), Madrid, Spain
3 Department of Clinical Medicine. Aarhus University, Aarhus, Denmark
4 Department of Cardiology. Aarhus University Hospital, Aarhus, Denmark
5 Department of Chemistry. University of Oxford., Oxford, United Kingdom
6 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES). , Madrid, Spain
7 CIC biomaGUNE., San Sebastian, Spain
8 Ikerbasque, Basque Foundation for Science. , Bilbao, Spain

Introduction

Atherosclerosis and its clinical complications are major constraints to living long and healthy lives. Therefore, a deeper understanding of the pathophysiology of the disease and tools capable of measuring disease activity are necessary. Phosphatidylcholine-specific phospholipase C (PC-PLC) and Sphingomyelinase are highly expressed in atherosclerosis lesions contributing to their progression. During this work, the enzyme-driven accumulation of Phosphatidylcholine (PC) and Sphingomyelin (SPH) iron oxide nanomicelles (IONM) in atherosclerotic plaques, is studied as a potential contrast agent.

Methods

PC-IONM and SPH-IONM, for T2 MR imaging, were synthesized in a two-step nanoemulsion synthetic protocol. Firstly, hydrophobic oleic-acid-coated Fe3O4 nanoparticles were prepared by thermal decomposition. Subsequently, using a nanoemulsion method, physiologically viable nanoparticles were made by encapsulation into micelles composed of amphiphilic PC or SPH molecules. The same protocol was used for the preparation of nanomicelles for fluorescence-based imaging except that 1mg of the fluorophore DilC18(5) was added to the nanoemulsion formulation.

After thorough sample characterization, the accumulation of the PC- and SPH-IONMs in atherosclerotic plaques was evaluated by MR in vivo and fluorescence ex vivo imaging, using atherosclerosis-prone ApoE knock out­ mice.

Results/Discussion

Hydrodynamic size distributions measured by dynamic light scattering (DLS) showed a narrow size distribution for all samples. Furthermore, relaxometric values yielded r2/r1 ratios suitable for T2-weighted contrast. PC-IONM and SPH-IONM showed aggregation when incubated with PC-PLC and Sphingomyelinase respectively. DLS showed an abrupt increase in their hydrodynamic size and core aggregation was observed by TEM imaging. Furthermore, the amount of phosphorous in the nanomicelles after the enzymatic reaction dropped, as shown by inductively coupled plasma mass spectrometry. Phosphorus is only present in the polar heads of the phospholipids in the nanomicelles, so its concentration is a direct measure of the cleavage effect that the enzymes have on their coating.

T2-MR in vivo imaging and fluoresce imaging showed accumulation of the micelles in the aortic arch of ApoE-/- mice. Inmmunohistochemistry showed a good correlation between IONM accumulation and enzyme expression.

Conclusions

We have fully characterized PC-IONM and SPH-IONM and demonstrated their selective physicochemical change in the presence of PC-PLC and sphingomyelinase respectively, two highly expressed enzymes in atherosclerosis lesions. Furthermore, T2-MR imaging and fluorescence imaging with the NM have shown the selective accumulation of this probe in the atherosclerotic plaque, making it a suitable probe for pathophysiology and activity characterization.

References

[1]        H. Li, L. Zhang, D. Yin, Y. Zhang, and J. Miao, “Targeting Phosphatidylcholine-Specific Phospholipase C for Atherogenesis Therapy,” Trends Cardiovasc. Med., vol. 20, no. 5, pp. 172–176, Jul. 2010.

[2]        J. Borén and K. J. Williams, “The central role of arterial retention of cholesterol-rich apolipoprotein-B-containing lipoproteins in the pathogenesis of atherosclerosis,” Curr. Opin. Lipidol., vol. 27, no. 5, pp. 473–483, Oct. 2016.

[3]        A. V. Lechuga-Vieco, H. Groult, J. Pellico, J. Mateo, J. A. Enríquez, J. Ruiz-Cabello, and F. Herranz, “Protein corona and phospholipase activity drive selective accumulation of nanomicelles in atherosclerotic plaques,” Nanomedicine Nanotechnology, Biol. Med., vol. 14, no. 3, pp. 643–650, Apr. 2018.

In vivo nanomicelles aggregation

Depiction of in vivo nanomicelles aggregation due to enzymatic degradation.

In vivo MR and fluorescence imaging
(a) In vivo MRI of the aorta an ApoE−/− mouse before and 24 h after injection of PC-IONM. NM accumulation denoted by white dot; (b) Histogram showing relative MRI signal change in aortic plaque areas identified in circular ROIs surrounding the lumen; (c) Ex vivo fluorescence image of C57BL/6 (control) and ApoE−/− mice aortas extracted 24 h after injection of Dil-PC-IONM.
Keywords: Atherosclerosis, MRI, Fluorescence, Nanomicelles, iron oxide nanoparticles
617

Smart liposomes for MRI-guided, light-triggered drug delivery (#132)

Friederike Reeßing1, 2, Marc Stuart2, Douwe Samplonius3, Rudi Dierckx1, Ben Feringa2, Wijnand Helfrich3, Wiktor Szymanski1, 2

1 University Medical Center Groningen, Department of Radiology, Groningen, Netherlands
2 University of Groningen, Stratingh Institute for Chemistry, Groningen, Netherlands
3 University Medical Center Groningen, Department of Surgical Oncolgy, Groningen, Netherlands

Introduction

Liposomes are well established as drug carriers in modern medicine. New, more selective and multimodal versions of these delivery systems are constantly being developed. Some of them can be tracked with different imaging techniques such as MRI. However, the common limitation is that generally only the localization but not the drug release can be monitored.1 Thus, our research focusses on the development of a liposomal MRI contrast agent (CA) responsive to light, envisioning the use of light-emitting targeting moieties accumulating in the disease tissue.2

Methods

We developed a smart CA,3 which - in its intact from - can be incorporated into liposomes. Photocleavage leads to the conversion of a macromolecular to a small, quickly tumbling contrast agent (Fig. 1A), causing a change in relaxivity.4–8 The photoactive core structure was synthesized via a Passerini multicomponent reaction.9 CryoTEM and an FFC relaxometry were used for the visualization of liposomes and acquisition of NMRD profiles. Visualization and quantification of Gd3+ was achieved by EDX spectroscopy and ICP-OES. Light-responsiveness was proven by UV-Vis analysis. Furthermore, we examined the effect of the formulation on different cell lines and finally proved the possibility of releasing cargo from the liposomes using calcein as a model compound.

Results/Discussion

Compound 1 was synthesized starting with the photoactive core structure, after which the anchoring group for liposomes and the Gd3+ ligand were introduced (Fig. 2A). 1 was successfully incorporated into liposomes with DOPC and the Gd3+ complex was formed. Upon irradiation  (λ = 400 nm) about 40% decrease in T1 relaxation rate was observed, with a substantial decrease already after 10 min (Fig. 2B). With progressing irradiation, the NMRD profile converges from the one of a macromolecular contrast agent to the one of a small molecule, following the same kinetics as observed in the UV Vis analysis of the photocleavage (Fig. 1C). The cargo release from the  liposomes upon irradiation was shown by a permeation assay monitoring the de-quenching of calcein fluorescence (Fig. 1D).10 The liposomal formulation did not exert cytotoxic effects on different cell lines neither before nor after pre-irradiation, showing the innocuousness of the photocleavage products (Fig. 1B).

Conclusions

An MRI CA has been designed, synthesized and evaluated in vitro for the use as a responsive CA for theranostic applications. Photo-activation leads to a substantial change in relaxation rate and content release and is free of toxic photo-products. This novel approach enables not only  imaging of the distribution of the drug delivery system, but also the cargo release from the carrier.

References

1.           Reeβing, F. & Szymanski, W. Following nanomedicine activation with magnetic resonance imaging: why, how, and what’s next? Curr. Opin. Biotechnol. 58, 9–18 (2019).

2.           Lammers, T., Aime, S., Hennink, W. E., Storm, G. & Kiessling, F. Theranostic Nanomedicine. Acc. Chem. Res. 44, 1029–1038 (2011).

3.           Reeßing, F., Stuart, C.A., Samplonius, D.F., Dierckx, R.A.J.O., Feringa, B.L., Helfrich, W. & Szymanski, W. Manuscript in preparation

4.           Wahsner, J., Gale, E. M., Rodríguez-Rodríguez, A. & Caravan, P. Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers. Chem. Rev. acs.chemrev.8b00363 (2018). doi:10.1021/acs.chemrev.8b00363

5.           Lacerda, S. & Tóth, É. Lanthanide Complexes in Molecular Magnetic Resonance Imaging and Theranostics. ChemMedChem 12, 883–894 (2017).

6.           Boros, E., Gale, E. M. & Caravan, P. MR imaging probes: design and applications. Dalt. Trans. 44, 4804–4818 (2015).

7.           Catanzaro, V. et al. A R 2p / R 1p Ratiometric Procedure to Assess Matrix Metalloproteinase-2 Activity by Magnetic Resonance Imaging. Angew. Chemie Int. Ed. 52, 3926–3930 (2013).

8.           Morrow, J. R. & Tóth, É. Next-Generation Magnetic Resonance Imaging Contrast Agents. Inorg. Chem. 56, 6029–6034 (2017).

9.           Szymański, W., Velema, W. A. & Feringa, B. L. Photocaging of Carboxylic Acids: A Modular Approach. Angew. Chemie Int. Ed. 53, 8682–8686 (2014).

10.        Shimanouchi, T. et al. Permeation of a β-heptapeptide derivative across phospholipid bilayers. Biochim. Biophys. Acta - Biomembr. 1768, 2726–2736 (2007).

 

Acknowledgement

The financial support from the Dutch Organization for Scientific Research (NWO VIDI grant no. 723.014.001 for W.S.) is gratefully acknowledged.

Figure 1
A) Schematic presentation of activation: The Gadolinium complex, incorporated into the liposomal bilayer, is released upon irradiation with λ = 400 nm light. B) The formulation does not exert any apparent cytotoxic effect on primary epithelial cell and HUVECs, neither before nor after photoactivation. Taxol and the cell medium were analyzed as positive and negative controls. C) The change in relaxivity (recorded at 10 MHz) follows the same kinetics as the decrease in absorbance at λ = 365 nm, representative for the photocleavage of the CA. D) Upon irradiation, calcein leaks out of the liposomes resulting in dilution and de-quenching of fluorescence.
Figure 2
A) Molecular structure of compound 1; B) NMRD profiles of intact liposomes and products of photocleavage after given irradiation times.
Keywords: theranostics, liposomes, MRI, responsive probes
618

Fluorinated paramagnetic contrast agents – Study of the paramagnetic effect from diverse lanthanide ions on fluorine relaxation times (#124)

Emilie Hequet1, Céline Henoumont1, Vera Djouana Kenfack1, Vincent Lemaur2, Roberto Lazzaroni2, Luce Vander Elst1, Robert N. Muller1, 3, Sophie Laurent1, 3

1 UMONS, NMR and Molecular Imaging Laboratory, Mons, Belgium
2 UMONS, Laboratory for Chemistry of Novel Materials, Mons, Belgium
3 CMMI, Center for Microscopy and Molecular Imaging, Gosselies, Belgium

Introduction

Medical imaging is a dynamic area of researches whose one of the goal is the elaboration of more efficient contrast agents (CA). Those agents need to be improved to optimize the detection of affected tissues such as cancers or tumours while decreasing the injected quantity of agents. The paramagnetic contrast agents containing fluorine atoms can be used both on proton and fluorine MRI.
This research field is therefore promising thanks to the ability to map the anatomy by 1H MRI and locate exactly the agents by 19F MRI.

Methods

One of the challenges in this domain is to synthesize a molecule containing several chemically equivalent fluorine atoms with short relaxation times to allow the record of 19F MR images in good conditions. In that aim, we propose to synthesize a CA containing a paramagnetic ion and nine chemically equivalent fluorine atoms by a cycloaddition reaction between two previously synthesized molecules. Initially, a derivative of DOTAGA macrocyclic ligand has been synthesized through a multistep synthesis. Diverse lanthanide ions have then been complexed in order to evaluate the more efficient to decrease the fluorine atoms relaxation time T1. Finally, a nonafluorinated compound has been synthesized and grafted on the DOTAGA derivative in order to obtain the final fluorinated complexes (figure 1).

Results/Discussion

The structure of the fluorinated paramagnetic contrast agents (Gd3+, Dy3+, Tb3+, Eu3+ complexes) were confirmed by mass spectrometry. Those fluorinated contrast agents were then characterized by 19F NMR where differences were observed on the fluorine relaxation times T1 and T2 depending on the lanthanide ion: Gd3+ induced a strong decrease of the relaxation times T1 and T2 whereas Eu3+ is nearly inefficient. On the other hand, Tb3+ and Dy3+ induced a moderate and appropriate decrease of each T1 and T2 (table 1). Moreover, no concentration dependence of T1 and T2 was observed for all the tested lanthanide ions, except for Tb3+ for which an important decrease of T1 and T2 was observed when the concentration increased. A ranking can then be made on the paramagnetic effect of each ion to fluorine atoms relaxation times based on their environment.

Conclusions

This study has shown the paramagnetic influence of several lanthanide ions on atoms situated close to them. Although the gadolinium ion has the highest paramagnetic effect, its influence can sometimes be too strong and decreases the relaxation times in a too significant way. An alternative can then be envisaged by the use of the dysprosium and terbium ions which allow to obtain appropriate relaxation times for clinical use.

Figure 1
Scheme of the target molecule
Table 1
Fluorine T1 and T2 relaxation times from the fluorinated molecule and the fluorinated complexes.
Keywords: Florine MRI, Lanthanide complex, Organic Chemistry, Proton MRI
619

Probing T1T2 interactions and their imaging implications through a thermally responsive nanoprobe (#83)

Juan Gallo1, Bethany Harriss2, Javier Hernandez-Gil2, Manuel Bañobre-Lopez1, Nicholas Long2

1 International Iberian Nanotechnology Laboratory, Life Sciences, Braga, Portugal
2 Imperial College London, Dept of Chemistry, London, United Kingdom

Introduction

The complex and specialised diagnostic process through magnetic resonance imaging (MRI) could be simplified with the implementation of dual T1T2 contrast agents. T1- and T2-weighted MR are compatible modalities, and co-acquisition of contrast enhanced images in both T1 and T2 will drastically reduce artefacts and provide double-checked results. To date, efforts in the development of dual MRI probes have provided inconsistent results.[1–4]

Methods

In this work a new probe in which the distance between paramagnetic and superparamagnetic moieties can be controlled externally and within a single probe, was thoroughly studied. We report the preparation and full characterisation of manganese doped magnetite (Fe3O4) nanoparticles coated with a SiO2 layer and further decorated with a custom modified gadolinium containing poly(N-isopropylacrylamide) (pNIPAM) shell (Scheme 1). This system was then fully characterised from a relaxometric point of view (1.41 T) and relaxivity values were translated to MR images at a clinical field strength (3.0 T).

Results/Discussion

A thorough series of nanoparticles incorporating a paramagnetic and a superparamagnetic moiety separated by a temperature sensitive polymer were prepared and studied. The effect of the incorporation of the paramagnetic species on the longitudinal and transversal relaxivities, was dependent on the minimum possible distance between moieties. Varying this minimum distance by means of a silica layer, optimal relaxivities as high as 24 and 732 mM-1 s-1 (r1 and r2 respectively) were obtained. While the effect of the temperature on the longitudinal relaxivity was the expected one (an increase in r1 was recorded for all samples, coming from the change in molecular correlation time, τR, as reported for this kind of polymeric systems[5]), the transversal relaxivity decreased in all cases.

Conclusions

A new probe in which the distance between para and superparamagnetic moieties can be controlled externally within a single probe, was thoroughly studied. Relaxivity changes produced by these samples, were in most cases unpredictable, but a closer look allowed to explain these results based on simple models. There is an optimal distance between para and superparamagnetic moieties in order to obtain enhanced r1/2 performance.

References

[1]      C.-C. C. Huang, C.-Y. Y. Tsai, H.-S. S. Sheu, K.-Y. Y. Chuang, C.-H. C.-H. H. Su, U.-S. S. Jeng, F.-Y. Y. Cheng, H.-Y. Y. Lei, C.-S. S. Yeh, C.-H. C.-H. H. Su, et al., ACS Nano 2011, 5, 3905–3916.

[2]      N. A. Keasberry, M. Bañobre-López, C. Wood, J. Graeme, J. Gallo, N. J. Long, Nanoscale 2015, 7, 16119–16128.

[3]      H. Yang, Y. Zhuang, Y. Sun, A. Dai, X. Shi, D. Wu, F. Li, H. Hu, S. Yang, Biomaterials 2011, 32, 4584–93.

[4]      K. H. Bae, Y. B. Kim, Y. Lee, J. Hwang, H. Park, T. G. Park, Bioconjug. Chem. 2010, 21, 505–12.

[5]      B. Jagadish, G. L. Brickert-Albrecht, G. S. Nichol, E. A. Mash, N. Raghunand, Tetrahedron Lett. 2011, 52, 2058–2061.

Acknowledgement

This work was partially supported by POCTEP (Operational Programme for Cross-border Cooperation Spain-Portugal), and by the P.O Norte CCDR-N/ON.2 programme. JG acknowledges the financial support from the Royal Society of Chemistry for a Researcher Mobility award. NJL acknowledges the EPSRC and Imperial College London for a PhD Scholarship (to BH).

Schematic representation of the dual thermoresponsive probe developed in this work
The distance between superparamagnetic and paramagnetic moieties can be modified externally within a single probe through temperature changes.
Keywords: MRI, magnetic nanoparticles, Gd chelates, Dual modal imaging, Thermoresponsive probes
620

Biodistribution and fate of fluorinated nanoparticles followed by 19F MRS and ICP-MS. (#145)

Daniel Padro1, Abraham Martín2, 3, Carlos Sanchez-Cano1, Monica Carril4, 3

1 CIC biomaGUNE, Donostia, Spain
2 Achucarro Basque Center for Neuroscience, Leioa, Spain
3 Ikerbasque, Basque Foundation for Science, Bilbao, Spain
4 Instituto Biofisika (CSIC-UPV/EHU), Leioa , Spain

Introduction

One of the main issues when using nanoparticles (NPs) as probes is to know their biodistribution and fate upon injection. This is controlled by the NPs’ shape, size, charge and nature. In case NPs get digested, the degradation products are likely to have different excretion paths. For inorganic NPs, ICP of ex vivo tissues is frequently used to assess NPs’ accumulation in organs, however, such technique is only providing information regarding one of the constituents of the NPs. Hence, an approach based on several techniques is required to have a full picture of the fate of each part of the NP.

Methods

Fluorinated ligands and fluorinated gold NPs (F-NPs) were prepared following a procedure previously reported by us, in which thiol ending fluorinated ligands are mixed with HAuCl4 and NaBH4. After purification, those NPs were fully characterised by TEM, UV-Vis, NMR and ICP-MS. For in vivo 19F MRS experiments, 5 mice were injected with so-obtained F-NPs and 19F MRS spectra were acquired every 20 minutes up to a maximum of 2 hours and a half. Subsequently, mice were sacrificed, organs extracted, lyophilised, digested and analysed by ICP-MS, looking for gold content. For ex vivo 19F MRS imaging, 2 mice were injected with fluorinated NPs and sacrificed 2 hours and a half after injection, organs were extracted after perfusion and 19F MRS spectra of each organ was acquired separately.

Results/Discussion

We prepared F-NPs of 3 nm (core diameter, TEM) and 18 nm (hydrodynamic diameter, 19F DOSY). 19F NMR spectrum shows a single narrow peak at -71.38 ppm and both T1 and T2 values are close to 1 s. F-NPs were injected (i.v.) and 19F MRS spectra were collected every 20 min. Signal increased with time, as NPs got distributed. Likewise, 2 mice were equally injected with F-NPs and sacrificed 2.5 h after injection. Organs were extracted and ex vivo 19F MRS spectra of them were acquired. Signal was mainly detected in liver, but also a bit in urine and kidneys. ICP-MS data showed that spleen accumulated the highest amount of gold per gram of organ, however no 19F MRS signal was detected there, which suggests that either NPs are too aggregated for 19F detection or the fluorinated ligand came off. In absolute numbers, the most loaded organ is liver, in which most of fluorine signal was detected. There was little gold in urine or kidney, suggesting that the 19F signal might come from unbound ligand.

Conclusions

Multiple labelling and the use of several techniques is key to achieve a greater understanding of the fate of NPs in vivo, which is needed to assess their value as tools for nanomedicine, such as their use as contrast agents. The data combination from 19F MRS and ICP-MS allows us to hypothesise about the fate of the components of our nanomaterial. Further experiments are currently being conducted to corroborate the findings presented herein.

References

O. Michelena et al. Chem. Commun. 2017, 53, 2447.

M. Carril et al. Nat. Commun. 2017, 8, 1542.

Acknowledgement

Parts of this work were funded by MINECO (CTQ2015-68413-R. MC acknowledges Ikerbasque for a Research Associate position. CSC acknowledges Diputacion de Guipozcoa for a Gizpuzkoa Fellowship.

F-NPs biodistribution by 19F MRS and ICP-MS
A) F-NPs structure and characterization by TEM and 19F NMR. B) In vivo 19F MRS and ICP-MS experiments.
Keywords: 19F MRS, fluorinated nanoparticles, biodistribution, ICP-MS
621

Synthesis of a bimodal contrast agent for magnetic resonance imaging and photoacoustic imaging (#238)

Marie Devreux1, Céline Henoumont1, Fabienne Dioury3, Sophie Laurent1, 2

1 UMons, NMR and molecular imaging, Mons, Belgium
2 CMMI, Charleroi, Belgium
3 CNAM, Paris, France

Introduction

One of the most used techniques to obtain anatomical information is magnetic resonance imaging (MRI). Although its high resolution, this method has a low sensitivity that can be solved by using a bimodal system where MRI is associated with a more sensitive technique, such as photoacoustic imaging (PAI). In this work, a bimodal contrast agent has been developed. The probe used for MRI is a Gd-PCTA derivative and that used for PAI is a chromophore ZW800-1, derivated from green indocyanine. The two probes have been covalently associated via a spacer based on L-lysine.

Methods

The synthesis starts with a lysine derivative. The protecting groups can be removed selectively to graft the different probes. Different syntheses have been performed to optimize the coupling reaction. The lysine has been functionalized with Gd-PCTA and then with ZW800-1 (figure 1). All of the synthesis intermediates have been characterized by 1H and 13C NMR and mass spectrometry.

Results/Discussion

The mass spectrum of the isolated compound is in agreement with the theoretical spectrum of the desired compound. This spectrum confirms the presence of the gadolinium complex. After the synthesis of the final product, Gd-PCTA-Lys (ZW800-1)OAl, relaxometry measurements were performed to highlight the efficacy of the MRI probe.

The NMRD profile shows a decrease of the relaxivity of the gadolinium complex after the linker coupling, which can be explained by the loss of a coordinated water molecule confirmed by 17O NMR. On the other hand, the coupling with the fluorophore causes a clear increase of the relaxivity which could result from π-stacking interactions confirmed by a modification of the rotational correlation time.

Conclusions

This synthesis allows to obtain a bimodal probe for MRI and photoacoustic imaging. In a close future, in vivo studies will be done and the grafting of specific biovectors will be optimized to prepare a specific probe for molecular imaging of inflammation.

References

(1)M. Port et al., Contrast Media Mol. Imaging, 1, 3, 121–127, 2006

(2)H. S. Choi et al.,Angew Chem Int Ed Engl., 6258–6263, 2011.

Acknowledgement

This work was performed with the financial support of the FNRS, the ARC, the Walloon Region (Gadolymph, Holocancer and Interreg projects), the Interuniversity Attraction Poles of the Belgian Federal Science Policy Office and the COST actions. Authors thank the Center for Microscopy and Molecular Imaging (CMMI, supported by European Regional Development Fund and Wallonia)

Synthesis of the bimodal probe
Synthetic scheme of the bimodal probe. (Figure 1)
NRMD profils
NMRD profiles at 310 K. The
straight lines correspond to the theoretical fitting
according to the theory of Solomon and
Bloembergen. (Figure 2)
Keywords: MRI, PAI, Contrast Agent, Bimodal Probe, Organic Synthesis
622

Long term fate and time dependent contribution to MRI relaxivity of 57Fe-based iron oxide nanoparticles as assessed by combined MRI and Mass Spectrometric Imaging (#219)

Max Masthoff1, Andre Beuker1, Rebecca Buchholz2, Lydia Wachsmuth1, Walter Heindel1, Uwe Karst2, Moritz Wildgruber1, Cornelius Faber1

1 Medical Faculty - University of Muenster - and University Hospital Muenster, Institute of Clinical Radiology, Muenster, North Rhine-Westphalia, Germany
2 University of Muenster, Institute for Inorganic and Analytical Chemistry, Muenster, North Rhine-Westphalia, Germany

Introduction

Iron oxide nanoparticles (ION) are common MRI contrast agents providing high sensitivity even to detect single labelled cells1, but signal reductions cannot easily be separated from those originating from endogenous iron. We aimed to investigate long term fate and time dependent contribution to MRI relaxivity of 57Fe-based iron oxide nanoparticles as assessed by combined MRI and Mass Spectrometric Imaging. We further investigated contribution of applied ION and endogenous iron sources to MRI T2 signal alterations in an inflammatory cell tracking model.

Methods

Healthy mice were injected with customized 57Fe-ION. For ION distribution T2 mapping of liver, spleen, kidneys and brain was performed on a 9.4T small-animal MRI 2h up to 90d after injection (n=30). To study ION contribution to T2 with regards to applied dose, we injected healthy mice with increasing ION dosage (n=21). Mice were sacrificed and organs extracted for laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to quantify 56Fe and 57Fe. To evaluate ION contribution to T2 in cell tracking, we induced local inflammation by s.c. injection of polyacrylamide-gel (pellet)2. T2 mapping of the pellet was performed as baseline, followed by i.v. injection of either 57Fe-ION or PBS as control (n=3 each). After 24h second MRI was performed followed by histology and LA-ICP-MS.

Results/Discussion

Combining 57Fe-ION MRI with LA-ICP-MS enabled to specifically assess and resolve local distribution in the long-term fate of applied ION as shown in liver, spleen, kidneys and brain (Fig.1). Most ION were internalized by macrophages in liver and spleen. 57Fe-signal was still found after 90d, but mainly relocated to endogenous iron stores especially in spleen (Fig.2), blood and, although low in the overall amount, within brain parenchyma (Fig.1d), indicating transfer of applied iron to endogenous sources.

A non-linear dependence of T2-relaxivity on increasing ION dosage was observed in the liver, most likely resulting from ION packing, location and state during metabolic processing. T2-relaxivity in the cell tracking model was mainly influenced by applied 57Fe-ION, which were found in adjacent inflammatory tissue representing invaded labelled cells, and not by endogenous iron sources. Thereby, the technique enabled for quantifiable, specific and validated MRI ION cell tracking.

Conclusions

Using 57Fe-ION we were able to show the long term fate of applied ION, showing transfer of applied iron to endogenous sources. Time and dose dependence of ION contribution to MRI signal was shown. Regarding cell tracking, the technique enabled to specifically attribute T2 signal alterations to migrated labelled cells and to distinguish from iron of other sources, facilitating quantification and validation of MRI ION cell tracking studies.

References

1.            Masthoff M, Gran S, Zhang X, et al. Temporal window for detection of inflammatory disease using dynamic cell tracking with time-lapse MRI. Scientific reports. Jun 22 2018;8(1):9563.

2.            Schmidt R, Nippe N, Strobel K, et al. Highly shifted proton MR imaging: cell tracking by using direct detection of paramagnetic compounds. Radiology. Sep 2014;272(3):785-795.

Acknowledgement

This study was supported by the German Research Foundation DFG through CRC1009 Z02 and Cluster of excellence cells in motion EXC1003, the Interdisciplinary Center for Clinical Research Münster core unit PIX, and the Medical Faculty Münster MedK program.

Fig.1: 57Fe-ION enable to study long-term fate of ION

MRI T2 and mass-spectrometric results of 57Fe and total iron (57Fe+56Fe) at 2h, 1d, 3d, 7d, 30d, 90d after 57Fe-ION injection are shown for a) liver, b) spleen, c) kidney, and d) brain. In the long term, 57Fe is mainly found in the spleen. Interestingly the brain shows increasing 57Fe in the long term.

