15th European Molecular Imaging Meeting
supported by:

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

Best of vEMIM Poster-Pitches | Cancer Imaging

   
Shortcut: BO-07
Date: Friday, 28 August, 2020, 3:30 p.m. - 5:00 p.m.
Session type: Spotlight Symposium

Contents

Abstract/Video opens by clicking at the talk title.

BO-07-01

Bleomycin plus ultrasound and microbubbles to treat feline oral squamous cell carcinoma, as a model for human head and neck cancer, preliminary results of the BUBBLEFISH Trial

Josanne S. de Maar1, Maurice M. J. M. Zandvliet2, Stefanie Veraa2, Mauricio Tobón Restrepo2, Chrit T. W. Moonen1, Roel Deckers1

1 University Medical Center Utrecht, Utrecht University, Imaging Division, Utrecht, Netherlands
2 Utrecht University, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht, Netherlands

Introduction

Most Head and Neck Squamous Cell Carcinoma patients(~60%) present with locally advanced disease, when primary surgery is seldom an option. Despite combination treatment, around half develops (often incurable) local recurrences. Improved local therapy is needed. Sonopermeation with ultrasound and microbubbles (USMB) can increase local efficacy of several drugs[1], particularly bleomycin[2,3]. Studying Feline Oral Squamous Cell Carcinoma (FOSCC) can bridge between preclinical and clinical research and these cats (with a life expectancy of only ±1 month) could benefit from a low-burden treatment.

Methods

A single-arm prospective study in cats with spontaneously arisen FOSCC without treatment options except for palliative care is currently ongoing. During general anaesthesia, the cats receive intravenous bleomycin (10.000 IU/m2) combined with USMB treatment of the oral tumour using intravenous injections of SonoVue (Bracco, conc. 1-5x108 bubbles/mL). USMB treatment is performed with an EPIQ5 (Philips) in Pulsed Wave doppler mode and US parameters were optimized during the treatment of the first two cats. Each cat is treated three times, once per week. Feasibility, adverse events and quality of life are monitored. Tumour response is evaluated by clinical (caliper) and ultrasound measurement of the tumour.

Results/Discussion

So far two feline patients, respectively with sublingual and right maxillary SCC, have been treated.
Safety Treatment was well tolerated. The only grade 3 adverse event (AE) was anorexia in patient 1. AE were considered to be related to anaesthesia (lethargy, mild vomiting, constipation, hypotension, hypothermia), comorbidity, or in case of patient 1 tumour progression (anorexia, drooling, grade 1 tumour bleeding, grade 2 soft tissue necrosis of the tongue). Quality of life did not change.
Contrast enhanced ultrasound (CEUS) In patient 2 CEUS was performed before and after sonopermeation and showed a clear increase of perfusion of part of the tumour, especially after sessions 2 and 3 performed at a low mechanical index (MI 0.3-0.4) (Fig. 1).
Tumour response Patient 1 progressed clinically (Fig. 2), developed necrosis of the tongue and was euthanized 46 days after the first session, while patient 2 had stable disease (Fig. 2) and is currently still alive 56 days after the first session.

Conclusions

After treating two feline patients, bleomycin combined with USMB on a clinical US system using EMA/FDA approved microbubbles seems a feasible palliative treatment for FOSCC patients. Preliminary results suggest that tumour perfusion increases after sonopermeation at MI levels corresponding to stable cavitation. Eventually the obtained safety and efficacy results should facilitate the translation of USMB treatment into the clinic.

AcknowledgmentWe thank the patient owners for participating in this study with their cat.
References
[1] Lammertink BHA, Bos C, Deckers R, Storm G, Moonen CTW, Escoffre J-M, 2015 ‘Sonochemotherapy: from bench to bedside’,Frontiers in Pharmacology, 6(138): 1-17.
[2] Iwanaga K, Tominaga K, Yamamoto K, Habu M, Maeda H, Akifusa S, Tsujisawa T, Okinaga T, Fukuda J, Nishihara T, 2007, ‘Local delivery system of cytotoxic agents to tumors by focused sonoporation’, Cancer Gene Therapy, 14: 354-363.
[3] Lamanauskas N, Novell A, Escoffre J-M, Venslauskas M, Šatkauskas S, Bouakaz A, 2013, ‘Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue® or BR14® microbubbles’, Journal of Drug Targeting, 21(4): 407-414.
Figure 1

Contrast enhanced ultrasound in patient 2, treatment 2. Left: Before sonopermeation only the deep part of the tumour is perfused. Right: After sonopermeation the tumour is also perfused superficially.

Figure 2
Left: Caliper measurements, percentages relative to baseline. Right: Ultrasound based tumour volume (0.5*length*width*width), percentages relative to baseline
Keywords: Sonopermeation, Contrast enhanced ultrasound, USMB, head and neck cancer, feline oral squamous cell carcinoma
BO-07-02

A dual click 18F-labeling strategy for pretargeted PET imaging of the tumor-targeting monoclonal antibody CC49

E. Johanna L. Stéen1, 2, Jesper T. Jørgensen2, 3, Kamilla Nørregaard2, 3, Raffaella Rossin5, Christoph Denk4, Martin Wilkovitsch4, Dennis Svatunek4, Klas Bratteby1, 2, Patricia E. Edem1, 2, 3, Claudia Kuntner6, Thomas Wanek6, Marc Robillard5, Jesper L. Kristensen1, Andreas Kjær2, 3, Hannes Mikula4, Matthias M. Herth1, 2

1 University of Copenhagen, Department of Drug Design and Pharmacology, Copenhagen, Denmark
2 Rigshospitalet, Department of Clinical Physiology, Nuclear Medicine & PET, Copenhagen, Denmark
3 University of Copenhagen, Department of Biomedical Sciences, Copenhagen, Denmark
4 Technische Universität Wien, Institute of Applied Synthetic Chemistry, Vienna, Austria
5 Tagworks Pharmaceuticals, Nijmegen, Netherlands
6 Austrian Institute of Technology, Health and Environment Department, Seibersdorf, Austria

Introduction

Pretargeted PET imaging using the ligation between a radiolabeled tetrazine (Tz) and a monoclonal antibody (mAb) modified with a trans-cyclooctene (TCO) allows for the use of short-lived radionuclides, such as fluorine-18.1,2 The objective of the present study was to develop a library of 18F-labeled tetrazines via indirect radiolabeling for subsequent evaluation in pretargeted PET imaging of the TAG-72 targeting mAb CC49 in murine colon carcinoma.3

Methods

A library of Tz-derivatives was obtained via Cu-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) between alkyne-modified Tz-derivatives and 18F/19F-fluorinated azides (Fig. 1). To assess their potential as PET tracers for pretargeting, a blocking assay was carried out in BALB/c mice bearing colon carcinoma LS174T xenografts. Here, the ability of each Tz to block a previously described 111In-labeled Tz was studied.1 The mice were pretreated with TCO-modified CC49 (100 µg, 6.7 nmol, ~7 TCO/mAb) 72 h prior to the administration of the non-radioactive Tz. After 2 h, the 111In-labeled Tz was injected and any potential blocking of TCO-moieties on the mAb in the tumor could be determined. Pretargeted PET/CT imaging was performed using the same tumor model and mAb as for the blocking assay.

Results/Discussion

Radiolabeling via the CuAAC provided 18F-labeled tetrazines in decay corrected radiochemical yields of up to 68% and high radiochemical purity (˃94%). The performances of the non-radioactive tetrazines in the blocking assay were correlated to parameters such as reaction kinetics and lipophilicity. In general, high rate constants (>200 M-1s-1) with a calculated distribution coefficient (clogD7.4) below 0.5 were favorable for in vivo pretargeting. Pretargeted PET imaging was performed with six 18F-labeled tetrazines from the library to assess the predictive ability of the assay. The best tetrazine in the blocking assay (99% blocking) also showed the highest tumor uptake (2.5 ± 1.0 % ID/g 1 h after tracer administration) in pretargeting studies using the 18F-labeled analog.