Fig.2: LA-ICP-MS quantitatively reveals local distribution changes of applied 57Fe over time

Quantitative LA-ICP-MS 57Fe distribution maps are shown for the spleen a) 3d, b) 30d c) 90d after i.v. injection of 57Fe-ION. LA-ICP-MS quantitatively reveals a transfer over time of applied 57Fe from the marginal zone of the spleen to the red pulp of the spleen, where endogenous iron stores can be found. In the long term more and more 57Fe is stored in these endogenous iron sources.

Keywords: MRI, Mass Spectrometry, Iron oxide nanoparticles
623

Improving the safety of Magnetic Resonance Imaging contrast agents via careful ligand design: new Mn(II) and Gd(III) complexes (#354)

Gyula Tircsó1, Zoltán Garda1, Kristóf Póta1, Ferenc K. Kálmán1, Do N. Quyen2, Zoltán Kovács3, Eva Jakab-Toth4, Imre Tóth1

1 University of Debrecen, Department of Physical Chemistry, Debrecen, Hungary
2 University of Texas Southwestern Medical Center, Department of Radiology, Dallas, Texas, United States of America
3 University of Texas Southwestern Medical Center, Advanced Imaging Research Center,, Dallas, Texas, United States of America
4 CNRS, Center of Molecular Biophysics, Orléans, France

Introduction

The link established between a rare but severe and potentially fatal fibrosis of the skin and internal organs that can arise in renally impaired patients called nephrogenic systemic fibrosis (NSF) and the use of gadolinium (Gd) based contrast agents (CA) in magnetic resonance imaging (MRI) pointed out that new agents possessing better in vivo stability is still very challenging. We believe this can be achieved either by improving the dissociation kinetic parameters of the Gd(III) complexes or by using biologically more accepted, possibly essential paramagnetic metal ions like Mn(II).

Methods

Two ligands (1,4-DO2MA and PCTMA) were synthesized based on cyclen (having 6 donor atoms for Mn(II) complexation) and pyclen (possessing 7 donor atoms for Gd(III) complexation) by using simple organic synthesis. Mn(II) and Gd(III) complexes of the ligands were prepared and their stability (by using pH-potentiometry and 1H-relaxometry), formation kinetics (1H-relaxometry and UV-vis spectrophotometry), rates of solvent exchange reactions (17O-NMR) and rates of dissociation (UV-vis spectrophotometry) were studied. The physico-chemical data obtained were compared with those obtained for the corresponding 1,4-DO2A (Mn(II)) and PCTA (Gd(III)) complexes.

Results/Discussion

The stability constants of the complexes formed with essential (Ca(II), Mg(II), Cu(II) and Zn(II)) and Mn(II) or Gd(III) were slightly higher compared to the parent metal chelates of acetate derivatives, which can be ascribed to the hyperconjugative electron donating effect of the methyl groups present on the side arms. The steric hindrance brought t by these methyl groups resulted in a slower formation and dissociation rates for the Mn(II) as well as for Gd(III) complexes, while the given structural modification had very little impact on the solvent exchange rates as well as relaxivities of the Mn(II) or Gd(III) complexes as compared to [Mn(1,4-DO2A)] and [Gd(PCTA)], respectively.[1, 2]

Conclusions

The replacement of the acetate pendant arms in 1,4-DO2A and PCTA with alpha-methyl acetate metal binding moieties (1,4-DO2MA and PCTMA) improves the dissociation kinetics of the Mn(II) and Gd(III) complexes, respectively. Altogether the 1,4-DO2MA chelator has some potential for Mn(II) ion complexation while the PCTMA ligand can be regarded as a very promising candidate for the development of stable, inert and high relaxivity Gd(III)-based MRI CA.

References

[1] Z. Garda, A. Forgács, Q. N. Do, F. K. Kálmán, S. Timári, I. Tóth, Zs. Baranyai, L. Tei, Z. Kovács, Gy. Tircsó, J. Inorg. Biochem. 2016, 163, 206-213.

[2] Gy. Tircsó, Z. Kovács, A.D. Sherry, Inorg. Chem., 2006, 45(23), 9269.

Acknowledgement

This research was funded by the GINOP-2.3.2-15-2016-00008 project supported by the EU and co-supported by the European Regional Development Fund. The authors are grateful for support granted by the Hungarian National Research, Development and Innovation Office (NKFIH K-120224 project) and for the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (Gy.T. and F.K.K.). The research was also supported by the ÚNKP-18-4 New National Excellence Program (Gy.T. and F.K.K.) of the Ministry of Human Capacities.

Figure 1. Structure of the ligands discussed in this work
Structure of the ligands mentioned in the text.
Keywords: Safety, Stability, inertnes, relaxation, agents
624

Cu-doped iron oxide nanoparticles with large longitudinal relaxivity; one-pot synthesis and in vivo tumour detection by T1 magnetic resonance imaging (#55)

Irene Fernández-Barahona1, 2, Lucía Gutiérrez3, Sabino Veintemillas4, Juan Pellico5, 6, Ignacio Rodríguez2, 6, Jesús Ruiz-Cabello2, 6, 7, Fernando Herranz1, 6

1 Instituto de Química Médica. Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
2 Departamento de Química Física II. Facultad de Farmacia. Universidad Complutense de Madrid, Madrid, Spain
3 Departamento de Química Analítica. Instituto de Nanociencia de Aragón. Univerisdad de Zaragoza, Instituto de Ciencia de Materiales de Aragón (ICMA-CSIC) and CIBER-BBN, Zaragoza, Spain
4 Instituto de Ciencia de Materiales de Madrid. Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain
5 Department of Chemistry. University of Oxford, Oxford, United Kingdom
6 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain
7 CIC biomaGUNE, Ikerbasque. Basque Foundation for Science, San Sebastián, Spain

Introduction

Iron oxide nanoparticles (IONPs) have been traditionally studied as a T2 contrast agent for MRI due to their superparamagnetic behaviour. Nevertheless, T1-based positive contrast, being much more advantageous for clinical applications, is still limited to gadolinium- or manganese-based imaging tools. Recently, we have shown how microwave-driven synthesis of citrate-coated IONPs renders large r1 values.1 In this work, we have studied the effect of Cu doping on the physicochemical, magnetic, relaxometric; and in vivo properties of IONPs in both angiography and targeted molecular imaging.

Methods

Synthesis of Cu-doped IONPs (CuNP) was performed following a microwave-driven protocol. A mixture of FeCl3·6H2O, citric acid trisodium salt and different quantities of CuCl2 was dissolved in water. Subsequently, hydrazine monohydrate was added and the mixture was introduced into the microwave. Samples were heated under vigorous stirring for 10 minutes at 120 °C. Once this step was completed, nanoparticles were purified through a gel filtration column.

After thorough sample characterisation, MR contrast agent capability of samples with improved relaxometric properties were tested in vivo in tumour-bearing mice. Specific tumour accumulation was achieved by conjugating the nanoparticles, via EDC and sulfo-NHS chemistry, to RGD peptide that specifically binds avb3 integrin.

Results/Discussion

Hydrodynamic size distributions measured by dynamic light scattering show a narrow size distribution for all samples. Zeta potential values are around -34 mV as expected for citrate coated nanoparticles. Core size of the nanoparticles, measured by STEM, shows a slight progression with amount of Cu-doping. Magnetisation curves for all samples show a superparamagnetic behaviour. Relaxometric properties of the samples were studied to assess how they were affected by Cu doping. Longitudinal relaxivity value improved for 1.7% mol (Cu1.7-NP) and 4% mol Cu doping (Cu4-NP) with respect to non-doped IONPs, while transversal relaxivity value was maintained low, yielding r2/r1 ratios suitable for T1-weighted contrast. Sample Cu4-NP was used in in vivo MR experiments as blood pool contrast agent for angiography and for targeted-tumour detection; proving its improved T1 contrast agent capability and its potential use in vivo for different biomedical applications.

Conclusions

We have fully characterised the Cu-doped nanoparticles and demonstrated the in vivo performance of this nanomaterial, both as a blood pool contrast agent and for targeted molecular imaging; key for further development and applications. The relaxometric values, the type of synthesis and the in vivo performance make Cu4-NP a remarkable candidate for future clinical translation.

References

1. Pellico, J. et al. One-Step Fast Synthesis of Nanoparticles for MRI: Coating Chemistry as the Key Variable Determining Positive or Negative Contrast. Langmuir 33, 10239–10247 (2017). doi:10.1021/acs.langmuir.7b01759

Acknowledgement

This study was supported by a grant from the Spanish Ministry for Economy and Competitiveness (MEyC) (SAF2016-79593-P) and Instituto de Salud Carlos III (DTS16/00059). L.G. received financial support from the Ramoń y Cajal subprogram (RYC-2014-15512)

Figure 1. Synthesis and Characterisation of Cu-NPs
a) Synthetic scheme for Cu1.7-NP, Cu4-NP and Cu28-NP; b) Hydrodynamic size distribution, measured by DLS; c) Core size measured by STEM (mean with standard deviation, N=30, P 0.01); d) Zeta potential for undoped iron oxide nanoparticles (IONP), Cu1.7-NP, Cu4-NP and Cu28-NP; e) FTIR spectra; and f) Thermogravimetric curves for Cu1.7-NP, Cu4-NP and Cu28-NP.
Figure 2. Relaxometric properties of CU-NPs and in vivo MRI
Relaxation rate against iron and copper concentration for a) Cu1.7-NP; b) Cu4-NP; c) Cu28-NP; d) T1 MRI body angiography in healthy mice; and e) MRI of breast tumour-bearing mice allografts 1h after i.v. injection of Cu4-NP and Cu4-NP-RGD. Left panels show normal contrast images, right panels obtained setting muscle intensity to zero to highlight possible increase in the tumour signal.
Keywords: iron oxide nanoparticles, T1-weighted MRI, Cu doping

PET/SPECT/CT & Multimodal | New Probes II

Session chair: Wiktor Szymansky (Groningen, Netherlands); Jason Lewis (New York, US)
 
Shortcut: PW13
Date: Thursday, 21 March, 2019, 12:45 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.

644

68Ga and MMAF dual-labelled single-chain variable fragments (scFv) for PET imaging of HER2 overexpressed tumors (#328)

Ruisi Fu1, 2, Marta Braga2, Laruence Carroll2, Ioanna Stamati3, Gokhan Yahioglu3, Eric Aboagye2, Philip Miller1

1 Imperial College London, Department of Chemistry, London, United Kingdom
2 Imperial College London, Department of Surgery and Cancer, London , United Kingdom
3 Antikor, Stevenage Bioscience Catalyst, Stevenage, United Kingdom

Introduction

Single-chain variable fragments (scFv) are small (28 kDa) antibody fragments that have been demonstrated to display excellent targeting and pharmacokinetics for both molecular imaging and drug delivery.1 Loaded antibody fragments with drug ‘pay-loads’ can, however, alter their pharmacokinetics.2  Herein, we aim to develop a novel 68Ga-radiolabelled scFv (TCT1067) and drug loaded 68Ga/Monomethyl auristatin F(MMAF) TCT1067 dual conjugate and assess their uptake and biodistribution in HER2 overexpressed cell lines and  in vivo tumor models.

Methods

In this study, a lysine enriched HER2 specific scFv TCT1067 was modified with either TCO-PEG4 or both MMAF-PEG2 and TCO-PEG4. The conjugates were characterized with mass spectrometry and surface plasmon resonance. A bioorthogonal inverse electron demand Diels–Alder (iEDDA) cycloaddition reaction between a 68Ga-tetrazine derivative ([68Ga]GaDOTA-Tz) and trans-cyclooctene (TCO) modified scFv’s was developed for the radiolabeling. Radiolabeling of the tetrazine derivative was achieved following a previously published method.3 The HPLC purified scFv conjugates labeled with tetrazine compound [68Ga]GaDOTA-Tz (scheme 1) at 37 °C for 30 min. The radiolabeling efficiency was confirmed using Reverse Phase HPLC (RP-HPLC) and size exclusion chromatography (SEC).

Results/Discussion

The Mass spectrometry results confirmed the conjugation of three to five TCO-PEG4 groups and three to seven MMAF’s per modified TCT1067. All the conjugates displayed low pM to low nM binding affinity toward HER2. The DOTA-tetrazine was labelled with 68Ga in high radiochemical incorporation (>90%). After solid phase extraction, a >99% radiochemical purity was achieved. The non-decay corrected radiochemical yield ranged 30-40%. The iEDDA reaction of [68Ga]GaDOTA-Tz and TCO functionalized TCT1067 was confirmed by size exclusion HPLC. In vitro uptake with HER2 positive SKOV3 cell lines showed high uptake with both TCO and MMAF/TCO dual conjugates. (Figure 1A, 1D) Uptake with unlabeled TCT1067 blocking showed that the high uptake values are specific to HER2. Uptake of the radiolabeled TCT1067 are low in HER2 negative cell lines (MDA-MB-231, MDA-MB-468) and following the same trend as the HER2 expression levels. (Figure 1A, 1C). There is no uptake of [68Ga]GaDOTA-Tz. (Figure 1B)

Conclusions

We have developed conjugation and labelling strategies for the preparation of novel HER2 specific scFv TCO and MMAF/TCO dual conjugates. We have demonstrated highly specific uptake of the 68Ga labelled and 68Ga/MMAF dual-labelled scFv conjugates in HER2 overexpressed cancer cells.  In vivo uptake studies with HER2 positive and negative xenograft are currently ongoing and will be presented in due course.

References

[1]    Fu, R. et al. ChemMedChem 2018 Sep 23.

[2]    Al-Saden, N. et al. Mol. Pharm. 2018 15 (8), 3383–3393

[3]    Evans, H. L. et al. ChemComm. 2014 50, 9557-9560,

Acknowledgement

Ruisi Fu gratefully acknowledges the award of a PhD scholarship from the China Scholarship Council and support from Department of Chemistry, Imperial College London.

Scheme 1

Radiolabeling between TCO modified TCT1067 and [68Ga]GaDOTA-Tz are achieved  in PBS/10% EtOH at 37°C for 30min.

Figure 1

(A)In vitro uptake of radiolabeled conjugates. The HER2 positive SKOV3 always has the highest uptake followed by MCF7, MDA-MB-231 and MDA-MB-468. (B)Uptake of [68Ga]GaDOTA-Tz and [68Ga]GaDOTA-TCT1067 in HER2 positive SKOV3 cells. (C)Western blot of p185-HER2 of cell lines. (D)Uptake of [68Ga]GaDOTA-TCT1067 and uptake of [68Ga]GaDOTA-TCT1067 with 10x unlabeled TCT1067 in HER2 positive SKOV3 cells.

Keywords: Single-chain variable fragments, immuno-PET, Ga-68, bioorthogonal click, HER2
646

AlF-18-Labeling of new AMPTA-based chelators for applications in Positron Emission Tomography Imaging (#467)

Lisa Russelli1, Jonathan Martinelli2, Francesco De Rose1, Michael Herz1, Wolfgang Weber1, Lorenzo Tei2, Calogero D'Alessandria1

1 Klinikum rechts der Isar der TUM, Nuclear Medicine Department, Munich, Bavaria, Germany
2 University of Piemonte Orientale, Department of Science and Technological Innovation, Alessandria, Italy

Introduction

Positron emission tomography (PET) is a non-invasive molecular imaging technology constantly expanding, with a high demand for specific antibody-derived imaging probes. The need to develop stable tracers based on temperature sensitive molecules leads us to design three chelators based on the structure of 2-aminomethylpiperidine with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, 2-AMPDA-NHB and 2-AMPDA-HB). The labelling with AlF-18 at room temperature would allow the use of these systems with heat sensitive biomolecules.

Methods

Three different AMPTA-based chelators were designed and synthetized, 2-AMPDA-HB, 2-AMPDA-NHB and 2-AMPTA. All the chelators were characterized by HPLC-MS analysis and NMR spectroscopy. The AlF-18 labeling reactions were performed at different pH (4 and 5) and temperature (rt, 37 and 80°C). The products were purified using Sep-Pak Alumina N Plus Light cartridges and eluted with a 0.9% NaCl solution. All the products were analyzed by radio-TLC and radio-HPLC and the stability of the tracers was investigated at 10, 30, 60, 120 and 240 minutes via incubation in three different solutions: human serum, PBS and 0.9% NaCl solutions.

Results/Discussion

For all chelators a radiolabeling efficiency between 50 and 78% was obtained at pH 5 and room temperature (50% 2-AMPDA-HB, 74% 2-AMPDA-NHB, 78% 2-AMPTA). A similar result was obtained at 37°C where the radiolabeling efficiency is 60% for 2-AMPDA-HB, 68% for 2-AMPDA-HB and 75% for 2-AMPTA. After the stability study good results were obtained on the chelators labeled at room temperature and pH 5. High stability in human serum was measured for the ligand 2-AMPDA-HB, with a 90% of AlF-18 complexed and 55% for the 2-AMPTA chelator after 120 min. In particular the stability after 240 minutes in human serum was respectively 68% of AlF-18 complexed for the chelator 2-AMPDA-HB and 33% for the 2-AMPTA.

Conclusions

In a preliminary screening of the reported chelators, promising results have been obtained in terms of radiolabeling efficiency at T-room and 37 °C, and good stability in physiological conditions. The selection of the best chelator will lead to the synthesis of the bifunctional derivate followed by the conjugation with temperature sensitive biomolecules, e.g. Fab fragments and/or nanobodies, the labeling with AlF-18 and then in vivo applications.

2-AMPTA derivatives chelators for the complexation of AlF-18.
Keywords: AlF-18 labeling, radiochemistry, chelators, PET imaging
647

111In-labeled VHH B9 for SPECT imaging of endogenous tumor hypoxia marker carbonic anhydrase IX (#77)

Sanne A M van Lith1, Fokko J Huizing2, Bianca A W Hoeben2, Martin Gotthardt1, Paul M P van Bergen en Henegouwen3, Johan Bussink2, Sandra Heskamp1

1 Radboudumc, Radiology and Nuclear Medicine, Nijmegen, Netherlands
2 Radboudumc, Radiation Oncology, Nijmegen, Netherlands
3 Utrecht University, Cell Biology, Utrecht, Netherlands

Introduction

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme that is upregulated by cells under hypoxic conditions. Hypoxia is present in the majority of solid tumors and is associated with poor outcome and radioresistance. Therefore, non-invasive imaging of CAIX could be of prognostic value and utilized for radiotherapy planning and treatment effect monitoring. The aim of this study was to validate and optimize SPECT imaging of CAIX expression in a head-and-neck squamous cell carcinoma model using an anti-CAIX variable domain of heavy chain antibody (VHH).

Methods

VHH B9 was conjugated with maleimide-DTPA and labeled with 111In. The binding affinity and internalization of [111In]InDTPA-B9 was analyzed using CAIX expressing SKRC-52 cells. Subsequently, a dose-escalation study was performed in athymic nude mice with subcutaneous SCCNij153 head-and-neck cancer xenografts. Targeting specificity was determined by blocking the specific uptake with unlabeled VHH B9, and by analyzing tumor uptake of an 111In-labeled irrelevant VHH R2. To reduce renal retention of [111In]InDTPA-B9, a subgroup of mice was co-injected with gelofusin. Tracer uptake at 4 hours after injection was determined by ex vivo radioactivity counting and SPECT/CT scans. Furthermore, autoradiography images of tumor sections were analyzed for correlation with CAIX immunohistochemistry.

Results/Discussion

DTPA-B9 was radiolabeled with 111In at a specific activity of 4 MBq/µg. [111In]InDTPA-B9 bound to SKRC-52 cells with high affinity (IC50 = 11.3 nm, Kd = 27.2 nM) and the internalization rate of the tracer was relatively low (Ke = 0.01). A protein dose of 5 µg resulted in the highest uptake in SCCNij153 tumors in vivo (1.05±0.14%ID/g), with tumor-to-blood and tumor-to-muscle ratios of 11.5 and 24.7, respectively (Figure 1). Unlabeled B9 reduced tumor uptake to 0.30±0.03%ID/g and irrelevant [111In]InDTPA-R2 showed tumor uptake of 0.20±0.14%ID/g. Gelofusin did not significantly alter renal retention of [111In]InDTPA-B9. Uptake of [111In]InDTPA-B9 in the SCCNij153 xenografts could be visualized with SPECT/CT and autoradiography (Figure 2). Furthermore immunohistochemistry and autoradiography images showed co-localization of [111In]InDTPA-B9 and CAIX expression.

Conclusions

[111In]InDTPA-B9 VHH shows specific targeting of CAIX-expression in head-and-neck cancer xenografts. In ongoing studies we will compare this tracer to other available CAIX tracers and we will assess its potential for treatment selection and monitoring of hypoxia responses to therapy.

Figure 1. In vivo dose-escalation
Biodistribution of [111In]InDTPA-B9 and negative control [111In]InDTPA-R2 in tumor, blood and muscle 4 hours post tracer injection.
Figure 2. SPECT and autoradiography
Uptake of [111In]InDTPA-B9 in a SCCNij153 tumor as visualized with (A) SPECT and (B) autoradiography. Note the similar spatial correlation of the signal in the two figures.  
Keywords: SPECT/CT imaging, Hypoxia, Carbonic anhydrase IX, VHH
648

Screening of small molecule compounds as PET tracers targeting oligomeric and aggregated α-synuclein (#455)

Sabrina Buss1, Kristina Herfert1, Laura Kuebler1, Andreas Maurer1, Maria Wahle1, Constanze Heinzel1, Felix Schmidt4, Andrei Leonov2, Sergey Ryazanov2, Armin Giese3, Christian Griesinger2, Bernd J. Pichler1

1 Eberhard Karls University , Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Tuebingen, Baden-Württemberg, Germany
2 Max Planck Institute for Biophysical Chemistry, Goettingen, Lower Saxony, Germany
3 Ludwig-Maximilians-University, Center for Neuropathology and Prion Research, Munich, Bavaria, Germany
4 MODAG GmbH, Wendelsheim, Bavaria, Germany

Introduction

Imaging α-synuclein (αSYN) pathology to distinguish synucleinopathies from other neurodegenerative disorders is challenging and relevant PET tracers are still missing. In our previous studies, we identified one compound, showing a very high affinity towards recombinant αSYN fibrils and good selectivity over Aß and tau. Although it showed favorable in vivo kinetics with fast washout from the brain after C-11 labeling, we identified one lipophilic metabolite in the brain, which was the demethylated form of the parent compound, confounding the in vivo quantification.

Methods

To test the metabolite and other derivatives, we performed a competition binding assay (CBA) and determined the binding affinity Ki. Saturation binding assays (SBA) and CBA were performed using recombinant human αSYN fibrils and a mixture of αSYN monomers, oligomers and fibrils. For SBA, fibrils were incubated with increasing concentrations (0.02nM–48nM) of the tritiated compound to obtain total binding. Non-specific binding was achieved using an excess of the cold compound. For CBA, fibrils were incubated with decreasing concentrations of competitor (1000nM–0.0002nM) in the presence of a fixed concentration of the tritiated lead compound (2nM). After vacuum filtration and washing, liquid scintillation counting was performed. Kd- and Ki-values were calculated using non-linear regression.

Results/Discussion

Our lead compound showed high affinity to pure αSYN fibrils (Kd<4nM) and to αSYN monomeric and oligomeric structures (Kd<5nM). Competition binding experiments revealed Ki-values between <0.1nM and1000 nM. We identified one compound with a very high competitive inhibition towards pure αSYN fibrils (Ki<0.1nM) as well as the mixture of αSYN monomers, oligomers and fibrils (Ki<0.1nM). Good competitive inhibition was seen for the identified lipophilic metabolite (Ki<1nM) and another compound (Ki<2nM) on αSYN fibrils.

Conclusions

In our screening assay, we identified three promising PET tracer candidates with a high affinity towards recombinant αSYN fibrils. In further experiments, we will test these compounds towards their selectivity over Aß and tau and evaluate their specificity and selectivity in human brain tissue with confirmed αSYN, Aß and tau pathology.

Keywords: alpha synuclein, PET tracer, synucleinopathies, fibrils
649

44Sc-AAZTA-PSMA: Synthesis, Characterization and Preclinical Evaluation of a new Pet Tracer (#442)

Ivan Hawala1, Simona Ghiani2, Dezso Szikra3, Gyorgy Trencsenyi3, Gabor Nagy3, Alessandro Maiocchi2, Silvio Aime1

1 Università degli studi di Torino, Dipartimento di Biotecnologie Molecolari e Scienze per la salute, Torino, Italy
2 Bracco Imaging Spa, Bracco Research Centre, Colleretto Giacosa (TO), Italy
3 Scanomed Ltd., Scanomed Ltd., Debrecen, Hungary

Introduction

PSMA is an extracellular hydrolase highly upregulated in metastatic and hormone-refractory prostate carcinomas1. Recently 44Sc (t1/2=3.97h) received much attention as potential radionuclide with favourable characteristics for PET imaging applications.

AAZTA was thoroughly studied as chelator for Gd3+ for MRI applications2. The excellent results of the equilibrium, kinetic, and labelling studies led to a preliminary assessment of the in vitro and in vivo behavior of 44Sc(AAZTA)- and two derivatives by assuming that the physicochemical properties of Sc(AAZTA) are maintained in its bioconjugates3.

Methods

The first aminoacid (Fmoc-L-2Nal-OH) was anchored on a 2-chlorotrytil chloride resin (loading: 1.63 mmol g-1). All solid phase synthesis steps were performed by standard Fmoc protocol. After the coupling of the ligand moiety, the fully protected peptide was cleaved from the resin as the next steps were carried out in solution. The best labelling condition for AAZTA-PSMA was found using HEPES buffer at pH 4: after 5 minutes reaction time at room temperature in the presence of low concentration of the ligand (1 µM), the RCP was >95% (Figure 1). Dynamic (0-90 min) and static (2.5 h) PET scans were acquired after the injection of AAZTA-PSMA (~18 MBq) in LNCaP tumour-bearing mice (n=5). A representative image is reported in Figure 2 showing high tumour uptake at 2.5 h from the administration.

Results/Discussion

The bioconjugate was synthesized combining solid phase peptide synthesis (SPPS) and solution chemistry and the desired high purity (97%) product was obtained with a chemical yield of 9%.

Subsequently, the radiolabelling with 44Sc was carried out using the radionuclide obtained from the irradiation of natural calcium target in cyclotron. Good radiochemical yields under mild conditions (pH 4, 298 K) were achieved.

Herein we report our preclinical results of a 44Sc-labeled PSMA binding motif based peptide for in vivo PET imaging of PSMA expression in a preclinical cancer model. Dynamic scans showed high tracer accumulation in the tumour regions already after 20 minutes and pharmacokinetic in different organs. Kidney is the main organ involved for the tracer clearance. Another focus of this work was also devoted to demonstrate that the ligand and spacer choice could has a strong impact on in vivo uptake of the probe.

Conclusions

The synthesis of AAZTA-PSMA consists of three main steps, namely i) the solid phase synthesis, ii) a solution chemistry step to carry out the PSMA binding motif conjugation and iii) a final purification by preparative HPLC.

This work have demonstrated the suitability of AAZTA-PSMA conjugate for the complexation with 44Sc and the feasibility of PETs images acquisitions for prostate cancer diagnosis.

References

1 Weineisen et al, EJNMMI Research 2014, 4:63

2 Manzoni et al, ChemMedChem 20127, 1084–1093

3 Nagy et al, AngewChemIntEd. 2017, 56, 1-6

Acknowledgement

All the radiolabellings and in vivo experiments were carried out by D.Szikra, G.Trencsényi, G.Nagy at Scanomed Ltd. (Debrecen, Hungary). The project is totally funded by Bracco Imaging Spa (Colleretto Giacosa, Italy).