Conclusions

In the present work we have developed a library of 18F-labeled tetrazines. Evaluation of the library revealed a tetrazine lead compound, which bound to the mAb at the tumor-site using high molar activity conditions. Current efforts are directed toward improving tumor-to-background ratios by using a clearing agent that can remove remaining mAb in the blood pool prior to injection of the 18F-labeled tetrazine.

Acknowledgment

The authors greatly acknowledge the H2020 project Click-it, under grant agreement no. 668532 for financial support.

References
[1] Rossin, R.; Verkerk, P. R.; van den Bosch, S. M.; Vulders, R. C. M.; Verel, I.; Lub, J.; Robillard, M. S. 2010, In Vivo Chemistry for Pretargeted Tumor Imaging in Live Mice. Angew. Chem. Int. Edit., 49 (19), 3375-3378.
[2] Denk, C.; Svatunek, D.; Filip, T.; Wanek, T.; Lumpi, D.; Frohlich, J.; Kuntner, C.; Mikula, H. 2014, Development of a F-18-Labeled Tetrazine with Favorable Pharmacokinetics for Bioorthogonal PET Imaging. Angew. Chem. Int. Edit., 53 (36), 9655-9659.
[3] Rossin, R.; van den Bosch, S. M.; Ten Hoeve, W.; Carvelli, M.; Versteegen, R. M.; Lub, J.; Robillard, M. S. 2013, Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems. Bioconjug. Chem., 24 (7), 1210-7.
Figure 1. Illustration of the dual click strategy for the 18F-labeling of CC49-TCO
A dual click strategy for the 18F-labeling of CC49-TCO in murine colon carcinoma. The CuAAC was performed between each tetrazine building block and a 18F-labeled azide synthon (click 1), respectively. The 18F-labeled click product was injected into tumor bearing mice, where it reacted with CC49-TCO that has accumulated at the tumor-site (click 2). 
Keywords: Pretargeting, immuno-PET, fluorine-18, click chemistry
BO-07-03

[18F]AZD2461 for PET imaging of PARP

Florian Guibbal2, 1, Samantha Hopkins2, Anna Pacelli2, Patrick Isenegger1, Michael Mosley2, Gemma Dias2, Julia Baguna-Torres2, Rebekka Hueting2, Véronique Gouverneur1, Bart Cornelissen2

1 Oxford University, Department of Chemistry, Oxford, United Kingdom
2 Oxford University, Department of Oncology, Oxford, United Kingdom

Introduction

Poly (ADP-ribose) polymerase (PARP) inhibitors have been intensively studied as cancer drugs. PET imaging of PARP would allow patient staging, therapy selection, and genotoxic therapy response evaluation. Olaparib resistance due to overexpression of P-glycoprotein and bone marrow toxicity are overcome in part by a structural variant: AZD2461. Here, we accessed the 18F-radiolabeled isotopologue of AZD2461, described as a poor substrate for P-glycoprotein as well as a PARP inhibitor with lower affinity towards PARP3. We also aim at investigating its potential for PARP targeting in vivo.

Methods

The arylboronate precursor of AZD2461 was synthesized using a modified previously described pathway. Automated copper-mediated 18F-fluorination was performed using an Eckert & Ziegler Modular Lab. [18F]KF was eluted from QMA cartridge with K222/K2C2O4/K2CO3 and dried with MeCN. After radiolabeling and deprotection, the radiotracer was isolated by semi-prep HPLC, collected in the dilution vial and formulated using a Sep-Pak C18 light cartridge in PBS 1X/DMSO (10%). Selectivity was assessed in PSN-1, PANC-1, CFPAC-1 and AsPC-1 cell lines by blocking [18F]AZD2461 with increasing amounts of cold, unlabeled PARP inhibitors to confirm PARP selectivity. Mice bearing tumour xenografts were injected with [18F]AZD2461, and PET images were acquired 1 h later.

Results/Discussion

AZD2461 protected arylboronate precursor was synthesized in 11 steps. Prep-HPLC allowed the recovery of the desired precursor in 99% chemical purity. Cell-free in vitro assays showed lower PARP-3 inhibition for AZD2461 (54 nM) than olaparib (6 nM) and a similar PARP-1/2 profile. The proto-deborylated AZD2461 shows a similar inhibitory profile as the native compound which underlines the importance of HPLC development methods. Starting from 20 GBq, radiolabeled AZD2461 was obtained with > 99% radiochemical purity and 3% ± 1% radiochemical yield. The specific activity obtained was up to 237 GBq/µmol. Co-injection of the tracer and the cold reference confirmed AZD2461 radiolabeling. [18F]AZD2461 was taken up in vivo in PARP1-expressing tumours and the highest uptake was observed for PSN-1 cells (7.34 ± 1.16%ID/g). In vitro blocking experiments showed a lesser ability of olaparib to reduce [18F]AZD2461 binding, indicating a difference in selectivity between olaparib and AZD2461.

Conclusions

PARP inhibitor AZD2461 was successfully radiofluorinated through fully automated copper-mediated aryl radiofluorination with excellent RCP and good radiochemical yield up to 4% and a specific activity up to 237 GBq/µmol molar activities. Taken together, we show the importance of screening PARP interactions profiles of radiolabelled PARP inhibitors for use as PET imaging agents.

AcknowledgmentThe authors would like to thank Dr. Thomas C. Wilson for fruitful discussion.
References
[1] Wilson TC, Xavier MA, Knight J, Verhoog S, Torres JB, Mosley M, Hopkins SL, Wallington S, Allen PD, Kersemans V, Hueting R, Smart S, Gouverneur V, Cornelissen B. 2019, "PET Imaging of PARP Expression Using 18F-Olaparib", J. Nucl. Med. 60(4), 504-510.
[2] O'Connor LO, Rulten SL, Cranston AN, Odedra R, Brown H, Jaspers JE, Jones L, Knights C, Evers B, Ting A, Bradbury RH, Pajic M, Rottenberg S, Jonkers J, Rudge D, Martin NMB, Caldecott KW, Lau A, O'Connor MJ. 2016, "The PARP Inhibitor AZD2461 Provides Insights into the Role of PARP3 Inhibition for Both Synthetic Lethality and Tolerability with Chemotherapy in Preclinical Models", Cancer Res. 76(20), 6084-6094.

PARP inhibitors and associated inhibiton profiles
(A) Structures and PARP1, 2 and 3 inhibitory profiles of AZD2461 1, proto-deborylated side-product 2 and olaparib 3. (B) In vitro PARP1-3 binding assay of compounds 1, 2 and 3 in PSN-1 pancreatic cells.
In vivo experiments with [18F]AZD2461 in pancreatic xenografted mouse model
(A)Biodistribution in xenograft-bearing mice with PSN-1, CFPAC-1, AsPC-1 and PANC-1 tumours, at 1h post injection of [18F]AZD2461. (B) Tumour uptake in PSN-1, CFPAC-1, AsPC-1 and PANC-1 xenografts 1h post injection (C) Western blot probing for PARP1, 2 and 3 in PSN-1, PANC-1, CFPAC-1 and AsPC-1 cell lines.
Keywords: PET, AZD2461, PARP, cancer, molecular imaging
BO-07-04

Simultaneous dual isotope PET-SPECT/CT imaging of a metastatic αvβ6 expressing syngeneic murine model of pancreatic cancer