Figure 1. Scandium 44 labelling of AAZTA-PSMA
Labelling of AAZTA-PSMA with 44Sc in 0.1 M HEPES buffer pH 4 at room temperature and different reaction time
Figure 2. Representative PET/MRI image
Static PET/MRI image of 44Sc-AAZTA-PSMA 2.5 h after injection
Keywords: PSMA, Scandium 44, PET, Synthesis, AAZTA
650

Development of 68Ga and 18F radiolabelled peptide probes for PET imaging of αvβintegrin expression in cancers and fibrotic diseases. (#395)

Juozas Domarkas1, 2, James Thompson2, 3, George Herbert1, 2, Benjamin P. Burke2, 4, Simon Hart3, Christopher Cawthorne1, 2, Alyn Morice3, Steve J. Archibald1, 2, 4

1 University of Hull, School of Life Sciences, Hull, United Kingdom
2 University of Hull, Positron Emission Tomography Research Centre, Hull, United Kingdom
3 Hull York Medical School, Hull, United Kingdom
4 University of Hull, School of Mathematics and Physical Sciences, Hull, United Kingdom

Introduction

Expression of integrin αvβis upregulated in multiple cancers and in injured epithelium, making it an attractive target for cancer and fibrotic phenotype imaging.1,2 In tracer development, small peptides occupy an intermediate position between small molecules (difficult to develop, fast PK) and antibodies (easy to develop, slow PK).We have modified an αvβbinding peptideto allow for its radiolabelling with either 68Ga,177Lu or 18F. A library of tracers, both imaging and theragnostic, was developed with variable PK properties to provide a better understanding of SAR requirements.

Methods

The peptide core was prepared by manual solid phase peptide synthesis and derivatised to contain DO3A, a Ga3+/ Lu3+chelator, or azide/ alkyne groups, for the introduction of fluorinated prosthetic groups via Cu(I) “click” cycloadditions. Pharmacokinetic (PK) variability was generated by use of rigid and/or hydrophilic linkers between the peptide and selected radiotag. Affinity to αvβ6 was assessed by hot ligand uptake and/or ELISA assays. 68Ga3+ and 177Lu3+ were introduced by labelling in HEPES pH 3.5 buffer (90oC, 10 min). 18F labelled prosthetic groups were prepared from tosylated precursors and K222/K18F complex (95oC, 10-20 min). LogD4.7 values were measured using a shake-flask method. Naïve and tumour bearing mice were used to acquire PET/CT images and to study biodistribution.

Results/Discussion

All studied tracers showed low or sub nM affinity specific to αvβ(ELISA). Derivatisation of the N-terminus had little effect on binding affinity, with metal chelation further increasing it. Study of 68Ga/ 177Lu tracers showed high hydrophilicity (LogD7.4= ca.-3). 68Ga3+ chelates were stable in human serum at 37oC for 3 h, (177Lu3+ chelate was stable up to 12 h), but showed ~ 50% degradation in mouse urine 90 minutes post-injection. PET/CT imaging using the 68Ga tracer showed fast renal clearance and blockable tumour uptake (BxPC3, αvβ6xenograft) in addition to gut and submandibular gland (natural αvβexpressing organs) uptake. Preliminary investigation of an example 18F derivative showed reduced hydrophilicity and renal clearance accompanied with increased gut uptake and lack of tumour uptake. Preparation and assessment of fluorinated derivatives with a range of linkers is ongoing.

Conclusions

ELISA and hot ligand cell binding assay revealed that the selected peptide can be functionalised on the N-terminus without a loss of αvβ6 affinity. In vivo PET/CT imaging showed that it is also sufficiently stable to biodistribute to the periphery and selectively retain within αvβexpressing tumours/organs. These results warrant extension of the study to a larger set of radiolabelled tracers for better understanding of optimal properties.

References

 

  1. W. Guo and F. Giancotti, Nat. Rev. Mol. Cell Biol., 2004, 5(10), 816-826.
  2. A. Tatler and G. Jenkins, Proc. Am. Thorac. Soc., 2012, 9(3), 130-136.
  3. H. Wan,ADMET & DMPK, 2016, 4(1), 1-22.
  4. J. Hsiao, Y. Chang, Y. Chen, S. Hsieh, K. Hsu, C. Wang, S. Tsai and Y. Jin, Head & Neck, 2010, 32(2), 160-72.

Acknowledgement

The authors would like to thank Dr. Assem Allam for his generous contribution to the University of Hull Positron Emission Research Centre.

Figure 1

The structure of αvβtargeted cyclopeptide with DOTA chelator for 68Ga/177Lu chelation and 18F conjugated prosthetics via triazole unit.

Figure 2
PET/CT images of 68Ga tracer in BxPC3 tumour-bearing NSG mice, without (left) and with prior blocking with αvβantibody for 24 hours (right).
Keywords: Gallium-68, Lutetium-177, Flourine-18, Integrin αvβ6, Peptide
651

Positron Emission Tomography Imaging of Chemokine Receptor CXCR4 using 68Ga and ­18F Radiolabelled Tetraazamacrocyclic Antagonists (#313)

Isaline Renard1, 2, Juozas Domarkas1, 2, Sophie Poty4, Noemi Perujo Holland1, 2, Benjamin P. Burke2, 3, Franck Denat4, Christine Goze4, Christopher Cawthorne1, 2, Stephen J. Archibald1, 2, 3

1 University of Hull, School of Life Sciences, Hull, United Kingdom
2 University of Hull, Positron Emission Tomography Research Centre, Hull, United Kingdom
3 University of Hull, School of Mathematics and Physical Sciences, Hull, United Kingdom
4 Université de Bourgogne, Institut de Chimie Moléculaire, Dijon, France

Introduction

C-X-C chemokine receptor 4 (CXCR4) plays an important role in the cross-talk between cancer cells and their environment. The over-expression of CXCR4 is known in over 23 different tumour types, and is associated with poor prognosis and increased risk of metastasis. The development of CXCR4 imaging agents for the non-invasive imaging of CXCR4 could improve disease staging and treatment planning. In this study, two derivatives of AMD3100 were synthesised to accommodate 68Ga using different linkers and chelators, and subsequently tested in vivo for their CXCR4 affinity and specificity.1,2

Methods

AMD3100 was functionalised on the central phenyl moiety with diamino-polyethylene glycol 3 (PEG3). This derivative was reacted with two chelators, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid (NODAGA). The AMD3100-based ligands were labelled with 68Ga.2

PET imaging studies were performed on SCID-beige mice implanted with CXCR4 over-expressing tumours. The subjects were injected with approximately 2 MBq of tracer (i.v.) and then underwent 66 min. dynamic PET scans. CXCR4 specificity was confirmed by administration of a blocking dose of either AMD3100 (5 mg/kg) or Cu2CB-Bicyclam3 (4 mg/kg).

Results/Discussion

The radiotracers [68Ga]Ga-1 and [68Ga]Ga-2 showed significantly different uptake in vivo. PET imaging studies for [68Ga]Ga-1 showed similar liver and tumour uptake, whereas [68Ga]Ga-2 showed greater accumulation in the liver compared to the tumour. Biodistribution studies using both [68Ga]Ga-1 and [68Ga]Ga-2 with blocking doses of Cu2CB-Bicyclam3 and AMD3100 confirmed CXCR4 specificity. Metabolism studies were performed in order to investigate in vivo stability and showed [68Ga]Ga-2 to stay intact while [68Ga]Ga-1 was metabolised.

Conclusions

Two radiotracers [68Ga]Ga-1 and [68Ga]Ga-2 were successfully synthesised and their affinity for CXCR4 was assessed in vivo. [68Ga]Ga-1 and [68Ga]Ga-2 showed specific, blockable uptake in CXCR4 over-expressing tumour models by biodistribution and PET imaging studies. [68Ga]Ga-2 metabolism studies demonstrated stability in vivo. Derivatives are being synthesised to include 18F, and preliminary in vitro and in vivo studies are being undertaken.

References

  1. U. M. Domanska, R. C. Kruizinga, W. B. Nagengast, H. Timmer-Bosscha, G. Huls, E. G. E. de Vries and A. M. E. Walenkamp, Eur. J. Cancer, 2013, 49, 219–230.
  2. S. Poty, E. Gourni, P. Désogère, F. Boschetti, C. Goze, H. R. Maecke and F. Denat, Bioconjug. Chem., 2016, 27, 752–761.
  3. A. Khan, G. Nicholson, J. Greenman, L. Madden, G. McRobbie, C. Pannecouque, E. De Clercq, R. Ullom, D. L. Maples, R. D. Maples, J. D. Silversides, T. J. Hubin and S. J. Archibald, J. Am. Chem. Soc., 2009, 131, 3416–3417.

Acknowledgement

The authors acknowledge the support of the Daisy Appeal with a fellowship to BPB, and would like to thank Dr Assem Allam for studentship funding to NPH and his generous contribution to the University of Hull PET Research Centre.

Figure 1.
Dynamic PET scan of CXCR4 over-expressing tumour bearing animals injected with approximately 2 MBq of [68Ga]Ga-2 (left), [68Ga]Ga-1 (middle) and [68Ga]Pentixafor (right).
Keywords: CXCR4, PET imaging, Gallium-68, Fluorine-18
652

Biodistribution of manganese in healthy mice using 52Mn (#324)

George Firth1, Julia Blower1, Jayanta Bordoloi1, Jesper Fonslet2, Philip Blower1

1 King's College London, School of Biomedical Engineering & Imaging Sciences, London, United Kingdom
2 Technical University of Denmark, The Hevesy Lab, Roskilde, Denmark

Introduction

52Mn (β+, t1/2 = 5.6 days, Iβ+ = 30%) is a positron emitting radionuclide that has the potential to non-invasively study manganese trafficking on a whole-body scale to better understand the role of manganese in biological processes and health in vivo. This could help elucidate potential changes to the transport of manganese in a range of pathological diseases, including diabetes and Alzheimer's disease. In this project we address the whole body distribution of 52Mn, administered intravenously as MnCl2 in healthy BALB/c mice. The data will be used in later studies as a healthy control.

Methods

[52Mn]MnCl2 was administered i.v. at time 0 h, a dynamic PET scan followed from 0-1 h and PET/CT scans were then performed at 4, 24, 48 and 72 h post injection. The mice (n = 3) were then euthanised at 96 h to provide ex vivo biodistribution data.

Results/Discussion

Fast blood clearance was observed initially with an estimated biological half-life of 1.35 min. Activity at 1 h was localised primarily to the abdominal organs with small amounts of radioactivity observed in the heart and joints. Retention of 52Mn varied over time for each organ, with activity associated with the salivary glands increasing over time. Prominent uptake at 96 h was seen in the liver (8.21 ± 0.62 %ID/g), pancreas (22.99 ± 3.44 %ID/g), salivary glands (9.25 ± 0.18 %ID/g) and kidneys (20.87 ± 3.11 %ID/g). Maximum intensity projection (MIP) PET images at 1 and 4h also demonstrated heart and intestine uptake which decreased over time as 52Mn was excreted via the faeces. Modest brain uptake (2.30 ± 0.24 %ID/g) was observed at 96 h.

Conclusions

The research described sets the foundation for the study of 52Mn trafficking using PET, and will be developed further to investigate the importance of manganese in a range of diseases.

References

1. Graves SA et al. Novel Preparation Methods of 52Mn for ImmunoPET Imaging. Bioconjug Chem. 2015;26:2118-2124.

2. Gawne P et al. Manganese-52: applications in cell radiolabelling and liposomal nanomedicine PET imaging using oxine(8-hydroxyquinoline) as an ionophore. Dalton Trans. 2018;47:9283-9293.

PET/CT images of 52Mn biodistribution at various time points

Figure 1. PET/CT images of 52Mn biodistribution at various time points: representative maximum intensity projection (MIP) and transverse slices of PET-CT images of a healthy BALB/c mouse injected with [52Mn]MnCl2 (1.5MBq, 100 μL) at 1 h, 4 h, 24 h, 48 h and 72 h post injection.

Keywords: Manganese-52, PET Metallomics, Manganese biodistribution
653

Imaging of apoptosis by PET/CT: a new F18-labeled Caspase-3 substrate (#279)

Federica Pisaneschi1, Brian J. Engel1, Seth T. Gammon1, Rajan Chaudhari2, Zhen Lu2, Hailing Yang2, Argentina Ornelas1, Victoria Yan1, Md Nasir Uddin1, Lindsay Kelderhouse1, Amer Najjar3, William Tong1, Shuxing Zhang2, David Piwnica-Worms1, Robert Bast2, Steven W. Millward1

1 UT MDAnderson Cancer Center, Cancer Systems Imaging, Houston, Texas, United States of America
2 UT MDAnderson Cancer Center, Department of Experimental Therapeutics, Houston, Texas, United States of America
3 UT MDAnderson Cancer Center, Department of Pediatrics - Research, Houston, Texas, United States of America

Introduction

Quantitative imaging of apoptosis in vivo could enable real-time monitoring of acute cell death pathologies such as traumatic brain injury (TBI) as well as the efficacy and toxicity of cancer chemotherapy and radiotherapy.  Here, we describe the development of a minimized F-18-labeled caspase-3 substrate, [18F]-TBD, for PET/CT imaging of apoptosis and its validation in a model of hepatotoxicity.

Methods

The minimized caspase 3 substrate,TBD, was designed by structural optimization of M808 irreversible caspase-3 inhibitor. [18F]-TBD was synthesized on the GE TracerLab automated module by click chemistry with [18F]-fluoroethylazide, using a TentaGel-derived azide stripping resin to remove the unreacted alkyne and enhance molar activity.1 [18F]-TBD was tested in vitro in cisplatin-treated ovarian cancer cells and in vivo in a Jo2 antibody-induced hepatotoxicity mouse model.

Results/Discussion

TBD showed a 14-fold improvement in substrate activity and significantly enhanced caspase selectivity relative to first-generation derivatives. Molecular modeling suggested that these improvements resulted from favorable interactions mediated by both the side chain of the P2 O-benzyl-threonine and the linker functionality between the scissile amide bond and the triazole. [18F]-TBD was obtained in 9.8±4.2% (n = 12) decay corrected end of synthesis radiochemical yield, 17.7-149.5 GBq/mmol (n = 12) molar activity and >99% radiochemical purity. The resulting radiotracer accumulated in cisplatin-treated ovarian cancer cells in a caspase- and cisplatin-dependent fashion. Using the Jo2 antibody-induced hepatotoxicity mouse model, [18F]-TBD showed 1.5-fold increased liver uptake in Jo2-treated mice compared with untreated controls. A chemical control tracer, [18F]-TBA, that could not be cleaved by caspase-3, showed no change in liver accumulation after induction of hepatocellular apoptosis.

Conclusions

These data suggest that [18F]-TBD imaging could provide an immediate pharmacodynamic readout of tumor apoptosis in vivo and enable rapid evaluation of treatment efficacy.2

References

1. Pisaneschi, F.; Kelderhouse, L. E.; Hardy, A.; Engel, B. J.; Mukhopadhyay, U.; Gonzalez-Lepera, C.; Gray, J. P.; Ornelas, A.; Takahashi, T. T.; Roberts, R. W.; Fiacco, S. V.; Piwnica-Worms, D.; Millward, S. W., Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers. Bioconjugate Chem. 2017, 28 (2), 583-589.

2. Engel, B. J.; Gammon, S. T.; Chaudhari, R.; Lu, Z.; Pisaneschi, F.; Yang, H.; Ornelas, A.; Yan, V.; Kelderhouse, L.; Najjar, A. M.; Tong, W. P.; Zhang, S.; Piwnica-Worms, D.; Bast, R. C.; Millward, S. W., Caspase-3 Substrates for Noninvasive Pharmacodynamic Imaging of Apoptosis by PET/CT. Bioconjugate Chem. 2018, 29 (9), 3180-3195.

Acknowledgement

This work was supported by UTMDACC startup funds (SWM), a UTMDACC Moonshot Knowledge Gap Pilot Project, 1R21CA181994-01 (SWM, ZL). MD Anderson’s Nuclear Magnetic Resonance Facility and Small Animal Imaging Facility (SAIF) are supported by the MD Anderson Cancer Center Support Grant CA016672 (Pisters).

[18F]-TBD accumulates and is retained in apoptotic livers of Jo2-treated mice.

a) Representative PET 7.5 min post i.v. injection of female athymic nude mice with [18F]-TBD (top) [18F]-TBA (bottom) in Jo2 treated (left) versus untreated (right) mice. b) Structures of [18F]-TBD and [18F]-TBA. c) Three-tissue compartment model used for dynamic PET analysis. d) Kinetic parameter k3 [18F]-TBD (left) or [18F]-TBA (right) treatment.

 

Keywords: apoptosis, caspase 3, PET imaging, click chemistry
654

Evaluation of novel 89Zr chelators and corresponding 89Zr-labeled immunoconjugates (#531)

Pierre Adumeau1, René Raavé2, Christian B. Jacobsen3, Gerwin Sandker2, Sandra Heskamp2, Otto Boerman2, Mark Rijpkema2, Floriane Mangin1, Michel Meyer1, Jean-Claude Chambron1, Mathieu Moreau1, Claire Bernhard1, Adrien Dubois1, Laurène Da Costa1, Victor Goncalves1, Franck Denat1

1 University of Burgundy, Institute of Molecular Chemistry of the University of Burgundy, Dijon, France
2 Radboud University Medical Center, Departement of Radiology and Nuclear Medicine, Nijmegen, Netherlands
3 Novo Nordisk A/S, Global Research Technologies, Måløv, Denmark

Introduction

For immunoPET with 89Zr, the current gold standard to label antibodies is desferrioxamine (DFO).1 However, preclinical studies have shown that the 89Zr-DFO complex is partly unstable in vivo, leading to 89Zr release and subsequent accumulation in mineral bone. This bone uptake may impede the detection of bone metastases, and hampers accurate estimation of the radiation dose to the bone marrow in dose planning for radioimmunotherapy. Therefore, there is a need for more stable 89Zr chelators.

Methods

We have synthesized new octacoordinating 89Zr-bifunctional chelating agents derivated from the DFO* chelator.2 These new chelators were synthesized by coupling different hydroxamic acid-bearing arms to DFO, followed by the introduction of an isothiocyanate moiety. The model antibody trastuzumab was conjugated to the NCS-derivated chelators and DFO-pPhe-NCS as a reference, and radiolabeled with 89Zr. The stability of the radiolabeled chelators and radiolabeled conjugates were evaluated in human plasma, and in PBS in presence of EDTA or DFO. The in vitro behavior of the most promising compounds was investigated more thoroughly using HER2-experessing SK-OV3 cells, and in vivo distribution was studied in mice with subcutaneous SK-OV3 xenografts by PET/CCT imaging and ex vivo tissue analysis.

Results/Discussion

The bifunctional chelators were conjugated efficiently to trastuzumab. Radiolabeling of the conjugates with 89Zr yielded the radioconjugates with high yield, purity and specific activity (RCY >95%, RCP >99%, SA >100 MBq/mg). When challenged with EDTA or DFO, the 89Zr-chelates and the corresponding radioconjugates displayed an improved stability compared to 89Zr-DFO and 89Zr-DFO-trastuzumab, with the best results obtained for the chelator dubbed cycloDFO*. The immunoreactive fraction and IC50 were similar for 89Zr-DFO-trastuzumab and 89Zr-cycloDFO*-trastuzumab. Internalisation after 2h was significantly higher for 89Zr-cycloDFO*-trastuzumab compared to 89Zr-DFO-trastuzumab. Accumulation of 89Zr in bone was significantly lower for 89Zr-DFO-cyclo*-trastuzumab compared to 89Zr-DFO-trastuzumab in knee (3.6 ± 0.4% vs 5.9 ±0.6%), femur (2.2 ± 0.2% vs 3.4 ± 0.3%), and sternum (3.5± 0.4% vs 4.5 ±0.4%) at 72 h after injection. Uptake in the SK-OV3 tumor was similar for both antibody conjugates.

Conclusions

The new 89Zr-chelators and the associated radioconjugates show improved in vitro stability compared to DFO and 89Zr-DFO-trastuzumab. The radioconjugate derivated from the more promising chelator, 89Zr-cycloDFO*-trastuzumab, demonstrated a better in vivo stability compared to 89Zr-DFO-trastuzumab. Therefore, less radiation exposure to bone marrow and improved bone metastasis detection could be achieved using cycloDFO*.

References

1 S. Heskamp, R. Raavé, O. Boerman, M. Rijpkema, V. Goncalves, F. Denat, Bioconjugate Chem., 2017, 28, 2211-2223.

2 D. Vugts, C. Klaver, C. Sewing, A. Poot, K. Adamzek; S. Huegli, C. Mari, G. Visser, I. Valverde, G. Gasser, T. Mindt, G. van Dongen, Eur. J. Nucl. Med. Mol. Imaging, 2017, 44, 286-295

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.

Keywords: ImmunoPET, 89Zr-chelates, DFO-derivatives, Trastuzumab

Multimodal Technologies

Session chair: Claudia Kuntner (Seibersdorf, Austria); Terry Jones (Wilmslow, UK)
 
Shortcut: PW14
Date: Thursday, 21 March, 2019, 12:45 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.

702

In vivo PET/CT imaging in zebrafish using 18F-FDG and 18F-NaF (#293)

Luiza R. Nazario1, 4, Jeroen Vierstraete2, Jan W. Bek2, Sara Neyt3, Niek V. Overberghe3, Rudi Dierckx1, Andy Willaert2, Cristina Maria M. Jeckel5, Rosane S. da Silva4, Janine Doorduin1, Erik de Vries1

1 University Medical Center Groningen, Department of Nuclear Medicine and Molecular Imaging, Groningen, Netherlands
2 Ghent University Hospital, Center for Medical Genetics Ghent, Ghent, Belgium
3 MOLECUBES NV, Ghent, Belgium
4 PUCRS, Laboratório de Neuroquímica e Psicofarmacologia, Porto Alegre, Brazil
5 PUCRS, Instituto do Cérebro do Rio Grande do Sul, Porto Alegre, Brazil

Introduction

Zebrafish are versatile in vivo model for preclinical research. For assessment of anatomical features, imaging techniques like computed tomography (CT) and magnetic resonance have been used in zebrafish [1-3]. The use of functional imaging techniques like Positron Emission Tomography (PET) in living zebrafish has not been reported yet, was only investigated ex vivo [4]. In larger fishes the uptake of 18F-FDG is similar to that in humans [5]. The aim of this study was to determine if PET/CT could be used to study functional processes in living zebrafish using the 18F-NaF and 18F-FDG.

Methods

Anesthetized (tricaine 0.1 g/L) zebrafish (n=3) were intraperitoneally injected with 10 µL of 18F-FDG or 18F-NaF (2-3 MBq). To determine the tracer’s distribution in the whole body, PET-CT imaging (β- and X-CUBE, MOLECUBES) was performed 30 and 60 min after 18F-FDG injection and 15, 30, 60 and 150 min after 18F-NaF injection. After tracer distribution in freely moving fish, each zebrafish were put in a 15 mL tube filled with tricaine solution and placed in the PET scanner (5 min scan with 3 animals simultaneously). CT images were obtained for anatomical reference. All data were reconstructed using the OSEM algorithm (voxel size: 400 µm), using 30 (18F-FDG) and 50 iterations (18F-NaF). To determinate the tracer uptake (SUV) in different regions PMOD software (version 3.808) was used.

Results/Discussion

After 30 and 60 min 18F-FDG uptake was observed in different areas, including encephalic area, heart, tail, digestive tract and kidney; with the highest uptake (SUV max) in the intestine area. After 15 min, 18F-NaF was mostly localized in the abdominal region (site of injection). After 30 min the 18F-NaF uptake in the bones, in the head region, vertebral column and fin started to increase until obvious bone uptake was observed after 150 min. The differences in 18F-NaF distribution time between zebrafish and humans can be due the fact that humans use bones more for support and thus have a different bone composition. For both tracer the intravenous injection could demonstrate a better and faster uptake, but this for zebrafish can be a challenge. Since zebrafish is already used to model a large number of diseases, this protocol can also be used to study various pathologies, by changing the radiotracer and the zebrafish disease model [6-7].

Conclusions

Despite the small size of the animal, reliable tracer administration and PET imaging in living zebrafish proved to be feasible and internal structures could be visualised. Further studies need to be done to complete this protocol, for example investigate different injection sites and uptake time.

References

1.KOBA W et al. MicroPET/SPECT/CT imaging of small animal models of disease. Am J Pathol, 2013.

2.CHENG KC et al. Whole-animal imaging, gene function, and the Zebrafish Phenome Project. Curr Opin Genet Dev, 2011.

3.KABLI S et al. Magnetic resonance microscopy of the adult zebrafish. Zebrafish, 2006.

4.DORSEMANS AC et al. Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled J Vis Exp,  2017.

5.BROWNING ZS et al. Using PET/CT imaging to characterize 18F-fluorodeoxyglucose utilization in fish. J Fish Dis, 2013.

6. MESHALKINA DA, et al. Adult zebrafish in CNS disease modeling: a tank that's half-full, not half-empty, and still filling. Lab Anim (NY). Oct 6;46(10):378-387. 2017

7. IDILLI AI, et al. Zebrafish in Translational Cancer Research: Insight into Leukemia, Melanoma, Glioma and Endocrine Tumor Biology. Genes (Basel). Sep 20;8(9). 2017

Keywords: Zebrafish, PET/CT, 18F-FDG, 18F-NaF
703

Monte Carlo simulation of a Total Body PET scanner based on the PENN PET (#392)

Eva Sousa1, 2, Nikos Efthimiou1, Alexander Turner2, Steve J. Archibald1

1 University of Hull, 1) PET Research Centre, Faculty of Health Sciences, Kingston upon Hull, United Kingdom
2 University of Hull, 2) School of Engineering & Computer Science, Big Data Analytics Research Center, Kingston upon Hull, United Kingdom

Introduction

Long axial PET scanners, referred as Total Body (TB), are gaining momentum. Two of the most popular originate from the EXPLORER project, but only recently has this been developed to give the first commercially available scanner of this type Their main area of focus will be kinetic and low dose imaging. Pediatric applications in have been proposed. In order to investigate the performance of such scanner geometry we used GATE Monte Carlo toolkit (v.7.2) to simulate the geometry of the PENN PET scanner, under different configurations with total axial length from 70 cm and 140 cm.

Methods

Using the Gate Monte Carlo simulation toolbox (V.7), two realistic scanner models of the Total Body PENN PET were developed, varying spacing between the modules to achieve axial FOV size of 70 cm (model a), and 140 cm (model b). In both models, each detector tile has 3.9 × 3.9 × 19 mm3 crystals of cerium doped lutetium yttrium orthosilicate (LYSO) coupled to a digital silicon photomultiplier (dSiPM) using as model the detectors developed by Philips. Each has an array of detector tiles, with 4 transaxial and 7 axial rows, and 23.22 cm of AFOV, each ring is formed by 18 modules, full scanners have 3 rings. Following the NEMA standard phantoms and sources were created to execute performance tests of deadtime and sensitivity, for these tests one ring was used.

 

Results/Discussion

Figure 1 shows the two geometries. When gaps are incorporated, the axial length increases, but at the same time between the gap information can be obtained only using large polar angles. The sensitivity of the equipment was estimated using a point source in the centre of the scanner. The absolute central sensitivity was found to be 20% without gaps and 13% when gaps are used. In addition, a polar angular limit in the theta angle was considered in order to reduce parallax error from the intense obligingness. The limit was 45 degrees. When that was applied the absolute sensitivity was reduced to 8% and 4.8% respectively. The use of a point source for the calculation of the sensitivity is not an appropriate method to compare the two scanners.

Conclusions

The development of such long scanners raises new issues regarding the appropriateness of the standard methods and techniques (NEMA), as they fail to exploit the benefits from the large coverage; Such as, concurrent imaging of distant locations, increased sensitivity and improved contrast. Therefore, we designed a long NEMA scatter fraction phantom (200 cm) in order to assess the equipment performance.

References

[1] Viswanath PV, Daube-Witherspoon ME, Schmall JP, Surti S, Werner M E, Muehllehner G, Geagan MJ, Perkins AE, Karp J S. Development of PET for Total Body Imaging. Acta Physica Polonica B. 49 (2017) n.º 10.

 

[2] Jan S, Santin G, Strul D, et. al. GATE: a simulation toolkit for PET and SPECT. Phys. Med. Biol. 49 (2004) 4543-4561

 

[3] NEMA NU 2-2012. Performance Measurements of Positron Emission Tomographs. National Electrical Manufacturers Association Rosslyn, VA. 2013.

Figure 1
PENN PET scanner simulation with with total axial length of 70 cm. 
Figure 2

PENN PET scanner with with total axial length of 140 cm.