Katie Dexter1, Nicholas F. Brown2, Julie Foster1, Roxana Kashani1, Juliette Chupin1, 3, Julie Cleaver1, Lauren Cutmore2, John F. Marshall2, Jane Sosabowski1

1 Queen Mary University of London, Centre for Biomarkers and Biotherapeutics, Barts Cancer Institute, London, United Kingdom
2 Queen Mary University of London, Centre for Tumour Biology, London, United Kingdom
3 Invicro, London, United Kingdom

Introduction

Integrin αvβ6 is expressed on approximately 90% of human pancreatic cancers and correlates with worse patient prognosis. Therefore αvβ6 is of great interest as a target for imaging and therapy in cancer [1].  Here we investigate the uptake of the αvβ6-specific 111In-DOTA-A20FMDV2 peptide and 18F-FDG using simultaneous SPECT-PET/CT imaging in αvβ6 high- and low-expressing primary and metastatic tumours of KPC derived cells lines. The KPC (KrasLSL.G12D/+;p53R172H/+;PdxCre) transgenic mouse is an established and clinically relevant immunocompetent model of pancreatic ductal adenocarcinoma [2].

Methods

KPC derived cell lines with either minimal or high αvβ6 expression were injected orthotopically into the pancreas of C57BL/6 mice. Tumour bearing mice with high (n=3) and low (n=3) αvβ6 expression were imaged at days 14-15 and 22 post-tumour inoculation. Healthy controls (n=3) were also included.
Mice were injected intravenously with 10-15 MBq of 111In-DOTA-A20FMDV2 3 hours prior to imaging and with 10-15 MBq of 18F-FDG 1 hour prior to imaging. 18F-FDG injections were performed under anaesthesia which was maintained during the uptake period, as was body temperature. The CT contrast agent gastrografin was used to opacify the intestinal tract to distinguish endogenous αvβ6 uptake in the gut from that of tumour. Mice underwent simultaneous SPECT-PET and CT in a MILabs VECTor6CTXUHR scanner.

Results/Discussion

All primary tumours and the majority of metastases in the high αvβ6 group were visualised via simultaneous 111In SPECT and 18F PET imaging.  This was validated through MRI and dissection. Co-localisation was evaluated in metastases using a qualitative scoring system.
111In and 18F co-localisation was seen in all primary tumours with high αvβ6 expression, whereas only 18F signal was detected in primary tumours with low αvβ6 expression.
In the high αvβ6 group, five metastases were investigated with three giving clear co-localisation and one showing moderate co-localisation. None of the metastases in the low αvβ6 group displayed 111In signal above background.
In the tumour-free controls no co-localisation was observed in the pancreatic area, although some clustering around the stomach and intestines was observed as would be expected due to endogenous αvβ6 expression.

Conclusions

Imaging with 111In-DOTA-A20FMDV2 and 18F-FDG in αvβ6 tumour bearing immune competent mice shows co-localisation of signal not only in the primary tumour but also in the majority of metastases. This indicates that metastases express αvβ6 and can be monitored using SPECT. We hypothesise that metastases without co-localisation have proportionally fewer αvβ6 receptors which we are investigating immunohistochemically.

References
[1] Reader, Claire S., et al., 2019, 'The integrin αvβ6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy.' The Journal of pathology
[2] Hingorani, Sunil R., et al., 2005, 'Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.'Cancer cell 7.5: 469-483.
Transaxial PET-SPECT/CT slice of a primary tumour with high αvβ6 expression
Transaxial PET-SPECT/CT slice of a metastasis with αvβ6 expression
Keywords: SPECT/CT, PET/CT, Pancreatic Cancer, Metastases, integrin αvβ6
BO-07-05

68GaNOTA-anti-MMR-Nb for PET/CT assessment of protumorigenic macrophages in patients with solid tumors: preliminary results of a phase I clinical trial