Keywords: Total Body PET, Monte Carlo Simulation, NEMA
704

Impact on image quality by concurrently scanning a NEMA-NU4 IQ phantom and a cylindrical phantom with Sedecal Argus preclinical PET scanner (#386)

Nikos Efthimiou1, Isaline Renard1, Steve J. Archibald1, Christopher Cawthorne1, 2, 3

1 University of Hull, PET Research Centre, Faculty of Health Sciences, University of Hull, Hul, United Kingdom
2 KU Leuven, Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, Leuven, Belgium
3 KU Leuven, MoSAIC- Molecular Small Animal Imaging Centre, Leuven, Belgium

Introduction

A common practice to increase the throughput in preclinical PET imaging is to concurrently scan multiple animals which can impact the quality and noise characteristics of the reconstructed images. Increased activity in the FOV leads to more random events getting detected, increasing the likelihood for scattering.

Methods

A NEMA phantom was acquired, solo and in combination with a 20 ml syringe, on a Sedecal Super Argus preclinical PET/CT scanner using the NEMA-NU4(2008) protocol. Due to the limited axial FOV, two bed positions (10 min each), were used. The scan of the single NEMA phantom was performed off axis, in the same position as the NEMA+syringe. The reconstruction was OSEM with 16 subsets and 2 iterations with randoms, scatter and PSF correction, as used for routine scans.

Results/Discussion

The uniformity analysis showed an increase in the %STDunif from 7% to 8.05%, attributed to the presence of an extra body in the FOV which increases the total attenuation which then reduced the number of detected events along some LORs, therefore increasing the noise. In terms of recovery coefficient (RC) it is shown that addition of the syringe reduced the spatial resolution of the scanner, especially for the smallest rods, more importantly increased the %STD of the axial profiles, with an impact on the 2mm rod. The spill over ratio (SOR) in air is significantly increased in the case of the two phantoms.

Conclusions

It was shown that when concurrently scanning two phantoms in the FOV the image quality degrades. The noise properties deteriorate and quantification accuracy is reduced. This has to be taken into account when an experiment is designed. A validated GATE simulation model will be used to further assess the nature of the resulted image quality degradations.

References

[1]. Siepel F.J., Van Lier M.G.J.T.B., Chen M. et. al. “Scanning multiple mice in a small-animal PET scanner: Influence on image quality”, Nucl Inst Meth Phys A (621), p.6075-610.

[2]. Herraiz J.L., Espana S., Vaquero J.J et. al. “FIRST: Fast Iterative Reconstruction Software for (PET) Tomography”, Phys Med Biol. 2006 Sep 21;51(18):4547-65

Figure 1

Recovery coefficients for the three thinner rods and a profile through the cold regions of the phantom (air and water), for the single NEMA and NEMA+syringe.

Keywords: NEMA, Small Animal, PET, Image quality, Contrast Recovery
705

Cardiac imaging of multiple mice using Simultaneous self-gated PET/MRI method (PolyGate). (#396)

Willy Gsell1, Cesar Molinos2, Carlos Correcher2, Christophe Deroose3, Uwe Himmelreich1, Michael Heidenreich2, Arno Nauerth2

1 University of Leuven, Biomedical MRI, Leuven, Belgium
2 Bruker Biospin, Ettlingen, Germany
3 University of Leuven, Nuclear Medicine and Molecular Imaging, Leuven, Belgium

Introduction

With depth of interaction (DOI) correction, it is now possible to have homogenous resolution across the entire field of view. Therefore, scanning multiple animals simultaneously became attractive to increase the throughput without compromising on the image quality. We recently demonstrated the possibility to use MRI information for retrospective gating of PET data (1). We hereby propose a new method with spatially resolved navigator (POLYGATE) enabling to extract the cardiac and respiratory motions of three separate mice and to retrospectively reconstruct cardiac gated PET data.

Methods

PET-MRI data were acquired on a BioSpec 70/30 MRI system equipped with a SiPM based PET insert (Bruker Biospin). Three C57BL/6 female mice were used in this study. Animals were not fasted for good myocardial FDG uptake. Anaesthesia was induced and maintained via inhalation of 2% Isoflurane in 100% oxygen. Mice were simultaneously scanned using the POLYGATE MRI protocol (TE/TR: 4/13.7ms, α: 20º, FOV: 70 x 30 mm, matrix172 x86 zero filled to 256 x128, 3 slice packages of 6 slices of 1mm thickness, 155 oversampling, acquisition time: 54 min 48s) combined with 60 min PET scan (i.v. injection of 8MBq of 18F-FDG 1hr before the start of the scan). The list-mode PET were rebinned according to the MRI based cardiac cycles to reconstruct 8 cardiac frames using MLEM reconstruction.

Results/Discussion

Respiration and cardiac motions were successfully retrieved from the spatially resolved navigator (heart rate: 489/405/444 and respiration rate: 63/75/48 bpm for mouse1/ mouse2 and mouse3 respectively) (fig. 1). This resulted in high quality MRI cine. The use of the MRI derived motion to the PET data enable us to resolved the right myocardium of each mice at end diastole (fig. 2). The application of the MRI derived cardiac motion to the unsynchronized PET data showed blurring of images with no apparent myocardial contraction similar to what will provide an ungated PET or MRI scan.

Conclusions

For the first time we were able to apply such PET/MRI method in 3 mice simultaneously with multi-slice scheme. End systole and diastole were resolved in each animals in both the MRI and PET data. While this prevail further work to evaluate the robustness of POLYGATE in diseased animals, one could expect a similar positive outcome as to the use of intragate compared to ECG- triggered acquisition (2,3).

References

  1. Retrospectively gated cardiac PET-MR imaging in rodents using MRI-based cardiac motion information. (#549). W. Gsell, A. Nauerth, C. Molinos, C. Correcher, A. J. Gonzalez, S. Sven, T. Greeb, R. Polo, B. Holvoet, C. M. Deroose, U. Himmelreich, M. Heidenreich. 13th annual meeting of the European Society for Molecular Imaging, ESMI. 20-23 March 2018.

  2. Bovens SM, te Boekhorst BC, den Ouden K, van de Kolk KW, Nauerth A, Nederhoff MG, Pasterkamp G, ten Hove M, van Echteld CJ. Evaluation of infarcted murine heart function: comparison of prospectively triggered with self-gated MRI. NMR Biomed. 2011 Apr;24(3):307-15.

  3. B. Hiba, N. Richard, H. Thibault, M. Janier. Cardiac and respiratory self-gated cine MRI in the mouse: comparison between radial and rectilinear techniques at 7 T. Magn. Reson. Med., 58 (2007), pp. 745.

Figure 1: PolyGate method
A: PolyGate sequence with spatially encoded navigator. The Fourier transform of the navigator signal provide a profile from which each animal can be identified (mouse1: blue, mouse2: purple and mouse3: orange). B: Corresponding navigator signal of 3 mice scanned simultaneously.
Figure 2: Self-gated reconstruction of PET and MRI
A: MRI and PET cine frames at end diastole (ED) and end systole (ES) for mouse1, 2 and 3. B: ED long axis view of mouse1 heart with corresponding line profile for ED and ES (C and D). MLEM reconstruction with 0.25mm isotropic resolution, 36 iterations and partial volume correction (iterative deconvolution method).
Keywords: PET/MRI, mouse, multiple animal, self-gated
706

Disease Development in Relapsing-remitting MOG 1-135/CFA induced EAE in Dark Agouti Rats: A preliminary Magnetic Resonance Imaging/Spectroscopy and mGluR5 PET Imaging Study (#247)

Frauke Conny Waschkies1, 2, Linjing Mu3, 4, Adrienne Herde3, Claudia Keller3, Simon M. Ametamey3, Roger Schibli3, Roland Martin5, Markus Rudin1

1 ETH and University of Zurich, Institute for Biomedical Engineering, Zurich, Switzerland
2 University Hospital Zurich, Visceral- and Transplantation Surgery, Zurich, Switzerland
3 ETH Zurich, Institute for Pharmaceutical Sciences, Zurich, Switzerland
4 University Hospital Zurich, Department of Nuclear Medicine, Zurich, Switzerland
5 University Hospital Zurich, Neuroimmunology and MS Research, Zurich, Switzerland

Introduction

Disease development in MOG1-125 /CFA induced EAE in Dark Agouti rats represents a reliable model of a relapsing-remitting multiple sclerosis (MS) phenotype. As changes in mGluR expression have been associated with processes involved in progressive damage in MS [1], and levels of mGluR5 expression have been found increased in reactive astrocytes in MS lesions [2], we were interested to study alterations in glutamatergic signalling using both metabolic MR readouts (1H MRS) and mGluR5 PET in the same animal. Metabolic readouts were complemented by conventional MRI assessing structural changes.

Methods

MR imaging was performed on a 94/30 Bruker BioSpec system, followed by mGluR5 PET with the ligand [18F]PSS232 in selected animals the next day on a Vista eXplore PET/CT scanner. 14 EAE-induced female DA rats and two controls were studied at baseline, during onset and peak and remission of EAE symptoms. Structural MRI included magnetization transfer (MT) and diffusion tensor imaging (DTI) and quantitative T2 mapping (qT2). The neurochemical profile from a 48uL volume placed in the pons was recorded using a 1H STEAM sequence. MRI data analysis was performed using Bruker Paravision v6.0.1 software, Matlab scripts, AEDES software and LCModel. Graphs and statistical analysis were generated with R v3.5.0. PET data were analysed with PMOD 3.8 software.

Results/Discussion

In contrast to a different EAE rat model [3], no prominent alterations either in qT2, MT ratio or DTI-derived markers for axonal & myelin damage have been found in brain of MOG1-125/CFA induced EAE in Dark Agouti rats. However, USPIO accumulation (Sinerem®) was observed in pons and cerebellum indicative of monocyte infiltration. 1H MRS STEAM volume was therefore placed in pons and Fig. 1 shows concentration of selected metabolites involved in inflammation and oxidative stress, as well as neuronal marker N-acetyl-aspartate (NAA) for the different disease stages. Typical SNR=8 was low as pons is not ideally suited for surface coil detection. However, Cramer-Rao lower bound (CRLB) values were good for glutamate and NAA revealing a trend for increased glutamate at EAE onset and a sign. decrease in NAA over the course of the disease. [18F]PSS232 PET revealed most prominent increase in mGluR5 expression at disease onset, and in pons, medulla & brainstem also at peak of the disease (Fig. 2).

Conclusions

Preliminary data indicate increase in glutamate signaling during onset of MOG1-125/CFA induced EAE, though changes in glutamate and mGluR5 receptor expression did not reach statistical significance. Decrease in NAA was noted throughout the disease course. Additional experiments will be required to substantiate these findings. Combination with structural MRI markers will allow a more comprehensive characterization of the disease process.

References

[1] Macrez et al. Lancet 2016 15: 1089-102

[2] Geurts et al. Brain 2003 126: 1755-1766

[3] Berger et al. NMR Biomed 2006 19: 101-107

Acknowledgement

Funding from CRPP Multiple Sclerosis is gratefully acknowledged. We thank Prof. Stefanie D. Krämer for her support for the animal studies.

Fig. 1: Quantitative analysis of selected metabolites/neurotransmitters obtained from 1H MRS in pons

Left: 1H STEAM voxel selected in pons. Right: Boxplots of 1H STEAM metabolite concentrations [mM] as determined from LCModel analysis using water-scaling with a reference spectrum, depicting individual baseline measures (blue), and during different stages of EAE at disease onset (yellow), peak of disease (gray) and during remission (red). Cramer Rao Lower Bounds (CRLBs) listed under each graph.

 

 

 

Fig. 2: mGlu5 in vivo PET.

Left: Color-coded PET images overlaid with the MRI-based ROI template in PMOD used for ROI analysis. Top: Sample baseline image before EAE induction. Bottom: Individual sample image from EAE onset. Right: Bar plots of [18F] PSS232 accumulation in selected ROIs relative to cerebellum in controls and EAE animals, respectively, at different stages of the disease, at onset, peak and during remission.

Keywords: multi-parametric Magnetic Resonance Imaging (MRI), Positron Emssion Tomography (PET), metabotropic glutamate receptor 5 (mGluR5) 1H MR Spectroscopy, Experimental Autoimmune Encephalomyelitis (EAE)
707

Ex vivo volumetry of mouse ischemic brain lesion: fixation bias (#512)

Sinisa Skokic1, Helena Justic1, Marina Dobrivojevic Radmilovic1, Christian Dullin2, Giuliana Tromba3, Srecko Gajovic1

1 University of Zagreb School of Medicine, Croatian Institute for Brain Research, Zagreb, Croatia
2 University Medical Center, Institute for Diagnostic and Interventional Radiology, Gottingen, Germany
3 Synchrotron Light Source Elettra, Trieste, Italy

Introduction

Sample preparation for ex vivo imaging (MRI, CT) in most cases involves tissue fixation. Historically, the choice of fixation protocol was dictated by the subsequent histological analysis, often resulting in poor imaging performance. Recent fixation protocols are developed with the goal of optimizing the imaging in terms of signal and contrast levels, but the known effect of tissue shrinkage/expansion is often neglected. It can, however, greatly impact the volumetry when pathology is present in the tissue. We evaluate this effect on ex vivo assessment of brain ischemic lesion in mice.

Methods

Several groups of C57Bl/6 albino mice (N = 2 to 5) underwent 60-minute middle cerebral artery occlusion. Two days after MCAO, the reference in vivo MRI scan was made, the animals sacrificed and their brains fixated applying different protocols, which can be grouped into ethanol-based (optimized for phase-contrast CT imaging) and formaldehyde-based fixation (optimized for histology, commonly used for ex vivo MRI).

The ex vivo samples were imaged with MRI (100x100x400 um resolution, T2w spin-echo), synchrotron (2.35/2.55 um isotropic, phase contrast imaging), and micro-CT (9 um isotropic). After the imaging, brains were stained with Nissl. The ischemic lesion, ipsilateral and contralateral hemispheres were manually segmented for the volumetric analysis.

Results/Discussion

The applied fixation protocols affected MRI, micro-CT and phase-contrast CT (SRµCT) image quality differently, with the best contrast (i.e. most morphological features preserved) for MRI imaging when using standard 4%-PFA fixation, and for SRµCT when using the Evaporation-of-Organic-Solvent (EOS) fixation. The visibility of the ischemic lesion varied greatly among different fixation protocols in all imaging modalities, resulting in a variation of detectable lesion size (measured relative to ipsilateral hemisphere) of up to 50% when compared with the reference in vivo MRI measurement. Moreover, the edema index showed even greater dissipation, ranging from as low as 28% to as high as 85% of the in vivo value. This apparent discrepancy is a direct result of uneven tissue shrinkage across the brain during fixation, caused by the different chemical composition of the diseased tissue compared to the healthy tissue and hence different chemical reaction to the fixation agent.

Conclusions

Tissue fixation protocols optimized for ex vivo imaging with either MRI or phase-contrast CT can deliver sufficient contrast for the needs of virtual histology on healthy tissue. However, chemical reactions between the fixating agent and the diseased tissue are different to those in the healthy tissue, resulting in an inherent fixation-induced bias when doing ex vivo volumetric evaluation of pathology, regardless of the analysis technique.

References

1. Dullin C et al. Functionalized synchrotron in-line phase-contrast computed tomography: a novel approach for simultaneous quantification of structural alterations and localization of barium-labelled alveolar macrophages within mouse lung samples, J Synchrotron Radiat. 2015  

2. Töpperwien M et al. Three-dimensional mouse brain cytoarchitecture revealed by laboratory-based x-ray phase-contrast tomography. Sci Rep. 2017.

Acknowledgement

This publication was co-financed by the European Union through the European Regional Development Fund, Operational Programme Competitiveness and Cohesion, grant agreement No. KK.01.1.1.01.0007, CoRE – Neuro, Croatian Science Foundation under the project IP-06-2016-1892 RepairStroke, and by Synchrotron Light Source ‘Elettra’ grant n.20170140. MRI scans were performed at the Laboratory for Regenerative Neuroscience – GlowLab, University of Zagreb, Croatia.

Keywords: MRI, synchrotron, fixation, volumetry, stroke
708

Synchronized PET/MRI at 9.4Tesla – A pictorial essay of different organs and disease models (#249)

Joachim Friske1, Viktoria Ehret1, Lubos Budinsky1, Katja Pinker-Domenig1, Vanessa Fröhlich1, Daniela Laimer-Gruber1, Anna S. Zacher1, Lukas Nics1, Markus Mitterhauser2, Wolfgang Wadsak1, Marcus Hacker1, Thomas Helbich1

1 Medical University of Vienna ‐ General Hospital of Vienna, Department of Biomedical Imaging and Image‐guided Therapy, Molecular Imaging Laboratory (PIL/EXPNUC), Vienna , Wien, Austria
2 LBI Applied Diagnostics, Vienna, Wien, Austria

Introduction

Synchronized PET/MRI has emerged as an exceptionally powerful and precise technique and visualizes complex interactions of different diseases in a clinical setting. The combination of several single parameters in a multiparametric (mp) imaging concept is necessary to maximize diagnostic accuracy and disease characterization [1-4]. With the development of a new PET insert which can be integrated in an MRI scanner synchronized mpPET/MRI can be introduced into field of preclinical imaging as well. This assay presents the potential of this new technique high lightening different disease models.

Methods

All mpPET/MRI measurements were performed using a horizontal bore 9.4T (BioSpec 94/30USR) system combined with a PET insert using a 1H volume coil (d=72 mm) (Bruker, Ettlingen, Germany). The PET insert consist of three rings of 8 octagonal orientated Silicon Photomultiplier (SiPMs) mounted inside the magnetic bore. Simultaneous PET and MRI scanning (with different sequences) is possible to acquire spatially and temporally registered images. Mice and rats were anesthetized using isoflurane (2‐3%) in air (2 l/min). Respiratory triggering was used to limit movement artifacts. Different preclinical organ and disease models were imaged using the concept of mpPET/MRI spanning the spectrum from different tumor, heart, vessel, and brain diseases.

Results/Discussion

In this pictorial assay we will present a variety of organ and disease models imaged with mpPET/MRI using different tracers. A spectrum of different tumor, heart, vessel, and brain diseases will be shown. mpPET/MRI is feasible without quality impairment of PET and MR images. Tumours and organs of interest are clearly visible on both imaging techniques (Figure 1 and 2). This offers the ability to localize tissue of interest more precisely and differentiate it more exact from the surrounding area. In addition mpPET/MRI allows to synergistically visualizing complex interactions of different diseases.

Conclusions

This pictorial assay presents a variety of organ and disease models imaged with mpPET/MRI using different tracers. The first results are promising. The FOV is large enough to image both, rats and mice using appropriate coils and hardware. Future application can combine quantitative MRI methods like CEST, BOLD, spectroscopy or diffusion with new PET radiotracers.

References

[1] Wehrl HF, Sauter AW, Divine MR, Pichler BJ. Combined PET/MR: a technology becomes mature. J Nucl Med. 2015 Feb;56(2):165-8. doi: 10.2967/jnumed.114.150318. Epub 2015 Jan 15. Review. PubMed PMID: 25593114.

[2] Pinker K, Stadlbauer A, Bogner W, Gruber S, Helbich TH. Molecular imaging of cancer: MR spectroscopy and beyond. Eur J Radiol. 2012 Mar;81(3):566-77. doi:10.1016/j.ejrad.2010.04.028. Epub 2010 Jun 4. Review. PubMed PMID: 20554145.

[3] Pinker K, Marino MA, Dr Meyer-Baese A, Helbich TH. Multiparametric andmolecular imaging of breast tumors with MRI and PET/MRI. Radiologe. 2016 Jul;56(7):612-21. doi: 10.1007/s00117-016-0129-3. Review. German. PubMed PMID:27364727.

[4] Sasser, T., Attarwala, A., Frederick, E., et al. General Considerations and Applications Strengths of Preclinical PET/MR in Oncology Research. Bruker BioSpin 04/2018

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). Study supported in part by a research grant from Bruker (Ettlingen, Germany).

Figure 1: FDG-PET/MRI of high aggressive breast cancer xenograph

Fused PET/MR image of 4T1 breast cancer xenograph in sagittal orientation. The tumor is marked by the red circle.

MRI Parameters: T2_Turbo_RARE TE:31.7ms TR:1000 ms Coronal SL:1 mm (350x116) RF:10 Fat Sat. Resolution ~0.3 mm in plain MT~5min

PET Parameters: [18F] FDG measurement time 10 min Reconstruction MLEM GPU 32x32 Number of Iterations 30 Spatial Resolution 0,5 mm

Figure 2: FDG-PET/MRI of medium aggressive brast cancer xenograph

Synchronized PET MRI of SKBR-3 breast cancer xenograph in axial orientation. The tumor is marked by the red circle.

MRI Parameters: T2_Turbo_RARE TE:24.6 ms TR:1803 ms Axial SL:1 mm Matrix: 300x256 RF:8 Fat Sat. Avg:6 Resolution ~0.14 mm in plain MT~10min

PET Paramters: [18F] FDG measurement time 10 min Reconstruction MLEM GPU 32x32 Number of Iterations 30 Spatial Resolution 0,5 mm

Keywords: PET, MR, PETMR, breast cancer

X-Ray based Imaging | Technology

Session chair: Benedicte Descamps (Gent, Belgium); Mónica Abella (Madrid, Spain)
 
Shortcut: PW15
Date: Thursday, 21 March, 2019, 12:45 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.

710

Particle Induced X-ray Emission (PIXE) for the development of new diagnostic tools. Elemental tissue imaging in prosthesis rejection cases (#274)

Esther Punzon-Quijorna1, Mitja Kelemen1, Primož Vavpetič1, Samo K. Fokter2, Primož Pelicon1

1 Jožef Stefan Institute (JSI), Department of Low and Medium Energy Physics F2. Microanalytical Center (MIC), Ljubljana, Slovenia
2 University Clinical Centre Maribor (UKC), Division of Surgery, Department of Orthopaedics, Maribor, Slovenia

Introduction

Recent reports on mass implant failures reveal insufficient understanding of physiological processes behind prosthesis failure [1-4]. Techniques applied in hospitals, such as X-ray and optical microscopies, do not fully explain the effects of implants in the body. Modern microscopy techniques, as energy dispersive X-ray analysis (EDX) and electron energy-loss spectroscopy (EELS), provide elemental distribution with high lateral resolution, however, they are unable to detect elements present at low concentrations.

 

Methods

This work is focused on investigating reasons of failures of hip prosthesis by application of Particle-Induced X-ray Emission (PIXE). This technique combines high elemental sensitivity (detection limit 0.1 ppm) and lateral resolution (600 nm). The proton microprobe of the 2 MV tandem accelerator available at JSI can feature the highest beam brightness at any tandem accelerator worldwide [5], which makes it especially suited for screening the effects of prosthesis degradation [6], [7]. Tissue samples are frozen and sliced (from 10 to 40-μm thickness). Tissue slices are scanned with a 3 MeV focused proton beam. Finally, the measured data files are treated with GUPIX software to achieve the elemental distribution maps, and quantification.

 

Results/Discussion

Micro-PIXE is an adequate technique to study the distribution and concentration of biologically active wear particles (such as Ti, Al, V, Cr, etc.) coming from prosthesis failures into the peri-prosthetic tissue, obtained by biopsy during hip-replacement surgery. The images, obtained with GUPIX software, show the pattern of elemental distribution in the tissue (from F to Ni) and allow the univocal identification of the different features observed with optical microscopy. This has permitted us to identify, for example, the white spots in the optical image as Ti particles. Figure 1 corresponds to a tissue sample obtained from a patient whose hip prosthesis failed due to a fracture of the prosthesis neck, which connects the head and the stem. Elemental quantification results, in ppm, are obtained from the spectrum fitting. Results allow estimating the particle sizes and the distribution paths into the tissue.

 

 

Conclusions

Micro-PIXE has been proved to suit well for element localization studies in biological tissues, by combining high elemental sensitivity, high lateral resolution and a quantification ability [8]. This multidisciplinary research project aims to combine efforts from Medicine and Physics to develop more complete diagnostic techniques. Results are expected to have an impact on everyday life of patients using or in need of hip replacements.

 

References


[1] Garellick, Göran, et al., The Swedish Hip Arthroplasty Register Annual Report 2014. www.shpr.se.

[2] Alison J Smith et al, Lancet 379: 1199–204, 2012.

[3] Fokter SK, et al., Acta Orthopaedica, 87(2), p.197-202, 2016.

[4] Kavalar R, et al., European Journal of Medical Research, 21:8, 2016.

[5] Primoz Pelicon et al., Nuclear Instruments and Methods in Physics Research B, vol. 332, pp. 229-233, 2014.

[6] Teresa Pinheiro et al., Modern Research and Educational Topics in Microscopy, p. 237-244, 2007.

[7] Fokter SK, et al., Journal of the Mechanical Behavior of Biomedical Materials, 69, p 107-114, 2017

[8] P. Vavpetič et al., Nuclear Instruments and Methods in Physics Research B, vol. 306, pp. 140-143, 2013.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 799182.

Optical and PIXE images from peri-prosthetic human tissue

Figure 1. a) Optical microscope image from the peri-prosthetic tissue analyzed. b) Elemental map distribution of Ti, obtained by PIXE, in the same area.

Keywords: life sciences, tissue imaging, diagnostic tools, medical imaging physics, nuclear microprobes
711

3D reconstruction of complex tissue based on differentiation of internal structures by novel contrast-enhanced μCT and AI-based auto classification (#251)

Tomonobu Ezure1, Satoshi Amano1, Kyoichi Matsuzaki2

1 Shiseido Co. LTD, Global Innovation Center, Yokohama, Japan
2 International University of Health and Welfare, Plastic Surgery, Tokyo, Japan

Introduction

Micro computed tomography (μCT) cannot readily distinguish internal structures of complex tissues even when contrast agents are used. Also, manual evaluation of large numbers of CT images is extremely laborious. Here, we established a new contrast-enhanced μCT method to differentially detect multiple internal structures of complex tissue by incorporating acetone pretreatment to optimize the distribution of contrast agent. We used an artificial intelligence (AI) deep learning system to classify structures automatically, and reconstructed the overall 3D structure on computer.

Methods

Study outline:  We chose skin as a representative complex tissue containing a variety of internal structures in abundant extracellular matrix (ECM: dermal layer) covered by epidermis. Skin samples (10 cheek skin) were taken from surplus skin excised during plastic surgery.

Contrast-enhanced μCT to distinguish internal structures:  Samples were pretreated or not pretreated with alcohol or acetone, and treated or not treated with contrast agents such as iodine and phosphotungstic acid (PTA), then analyzed by μCT (D200RSS270; Comscan Techno) [1].

Auto classification:  Individual structures in each μCT image of skin were auto-classified by an artificial intelligence (AI)-based deep-learning system (Dragonfly) and an overall 3D image was reconstructed [2].

Results/Discussion

Differentiation of internal structures of skin by μCT:  Skin contains many internal structures (sweat gland, sebaceous gland, hair muscle, hair follicle), but μCT without contrast agent could not detect most of them (Fig. 1a). We found that pretreatment with acetone followed by treatment with iodine as a contrast agent enabled each of these internal structures to be successfully differentiated by μCT (Fig. 1b,c).

AI auto classification is effective to process large-scale μCT image data:  The AI deep learning system was trained with a set of μCT images, and then used to auto-classify internal structures in serial image data obtained by μCT. The 3D skin structure was reconstructed in the computer from these data (Fig. 2a). The 3D reconstruction can be manipulated as desired for “computer anatomy” to explore the skin, as well as to isolate and analyze all of the internal structures and their relationships (Fig. 2b,c).

Conclusions

We established a new contrast-enhanced μCT method with acetone pretreatment to optimize the distribution of contrast agent, and AI auto classification to identify individual internal structures in a complex tissue (skin), followed by 3D tissue reconstruction in the computer. This is a breakthrough in tissue structure analysis, enabling precise and flexible analysis of a complex tissue containing multiple internal structures on the computer.

References

[1] Hafez A, Squires R, Pedracini A, Joshi A, Seegmiller RE, Oxford JT, Col11a1 Regulates Bone Microarchitecture during Embryonic Development. J. Dev. Biol. 3, (2015),158

[2] Heidrich A, Schmidt J, Zimmermann J, Saluz HP, Automated Segmentation and Object Classification of CT Images: Application to In Vivo Molecular Imaging of Avian Embryos. Int. J. Biomed. Imaging 2013, (2013), 508474

Effects of acetone pretreatment and iodine treatment on μCT visualization of internal structures
(a) Untreated skin, (b) iodine-treated skin: fat can be detected, (c) acetone-pretreated, iodine-treated skin: sweat glands (SG) (representative internal structure) can be distinguished from fat and the dermal layer.
3D reconstructed skin (a) can be analyzed freely on the computer.