Odrade Gondry1, 2, Catarina Xavier1, 2, Johannes Heemskerk2, Nick Devoogdt1, Hendrik Everaert2, Karine Breckpot5, Lore De Coster3, Christel Fontaine3, Bart Neyns3, Denis Schallier3, Sofie Joris3, Sandrine Aspeslagh3, Ilse Vaneycken2, Kiavash Movahedi4, Geert Raes4, Jo Van Ginderachter4, Tony Lahoutte1, 2, Vicky Caveliers1, 2, Marleen Keyaerts1, 2

1 Vrije Universiteit Brussel, In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Brussels, Belgium
2 Universitair Ziekenhuis Brussel, Nuclear Medicine Department (NUCG), Brussels, Belgium
3 Universitair Ziekenhuis Brussel, Department of Medical Oncology, Brussels, Belgium
4 Vrije Universiteit Brussel, Laboratory of Cellular and Molecular Immunology (CMIM), Brussels, Belgium
5 Vrije Universiteit Brussel, Laboratory for Molecular and Cellular Therapy, Brussels, Belgium

Introduction

Tumor Associated Macrophages (TAM) play a role in shaping the tumor micro-environment (TME). An imbalance between pro-inflammatory and immunosuppressive, protumorigenic TAMs can support tumor growth and induce resistance to therapy. The presence of protumorigenic TAMs can be assessed by immunohistochemistry (IHC), but due to tumor heterogeneity and locations unable to take biopsies, the use of whole-body PET/CT is wanted. That is why a new PET-tracer is developed, a 68Ga labeled nanobody (Nb) targeting macrophage mannose receptor (MMR), expressed by the protumorigenic TAMs.

Methods

Patients (pts) with a solid tumor of at least 10 mm, ECOG score of 2 or lower and a good renal and hepatic function are eligible for inclusion in this phase I study. Safety is assessed using clinical examination and blood sampling before and 3h post injection (p.i.). Biodistribution and dosimetry is assessed using multiple blood samples for blood activity assessment and using PET/CT scans at 10, 90 and 150 min p.i. Blood and urine is assessed for metabolites. Cytokines are measured before and up to 24h p.i. to exclude macrophage activation.

Results/Discussion

Up to today, two pts were injected with 174MBq and 132MBq of Nb. Both were lung cancer patients treated with immunotherapy without complete radiological response. No adverse events (AE) were recorded, confirming safety in the first two subjects. Biodistribution analysis showed rapid blood clearance with <10% in total blood volume at 3h p.i. (fig. 1). PET/CT showed high tracer uptake in liver, spleen, adrenals and kidneys, as was expected from preclinical data. Other organs showed a very low background activity, with good potential to assess tumor lesions in such parts of the body. No uptake in tumor lesions was seen, but correlation to IHC was not available for these patients. Full biodistribution and dosimetry analysis is ongoing and will be presented at the meeting. Four additional patients will be recruited in the coming weeks.

Conclusions

68Ga-MMR-Nb PET/CT was well tolerated, without AE reported in the first two patients. The biodistribution is favorable, with the highest uptake in the kidneys, liver, spleen and adrenals. Further assessment in 4 pts is needed to complete the phase I study. A phase II study to compare with IHC is subsequently planned in a seamless phase I/II design.

Acknowledgment

We would like to thank Yasmine De Maeyer, Sonja Van den Block and Gratienne Van Holsbeeck for their contribution to the study. Also Kom op tegen Kanker and FWO to make our project possible.

References
[1] Xavier, C et al. 2019 'Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages', Mol imaging Biol. Oct;21(5):898-906.
[2] Keyaerts, M et al. 2016 ‘Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma', J Nucl Med. Jan;57(1):27–33.
[3] Blykers, A et al. 2015 ‘PET Imaging of Macrophage Mannose Receptor-Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments', J Nucl Med. Aug;56(8):1265-71. 
[4] Movahedi, K et al. 2012 ‘Nanobody-based targeting of the macrophage mannose receptor for effective in vivo imaging of tumor-associated macrophages', Cancer Res. Aug 15;72(16):4165-77.
Figure 1: Blood Clearance Curve MMR

Several bloodsamples were taken to evaluate the biodistribution. The analysis showed rapid blood clearance with <10% in total blood volume at 3h p.i. The curve starts at >100%, due to the use of a standardized formula to calculate the total blood volume. Underestimating the total blood volume in this specific patient. 