As examples, (b) sweat glands are isolated and ranked by volume, and (c) sebaceous glands are ranked by number of lobules.

Keywords: μCT, Skin, AI, Contrast agent, 3D
712

Dose and radiosafety of repeated whole-body high-resolution micro-CT protocols for monitoring lung metastasis, inflammation and fibrosis (#36)

Kaat Dekoster1, Nathalie Berghen2, Eyra Marien3, Amy Hillen1, Jens Wouters1, Jasmine Deferme1, Thibault Vosselman1, Eline Tiest1, Jérémie Dabin4, Marleen Lox5, Ellen De Langhe2, Ria Bogaerts3, Marc F Hoylaerts5, Rik Lories2, Greetje Vande Velde1

1 KU Leuven, Biomedical MRI, Department of Imaging and pathology, Leuven, Belgium
2 KU Leuven, Skeletal Biology and Engineering Research Center, Leuven, Belgium
3 KU Leuven, Department of Oncology, Leuven, Belgium
4 SCK-CEN, Research in Dosimetric Applications, Mol, Belgium
5 KU Leuven, Centre for Molecular and Vascular Biology, Leuven, Belgium

Introduction

In vivo µCT is excellent for longitudinal lung imaging, but it exposes the animal to repeated low x-ray doses, further increased by the longer scanning times needed for respiratory gating. Although X-ray doses in µCT are an order of magnitude lower than the high dose that can result in inflammation, fibrosis and malignancies, its potential effects are unknown. To rule out potential confounding factors when implementing µCT for longitudinal studies of mouse lung disease, we measured radiation dose and effects of repeated whole-body 4D and 3D µCT on the most radiosensitive cells.

Methods

Mice with induced lung metastasis1, lung inflammation and fibrosis2 and sham-induced healthy controls were either scanned at baseline and weekly for 4 weeks, or only at baseline and endpoint or exposed to anesthesia only (scan-related stress), with 4D respiratory gated or 3D expiration weighted high-resolution whole-body µCT (SkyScan 1278). We evaluated four µCT-derived lung biomarkers: total lung volume, aerated lung volume, non-aerated lung volume and mean lung density2. Lung metastasis burden was quantified by bioluminescence imaging (BLI, IVIS Spectrum). At endpoint, mice were sacrificed, blood was obtained by cardiac punction and mixed with sodium citrate for complete blood cell counts (Cell-dyn 3700). Lungs were isolated for ex vivo standard histochemical and histological analysis.

Results/Discussion

Weekly scanning with 4D respiratory-gated µCT (700 mGy/scan) did not significantly alter the metastatic load or host response, demonstrated by BLI and all four µCT-derived lung disease biomarkers. We found a small but significant decrease in platelet counts and absolute numbers of white blood cells (mainly circulating lymphocytes) in the lung metastasis model. Weekly scanned bleomycin-induced and healthy animals showed the same trend, indicating a disease-independent effect of cumulated x-ray dose on hematopoiesis. Optimizing a 3D respiration-weighted µCT (233 mGy/scan), lung image quality was maintained similarly high as for the 4D respiratory-gated protocol while platelet, white blood cell and lymphocyte counts did not decrease after weekly µCT-scanning. This indicated that the 3D-protocol had no effect on the bone marrow compartment. Detailed ex vivo analyses confirmed the imaging-findings and revealed no further potential radiotoxic damage to the lungs for both 4D and 3D µCT.

Conclusions

We could not observe any side effects of µCT-associated radiation on lung diseases involving rapidly dividing cells and host response, while delivering high-quality 4D and 3D µCT data. With repeated 4D scans, we operate at the upper safety limits of cumulated whole-body x-ray exposure, but 3D µCT is an excellent and safe technique to monitor disease processes without concerns for radiotoxicity when carefully designing scan protocols.

References

1. Marien, E., Hillen, A., Vanderhoydonc, F., Swinnen, J.V. & Vande Velde, G. Longitudinal microcomputed tomography-derived biomarkers for lung metastasis detection in a syngeneic mouse model: added value to bioluminescence imaging. Laboratory Investigation 97, 24-33 (2017).

2. Vande Velde, G. et al. Longitudinal micro-CT provides biomarkers of lung disease and therapy in preclinical models, thereby revealing compensatory changes in lung volume. Dis Model Mech 9, 91-98 (2016).

Acknowledgement

This research was supported by KU Leuven IF research grants C24/17/061 and StG/15/024.

Keywords: Micro-computed tomography, Radiosafety, Lung metastasis, Lung inflammation & fibrosis
713

What’s the role of µCT imaging in pulmonary fibrosis drug discovery? Side by side comparison between histology and µCT (#230)

Laura Mecozzi1, Francesca Ruscitti2, Roberta Ciccimarra3, Valeria Bertani3, Francesca Ravanetti3, Nicola Sverzellati1, Valentina Menozzi3, Gino Villetti2, Maurizio Civelli2, Franco Fabio Stellari2

1 University of Parma, Department of Medicine and Surgery, Parma, Italy
2 Chiesi Farmaceutici S.p.A., Corporate Pre-Clinical R&D, Parma, Italy
3 University of Parma, Department of Veterinary Science, Parma, Italy

Introduction

MicroCT (μCT) has been recently proposed as a powerful tool to evaluate pharmacological efficacy and pathobiology of lung fibrosis in bleomycin (BLM)-induced murine models [1]. In this study, the staging of pulmonary fibrosis and the efficacy of Nintedanib (NINT) treatment (FDA approved drug) have been evaluated by correlating histological and biochemical parameters (hydroxyproline (HYP) content) associated to fibrotic lesions with different degrees of aeration in lung compartments measured by in vivo μCT.

Methods

C57BL/6 mice received a double BLM oropharyngeal administration (at days 0 and 4) to induce lung fibrosis. A group of mice was treated with NINT (60 mg/kg) for 2 weeks starting at day 7. At day 21 μCT scans were acquired (Quantum GX PerkinElmer, Inc., Waltham, MA) and lungs were isolated for ex vivo measurements, assigning the left lobe to histology and right ones to HYP determination. The reconstructed 3D μCT datasets (50μm voxel size) were linearly rescaled into Hounsfield Units (HU) and analyzed using Analyze software (version 12.0, PerkinElmer). For quantitative assessment, HU clinical ranges were applied to the segmented lung area [2] and the resulting normally and poorly aerated regions compared to moderate and severe fibrotic lesions as defined by Ashcroft score.

Results/Discussion

Saline, BLM and NINT groups were evaluated either by μCT or ex vivo analysis to detect pulmonary fibrotic lesions, whose severity has been reduced in response to NINT treatment (%poorly aerated tissue and Ashcroft score inhibition of 51% and 37%, respectively). The concordance among μCT data (% poorly aerated tissue), Ashcroft score and HYP content was 67% and 72% for semiautomatic and manual 3D lung segmentation, respectively. A side by side comparison between histological slices (gold standard) and the corresponding μCT sections (manually segmented) highlighted a good match, resulting in a R2=0.9.

Conclusions

Good concordance has been found among three different technologies, but histology and biochemical determination described only a snapshot of the pathology. μCT imaging can provide tridimensional predictive measurements describing the whole lesions distribution. μCT needs to be considered as a reliable and useful tool to investigate lung fibrosis progression and drug efficacy evaluation.

References

  1. Ruscitti F. et al. ’Longitudinal assessment of bleomycin-induced lung fibrosis by micro-CT correlates with histological evaluation in mice’, Multidisciplinary Respiratory Medicine,12:8 (2017)
  2. Gattinoni L. et al. ‘What has Computed Tomography taught us about the acute respiratory distress syndrome?’ Am J Respir Crit Care Med 164(9):1701, 2001
Figure1. Automatic vs manual segmentation

3D lung renderings obtained with semi-automatic (left) and manual (right) segmentation

Figure2. Side by side comparison histology-microCT
‘Aligned’ histological and microtomographic slices. The plot highlights the very good correlation found between ex vivo and in vivo parameters.
Keywords: MicroCT, Pulmonary Fibrosis, Drug Discovery, Histology
714

Dynamic in vivo synchrotron lung microscopy: towards the alveolar scale. (#462)

Luca Fardin1, 2, Alberto Mittone1, Francesca Di Lillo1, Anders Larsson2, Alberto Bravin1, Sam Bayat3

1 European Synchrotron Radiation Facility, Grenoble, France
2 Uppsala University , Hedenstierna Laboratory, Uppsala, Sweden
3 Grenoble University Hospital Center, Department of Clinical Physiology, Grenoble, France

Introduction

Micrometric-resolution x-ray imaging of moving organs such as the heart and lungs is highly challenging due to motion artifacts. In the lung, this limitation has hindered the investigation of disease processes at the alveolar level. At the biomedical beamline (ID17) of the European Synchrotron Radiation Facility, a time-resolved tomographic technique was developed, which allows imaging the lungs in vivo down to the alveolar scale, opening the way to the investigation of the micromechanics of the lung parenchyma in a physiological model.

Methods

The experiment was performed on 9 anesthetized, muscle-relaxed and mechanically ventilated adult rats. Projection images were acquired at a constant frame rate at time resolution down to 2 ms using a PCO edge 5.5 camera, coupled with two different optics determining a final pixel size of ~6 µm and 11 µm, respectively. During imaging, animals were either under apnoeic oxygenation or under controlled ventilation using a custom-made mechanical ventilator. To synchronize the image reconstruction with the parenchymal motion, the ECG and respiratory pressure signals were also registered. Images were post-reconstructed by sorting image projections based on the respiratory and cardiac activity phases.

Results/Discussion

This post-gated reconstruction technique allowed obtaining dynamic volumetric images while keeping motion artefacts low (Figures 1 & 2). Images were obtained at different phases within the cardiac cycle. Figure 1 shows a tomographic slice acquired at 11 µm pixel size, under controlled breathing. Figure 2 shows a tomographic image acquired at 6 µm pixel size during apnoea.

Conclusions

A time resolved lung tomographic (4D) technique was developed, allowing to 1) minimize motion artefact; 2) achieve the time resolution required to resolve the motion down to the alveolar scale. This imaging technique could be a valuable tool to investigate the lung micromechanics in fields ranging from aerosol deposition to mechanical ventilation-associated lung injury.

Acknowledgement

This work was supported by the Swedish Research Council (K2015-99X-22731-01-04) and the Swedish Heart and Lung foundation (2017-0531)

Figure 1
Tomographic image of rat lung acquired with a pixel size of 11mm during mechanical ventilation.
Figure 2
Tomographic image of rat lung acquired with a pixel size of 6mm during apnoea.
Keywords: microto-mography, lung imaging, time-resolved
715

Automatic quality control and recalibration for micro-CT without user intervention (#177)

Bert Vandeghinste1, Roel Van Holen1

1 Molecules NV, Gent, Belgium

Introduction

In preclinical imaging, it is necessary to have reliable systems with known performance characteristics. Although major defects are easy noticeable, more subtle issues may remain hidden. Ideally, any end-user should be able to characterize uniformity, contrast and spatial resolution and, when needed, be able to recalibrate the system without any specialised knowledge. Some automated methods exist for clinical CT on eg. CATPHAN phantoms [1], but is currently non-existent in preclinical in-vivo micro-CT.

Methods

A rat-size CT QC phantom was specifically developed for Molecubes and produced at Phantom Labs, USA. It exists of 4 specialised sections and ends on both sides with registration markers for automatic registration. Image uniformity is calculated from a uniform PMMA section according to IEC specifications [1]. Spatial resolution is measured with a Fourier-based analytical approach based on [2]. Low contrast detectability is determined from 9 low-contrast spheres with a NPWE model observer [3]. The automated spatial resolution method is validated against measurements of the QRM bar phantom. All data is reported to the user and kept historically over time. If the results deviate, the system is geometrically recalibrated with an automated image-based method based on [4].

Results/Discussion

Fig. 1 illustrates the different phantom sections recorded with general protocols on the X-CUBE micro-CT system. Total time for all data acquisitions is less than 5 minutes, after which the CT system can be used again for other scans. Iterative reconstructions and image processing takes up to 15 minutes before a report is shown to the user (Fig. 2). Manually acquiring all data and processing all results would take much longer and would be prone to errors. The automated spatial resolution measurements agree with results obtainable from the QRM bar phantom. Further analysis will be necessary to determine the variability over time.

Conclusions

We demonstrate that it is possible to automatically characterize a micro-CT system and evaluate the necessity of recalibration. This enables daily quality control and should improve the day-to-day use of molecubes micro-CT scanners. This will be useful for end-users to guide them in developing their own custom protocols.

References

[1] P. Nowik, R. Bujila, G. Poludniowski, and A. Fransson, “Quality control of CT systems by automated monitoring of key performance indicators: a two-year study.,” Journal of Applied Clinical Medical Physics, vol. 16, no. 4, pp. 254–265, Jul. 2015.

[2] S. N. Friedman, G. S. K. Fung, J. H. Siewerdsen, and B. M. W. Tsui, “A simple approach to measure computed tomography (CT) modulation transfer function (MTF) and noise-power spectrum (NPS) using the American College of Radiology (ACR) accreditation phantom,” Med. Phys., vol. 40, no. 5, p. 051907, 2013.

[3] I. Hernandez-Giron, J. Geleijns, A. Calzado, and W. J. H. Veldkamp, “Automated assessment of low contrast sensitivity for CT systems using a model observer,” Med Phys, vol. 38, no. 1, p. S25, 2011.

[4] A. Kingston, A. Sakellariou, T. Varslot, G. Myers, and A. Sheppard, “Reliable automatic alignment of tomographic projection data by passive auto-focus,” Med Phys, vol. 38, no. 9, p. 4934, 2011.

Fig. 1: The different phantom sections acquired.
From left to right: low contrast detectability from 9 spheres, spatial resolution, uniformity.
Fig. 2: sample report

Sample of what is reported to the end-user. Spatial resolution is reported as MTF, while low contrast detectability is shown as area under the ROC curves.

Keywords: CT, quality control, calibration
716

Improvement of the image quality of a commercial CT system by iterative volume reconstruction (#566)

Martin Pichotka1, 4, Moritz Weight3, Karla D. Palma-Alejandro2, Ludek Sefc2

1 University Freiburg Medical Centre, Department of Radiation Oncology, Freiburg, Germany
2 First Faculty of Medicine, Charles University, Center for Advanced Preclinical Imaging (CAPI), Praha 2, Czech Republic
3 SpeCTive GmbH, Freiburg, Germany
4 CTU Prague, Institute of Experimental and Applied Physics, Praha 2, Czech Republic

Introduction

The high cost of purchase of pre-clinical imaging systems often leads to long life cycles of such machines. Whereas hardware upgrades are typically rather costly, the employment of improved image processing methods allows for incremental and affordable quality improvement during a machine's life time.
The exponential increase of computation power available in commodity hardware in particular by now allows us to employ advanced iterative volume reconstruction in CT, providing significant quality improvement of imaging results and dynamical trade-off between scanning time and image quality.
 

Methods

In the current investigation, we modify the data processing chain of an Albira small animal PET/SPECT/CT system in order to obtain unprocessed detector data. These data are consecutively processed using the speCTive COBRA iterative reconstruction software. This tool, which was originally intended to be used with photon-counting pixel detectors, features a highly parallelized and fully 3D implementation of a distance-driven raytracer. Following initialization by back-projection the volume is updated in OSEM by subsets of multiples of 90 degrees spaced sets projections, allowing significant speed-up due to exploitation of symmetries. Dynamic refinement of the subset size allows optimization between image quality and speed of convergence.

Results/Discussion

Comparative evaluation was performed for typical CT acquisition parameters used in pre-clinical imaging. By design, reconstruction of the entire volume can only be performed by FBP in the Albira manufacturers software. Additionally, part of the volume can be reconstructed in higher resolution using the iterative advanced reconstruction module.
Therefore one first advantage of COBRA is the possibility of reconstructing the entire volume at high resolution. Also, opposite to FBP, COBRA performs well under low angular sampling, therefore, giving reasonable preview quality in a fast preliminary scan.
 

Conclusions

Using a state-of-the-art multi-modal SPECT/PET/CT scanner we achieved a significant improvement of the CT reconstruction quality by alternative data processing.
Given full control over all relevant parameters of the reconstruction, we are able to efficiently tune our volumetric results.
In a next step we will use the same data processing chain to reconstruct SPECT data from our system and to fuse the result into a multi-modal dataset.
 

References

  1. B. De Man and S. Basu, "Distance-driven projection and backprojection," 2002 IEEE Nuclear Science Symposium Conference Record, Norfolk, VA, USA, 2002, pp. 1477-1480 vol.3. DOI:10.1109/NSSMIC.2002.1239600
  2. M. P. Pichotka: “Iterative CBCT reconstruction-algorithms for a spectroscopic Medipix-Micro-CT, Albert-Ludwigs-Universität Freiburg, Diss., 2014.

  3. Sánchez, F. , Orero, A. , Soriano, A. , Correcher, C. , Conde, P. , González, A. , Hernández, L. , Moliner, L. , Rodríguez‐Alvarez, M. J., Vidal, L. F., Benlloch, J. M., Chapman, S. E. and Leevy, W. M., ALBIRA: A small animal PET/SPECT/CT imaging system. Med. Phys., 2013. DOI:10.1118/1.4800798

Acknowledgement

This work was supported by MEYS CR (LM2015062 Czech BioImaging).

Postprocessing of CT image
Comparison of image quality obtained by a commertial SW and the new  iterative volume reconstruction.
Keywords: image reconstruction, computed tomography, SPECT

Mass Spectrometry | Technology

Session chair: John Fletcher (Gothenburg, Sweden); Joanna Polanska (Gliwice, Poland)
 
Shortcut: PW16
Date: Thursday, 21 March, 2019, 12:45 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.

720

New Concepts for Multimodal iImaging – Elemental and Molecular Information from Tissue Sections (#495)

Martina Marchetti-Deschmann1, Matthias Holzlechner1, Maximilian Bonta1, Anna Turyanskaya2, Karin Wieland1, Bernhard Lendl1, Uwe Karst3, Martin Wiemann4, Christina Streli2, Andreas Limbeck1

1 TU Wien, Institute of Chemical Technologies and Analytics, Vienna, Austria
2 TU Wien, Atominstitut, Vienna, Austria
3 University of Münster, 1Institute of Inorganic and Analytical Chemistry, Münster, Germany
4 IBE R&D Institute for Lung Health gGmbH, Münster, Germany

Introduction

We combine imaging technologies like Mass Spectrometry imaging (MSI), Fourier-Transform Infrared (FT-IR), µX-ray fluorescence (µ-XRF) and laser ablation inductive coupled plasma (LA-ICP) MS. MSI provides localized information on intact molecules, FT-IR molecular class info, LA-ICP-MS quantitative, elemental distributions and µ-XRF imaging shows spatial context of light elements without destroying samples. Combining such approaches allows unbiased, novel insights into molecule/element co-localization providing data not accessible so far as demonstrated for Si-nanoparticles in lung tissue.

Methods

Mesothelioma samples treated with Pt-containing anti-cancer drugs were used for lipid analysis after applying matrix by sublimation. A MALDI TOF/RTOF system (UltrafleXtreme) and a LA-ICP-MS (Thermo iCAP Qc quadrupole ICP-MS) were used for analysis. Fleximaging 4.0 (Bruker Daltonics) and ImageLab (Epina) were used for data analysis. Chicken toes purchased from a local market were embedded and cryosectioned at 10 µm. A MALDI qTOF system (Synapt G2 HDMS) was used for lipid analysis and non-destructive elemental imaging was carried out on a µ-XRF spectrometer (home-built). Unmodified and modified SiO2-nanoparticles were administered to rats and cryo-sections of lung were subjected to fluorescence microscopy (Olympus IX51), MALDI-MSI (Synapt G2 HDMS), FT-IR (Hyperion 3000) and LA-ICP MS.

Results/Discussion

We present methodologies allowing molecular and elemental image analyses on one tissue section. Robust workflows allow non-destructive elemental µXRF imaging of representative elements for bone together with lipid distributions in soft tissue (cartilage, blood vessels, periosteum) by MALDI MSI.

Latter was also combined with LA-ICP-MS to gather quantitative elemental info. We aimed at lipid detection from the same tissue section while preserving quantitative, elemental distributions for P, S and Pt. We present molecular data significantly correlating with high Pt concentrations in human malignant pleural mesothelioma samples after administration of individuals with platinum containing cytostatic drugs.

Ultimately the combination of LA-ICP-MS, MALDI MSI andwith FT-IR imaging is presented for analyses of lung tissue after administering animals with SiO2nanoparticles of different kinds. Results are correlated to inflammation markers.

Conclusions

We show that new concepts for multimodal imaging allow to gather unbiased, novel insights into molecule and element co-localization providing biological data not accessible by any other methodology at so far.

References

  1. Svirkova A, Turyanskaya A, Perneczky L, Streli C, Marchetti-Deschmann M. Multimodal imaging of undecalcified tissue sections by MALDI MS and µXRF. Analyst 2018;143:2587-95

  2. Holzlechner M, Bonta M, Lohninger H, Limbeck A, Marchetti-Deschmann M. Multi-sensor Imaging - from sample preparation to integrated multimodal interpretation of LA-ICP-MS and MALDI MS imaging data. Anal Chem 2018;90(15):8831-37.

  3. Großgarten M, Holzlechner M, Vennemann A, Balbekova A, Wieland K, Sperling M, Lendl B, Marchetti-Deschmann M, Karst U, Wienmann M. Phosphonate Coating of SiO2 Nanoparticles Abrogates Inflammatory Effects and Local Changes of the Lipid Composition in the Rat Lung: A Complementary Bioimaging Study. Particle Fibre Toxicology 2018;15(1):31.

Acknowledgement

The authors thank TU Wien for funding the doctoral program MEIBio (Molecular and Elemental Imaging in Biosciences) and COST BM1104 (Mass Spectrometry Imaging: New Tools for Health Care Research) for financial support.

Keywords: multimodal imaging, mass spectrometry, µXRF, Laser Ablation ICP MS, FT IR
721

New tools for intraoperative assessment of surgically resected tissue specimens (#493)

Tiffany Porta Siegel1, Pierre-Maxence Vaysse1, 2, Lieke Lamont1, Heike I. Grabsch3, 4, 5, Steven W. Olde Damink2, 6, Ron M. Heeren1

1 Maastricht University, Division of Imaging Mass Spectrometry / M4I Institute,, Maastricht , Netherlands
2 Maastricht University Medical Centre, Department of Surgery, Maastricht , Netherlands
3 Maastricht University Medical Centre, Department of Pathology, Maastricht, Netherlands
4 Maastricht University Medical Centre, GROW School for Oncology and Developmental Biology, Maatsricht, Netherlands
5 Leeds Institute of Cancer and Pathology University of Leeds, Section of Pathology and Tumour Biology, Leeds, United Kingdom
6 Maastricht University, NUTRIM school of Nutrition and Translational Research in Metabolism, Maastricht, Netherlands

Introduction

In recent year, the expansion of ambient ionization mass spectrometry techniques has provided a large varieties of tools that are foreseen to revolutionize tissue diagnostics dring surgery, both for in vivo and ex vivo tissue analysis. Here, we discuss the possibilities of rapid evaporative ionisation MS (REIMS) [1] and desorption electrospray ionisation (DESI) MS imaging [2] for the classification of tissue from patients undergoing surgery for colorectal liver metastasis (CRLM), provided for this study by the Department of Pathology of MUMC+.

Methods

Experiments were conducted on resected specimens from patients who underwent oncological surgery. Samples were selected by the pathologist after diagnostics routine for mass spectrometric analysis. Experiments were conducted on a Xevo-G2-XS QTOF system (Waters) fitted with a prototype REIMS source, operated in negative ion mode. The flexible tubing of the electrosurgical sampling tool was connected to the source, and vapor generated was transferred using a Venturi pump. Molecules were ionized at the surface of a heated impactor surface. The resulting REIMS data were analyzed using multivariate statistical tools to build the statistical model. DESI-MSI experiments were performed on the same evo-G2-XS QTOF system, and also on a triple quadrupole instrument for targeted analysis of lipids.

Results/Discussion

Tissue-specific molecular fingerprints were collected from non-tumor and tumor regions with the electrocautery-REIMS system, and each data points were annotated by the pathologist. Spectral differences were observed and based on tissue-specific phospholipids pattern in the mass range m/z 600–900 (detected in negative ion mode). An identification algorithm combining principal component analysis (PCA) and linear discriminant analysis (LDA) was used to process the REIMS data generated and build a classification model for real-time recognition. Our overall classification rate of above 99% for prediction between tumor versus non tumor tissue types.

Next to the REIMS analysis, the spatial analysis of tissue sections from the same specimens employing DESI-MSI allows a detailed analysis of the resection margin region and characterisation of molecular markers that could be used for better definition of the resection margin.

 

Conclusions

We present several approaches based on ambient mass spectrometry (imaging) for molecular profiling for rapid and in situ tissue analysis. These approaches presented excellent performance for tissue evaluation and diagnosis of tumor during intraoperative procedure - bot hin vivo and ex vivo. This demonstrates the great potential of these approaches to improve patients's management and ultimatelly outcome.

References

[1] Edward R. St John, Breast Cancer Res. 2017; 19: 59. doi: 10.1186/s13058-017-0845-2

[2] Livia S. Eberlin, PLoS Med. 2016 Aug; 13(8): e1002108. doi: 10.1371/journal.pmed.1002108

Acknowledgement

This work was supported by the Dutch Province of Limburg through the LINK program.

Keywords: mass spetrometry imaging, intraoperative diagnostics, tumor margin assessment, molecular profile
722

Revealing the regional localization and retention of inhaled drugs in the lung by mass spectrometry imaging (MSI) (#355)

Gregory Hamm1, Erica Bäckström2, Mikael Brülls3, Richard Goodwin1, Markus Fridén2

1 AstraZeneca, Pathology Sciences, Drug Safety & Metabolism IMED Biotech Unit,, cambridge, United Kingdom
2 AstraZeneca, Drug Metabolism and Pharmacokinetics, Respiratory, Inflammation and Autoimmunity IMED Biotech Unit, Gothenburg, Sweden
3 AstraZeneca, Early Product Development, Pharmaceutical Sciences, IMED Biotech Unit, Gothenburg, Sweden

Introduction

Treatment of respiratory disease with drug delivered via inhalation is generally held as being beneficial as it provides direct access to the lung target site with a minimum systemic exposure. There is however only limited information of the regional localization of the drug retention following inhalation especially for combination therapies. Mass spectrometry imaging (MSI) appears as a game changer to understand local drug delivery in lung thanks to its high spatial resolution and multiplexing capabilities.

Methods

Here, we investigated the distribution and retention of different chemotypes after dually cassette administration in rat lungs by MSI. Salmeterol, salbutamol and fluticasone propionate (FP) were administered via nebulisation followed by intravenous (IV) injection of d3-salmeterol, d3-salbutamol and d3-FP to the same rat. Samples of lung tissue and plasma were obtained at 2 and 30 minutes after administration and the distribution and retention of drugs in the lungs were imaged with negative mode Desorption Electrospray Ionisation (DESI) at 70 and 30 μm of spatial resolution.

Results/Discussion

All three compounds were distributed in a patchy manner to the entire lung directly after administration via inhalation (fig 1) whereas IV delivery resulted in a more homogeneous distribution. At 30 minutes were inhaled salmeterol and salbutamol preferentially retained in bronchiolar tissue whereas fluticasone propionate (FP) was retained in all regions of the lungs with a local accumulation through alveolar space. Spatial segmentation of generated MSI data enabled the direct comparison of relative abundances of each species in lung sub-compartment as peripheral v bronchiolar regions. The present study clearly demonstrate that label-free molecular imaging can be applied to gain knowledge of the different retention patterns of drug of diverse chemotypes.

Conclusions

This MSI-based framework for quantifying regional and histological lung retention is required for tailoring inhaled drug molecules and formulations to target the diseased sub-structures and it has the potential to accelerate discovery and development of local and more precise treatments of respiratory disease.