Keywords: Macrophages, Nanobodies, Phase I, PET/CT, macrophage mannose receptor (MMR)
BO-07-06

Image-guided Boron Neutron Capture Therapy: Preliminary results

Ghadir Kalot1, Amélie Godard2, Simon Coninx3, Ulli Köster4, Ewen Bodio2, Vincent Motto-Ros5, Jean Luc Coll1, Benoit Busser1, 6, Rachel Auzély3, Christine Goze2, Lucie Sancey1

1 Université Grenoble Alpes, Institute for Advanced Biosciences, INSERM U1209, CNRS UMR 5309, La Tronche, France
2 Université de Bourgogne, Institut de Chimie Moléculaire , CNRS UMR 6302, Dijon, France
3 Université Grenoble Alpes, Centre de Recherches sur les Macromolécules Végétales, CNRS, Grenoble, France
4 Institut Laue Langevin, Grenoble, France
5 Institut Lumière Matière, Lyon, France
6 CHU Grenoble Alpes, Grenoble, France

Introduction

Boron neutron capture therapy (BNCT) is a targeted form of radiotherapy. It relies on the sufficient and selective 10B accumulation at the tumor site, which is followed by a neutron beam irradiation. This later induces nuclear capture reactions in the 10B-rich tissues leading to their destruction.
This therapy is still limited, in particular due to the suboptimal distribution of the FDA-approved 10B-containing compounds. Thus, there is a potential for developing innovative theranostic carriers able to deliver ideal 10B concentrations to the tumor site and to be easily monitored in vivo.

Methods

Aza-BODIPY, a fluorescent probe for optical imaging in the NIR, was linked to the clinically available 10B-containing-compound, 10B-BSH. The obtained complex was either encapsulated in a biocompatible hyaluronic acid-based nanogel or evaluated as a free compound.
In vitro, both formulations were assessed for their internalization in 2D and 3D glioblastoma models. In vivo, biodistribution assays were conducted on glioblastoma-bearing mice, using optical imaging.
To estimate their potential as BNCT agents, an in ovo glioblastoma model was developed. Optical imaging was used to monitor the tumoral uptake of these formulations, prior to the neutron beam exposure at the Institut Laue-Langevin. Finally, tumors were collected, weighed and analyzed using LIBS elemental imaging technique.

Results/Discussion

Efficient cell internalization was observed for both formulations. A faster and a deeper penetration was detected for the free compound, in 2D and 3D glioblastoma models respectively. In vivo, both formulations showed a favorable tumoral accumulation due to the enhanced permeability and retention effect.
Regarding BNCT, in ovo optical imaging played an important role in determining the optimal tumoral accumulation, thus the ideal time to perform neutron beam exposure. Preliminary data showed a reduction in tumor sizes in the eggs incubated with the theranostic formulations followed by neutron irradiation, which was not the case for eggs incubated with the BNCT gold standard treatment “BSH”. LIBS elemental imaging further explained the obtained data as boron was only detected in the tumors incubated with the theranostic formulations.

Conclusions

The use of theranostic formulations is key in defining the neutron exposure protocol. Promising preliminary data regarding the tumor sizes were obtained, highlighting the potential of these novel formulations as BNCT agents. Experiments are ongoing to optimize the chemical formulations and the biological models in order to validate the results.