Whole lung imaging by DESI-MSI in negative detection mode at 70 µm spatial resolution.
(a) H&E stain images from 2 (top) and 30 (bottom) min after administration via inhaled nebulization. Molecular images of endogenous (b) Phosphatidylinositol (38:5) (PI) and inhaled (c) salmeterol, (d) salbutamol and (e) FP.
Keywords: Pharmacokinetics, respiratory, mass spectrometry, imaging
723

Universal sample preparation for multimodal tissue analysis (#464)

Andreas Dannhorn1, Emine Kazanc1, Stephanie Ling2, Gregory Hamm2, Gareth Maglennon2, John Swales2, Nicole Strittmatter2, Chelsea Nikula3, Evdoxia Karali4, George Poulogannis4, Josephine Bunch3, Richard Goodwin2, Zoltan Takats1

1 Imperial College London, Surgery and Cancer, London, United Kingdom
2 AstraZeneca, Pathology, Drug Safety and Metabolism, IMED Biotech Unit, Cambridge, United Kingdom
3 National Physical Laboratory, National Centre of Excellence in Mass Spectrometry Imaging (NiCE-MSI), Teddington, United Kingdom
4 The Institute of Cancer Research , London, United Kingdom

Introduction

Cryo-sectioning of tissue specimens can be time-consuming and tedious, especially when numerous small tissues need to be prepared. Most reported embedding protocols were adopted from histological workflows. However, mass spectrometry imaging (MSI) has additional requirements that need to be considered such as preservation of microscopic tissue structures to accurately elucidate metabolite distributions. We aimed to create a universal sample preparation protocol suited for a broad range of specimen and compatibility with multimodal analysis, allowing to study the systems biology of tissues.

Methods

A systematic evaluation of all steps, from medium preparation over sectioning and MSI experiments to post-MSI stains, was performed by embedding multiple rat tissues in each medium. Chemical background as well as analyte delocalization were evaluated by multimodal MSI analysis. Effects on immunochemical analysis of proteins were performed by multiplexed analysis through imaging mass cytometry (IMC) and immunohistochemistry (IHC). The effects on RNA stability were evaluated through RNA in-situ hybridization (RNA-ISH). The integrity of DNA and RNA were confirmed by analysis of tissue extracts analyzed by micro-capillary electrophoresis. The impact of the embedding on the quality of tissue sections and histological stains were evaluated through conventional hematoxylin and eosin (H&E) stains

Results/Discussion

The results of the systematic evaluation of handling, sectioning and background interference of the evaluated media are summarized in table 1. Hydroxypropyl-methylcellulose (HPMC) was favored during the evaluation process, as it is easy to section, beside a poor tissue adherence, did not to leak any chemical background or interfere with H&E staining. Addition of polyvinylpyrrolidon (PVP) to the HPMC hydrogel was found to improve tissue adherence significantly without compromising on the benefits of HPMC. Embedding in this medium, which is liquid at 0˚C, followed by snap freezing of prepared molds was found to limit the delocalization analytes to a minimum. The embedding improved also the quality of the obtained tissue sections by preserving tissue morphology comparable to OCT, the histological gold standard for fresh-frozen tissues. Embedded samples showed highly reproducible results for DNA and RNA extraction and showed no embedding related degradation of either bio-macromolecule.

Conclusions

The newly developed medium increases sample throughput and quality whilst enabling uncompromised multimodal analysis of tissue sections. The superior preservation of microscopic tissue structures ensures for high accuracy of determined analyte distributions, from small metabolites over intact proteins to DNA and RNA, without compromising any orthogonal techniques used for tissue analysis or interfering with subsequent histological stains.

Table 1
Summary of sectioning properties for the evaluated embedding media. Attributes for viscosity and tissue adherence were chosen subjectively in lack of objective measuring criteria. The classification categories were poor, acceptable, good, very good, HPMA=N-(hydroxypropyl)-methacrylamide, Na-CMC=sodium carboxymethylcellulose, HPMC=(hydroxypropyl-)methylcellulose, PVP=polyvinylpyrrolidone
Keywords: Mass spectrometry imaging, imaging mass cytometry, RNA-ISH, Tissue analysis
724

Imaging of prostaglandins by mass spectrometry imaging (#420)

Kyle Duncan1, Ru Fang1, Jia Yuan2, Rosalie Chu3, Sudhansu Dey2, Kristin Burnum-Johnson3, Ingela Lanekoff1

1 Uppsala University, Dept. of Chemistry-BMC, Uppsala, Sweden
2 Cincinnati Childrens Hospital Medical Center, Division of Reproductive Sciences, Cincinnati, Ohio, United States of America
3 Pacific Northwest National Laboratory, Biological Sciences Division, Richland, Washington, United States of America

Introduction

Prostaglandins are an important class of lipid biomolecules that are essential in many biological processes, including inflammation and successful pregnancy. Despite a high bioactivity, physiological concentrations are typically low, and there are few means to obtain their spatial distribution in tissue. Here, we demonstrate the first method for imaging prostaglandins using mass spectrometry imaging (MSI). Specifically, we employ nanospray desorption electrospray ionization (nano-DESI) MSI with added silver ions to image prostaglandin species directly in cryo sectioned mouse uterine tissue.

Methods

Nano-DESI MSI was performed with a custom built sampling apparatus connected to a Q-Exactive Orbitrap mass spectrometer. 10 ppm Ag+ was added to the nano-DESI solvent (containing acetonitrile and methanol, 9:1, and deuterated prostaglandin (PG) species as internal standards) to facilitate formation of [PG+Ag]+ ions. Ion images were generated from Wnt5ad/d rat uterine tissue, size ~ 2 mm in diameter, with higher spatial resolution using oversampling. Oversampling was performed using a 90 µm fused silica primary capillary stepping 30 µm along between each line scan along the y-axis. The sample was moved along the x-axis at 10 µm s-1, and as a result the generated ion images had an approximate pixel size of 30 µm x 10 µm.

Results/Discussion

Nano-DESI MSI with silver doped solvent was employed to image PG species directly from mouse uterine tissue sections. The solvent also contained isotopically labeled PG species as internal standards to enable generation of quantitative ion images. Generally, all PG species were detected between 50 and 300 nM across the issue. Three unexpected PG isomer classes, dimethyl-PGE2, PGA1 and PGJ2, were identified in the tissue. These understudied PG species all localize to the same cellular regions of the mouse uterine tissue sections as the well characterized PG species, PGE2 and PGF2a. Specifically, all detected PG species localize to the luminal epithelium and glandular epithelium of Wnt5ad/d mouse uterine sections at day 4 of pregnancy prior to embryo attachment. The cellular regions of the luminal epithelium are only 100-300 µm in diameter, and could be resolved in this study by use of oversampling to increase the spatial resolution.

Conclusions

This is the first study detailing the specific localization and quantity of prostaglandin species in biological tissue sections. The presented results successfully demonstrate quantitative nano-DESI MSI of [PG+Ag]+ species directly from biological tissue without additional sample preparation. The described approach will be extremely valuable for future studies investigating the role of PG species in biological systems.

References

Duncan K.D. et al. Anal. Chem. 2018, 90, 7246

Acknowledgement

Funding from the Swedish Research Counsil and the Swedish Foundation for Strategic Research.

Keywords: mass spectrometry imaging, prostaglandins, uterine tissue
725

Multimodal mass spectrometry imaging for assessment of response and resistance to Oncology combination therapies (#400)

Stephanie Ling1, Gregory Hamm1, Maria P. Serra1, Scott Hoffman1, Urszula Polanska2, Alex Dexter3, Rory Steven3, Simon Barry2, Josephine Bunch3, Richard Goodwin1

1 AstraZeneca, Pathology, IMED Drug Safety Metabolism, Cambridge, United Kingdom
2 AstraZeneca, IMED Oncology, Cambridge, United Kingdom
3 National Physics Laboratory, National Centre of Excellence in Mass Spectrometry Imaging, Teddington, United Kingdom

Introduction

Tumour Microenvironment Heterogeneity impacts drug delivery, metabolism and resistance and drug interventions in turn regulate tumour metabolism. Analysis of impact of tumour metabolic heterogeneity in tissue has previously been challenging. Combination of multimodal Mass Spectrometry Imaging and Imaging Mass Cytometry (IMC) for comparison of the monotherapy and combination efficacy of a combination therapy targeting the PI3K-AKT-mTOR pathway enabled identification of endogenous metabolite PK/PD biomarkers and gave new insight into drug mechanism of action, response and resistance.

Methods

Mass Spectrometry Imaging of fresh frozen tissue sections was used to evaluate drug delivery and potential regional accumulation of compound and targeted analysis of molecular biomarkers were used to assess predicted changes in glucose uptake, inflammatory processes and cholesterol biosynthesis. Untargeted analysis including statistical identification of novel discriminative metabolites between samples and database search for metabolite biomarker identification.

These tissues were then fixed and stained for >35 biomarkers concurrently with subcellular resolution by CyTOF Imaging Mass Cytometry for simultaneous metabolomic and proteomic analysis of the exact same tissue section to identify the cell types and phenotypes underlying the differential metabolic states and responses observed.

Results/Discussion

Although it was not possible to define any differential tumour regions based on H&E, unsupervised clustering based segmentation of the metabolomic MSI data enabled identification of distinct tissue regions based on similar changes in metabolite profile. These revealed ‘hotspots’ of potential resistance within tumour microenvironment.

Sequential IMC revealed the cell types and phenotypes associated with resistance to PI3K/mTOR combination therapy. Automated phenotyping enabled rapid identification of differences between treatment groups and in resistance hotspots including lack of inhibition of PI3K signalling, lower vascularisation and higher proliferation. In addition, these AI-driven approaches identified subtle multi-parameter phenotypes that could not have been characterized using traditional methodologies such as differences in immune infiltrates and increased EMT.

Conclusions

MSI metabolomics analysis of metabolic phenotypic response enables detection of greater heterogeneity in tumour response than visible by traditional pathology methods such as H&E. Combining this with biomarker information from IMC is revolutionizing our understanding of the complex ‘multisurface’ microenvironment to drive understanding of the impact of tumour heterogeneity on drug efficacy in vivo and in patients.

Keywords: Multimodal, Mass Spectrometry Imaging, Imaging Mass Cytometry, Immunooncology, Metabolism

Understanding Tumour Biology

Session chair: Filip Bochner (Zurich, Switzerland); Frauke Alves (Göttingen, Germany)
 
Shortcut: PW17
Date: Thursday, 21 March, 2019, 12:45 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.

810

The accumulation of tumor-derived exosomes changes the immune cell composition in target tissue of metastasis (#125)

Mirjam Gerwing1, Vanessa Kocman1, Anne Helfen1, Miriam Stölting1, Lilo Greune2, Alexander Schmidt2, Walter Heindel1, Moritz Wildgruber1, 4, Michel Eisenblätter1, 3

1 University of Muenster, Institute of Clinical Radiology, Muenster, North Rhine-Westphalia, Germany
2 University Muenster, ZMBE, Institute of Infectiology, Muenster, North Rhine-Westphalia, Germany
3 King's College London, Richard Dimbleby Department of Cancer Research, Randall Division & Division of Cancer Studies, London, United Kingdom
4 DFG Cluster of Excellence, EXC 1003 "Cells in Motion", Muenster, North Rhine-Westphalia, Germany

Introduction

Exosomes, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators in intercellular communication [1]. The purpose of this project was to assess the in vivo biodistribution of exosomes from highly malignant breast cancer cells in comparison to exosomes from the serum of healthy mice, and their effect on the immune cell infiltrate in target organs of metastasis, by means of molecular imaging.

Methods

Exosomes were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was checked by electron microscopy and western blotting. After labeling with the fluorescent dye DiR (750/780 nm), exosomes were injected i.v. into healthy BALB/c mice and their distribution was assessed using fluorescence-reflectance imaging (FRI). After ex vivo imaging of the organs, the lungs were stained for FACS analysis of granulocytes, T- and B-cells to identify changes in the immune cell content. 

Results/Discussion

The assessment of the in vivo distribution of DiR-labeled exosomes with FRI showed exosomes from highly malignant 4T1 cells, in comparison to exosomes from the serum of healthy BALB/c mice, to preferentially accumulate in the target organs of metastasis, in this case lung, liver and spine (tumor-exosomes vs. serum-exosomes: lung 18.6 vs. 10.4, p=0.01; liver 72.2 vs. 56.5, p=0.02; spine 5.1 vs. 3.5, p<0.01). Furthermore, an increase of cytotoxic CD8+T-cells and a decrease of CD4+T-helper cells was identified in the lung.

Conclusions

Exosome accumulation changes the immune cell composition in target-organs of metastasis, and can be visualized by FRI.

References

[1] Xu R. et al., Extracellular vesicles in cancer- implications for future improvements in cancer care. 2018. Nat Rev Clin Oncol. 15:617-638.

In vivo distribution of exosomes
Assessment of the in vivo distribution of tumor-derived, DiR-labelled exosomes with FRI shows accumulation in the liver, spleen and spine. 
Keywords: Exosomes, Premetastatic niche, Cancer, FRI
811

Tumor Neovasculature-On-A-Chip for Exploring Prostate-Specific Membrane Antigen Biology (#498)

Magdalena Skubal1, 2, Vladimir Ponomarev1, 3, Jan Grimm1, 3

1 Memorial Sloan Kettering Cancer Center, Department of Radiology, New York, New York, United States of America
2 Memorial Sloan Kettering Cancer Center, Program in Molecular Pharmacology, New York, New York, United States of America
3 Weill Cornell Medical College, Department of Radiology, New York, New York, United States of America

Introduction

Elevated expression level of prostate-specific membrane antigen (PSMA­­­) is characteristic for prostate cancer. PSMA overexpression is associated with cancer progression, metastasis, and is correlated with poor prognosis in prostate cancer patients (1). Interestingly, elevated levels of PSMA are also found on the vasculature of the variety of solid tumors (2). Although the presence of PSMA in tumor-associated neovasculature was described nearly two decades ago, its biological role in this context remains elusive. 

Methods

We have established an innovative bioengineering approach to investigate PSMA-dependent mechanisms involved in formation of tumor neovasculature. Collagen-basedon-chip microfluidic system allows us to create a tumor with microenvironment in vitro, where cells are allowed to migrate and self-organize into three-dimensional structures similar to that found in vivo. In specific, by growing neovasculature-on-a-chip we are able to control extracellular microenvironment, monitor a real time changes in vessels microarchitecture and alter the interaction of tumor and endothelial cells.

Results/Discussion

On-chip assays in combination with high-resolution confocal microscopy indicated that primary human endothelial cells with elevated expression levels of PSMA were more likely to sprout and form vessel-like structures in comparison to control endothelial cells lacking PSMA expression. Our preliminary data confirmed that PSMA expressing cells can be activated to sprout by the cytokines secreted from the tumor and that PSMA inhibitors successfully block the cytokine-induced PSMA activation. 

Conclusions

Elevated levels of PSMA favor tumor neovasculature formation. Organ-on-a-chip system bridges the gap between in vitro and in vivo models and allows a precise study of cellular and molecular mechanisms driving PSMA-dependent tumorigenesis. 

References

(1) Kaittanis C, Andreou C, Hieronymus H, Mao N, Foss CA, Eiber M, Weirich G, Panchal P, Gopalan A, Zurita J, Achilefu S, Chiosis G, Ponomarev V, Schwaiger M, Carver BS, Pomper MG, Grimm J. Prostate-specific membrane antigen cleavage of vitamin B9 stimulates oncogenic signaling through metabotropic glutamate receptors. J Exp Med. 2018. PubMed PMID: 29141866.

(2) Liu H, Moy P, Kim S, Xia Y, Rajasekaran A, Navarro V, Knudsen B, Bander NH. Monoclonal antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor vascular endothelium. Cancer Res. 1997. PubMed PMID: 9288760

Dr. Grimm has a financial interest in the manufacturer of the technology on which this scientific research is based.

 

Acknowledgement

This scientific research was supported by the National Institutes of Health (Grant R01 CA212379-02).

Keywords: PSMA, tumor, neoangiogenesis
812

Metabolic phenotyping of cellular senescence using hyperpolarized 13C-MRS and 1H-NMR (#264)

Marie-Aline Neveu1, Christoph Trautwein1, Benyuan Zhou1, Marcel A. Krueger1, Lars Zender2, Andreas M. Schmid1, Bernd J. Pichler1

1 Eberhard Karls University Tuebingen, Department of Preclinical Imaging and Radiopharmacy, Werner Sieming Imaging Center, Tuebingen, Baden-Württemberg, Germany
2 University Hospital Tuebingen, Department of Internal Medicine VIII, Tuebingen, Baden-Württemberg, Germany

Introduction

Senescence, a stable state of cell cycle arrest induced by various stress factors, is considered as double edged sword in tumor biology. While it can be seen as a stable end-point for the disease, still little is known about detailed mechanisms during therapy. The investigation of tumor cell metabolism pre- versus post-senescence induction may therefore help to gain a deeper understanding and develop in vivo markers. The aim of the study was to characterize metabolic changes in different senescent cancer cell models using in vitro hyperpolarized 1-13C-pyruvate MRS and ex vivo 1H-NMR.

Methods

Senescence was induced in hepatocellular carcinoma cells (HCT116) by doxorubicin, in a liver progenitor cell line (HRas) by p53-reactivation and in a liver carcinoma cell line (AMP19) by a ribosomal checkpoint inhibitor (RCI).

Hyperpolarized MRS experiments were carried out on a 7T imaging instrument (Bruker Biospec). To monitor pyruvate metabolism, hyperpolarized 1-13C-pyruvate, prepared using a SpinLab polarizer (General Electric), was injected in untreated and treated cell suspensions (n=3-6). For 1H NMR-based metabolomics, control and senescent cells (six replicates each) were extracted with a two-phase solvent system. Polar extracts were measured with a 5 mm room temperature TXI probe on a 600 MHz spectrometer (Bruker BioSpin Avance III HD) and statistically analyzed.

Results/Discussion

The administration of hyperpolarized 1-13C-pyruvate led to a significant decreased lactate production in RCI treated AMP19 cells compared to control cells (p=0,0201). However, alanine production was not modified. Interestingly, lactate and alanine metabolism weren’t impaired in senescent HCT116 cells compared to controls. These findings are supported by first results from the ex vivo 1H NMR data. Here, a direct comparison of metabolite ratios for cell growth (phosphocholine/glycerophosphocholine), tumor metabolism (lactate/alanine) and energy storage (phosphocreatine/creatine) was performed. A 2-fold lactate/alanine increase and 3-fold phosphocholine/glycerophosphocholine decrease was identified for HRas compared to HCT116 between control and senescense. By contrast, ratios of energy storage were similar between the two cell lines and applied therapy. Detailed analysis of all polar metabolites and lipid phases are pending.

Conclusions

Our first data suggests that metabolism during the process of senescence is influenced by treatment type as well as cell genotype. RCI inhibition and p53-reactivation impaired lactate production in AMP19 and HRas progenitor cells, respectively, while doxorubicin didn’t in HCT116. Furthermore, cell growth markers remained elevated in senescent HCT116 cells. Further investigations of lipid metabolites and sampling time points will be required.

Keywords: Cancer, Hyperpolarized 13C-MRS, 1H-NMR, Senescence
813

Homo- and Heteroassociations Drive Activation of ErbB3 (#18)

Timea Varadi1, 2, Magdalena Schneider2, Eva Sevcsik2, Dominik Kiesenhofer2, Gyula Batta1, Tamás Kovács1, Rene Platzer3, Johannes Huppa3, Gerhard Schütz2, Mario Brameshuber2, Peter Nagy1

1 University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology, Debrecen, Hungary
2 Vienna University of Technology, Institute of Applied Physics, Biophysics, Vienna, Austria
3 Medical University of Vienna, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectology and Immunology, Vienna, Austria

Introduction

The four ErbB receptors constitute a family of transmembrane proteins standing in the focus of interest of basic researchers and clinicians. Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. ErbB2/3 heterodimers formed upon binding of heregulin to ErbB3 constitute the most potent oncogenic unit capable of strong activation of both the MAPK and PI3K pathways.

Methods

Single molecule microscopy: Here we used a technique named “thinning out clusters while conserving stoichiometry of labeling” (TOCCSL) to measure the homo- and heterodimerization of ErbB3 in the presence or absence of ErbB2 coexpression in quiescent and heregulin-stimulated cells. An Axiovert 200 microscope was used for illuminating samples in objective-based total internal reflection (TIR) configuration. Samples were bleached with a power density of 5 kW/cm².

FRAP experiments: FRAP experiments were carried out similarly to what was described in the single molecule microscopy section. In order to avoid bleaching of dyes during the recovery phase, the laser power for the pre- and post-bleach images was reduced to ~ 30 W/cm².

Results/Discussion

Our results show that monomeric, inactive ErbB3 undergoes heregulin-induced homodimerization both in the absence or presence of ErbB2 coexpression. Heregulin also induced the formation of ErbB2/3 heterodimers. Based on the mobility of dimers and correlations with the effect of heregulin we identified a slowly moving dimeric species of ErbB3 which seems to correspond to molecular, active dimers. Pertuzumab, an antibody binding to the dimerization arm of ErbB2, blocked heregulin-induced heterodimerization, while it did not block the effect of the growth factor on homodimers of ErbB2, and even increased the fractional presence of ErbB3 homodimers in unstimulated cells. The presented results make a connection between cell and structural biological properties of ErbB2/3 homo- and heterointeractions. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced dimers which not only differ in their stoichiometry, but also in the distance between subunits.

Conclusions

ErbB3 is mostly monomeric in quiescent cells and forms homodimers upon HRG activation. Coexpression of ErbB2 induced only subtle alterations in the homodimerization of ErbB3. Pertuzumab vanished the formation of heteromers in both cell lines. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced dimers which not only differ in their stoichiometry, but also in the distance between subunits.

References

1. Citri, A., and Yarden, Y. (2006) Nat Rev Mol Cell Biol 7, 505-516.

2. Brameshuber, M. and G.J. Schutz (2012) Methods Enzymol 505, 159-86.

3. Moertelmaier, M., M. Brameshuber, M. Linimeier, G. J. Schutz, and H. Stockinger (2005) Applied Physics Letters 87, 263903-263903.

Acknowledgement

This work has been supported by research grants from the National Research, Development and Innovation Office, Hungary (K120302, GINOP-2.3.2-15-2016-00020, GINOP-2.3.2-15-2016-00044).

Keywords: receptor tyrosine kinases, ErbB2, ErbB3, single molecule fluorescence microscopy, fluorescence recovery after photobleaching
814

Pan Cancer analysis of similarities and differences in hormonal cancers and environmental cancers using MALDI-MSI. (#472)

Katarzyna Frątczak1, Marta Gawin2, Monika Pietrowska2, Mykola Chekan2, Piotr Widłak2, Joanna Polanska1

1 Silesian University of Technology, The Faculty of Automatic Control, Electronics and Computer Science, Data Mining Group, Gliwice, Poland
2 Maria Sklodowska-Curie Institute - Oncology Center, Gliwice Branch, Gliwice, Poland

Introduction

The Pan Cancer analysis is a comprehensive molecular analysis of diverse cancer types. It aims to examine similarities and differences of cancers related to different genomic behaviours. Current research allows to show similarities between cancers with similar factors determining the process of carcinogenesis, including environmental and hormonal factors. This work aims to compare molecular behaviour between 6 different types of cancer with different origins (head and neck, colon, stomach - environmental cancers; testis, thyroid, prostate - hormonal cancers).

Methods

For each of the six types of tissue, biological samples were collected from 10 patients, including cancer sample and healthy control. All tissue specimens were analysed by MALDI mass spectrometry imaging. Spatially distributed spectra were than preprocessed (spectra resampling, baseline correction, PAFFT alignment, TIC normalization, Gaussian mixture modelling, peptide abundance estimation). In order to keep cancer differences independent from differences in healthy tissue, the comparative analysis was made on the fold change calculated for each type of cancer. The relative measure of similarity between cancers was calculated for 80 features (fold-change) with the highest variance. The value of differentiation was estimated with the eta-square effect size.

Results/Discussion

Analysis of spectral similarity showed that hormonal cancers are more similar to each other than environmental cancers. In the case of hormonal cancers, testicular and prostate cancer are the most similar (93%), while thyroid cancer is similar to them at medium-level 88%. The most similar in environmental cancers are colon and stomach (94%). Complex environmental factors related to head and neck cancer cause a lower similarity value (83%). The analysis of eta-square showed that hormonal cancers have much less features with at least a small effect (184) compared to the environmental cancer (550), but they also have more features with a large effect (724-hormonal, 403-enviromental). The very high similarity of colon and stomach cancer and their relatively low dissimilarity to head and neck cancer at a similar level, results in a lower number of features with a large differential effect in environmental cancers compared to hormonal cancers.

Conclusions

This analysis demonstrates that hormonal cancers are more similar to each other than environmental cancers. A different molecular profile of testicular and prostate cancer, resulting from its occurrence only in men, influences their differentiation from thyroid cancer. For head and neck cancers, complex environmental factors (smoking, alcohol, diet, inflammations) affect its different profile.

Acknowledgement

The work was financially supported by National Science Centre, Poland NCN BiTIMS grant no 2015/19/B/ST6/01736 (JP) and BKM grant no 02/010/BKM18/0136 (KF)

Keywords: Pan Cancer, MALDI-MSI, tumour biology
815

Quantifying collagen alignment in the uninvolved human colon mucosa 10 cm and 20 cm away from the malignant tumor (#428)

Sanja Z. Despotovic1, Novica Milicevic1, Zivana Milicevic1, Aleksandar Krmpot2, Mihailo Rabasovic2, Aleksandra Pavlovic3, Vladimir Zivanovic3

1 University of Belgrade, School of Medicine, Insititute of Histology and embryology, Belgrade, Serbia
2 University of Belgrade, Institut of Physics, Belgrade, Serbia
3 University of Belgrade, KBC "Dr Dragiša Mišović-Dedinje", Belgrade, Serbia

Introduction

We demonstrated the structural and cellular alterations (reduced cellularity, decreased representation of pericryptal myofibroblasts, increased number of newly formed blood and lypmhatic vessels) of the lamina propria of the mucosa 10 cm and 20 cm away from the rectal adenocarcinoma. Our study also provided indications  that the representations of collagen fibers in the colon lamina propria could be affected. The aim of our study was to investigate the organization of collagen fibers in the lamina propria of the rectal mucosa in the remote surrounding of the malignant tumor.

Methods

Tissue samples of colonic mucosa 10 cm and 20 cm away from the malignant tumor were endoscopically collected from 10 patients with adenocarcinoma. The colonic samples taken from 10 healthy persons were used as control. For collagen fibers detection, SHG imaging on two photon excitation microscope was used on unlabeled colon tissue samples. The laser wavelenght was 840 nm. The SHG was selected by narrow bandpass filter at 420 nm. The average laser power on the sample was 30mW and the peak laser power was 2.5 kW. To calculate anisotropy parameter beta, which we used to quantify collagen fiber alignment), images of colon tissue samples were taken with the polarizer oriented parallel and perpendicular to the laser polarization. Parameter beta was calculated: β=(Ipar-Iorth)/(Ipar+Iorth).

Results/Discussion

Mean values of anisotropy parameter beta for the collagen fibers 10 cm and 20 cm away from the cancer (0.29 ± 0.08 and 0.27 ± 0.07) were significantly decreased (fibers were less organized) compared with collagen fibers in healthy mucosa (0.49 ± 0.06).

Accumulating evidence are showing that collagen fibers have a great impact on tumor progression (1, 2). The representation and alignment of collagen fibers play an important role in regulating tension in cancer microenvironmenet and migration of cancer cells (3). In previous study, using Masson trichrome staining, we demonstrated decreased representation of collagen fibers 10 cm and 20 cm away from colon cancer. Powerful tool for visualizing collagen fibers is SHG imaging on two photon excitation microscopy, and a great advantage is that label-free tissue can be analyzed. Using polarizer, on the same microscope, we demonstrated increased anisotropy of collagen fibers 10 cm and 20 cm away from the cancer. 

 

Conclusions

There is disorganization of collagen fibers in the unaffected lamina propria 10 cm and 20 cm away from the colon cancer.

Figure 1
Values of β coefficient presented as an image. Lamina propria of healthy person (A) and lamina propra 10 cm away from cancer (B). For values of β coefficent close to 0 (disorganized collagen) pixel are represented with red. For values of β coefficient closer to 1 (orderly organized collagen) pixels are represented with green.
Scale bar for β coefficient values
For β values close to 0 (disorganized collagen) pixels are represented as red. For β values close to 1 (orderly organized collagen) pixels are represented as green.
Keywords: collagen, colon, cancer, shg
816

A multiparametric approach towards metabolic fingerprinting of murine breast cancer cells (#204)

Sabrina H. L. Hoffmann1, Marie-Aline Neveu1, Mohamed A. Jarboui1, Christoph Trautwein1, Christina S. Wong2, Jaclyn E. Sceneay2, Andreas Möller2, Bernd J. Pichler1, Christoph M. Griessinger1

1 Eberhard Karls University Tübingen, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Germany
2 QIMR Berghofer MedicalResearch Institute, Tumour Microenvironment Laboratory, Brisbane, Australia

Introduction

Cancerogenesis is often accompanied by reprogramming of the cellular energy metabolism to sustain proliferation1. Metabolic alterations may arise as a consequence of mutations in oncogenes and result in the exhibition of a metabolic phenotype like the prevalent glycolytic phenotype. Further, metabolic change can serve as driver for tumor metastasis. Here, we examined metabolic and proteomic changes between the non-metastatic MMVT-PyMT-derived breast cancer cell line S2WTP3 and its potentially metastatic progeny line ML1B1B1 to unravel metabolic drivers for metastasis in vitro and in vivo.

Methods

[18F]FDG uptake was measured in S2WTP3 and MLB1B1 cell suspensions by γ-counting in vitro; to assess the real-time metabolism of pyruvate, 13C magnetic resonance spectroscopy (MRS) with hyperpolarized [1-13C]pyruvate was performed. 1H NMR metabolomic fingerprinting was executed on ether-extracted polar metabolites from S2WTP3 and ML1B1B1 cells. Untargeted proteomic analysis of cellular extracts was performed by LC-MS and utilized to correlate metabolic and proteomic discrepancies between S2WTP3 and ML1B1B1. To assess in vivo glucose metabolism, S2WTP3 or ML1B1B1 allograft tumors were inoculated orthotopically into 6-8 week old female C57BL/6 mice and dynamic [18F]FDG PET scans were acquired.

Results/Discussion

While a significantly enhanced in vitro [18F]FDG uptake was detected in ML1B1B1 in comparison to S2WTP3 cell suspensions (0.80 ± 0.03 % vs. 2.81 ± 0.05 %, n=5, p<0.0001), no significant differences in the in vivo [18F]FDG uptake was observed between allograft tumors (9.40 ± 0.54 %ID/cm³ in ML1B1B1 vs. 9.11 ± 0.83 %ID/cm³ in S2WTP3, n=5). Interestingly, a non-significant trend towards a higher conversion of [1-13C]pyruvate to [1-13C]lactate was observed in S2WTP3 cells in comparison to ML1B1B1 cells in 13C MRS. Concordantly, 1H NMR metabolomic fingerprinting showed significantly higher intensities of certain nucleotide species in ML1B1B1 compared to S2WTP3 cells pointing towards enhanced activity of the pentose phosphate and hexosamine biosynthetic pathway. Proteomic ontology analysis further revealed higher abundance of proteins related to epithelial-to-mesenchymal transition (EMT) in ML1B1B1 cells compared to S2WTP3 cells and significantly higher abundances of RAS family oncoproteins.

Conclusions

Differences in glucose metabolism could be detected in vitro between the cell lines but not confirmed in vivo. Further, metabolic examinations pointed towards shunting of glucose-derived carbons towards biosynthetic pathways in ML1B1B1 cells compared to S2WTP3 cells. Proteomic analysis revealed RAS and AKT as possible oncogenic drivers of the phenotype of ML1B1B1 cells although other potential drivers could not be excluded.

References

1 Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of Cancer: The Next Generation. Cell 144, 646-674.

Keywords: Breast cancer, multi-omics analysis, metabolic fingerprinting
817

Light Sheet Fluorescence Microscopy Imaging of promyelocytic leukemia protein physiologic effects on the U87MG Glioblastoma cell line (#383)

Maria Tampakaki1, 2, Mariam-Eleni Oraiopoulou1, Stylianos E. Psycharakis3, Eleftheria Tzamali1, Vangelis Sakkalis1, Joseph Papamatheakis4, 5, Giannis Zacharakis3

1 Foundation for Research and Technology Hellas, Institute of Computer Science , Heraklion, Greece
2 University of Crete, Department of Medicine, Heraklion, Greece
3 Foundation for Research and Technology-Hellas, Institute of Electronic Structure and Laser, Heraklion, Greece
4 Foundation for Research and Technology-Hellas, Institute of Molecular Biology and Biotechnology, Heraklion, Greece
5 University of Crete, Department of Biology, Heraklion, Greece

Introduction

The promyelocytic leukemia protein (PML) is a tumour suppressor with multiple cellular functions regarding the modulation of apoptosis, growth inhibition and migration suppression [1]. Modifications in PML expression have been associated with various types of cancer [2], [3]. In Glioblastoma (GB), PML has been shown to decelerate the cell cycle and proliferation [4], while it induces migration [5]. In order to study further the effects of PML in GB, we present in vitro results of the physiology of GB-PML expressing cells, measured with Optical and Light Sheet Fluorescence Microscopy (LSFM).

Methods

The well-established human GB cell line U87MG was used in this work. The cells were lentivirusly transfected with the PML protein, which was tagged with the red fluorescent protein mCherry. The expression of both proteins in U87MG cells was induced by the presence of doxycycline (DOX). 3D spheroids were generated using the hanging drop technique. The invasive capacity of the cells was assessed by cultivating the spheroids in an extracellular matrix-like substrate. Images were captured every 24h using optical microscopy with fixed acquisition parameters to monitor the growth pattern and the invasive capacity of both the transfected and the control spheroids. In order to visualize the PML expression and the cell death pattern of both spheroid types, LSFM was used with the Draq7 nuclear dye.

Results/Discussion

U87MG-PML cells exhibited significant differences compared to the control U87MG cell line regarding the growth and migration properties. The U87MG-PML cells generated smaller spheroids, indicating lower proliferative rate. This is in accordance with previous reports showing that high levels of PML in primary GB cultures inhibit proliferation [4], whereas loss of PML in neural stem cells leads to increased neurosphere diameter [5]. The invasive pattern was common in both cell lines adopting the typical starburst morphology [6], however, they presented different migration dynamics in terms of both the expansion and the cellularity of the invasive rim.

The LSFM scans showed that the PML protein was uniformly distributed in the core of the invasive and the non-invasive spheroids and the death pattern was not considerably affected by the presence of the antibiotic in either the induced or the non-induced PML condition.

Conclusions

GB expansion is attributed to both excessive proliferation and local spreading. Our results are in line with previous findings highlighting the dual role of PML in both PML-driven growth inhibition [4] and altered migration dynamics [5]. Unlike other therapeutic purposes, PML can serve as a therapeutic target aiming at eliminating multiple sub-clones depending on their proliferative and/or invasive phenotype within the heterogeneous GB tumour.

References

[1]         R. H. Chen, Y. R. Lee, and W. C. Yuan, “The role of PML ubiquitination in human malignancies.,” J. Biomed. Sci., vol. 19, p. 81, 2012.

[2]         N. Martín-Martín et al., “Stratification and therapeutic potential of PML in metastatic breast cancer,” Nat. Commun., vol. 7, 2016.

[3]         H. E. Lee et al., “Loss of promyelocytic leukemia protein in human gastric cancers,” Cancer Lett., vol. 247, no. 1–2, pp. 103–109, 2007.

[4]         A. Iwanami et al., “PML mediates glioblastoma resistance to mammalian target of rapamycin (mTOR)-targeted therapies,” Proc. Natl. Acad. Sci., vol. 110, no. 11, pp. 4339–4344, 2013.

[5]         V. Amodeo et al., “A PML/Slit Axis Controls Physiological Cell Migration and Cancer Invasion in the CNS,” Cell Rep., vol. 20, no. 2, pp. 411–426, 2017.

[6]         M.-E. Oraiopoulou et al., “Integrating in vitro experiments with in silico approaches for Glioblastoma invasion: the role of cell-to-cell adhesion heterogeneity,” Sci. Rep., vol. 8, no. 1, p. 16200, 2018.

Acknowledgement

Authors would like to thank Evangelos Liapis and Eirini Gonianaki for all the help they provided. This work was supported by the project “BIOIMAGING-GR” (MIS5002755) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).

Keywords: LSFM, Glioblastoma, PML, U87MG, Invasion

Imaging to Diagnose Cancer

Session chair: Katharina Lückerath (Los Angeles, US); Eric Kaijzel (Leiden, Netherlands)
 
Shortcut: PW18
Date: Thursday, 21 March, 2019, 12:45 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.

820

Comparison of [18F]DMFB and [18F]DMPY2 as PET imaging agents for malignant melanoma: Radiosynthesis and preclinical evaluation in small animal models (#343)

Ayoung Pyo1, Dong-Yeon Kim1, Jung-Joon Min1

1 Chonnam National University Hwasun Hospital, Nuclear Medicine, Hwasun, Republic of Korea

Introduction

Malignant melanoma is one of the most mortal cancers because of its very aggressiveness and high metastatic potential. Thus, early detection is very important to improve therapeutic outcome and survival of patients. In this study, we synthesized novel 18F labeled benzamide derivatives (N-(2-(dimethylamino)ethyl)-4-[18F]fluorobenzamide; [18F]DMFB and N-(2-(dimethylamino)ethyl)-5-[18F]fluoropicolinamide; [18F]DMPY2). We evaluated and compared radiochemical and biological characteristics of 18F labeled probes in B16F10 (mouse melanoma) -bearing subcutaneous and metastasis models.

Methods

[18F]DMFB was synthesized from N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) via two steps. [18F]DMPY2 was synthesized from 5-bromo-N-(2-(dimethylamino)ethyl)pyridine-2-carboxamide as the precursor. At the end of reaction, the mixture was purified by using HPLC. Biodistibution and microPET studies were performed at different time points after i.v. injection of each compound (7.4 MBq) in B16F10 primary and metastasis mouse models. The static images at 30 and 60 min were acquired for 10 min.

Results/Discussion

Radiochemical yields of [18F]DMFB and [18F]DMPY2 was approximately 10~15%. In biodistribution studies, both agents accumulated and retained in tumor from 10 to 120 min. Tumor uptake of [18F]DMPY2 (10, 30, 60 and 120 min % ID/g: 9.2, 15.8, 24.8, 10.6) was higher than [18F]DMFB (10, 30, 60 and 120 min % ID/g: 9.24, 10.8, 13.0, 10.6). MicroPET study clearly demonstrated that [18F]DMFB and [18F]DMPY2 accumulated in tumor specifically at 10 min after i.v. injection and tumor was clearly visible with high tumor-to-background ratio. Consistent with biodistribution study, [18F]DMFB has lower tumoral uptake in B16F10 tumors than [18F]DMPY2. B16F10 lung / lymph node metastasis lesions were clearly visible after injection of [18F]DMPY2 and [18F]DMFB.

Conclusions

[18F]DMPY2 demonstrated malignant melanoma with higher tumor uptake than [18F]DMFB in B16F10 tumor bearing mice. [18F]DMFB and [18F]DMPY2 successfully visualized lesions in B16F10 lung / lymph node metastasis models. [18F]DMPY2 might have a potential to be utilized as a novel melanoma imaging agent for PET.

References

Ren G, Miao Z, Liu H, et al. Melanin-targeted preclinical PET imaging of melanoma metastasis. J Nucl Med. 2009;50:1692-1699

Acknowledgement

This research was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2017R1D1A1B03029055 and 2018R1A6A3A01012344), and by the Pioneer Research Center Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning (2015M3C1A3056410).

Figure 1
MicroPET images of (A) [18F]DMFB and (B) [18F]DMPY2 in B16F10 subcutaneous tumor model at 30, 60 min after i.v. injection. (C) MicroPET quantification of tumors at 60 min after i.v. injection of [18F]DMFB and [18F]DMPY2
Keywords: malignant melanoma, metastasis, 18F-labeled benzamide derivative, PET, molecular imaging
821

Improved Diagnostic Performance by Using Quantitative Multiparametric Breast Ultrasound (#102)

Panagiotis Kapetas1, Paola Clauser1, Ramona Woitek1, 2, Georg Wengert1, Mathias Lazar1, Katja Pinker1, 3, Maria Bernathova1, Thomas Helbich1, Pascal Baltzer1

1 Medical University of Vienna, Department of Biomedical Imaging and Image-guided Therapy, Vienna, Wien, Austria
2 University of Cambridge, Department of Radiology, Cambridge, United Kingdom
3 Memorial Sloan-Kettering Cancer Center, Molecular Imaging and Therapy Service, New York, New York, United States of America

Introduction

Ultrasound (US) can provide both morphologic and functional information about tissue. Different functional US modalities include elastography, Doppler and Contrast enhanced US (CEUS). These offer quantitative parameters that may serve as imaging biomarkers. Until now, the added value of combining different quantitative US modalities (multiparametric US- mpUS) has not been explored in detail (1-5).

The aim of this study was to evaluate quantitative mpUS of the breast for the differentiation of benign and malignant lesions and investigate a possible variation according to the reader experience

Methods

124 patients, each with one biopsy-proven breast lesion were included in this prospective, IRB-approved study. Each lesion was examined with B-mode US, elastography (Virtual Touch IQ-VTIQ), Doppler US and CEUS. Different quantitative parameters were recorded for each modality. Using ROC curve analysis, the quantitative parameter with the best diagnostic performance for each modality was chosen. 4 readers (2 experienced breast radiologists and 2 residents) independently evaluated each lesion and assigned a BI-RADS score to it. The BI-RADS scores of all readers were then combined with the quantitative parameters. Overall, we assessed 8 different modality combinations. The diagnostic performance of mpUS was evaluated with descriptive statistics. Histology served as the reference standard. 

Results/Discussion

59 lesions were benign and 65 malignant. SWVmax, RI and mTTl (mean transit time) showed the highest diagnostic performance for VTIQ elastography, Doppler and CEUS respectively. MpUS with three parameters (B-mode, VTIQ and CEUS) showed the highest diagnostic performance irrespective of the experience level of the readers (averaged AUC 0.812 vs. 0.683 for B-mode US, p-value 0.0001), while the combination of B mode, VTIQ and Doppler US the second best (averaged AUC 0.789, p-value 0.0001). All other combinations (with 2, 3 or 4 parameters) showed a lower AUC. The combination of B-mode with VTIQ significantly improved the performance only for less experienced readers (p-value <0.05). MpUS with B-mode, VTIQ and CEUS was able to significantly reduce the number of false positive biopsy recommendations (p<0.0001).

Conclusions

Quantitative breast mpUS with three parameters (B-mode US, VTIQ elastography and CEUS) significantly improves the diagnostic performance of B-mode US alone, irrespective of the experience level of the examiner and offers quantitative parameters that may be used as imaging biomarkers for the differentiation of benign from malignant breast lesions.

References

1. Kapetas et al., Acta Radiol 2017 Feb;58(2):140-147

2. Özdemir et al., J Ultrasound Med 2001 Oct;20(10):1091-101; quiz 1102

3. Saracco et al., Acta Radiol 2012 May 1;53(4):382-8

4. Cho et al., Radiology 2012 Jan;262(1):80-90

5. Lee et al., Radiology 2017 Nov;285(2):660-669

Keywords: Breast cancer, ultrasound, elastography, Doppler, contrast-enhanced ultrasound
822

[64Cu]Cu-GluCAB: an in vivo albumin-binding radiopharmaceutical for tumour targeting and cancer diagnosis (#141)

Cathryn Driver1, Thomas Le Bihan2, Jan Rijn Zeevaart1

1 NECSA, Radiochemistry, Pretoria, South Africa
2 University of Bretagne Occidentale, CEMCA, Brest, France

Introduction

A new radiopharmaceutical, [64Cu]Cu-GluCAB1, developed by Necsa for targeted cancer diagnosis comprises a copper-64 radiolabeled, glucose-functionalised macrocycle with a maleimide moiety that binds in vivo to circulating albumin2. Binding to albumin increases the compounds biological half-life and allows for tumour targeting through the EPR effect.3 The aim of this investigation was to determine in vivo binding of the GluCAB-precursor to albumin and to determine the tumour targeting capabilities of the [64Cu]Cu-GluCAB1 using microPET/CT imaging.

Methods

The GluCAB precursor with a maleimide moiety (experimental compound) and without (control) was radiolabeled with 64Cu in 0.01M PBS (pH 7.4) at 45°C and purified on a C-18 SPE cartridge. The percentage labeling efficiency was determined by radio-HPLC. E0771 breast cancer cell allograft C57/BL6 mice (n=5) were injected intravenously with 0.1 mL (8 ± 2.5 MBq/animal) of [64Cu]Cu-GluCAB precursor (or control) and imaged using microPET/CT at 1, 2, 6 and 24 h post-injection (p.i). Time-activity-curves were drawn from the microPET/CT images to determine the uptake of the compound in various organs over the 24h time period and the final biodistribution of the compound was determined ex vivo after 24h.   

Results/Discussion

[64Cu]Cu-GluCAB and its control was obtained with 97% radio-labeling efficiency. The mice exhibited no side-effects following compound injection. Both compounds in the allograft C57/BL6 mice showed high radioactivity in the liver, intestines and bladder due to hepatobiliary and renal excretion. After 24 h, the control compound was completely excreted and almost no radioactivity (0.71 ± 0.32 %ID/g) was found in the plasma while the [64Cu]Cu-GluCAB precursor showed high levels of retained radioactivity in the heart from 1-6 p.i and after 24h the %ID/g in the plasma was 9.99 ± 2.7 (p<0.001). The uptake in the E0771 tumours was clearly visible from 1-6 p.i and after 24h the radioactivity was 4.67 ± 1.49 %ID/g (significantly higher (p<0.05) than the control group 1.39 ± 0.91 %ID/g).

Conclusions

The [64Cu]Cu-GluCAB precursor binds to circulating albumin as indicated by higher radioactivity (approx 10x more than the control group) presenting in the plasma. The tumour uptake of [64Cu]Cu-GluCAB is clearly seen between 1 and 6h p.i on the microPET/CT images and after 24h the activity retained in the tumour is approx. 3 x higher than in the control group. [64Cu]Cu-GluCAB therefore has potential as a new cancer diagnostic agent

References

  1. C. H. S. Driver, J. R Zeevaart, M. I. Parker, R. Hunter, 2016, WO2016046793 A3
  2. M. Muller, H. Struthers, C. Winiger, K. Zhernosekov, R. Schibli, J. Nucl. Med. 2013, 54, 124-131
  3. Maeda, H. Adv. Drug Deliv. Rev. 2001, 46, 169-185

 

Acknowledgement

With thanks to Necsa and BGM Pharmaceuticals for funding; Biljana Marjanovic-Painter (Necsa) for MS analysis; Cor Bester, Delene Van Wyk, Thomas Ebenhan, Janke Kleynhans and Axim personnel for assistance with animal studies and microPET-CT imaging

64Cu-GluCAB microPET/CT imaging
Maximum Intensity Projection microPET images aquired at 6h post-injection of E0771 breast cancer allograft C57BL/6 mice injected intravenously with a) 64Cu-GluCAB control and b)64Cu-GluCAB experimental radiopharmaceutical
Keywords: copper-64, radiopharmaceutical, EPR Effect, albumin binding, tumour targeting
823

Micro-Computed Tomography of a murine non-alcoholic fatty liver disease model with liver-specific knockout of the mTOR pathway (#388)

Diana Möckel1, Andreas Kroh2, Jeanette Walter2, Thorsten Cramer2, Ulf Peter Neumann2, Twan Lammers1

1 RWTH Aachen, Institute for Experimental Molecular Imaging, Aachen, North Rhine-Westphalia, Germany
2 RWTH Aachen University Clinic, Department of Visceral and Transplantation Surgery, Aachen, North Rhine-Westphalia, Germany

Introduction

Contrast enhanced micro-computed tomography (µCT) represents a potential technique for detection and staging progression of murine non-alcoholic fatty liver disease (NAFLD). One objective of this study was to compare different X-ray contrast agents in their ability to detect fatty liver disease in a non-alcoholic steatohepatitis hepatocellular carcinoma (NASH-HCC) mouse model. Another aim was to compare the amount of liver tumors in mice with liver-specific knockout of the mTOR pathway (mammalian target of rapamycin) and control mice. The mTOR pathway is a key regulator of cellular metabolism.

Methods

To induce NASH-HCC neonatal mice are treated with 7,12-Dimethylbenzo[a]anthracen (DMBA) and fed a high-fat, high-fructose and high-cholesterine diet (Western diet) in 6 week old C57BL/6 mice and control mice of same age and sex for 30 weeks with or without mTOR knockout (animals were provided by the clinic for general, visceral and transplant surgery, University Clinic Aachen). Micro-CT measurements were obtained using a dual energy gantry-based flat-panel micro-computed tomography scanner (TomoScope 30s Duo; CT Imaging, Erlangen, Germany). The contrast agent (Imeron 400 MCT, ExiTron nano 6000, Mvivo™ BIS or Fenestra® LC) was administered via tail vein IV injection followed by acquisition of µCT measurements post injection.

Results/Discussion

In this model only mice that are fed the Western diet develop NASH and subsequently NASH-derived HCC. In control mice, all contrast agents provide µCT contrast enhancement (CE) in the mouse liver and the ability to delineate liver blood vessels through a negative contrast enhancement effect (Fig.1 a,b). In Nash mice, less liver CE for all contrast agents (Fig.1 c, d) and Fenestra® LC remained in liver blood vessels 24 hours after injection suggesting that the hepatobiliary system of the compromised NASH mouse liver could not take up the triglycerides of the Fenestra emulsion (Fig.1 c,d). In control mice and in Nash mice, Mvivo™ BIS provides a stronger spleenic and spleen to liver CT enhancement ratio (Fig.1 b, d). Non-invasive μCT-based quantification of the liver volume, amount of tumors, tumor volume and fat ratio in all groups has shown high values for Nash HCC mice and DMBA treatment and highest values for Nash HCC mice with DMBA treatment and mTOR knockout.

Conclusions

The use of the Nash HCC model in combination with the liver-specific mTOR knockout reveals the effect of the mTOR pathway to the tumor promotion in vivo. A careful selection of the employed contrast agents is essential in order to achieve an acceptable CE in the organs of interest. Further work with these contrast agents will need to be done also regarding their pharmacokinetics and toxicological side-effects.

References

[1] Ehling J, Bartneck M, Wei X, Gremse F, Fech V, Möckel D, Baeck C, Hittatiya K, Eulberg D, Luedde T, Kiessling F, Trautwein C, Lammers T, and Tacke F. CCL2-dependent infiltrating macrophages promote angiogenesis in progressive liver fibrosis. Gut. 2014 Dec;63(12):1960-1971. 

[2] Ehling J, Theek B, Gremse F, Baetke S, Möckel D, Maynard J, Ricketts S, Grüll H, Neeman M, Knuechel R, Lederle W, Kiessling F, and Lammers T. Micro-CT imaging of tumor angiogenesis: quantitative measures describing micromorphology and vascularization. Am J Pathol. 2014;184(2):431-41. 

[3] Gremse, F. et al. Theranostics. 1;6(3):328-41(2016).

Acknowledgement

This work was supported by the START program of the medical faculty RWTH Aachen.

Micro-Computed Tomography of Nash HCC and control mice.
Visualization of the liver in Nash induced mice and control mice by in vivo μCT using different x-ray contrast agents resulting in a spatial resolution of 35μm voxel side length showing 2D cross-sectional images in transversal plane (a,c) and coronal plane (b,d). A CT scanner is shown in Fig. 1e. Representative images of 3D volume renderings (f) and 2D cross-sectional images of liver tumors (g).
Keywords: micro-computed tomography, x-ray contrast agents, hepatocellular carcinoma, Nash-HCC
824

Ultrasonography of spontaneous intestine tumor and spleen evaluation in MSH2LC transgenic mice (#31)

Juan Antonio Cámara Serrano1, Anna Pujol Esclusa1, Jordi Martinez Quintanilla2, Irene Chicote Ramos2, Hector Garcia Palmer2

1 Vall d´Hebron Institute of Research, Preclinical Imaging Platform, Barcelona, Spain
2 Vall d´Hebron Institute of Oncology, Stem Cells and Cancer Group, Barcelona, Spain

Introduction

Development of transgenic animals led to mimic different neoplasia in mice that permitted to test potential drugs reducing clinical trials and accelerating effective drug development. Spontaneous intraabdominal tumors are sometimes difficult to find and follow due to their location and random appearance timing. Imaging technologies reduce the number of animals and improve the refinement of the experiments.

Here we present a study that uses the ultrasonography to evaluate tumor presence in unharmed animals and the affectation of spleen size during the neoplasia.

Methods

173 homozygote animals were included in the experiment (96 females and 77 males) with a mean age of 280 days. Three animals were excluded after finding other intraabdominal pathologies during the exam.

Ultrasonography was done with a 12Mhz linear probe. Animals were anesthetized and hair clipped. Exam started with measure of spleen width in transversal view. Then, complete abdominal exploration was performed, looking for tumors, increases of intestinal walls or obstruction signals. Measurements of tumors were obtained in three axis, in transversal and longitudinal views. Animals were placed in their cages again for recovery.

Statistical analysis included spleen width, tumor volume and animal sex and age. Comparisons between all the parameters were performed with Sigmaplot© Systat software.

Results/Discussion

Incidence of pathology was 34.35% (56% males). Technique sensitivity was 74.19% while specificity reached 95.65%. Mean volume of tumor was 60.98mm3 and spleen diameter 2.88 in negative animals. Minimal detectable tumor volume was 2.76mm3.

Animal sex did not affect spleen width or tumor volume, while age was direct and positively related to spleen diameter but not to tumor volume. Negative animals had a thinner spleen than positive ones. Tumor volume and spleen width had a positive correlation. Spleen diameters were always measured in longitudinal way and at the vascular ilium to ensure an anatomically defined landmark.

Even though these mutants have a 100% of tumor incidence, age of tumor arising should be consider for experimental timings and ultrasound help to know this key point. Comparing to referenced papers, in this study it has not been necessary to make animal necropsies in order to measure tumor volume. This could be a key point if using this protocol for drug evaluation.

Conclusions

Ultrasonography can be a profitable tool for tumor evaluation in spontaneous intestinal tumor models. Tridimensional studies can be performed and tumor volume is suitable as a parameter for neoplasia development. Spleen width can be used as a tool for fast “positive/negative” screenings.

References

Hunga, K.E. et al. Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment. PNAS. 2010 January ; 107(4): 1565-1570.

Kucherlapati, M.H. et al. An Msh2 Conditional Knockout Mouse for Studying Intestinal Cancer and Testing Anti-cancer Agents. Gastroenterology. 2010 March ; 138(3): 993–1002.

Hodgson A1, Wier EM1, Fu K1, Sun X1, Wan F2,3. Ultrasound imaging of splenomegaly as a proxy to monitor colon tumor development in Apc(min716/+) mice. Cancer Med. 2016 Sep;5(9):2469-76.

Acknowledgement

Authors will like to thank animal facility staff for their help during the procedures.

Figure 1
B-mode images of positive (A) and negative (B) spleens. C: significative diferences between groups. D: linear correlation between tumor volume and spleen diameter.
Figure 2
Examples of intestinal tumors in longitudinal (A) and transversal (B) views. Image C shows measuring methodology.
Keywords: Ultrasound, mice, tumor, intestine, spleen
825

Non Invasive High Frequency Ultrasound for the study of mice models of thyroid cancer.  (#372)

Adelaide Greco1, 3, Luigi Auletta2, Sandra Albanese3, Giovanni Della Valle5, Francesca Maria Orlandella2, Giuliana Salvatore4, 2, Leonardo Meomartino6, Marcello Mancini3

1 University of Naples Federico II, Department of Advanced Biomedical Science, Naples, Italy
2 IRCCS, SDN, Naples, Italy
3 CNR, Institute of Biostructures and Bioimaging, Naples, Italy
4 Università di Napoli Parthenope, (4) Dipartimento di Scienze Motorie e del Benessere, Naples, Italy
5 University of naples Federico II, (5) Dipartimento di Medicina Veterinaria e Produzioni animali, Naples, Italy
6 University of Naples Federico II, (6) Centro Interdipartimentale di Radiologia Veterinaria, Naples, Italy

Introduction

Thyroid cancer represents one of the most common tumors among endocrine malignancies1; animal models represent valuable tools to resemble human pathologies, to refine techniques of diagnosis and to establish new therapies. High Frequency Ultrasound (HFUS)2 and Contrast-enhanced ultrasound (CEUS) could be used to characterize mice models of thyroid cancer. Moreover HFUS could help to inject directly tumor cells into specific sites, avoiding surgery4. We focused on studies from our laboratory for the generation of a mouse model of thyroid cancer and for their characterization with HFUS and CEUS.

Methods

Our studies have been conducted since 2009 with an High Frequency Ultrasound (HFUS) equipment. To evaluate if HFUS could be useful in the evaluation of thyroid, 10 normal C57BL/6 mice, 8 C57BL/6 mice with thyroid hyperplasia and 22 Tg-TRK-T1 transgenic mice were used. To evaluate angiogenesis, Contrast-Enhanced Ultrasound (CEUS) targeted with anti-VEGFR-2 monoclonal antibody and isotype control antibody were used. CEUS was performed in 7 mice with thyroid carcinoma, 5 with hyperplasia and 4 normal. To generate an orthotopic mice model of thyroid cancer guide by ultrasound, 20 mice underwent to a surgical orthotopic implantation of follicular carcinoma cell line in the right thyroid gland and 20 mice using HFUS-guided system. Results of the 3 protocols were confirmed by histology.

Results/Discussion

High Frequency Ultrasound has the ability to detect structures as small as 30 μm, a property that has been exploited for thyroid morphology evaluation in mice. Contrast-enhanced ultrasound is an emerging imaging strategy that combines ultrasound technology with contrast agents (CA) to target specific molecular markers of disease.

In our laboratory we used HFUS for a precise placement of tumor cells in echoguided orthotopic thyroid model, for morphological characterization of thyroid gland and in vivo quantification of VEGFR-2 expression in thyroid tumors3.

Conclusions

demonstrated a high specificity and sensitivity of HFUS in predicting the malignancy of the thyroid nodules. Furthermore, the possibility of using CEUS to quantify, in vivo and in a non invasive way, the expression of VEGFR-2 in thyroid gland, and to better differentiate benign from malign thyroid features. Finally we establish a new protocol to perform an alternative, less invasive and more reproducible mice model of orthotopic thyroid cancer.

References

1.Kitahara, C. M.; Sosa, J. A., The changing incidence of thyroid cancer. Nature reviews. Endocrinology 2016, 12, (11), 646-653

2.Mancini, M.; Vergara, E.; Salvatore, G.; Greco, A.; Troncone, G.; Affuso, A.; Liuzzi, R.; Salerno, P.; Scotto di Santolo, M.; Santoro, M.; Brunetti, A.; Salvatore, M., Morphological ultrasound microimaging of thyroid in living mice. Endocrinology 2009, 150, (10), 4810-5.

3.Mancini, M.; Greco, A.; Salvatore, G.; Liuzzi, R.; Di Maro, G.; Vergara, E.; Chiappetta, G.; Pasquinelli, R.; Brunetti, A.; Salvatore, M., Imaging of thyroid tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice. BMC medical imaging 20 3, 13, 31.

4.Greco, A.; Albanese, S.; Auletta, L.; Mirabelli, P.; Zannetti, A.; D'Alterio, C.; Di Maro, G.; Orlandella, F. M.; Salvatore, G.; Soricelli, A.; Salvatore, M., High-Frequency Ultrasound-Guided Injection for the Generation of a Novel Orthotopic Mouse Model of Human Thyroid Carcinoma. Thyroid : official journal of the American Thyroid Association 2016, 26, (4), 552-8.

Keywords: Thyroid, High Frequency Ultrasound, Mice, Contrast-enhanced ultrasound

Brain: Structure, Function, Networks

Session chair: Albrecht Stroh (Mainz, Germany); Valerio Zerbi (Zurich, Switzerland)
 
Shortcut: PW19
Date: Thursday, 21 March, 2019, 12:45 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.

901

Investigating the Biological Basis of the BOLD fMRI Signal in Mice (#463)

Zhiva Skachokova1, Felix Schlegel1, Yaroslav Sych2, Aileen Schroeter1, Markus Rudin1

1 ETHZ, Institute for Biomedical Engineering, Zurich, Zürich, Switzerland
2 University of Zurich, Brain Research Institute, Zurich, Switzerland

Introduction

Functional magnetic resonance imaging (fMRI) is a technique widely used in both research and clinical practice. At the same time, the biological mechanisms underlying the generation of blood oxygen-level dependant (BOLD) fMRI signal remains unclear. The relationship between neuronal activity and local blood flow is not always straightforward, resulting in a debate in the field. Astroglial cells are an important elment of the neurovascular unit, and it has been porposed that release of vascoactive substances by atsrocytes couples neuronal activity to changes in cerebral blood flow.

Methods

In order to investigate this question further, we measured calcium activity of astrocytes and neurons in the mouse somatosensory cortex with the use of genetically encoded calcium indicators (GECIs), while simultaneously performing fMRI during electrical hindpaw stimulation, under varying stimulation amplitude and frequency, and two different anaesthesia condition.

Results/Discussion

Our results point out that astroglial activity time course is better correlated with the BOLD signal time course generation, as compared to neurons, during mild stimulation and under isoflurane anaesthesia. We also compared the correlation between BOLD and astroglial and neuronal calcium activity under different anaesthesia, and during contralateral and ipsilateral hindpaw stimulation.

Conclusions

This multimodal approach allows us to study neurovascular coupling in the intact brain, and our data reveals an important role of astrocytes in blood flow control. This could potentially help the better interpretation of fMRI data in health and disease.

References

Schulz et al. Nat Methods 2012

Schlegel et al. Nat Protocols 2018

Stobart et al. Neuron 2018

 

Acknowledgement

VVF, University of Zurich/ ETH Zurich.

Opto-fMRI signal in responce to el. hindpaw stimulation
Simultaneous measurement of astroglial and neuronal calcium fluorescent traces, and BOLD time course form the right hindpaw cortical area during el. hindpaw stimulation (3Hz, 0.75mA, average of 10 blocks, n=6 mice).
Keywords: BOLD, neurovascular coupling, optofMRI
902

fMRI reveals mitigation of cerebrovascular dysfunction by bradykinin receptor 1 and 2 inhibitor noscapine in a mouse model of cerebral amyloidosis (#60)

Ruiqing Ni1, Diana Kindler1, Rebecca Waag1, Marie Rouault1, Markus Rudin1, Giovanni Camici2, Luca Liberale2, Luka Kulic3, Jan Klohs1

1 ETH Zurich & University of Zurich, Biomedical Engineering, Zurich, Switzerland
2 University of Zurich , Center for Molecular Cardiology, Zurich, Switzerland
3 University of Zurich , Institute for Regenerative Medicine - IREM, Zurich, Switzerland

Introduction

Functional magnetic resonance imaging (fMRI) techniques can be used to assess cerebrovascular dysfunction in Alzheimer’s disease, an important and early contributor to pathology. We hypothesized that bradykinin receptors inhibition alleviates the vascular dysfunction in a transgenic arcAβ mouse model of cerebral amyloidosis and that fMRI techniques can be used to monitor the treatment response.

Methods

Using an enzyme immunoassay we detected increased bradykinin plasma levels in arcAβ mice as compared to non-transgenic littermates. Transgenic arcAβ mice, and non-transgenic littermates of 14 months-of-age were either treated with the bradykinin receptor 1 and 2 blocker noscapine or received normal drinking water as control over three months (n = 8-11/group) and all mice were assessed using fMRI at the end of the treatment period.

Results/Discussion

Perfusion MRI using an arterial spin labeling technique showed regional hypoperfusion in arcAβ compared to non-transgenic controls, which was alleviated by noscapine treatment. Similarly, measuring cerebral blood volume changes upon pharmacological stimulation using vessel dilator acetazolamide revealed recovery of regional impairment of cerebral vascular reactivity in arcAβ mice upon noscapine treatment. In addition, we assessed with immunohistochemistry beta-amyloid (Aβ) and inflammation levels in brain sections. Staining for Aβ deposition (6E10) and related microgliosis (Iba1) in the cortex and hippocampus were found comparable between noscapine-treated and untreated arcAβ mice.

Conclusions

In summary, bradykinin receptors blockage recovered cerebral vascular dysfunction but did not alter microgliosis and Aβ levels. fMRI method revealed the functional deficit in disease condition and was a useful tool to monitor the treatment response.

Keywords: Alzheimer's disease, magnetic resonance imaging, disease model, neuroimaging
903

Longitudinal in vivo MRI assessment of functional connectivity reveals early pathological hypersynchronisation of brain networks in the inducible Tet-off APP mouse model of Alzheimer’s disease (#434)

Inès Ben-Nejma1, Aneta J. Keliris1, Jasmijn Daans2, Peter Ponsaerts2, Marleen Verhoye1, Georgios A. Keliris1, Annemie Van der Linden1

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

Introduction

Soluble amyloid β (sAβ) before plaque deposition has been shown to be synaptotoxic and involved in pathological hypersynchronisation of brain networks by using resting state functional MRI (rsfMRI) in different mouse models overexpressing APP from birth1. However, the impact of protein overexpression during brain development may cause additional phenotypes unrelated to AD. To address this, we investigated by rsfMRI whether sAβ causes synaptic deficits and impairment of networks before plaque formation in Tet-off APP mice, in which the APP overexpression was switched on in adulthood2.

Methods

Tet-off APP mice and control littermates (WT micetTA mice, as a control for promoter-induced changes) have been treated with DOX to suppress transgenic Aβ production from 3 days up to 3 months of age. The animals were longitudinally scanned on a 9.4T MR system at week 0, 8, 16 and 28 post DOX treatment. The data acquisition was performed using a mixture of medetomidine (0.05mg/kg s.c. bolus; 0.1mg/kg/h s.c. infusion) and a low dose of isoflurane (0.4%). Each imaging session consisted of a rsfMRI scan (2D GE-EPI; TR/TE 2000/17ms; 300 repetitions; 12 coronal slices; 0.208x0.313x0.4 mm³) and a T2-weighted 3D anatomical scan (RARE, TR/TE 1800/42ms; RARE factor 16; 0.078x0.078x0.078 mm³). Samples for ex vivo analyses were collected at 8 and 28 weeks post DOX treatment.

Results/Discussion

Region-of-interest (ROI) based Functional Connectivity (FC) analysis showed a pathological hypersynchronisation in the Tet-off APP mice compared to the controls at 8 weeks post DOX treatment (Figure 1B). Ex vivo analyses performed at this time point confirmed the absence of amyloid plaques and showed a 20-fold increase of total sAβ levels in the Tet-off APP mice compared to the controls. Furthermore, a pathological hyposynchronisation was observed in the Tet-off APP mice compared to controls at 28 weeks post DOX treatment (Figure 1D) and Tet-off APP mice presented amyloid plaques in the cortex and the hippocampus. The hyper- and hyposynchronisation were mainly observed in regions within the default mode-like network (hippocampus, frontal cortex and parietal cortex), which is known to be affected in AD3. Moreover, no significant FC differences were found between the 2 control groups. Further analyses (quantitative ex vivo evaluation and 3D volume changes) are ongoing.

Conclusions

Our results indicate that abnormalities in neuronal network FC, driven by the sAβ synaptotoxicity, also occur when APP expression is switched-on in adulthood, precluding that these effects were caused during development. Thus, the combination of this controllable APP expression model with early in-vivo (rsfMRI) readout for soluble Aβ toxicity sets the stage for future Aβ targeting preventative treatment studies.

References

  1. Shah D, et al., (2016) Early pathologic amyloid induces hypersynchrony of BOLD resting-state networks in transgenic mice and provides an early therapeutic window before amyloid plaque deposition. Alzheimers Dement. 12, 964–976. doi: 10.1016/j.jalz.2016.03.010
  2. Jankowsky JL, et al.(2005) Persistent amyloidosis following suppression of Ab production in a transgenic model of Alzheimer disease. PLoS Med2(12):e355.
  3. Sheline YI, Raichle ME (2013) Resting state functional connectivity in preclinical Alzheimer’s disease. Biol Psychiatry74:340–347

Acknowledgement

This research was supported by the Fund for Scientific Research Flanders (FWO) (grant agreement G067515N).

Figure 1.
Functional connectivity differences between WT and Tet-off APP mice at different time point post DOX treatment. A-D. Averagez-functional connectivity (zFC) matrices for WT (top) and Tet-Off APP mice (bottom) at each time point. The color of each square indicates the correlation coefficient (transformed to Fisher z-score) between each pair of ROIs. * p<.05.
Keywords: Alzheimer's disease, resting-state fMRI, Functional Connectivity, Hypersynchronisation, Tet-Off APP mouse model
904

Changes in brain-wide neural activation patterns caused by hippocampal disinhibition in awake rats – a functional SPECT-imaging study (#250)

Stuart Williams1, Rebecca Hock1, Anja M. Oelschlegel2, 3, Jürgen Goldschmidt2, Tobias Bast1

1 University of Nottingham, School of psychology, Nottingham, United Kingdom
2 Department of Systems Physiology of Learning, Leibniz Institute for Neurobiology, Magdeburg, Germany
3 Institute of Anatomy, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany

Introduction

Hippocampal metabolic hyperactivity and neural disinhibition have been associated with early stage schizophrenia and age-related cognitive decline (1). Regional neural disinhibition may also cause activation changes in projection sites, which may contribute to cognitive impairments caused by hippocampal disinhibition (2; 3). We here examined the brain-wide activation changes caused by hippocampal disinhibition, using SPECT-imaging of cerebral blood flow (CBF) based on protocols with continuous intravenous blood-flow tracer injections in awake unrestrained rats (4).

Methods

We combined ventral hippocampal picrotoxin (GABA-A antagonist) infusions (3) with SPECT-imaging of CBF (4). Awake unrestrained rats pre-implanted with hippocampal guide cannulae and external jugular vein catheters received hippocampal picrotoxin / saline infusions, followed 10 min later by continuous intravenous injections of the flow tracer 99mTcHMPAO for another 10 min. The distribution of the trapped 99mTc was then mapped in anesthetized animals in a SPECT/CT scanner. Each rat received picrotoxin and saline infusions in a within-subjects design. SPECT data were co-registered to CT scans acquired in the same session, aligned to an MR brain template and global-mean normalised. Brain-wide activation changes after disinhibition and control were compared using voxel-wise paired t-tests.

Results/Discussion

99mTcHMPAO is a lipophilic tracer that is trapped in the brain after crossing the blood-brain barrier. The 99mTc-distribution as determined under anaesthesia represents the spatial pattern of the average CBF during tracer-injection in the awake state. Picrotoxin increased CBF around the infusion site in the ventral hippocampus, whereas CBF in the dorsal hippocampus was markedly decreased. This may resemble the posterior hippocampal hypoactivation reported alongside anterior hippocampal hyperactivity in schizophrenia (5) and suggests both changes may be caused by hippocampal disinhibition. Importantly, ventral hippocampal disinhibition caused marked extra-hippocampal changes in neocortical and subcortical sites. This included marked activation of medial prefrontal cortex and lateral septum consistent with strong hippocampal projections to these sites (6), and less pronounced changes in other sites, including ventral striatum activation, and deactivation of amygdala and piriform cortex.

Conclusions

CBF-SPECT enabled us to study brain-wide activation changes upon ventral hippocampal disinhibition without confounding effects of anaesthetics. The wide-spread, local and distal, activation changes revealed in our study may contribute to cognitive and behavioural changes, including memory and attentional impairments and hyper-locomotion, caused by ventral hippocampal disinhibition (3).

References

1 Heckers & Konradi, 2015, Schizophr Res

2 Bast et al, 2017, Br J Pharmacol

3 McGarrity et al, 2017,Cereb Cortex

4 Kolodziej et al, 2014, Neuroimage

5 Ragland et al, 2017, Neuroimage Clin

6 Petrovich et al,2001,Brain Res Rev

Keywords: brain-wide neural activation patterns, SPECT-imaging, hippocampal disinhibition, rats, awake
905

Imaging Optogenetic Activation of Two Distinct Neuronal Circuits in Central Amygdala via fMRI Give Insight Into Their Role in Central Nociception (#367)

Isabel Wank1, Pinelopi Pliota2, Wulf Haubensak2, Andreas Hess1

1 University of Erlangen-Nuremberg, Institute of Pharmacology, Erlangen, Bavaria, Germany
2 Vienna Biocenter, Research Institute of Molecular Pathology, Vienna, Wien, Austria

Introduction

Optogenetics provide a powerful tool to decipher functions of distinct cell-types. Here, this method was combined with BOLD fMRI (ofMRI) to investigate in detail the effect of the selective activation of two interacting but supposedly opposing neuronal populations of the lateral central amygdala (CEl) in-vivo, which have shown to be the major player for the output function of the CEl [1]. The impact of the activation of either PKCδ- or somatostatin (SST)-expressing neurons here in particular on central nociception was measured via classical fMRI stimulus-based and resting state (RS) paradigm.

Methods

♂ mice (PKCδ::cre & SST::cre) were injected at CEl with:

  • PKCδ::GFP - AAV2/5.EF1a.DIO.GFP.WPRE (GFP-PKC; controls)
  • PKCδ::ChR2 - AAV2/5.hsyn.hChR2(H134R).eYFP.WPRE (PKC)
  • SST::GFP - AAV2/5.EF1a.DIO.GFP.WPRE (GFP-SST; controls)
  • SST::ChR2 - AAV2/5.hsyn.hChR2(H134R).eYFP.WPRE (SST).

OfMRI was performed on a 4.7T Bruker Biospec with standard EPI [2] with a dedicated optogenetic compatible 2x2 array coil.

2 stimuli were presented alternatingly:

  • noxious heat (50°C; 20sec, hind paw)
  • laser (473nm; 10mW; 10Hz; 20sec).

Every 2nd heat stimulus was combined with laser stimulation.

OfMRI-Analysis:

  • GLM (stimulus only),
  • brain structure identification [3],
  • graph-theory [4].

Influence of the optogenetic stimulation on pain perception was evaluated using functional connectivity (FC)-based network analysis.

Results/Discussion

Pure laser activation of the 2 CEl cell populations was found to drive distinct brain regions. PKCδ reduced FC within contralateral sensory cortex (cxS), thalamus (Th) and extensively within brainstem (Bs) and cerebellum (Cer). The ipsilateral amygdala (Am) showed strong negative correlations with limbic system (Ls) and cxS (Fig1 l). Activation of SST reduced FC within ipsilateral cxS/association cortex and the Ls. Am was activated in both hemispheres and showed strong negative correlations with Bs and cxS (Fig1 r).

Comparing heat-only to heat-laser-condition (Fig2), the influence of both populations on nociception was revealed: activation of PKCδ reduced FC within Th, and between Th, cxS and Ls, with a slight lateralization (contra). Contrary for SST activation differences in heat processing were mainly reflected in enhanced FC within cxS and Cer and reduced FC within ipsilateral cxS and motor cortex as well as within the Bs. Results were backed up by RS findings.

Conclusions

By combining optogenetics with fMRI, the influence of the selective activation of two neuronal populations within CEl on CNS heat processing and RS networks could be investigated.
PCKδ-expressing neurons seem to regulate thalamus and higher-order brain regions (esp. cortex).
SST was found to act mainly via controlling the brainstem and starting therefrom to thalamic, limbic and cortical regions.

References

[1] Haubensak, Wulf, et al. "Genetic dissection of an amygdala microcircuit that gates conditioned fear." Nature 468.7321 (2010): 270.

[2] Hess, Andreas, et al. "Blockade of TNF-α rapidly inhibits pain responses in the central nervous system." Proceedings of the National Academy of Sciences 108.9 (2011): 3731-3736.

[3]Franklin KBJ, Paxinos G. The Mouse Brain in stereotaxic coordinates. Academic Press, New York, 3 Ed. 2008

[4] Sporns O, Chialvo DR, Kaiser M, Hilgetag CC. Organization, development and function of complex brain networks. Trends in cognitive sciences 2004;8(9):418-425.

Acknowledgement

BMBF (NEUROIMPA, TP4 01EC1403C)

Brain networks influenced by optogenetic laser activation of PKCδ+- or somatostatin+-cells in CEl

Shown are 5 different subnets of brain regions: thalamus, sensory/association cortex, amygdala, limbic system and brainstem.

Whereas positive correlations were found to be quite similar between PKCδ and SST, negative correlations revealed strong control of the brainstem by SST, reaching out to thalamus, cortex and the limbic system.

PKCδ showed greater influence of the amygdala on cortical regions.
Modulation of pain perception by two different CEl circuits

Comparison of heat-only and heat-laser-condition.

Differences in PKCδ heat processing were found within thalamus, and between thalamus, sensory cortex and hypothalamus, with slight lateralization (contra), reflected by reduced (blue) FC compared to controls. SST showed enhanced (red) FC within cortex and cerebellum, and reduced FC within (ipsi) sensory and motor cortex as well as within brainstem.

Keywords: nociception, graph theory, optogenetics, amygdala circuits, fMRI
906

Changes in resting state networks and estimation of severity in a mouse model of inflammatory bowl disease (IBD) (#483)

Martin Meier1, Christine Häger2, Nora Weegh2, Ekkehard Küstermann3

1 Hannover Medical School, ZTL Imaging Center, Hannover, Lower Saxony, Germany
2 Hannover Medical School, ZTL, Institute for Laboratory Animal Science, Hannover, Lower Saxony, Germany
3 University of Bremen, Department of Chemistry (FB 2), in-vivo MR group, , Bremen, Bremen, Germany

Introduction

Magnetic resonance imaging is an established diagnostic tool in the assessment of inflammatory bowel disease (IBD). Here we use a mouse model [1] to monitor neuronal activity and connectivity of brain regions that might reflect exposure to acute or chronic stress situations. We analyze whether changes in resting state activity correlate with the burden of our IBD model. This study is aiming at: 1. define and validate stress markers detectable with advanced imaging methods. 2. create standardized methods for severity assessment. 3. Create scales for the classification of severity grades.

Methods

For rsfMRI at baseline and after stress induction EPI-based BOLD measurements were performed and an established analysis protocol was used. Reorientation, normalization and smoothing was carried out using probabilistic independent component analysis (ICA) [Beckmann 2004] as implemented in MELODIC Version 3.14, part of FSL (FMRIB's Software Library, www.fmrib.ox.ac.uk/fsl). Pre-processed data were whitened and projected into a 40-dimensional subspace using Principal Component Analysis. The whitened observations were decomposed into sets of vectors by using a fixed-point iteration technique [Hyvärinen 1999]. Estimated Component maps were divided by the standard deviation of the residual noise and thresholded by fitting a mixture model to the histogram of intensity values [Beckmann 2004].

Results/Discussion

Statistically secured network differences between timepoints (BL vs d11) were found in motor cortex, retrosplenial cortex and medial hypothalamus. Spontaneous low frequency fMRI fluctuations measured during resting-state have been shown to provide important information on the functional organization of the brain. Only a handful of resting-state fMRI studies have been conducted in rodent models. The major concern in rodents and in animal studies in general is the use of anesthesia. The ideal but very challenging approach – especially for longitudinal investigations –would be awake animal brain rsfMRI. However, [Liang et al.] showed that even though local functional properties were reorganized under isoflurane anesthesia in rats, global topological features were preserved. This opens a very important pathway towards combinations of findings at the functional connectivity level with knowledge gained from structural investigations for revealing the full microbiome-gut-brain axis “image”.

Conclusions

There is interesting correlation between morphological and rsfMRI findings. These biomarkers might allow for the investigation of scales for the classification of severity grades and elaboration of minimal distress conditions. rsfMRI might provide a robust method for measuring longterm changes. We tested a non-invasive functional imaging approach to characterize network changes during disease progression in a mouse model of IBD.

References

[1]Inflamm Bowel Dis. 2013 Jan;19(1):185-93. doi: 10.1002/ibd.23006
[Hyvärinen 1999] A. Hyvärinen. Fast and Robust Fixed-Point Algorithms for Independent Component Analysis. IEEE Transactions on Neural Networks 10(3):626-634, 1999.
[Beckmann 2004] C.F. Beckmann and S.M. Smith. Probabilistic Independent Component Analysis for Functional Magnetic Resonance Imaging. IEEE Transactions on Medical Imaging 23(2):137-152 2004.
[Liang, Z.], Liu, X., Zhang, N., 2015. Dynamic resting state functional connectivity in awake and anesthetized rodents. Neuroimage 104, 89–99.

Acknowledgement

The authors extend gratitude to C. Bergen for his technical assistance. This work is supported by the DFG FOR2591 grant agreement and by the cluster of excellence REBIRTH .

Animal model

DSS induced colitis with titrated stress through restraining

Functional results
Statistcally secured network differences in three brain regions
Keywords: IBD, resting state, mouse model, severity, biomarker
907

Evaluation of hemodynamic and molecular-level resting-state functional connectivity changes in a rat model of Parkinson’s disease using simultaneous [11C]raclopride PET/ fMRI (#573)

Laura Kuebler1, Tudor M. Ionescu1, Mario Amend1, Sabrina Buss1, Andreas Maurer1, Deniz Kirik2, Bernd J. Pichler1, Kristina Herfert1

1 Eberhard Karls University of Tuebingen, Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Tuebingen, Germany
2 Lund University, Department of Experimental Medical Science, Brain Repair and Imaging in Neural Systems, Lund, Sweden

Introduction

The diagnosis of Parkinson’s disease remains challenging as no biomarker is available yet. Resting-state functional connectivity (rs-FC) acquired by BOLD-fMRI is widely used to study functional changes in the brain. [11C]Raclopride (RAC), a PET tracer binding to dopamin-D2-receptor, may offer a complementary approach to acquire FC on a molecular level. Using simultaneous PET/fMRI, we aimed to compute molecular-level FC from RAC in healthy rats, investigated reliability (REL) and compared it to the fMRI-derived rs-FC. Then, we evaluated rs-FC in an α-synuclein (αSYN) overexpression rat model.

Methods

To test REL, 8 healthy rats were scanned with a 17 week retest interval. Simultaneous 100-min RAC PET/fMRI scans were acquired from anesthetized (1.3% isoflurane/air) rats. An EPI-BOLD sequence (TR=2.5s, TE=18ms) was used for fMRI. RAC was applied using bolus-infusion (1mL/min bolus+15µL/min infusion, Kbol=34min). After preprocessing, pairwise rs-FC was computed and group-level mean correlation matrices were generated. Between- and within-scan REL was calculated using Pearson’s r. Dice coefficient was used to compare strongest correlations in RAC PET and fMRI-derived outputs following sparsity thresholding (10-20%). In addition, 12 rats were injected with an adeno-associated virus expressing human αSYN into the right substantia nigra, scanned 5 months later and compared to 13 healthy rats.

Results/Discussion

RAC PET-derived rs-FC revealed excellent group-level REL of 0.79 for between-scan and 0.80 for within-scan REL. The correlation matrices displayed connections between caudate putamen (CPu) and several posterior and anterior dopaminergic regions (e.g.: cingulate cortex (Cg), thalamus (Th)). fMRI-derived rs-FC yielded Pearson’s r values of 0.93 for between-scan and of 0.98 within-scan REL. Comparison between PET and fMRI outputs showed similar clusters of connectivity between CPu and nucleus accumbens as well as Cg and prefrontal cortex. Quantification using Dice coefficient showed an overlap of 0.54-0.59 depending on the sparsity threshold.

In the αSYN group, RAC rs-FC revealed decreased connectivity between the CPu and the midbrain and superior colliculus (SC) as well as between the Th and the midbrain and SC compared to the healthy rats. fMRI-derived rs-FC showed decreased anterior-posterior long-distance connectivity and increased short-distance connectivity in the αSYN group.

Conclusions

We report for the first time the evaluation of molecular-level FC from RAC showing stable REL acquired by simultaneous RAC-PET/BOLD-fMRI. Furthermore, RAC PET-derived rs-FC revealed differences in the connectivity of dopaminergic regions in αSYN overexpressing rats. Our simultaneous acquired data showed different rs-FC patterns for PET and fMRI which may provide complementary information on functional and molecular-level.

Keywords: α-synuclein, functional connectivty, Simultaneous PET/fMRI