AcknowledgmentThe Authors acknowledge the following agencies for funding support: FRM ECO201806006861, ANR JCJC “SPID” ANR-16-CE07-0020, Project JCJC “WazaBY” ANR-18-CE18-0012, CNRS # 2015-9205AAO033S04139, MITI for the project BREVET-ISOTOP, France Life Imaging Thera-BODIPY, and the GEFLUC. The Authors acknowledge also the Optical Imaging platform OPTIMAL (Grenoble France) and the elemental imaging company Ablatom (Lyon, France) for technical collaboration.
Figure 1:

(A) Biodistribution of the nanogel formulation in a glioblastoma-bearing chicken egg, obtained using optical imaging. (B) Exposure of a glioblastoma-bearing chicken egg to the PF1b neutron beam line at the Institut Laue-Langevin. (C) LIBS elemental imaging of a collected tumor: Boron is presented in green, phosphorus is presented in blue.

Keywords: Theranostic probes, Optical imaging fluorophores, Boron vectorization
BO-07-07

Multi-modal PET and MR imaging in the Hen’s egg test chorioallantoic membrane (HET-CAM) model for initial in vivo testing of target-specific radioligands

Gordon Winter1, Andrea B. F. Koch1, Jessica Loeffler1, 3, Christoph Solbach1, Hao Li3, Gerhard Glatting1, Ambros J. Beer1, Volker Rasche2, 3

1 Universität Ulm, Nuclear Medicine, Ulm, Germany
2 Universität Ulm, Internal Medicine II, Ulm, Germany
3 Universität Ulm, Core Facility Small Animal Imaging, Ulm, Germany

Introduction

Non-invasive testing of the biodistribution is important in drug development. In contrast to animal studies, the Hen’s egg test chorioallantoic membrane (HET-CAM) model do not rise legal authorization issues if sacrificed before hatching, and thus is an attractive alternative for initial biodistribution studies regarding the 3R principle in animal welfare. Due to an undeveloped immune response, vascularized tumor xenografts can be established on the CAM. The applicability of the HET-CAM tumor model for biodistribution assessment of radiolabeled compounds after systemic injection was tested.

Methods

After six days of incubation at 37.8°C, silicone rings were placed on the membrane of the opened chicken eggs (n=20). Xenografts were established using the PCa cell lines LNCaP C4-2 (1x106 cells; PSMA+) and PC-3 (7.5x105 cells; PSMA-) with Matrigel (40%, v/v). MR and PET imaging was performed starting on day 12. High-resolution anatomical imaging was provided by using a small animal MR (BioSpin 117/16, Bruker) based on the protocol of Zuo et al. [1, 2]. 150µl of [68Ga]PSMA-11 solution was injected intravenously followed by a dynamic 60min PET scan (Focus 120, Concorde Microsystems Inc.). Tumors have been excised after measurement for quantitative assessment by gamma counting (COBRA II, Perkin Elmer). MRI and PET data were fused by fiducial registration using the 3Dslicer software [3].

Results/Discussion

MR imaging was successful in all (n=20) chicken embryos and detailed visualization of the tumor growth could be provided.  A successful injection into a CAM vein was achieved for 65% of the eggs. Accumulation of [68Ga]PSMA-11 was observed in the PSMA-positive tumor and chick embryo in the PET images (Fig. 1). In direct comparison, the PC-3 tumors showed lower accumulation of [68Ga]PSMA-11 as the LNCaP C4-2 tumors: 0.26 ± 0.71 %ID (LNCaP C4-2) vs. 0.07±0.05 %ID (PC-3); (n=20) yielding a ratio of 4.2±4.0 (PSMA-specific/non-specific). In the longitudinal PET measurement, the LNCaP C4-2 activity increased over time by 8.5±2.6 % due to progressing accumulation of the [68Ga]PSMA-11 in the PSMA positive tumor (Fig.2).

The suggested technique appears as an efficient approach to initial biodistribution assessment under almost in vivo conditions only limited by the only short time available for tumor growth monitoring and technical challenges of the injection into the rather small CAM veins.

Conclusions

The successful use of the HET-CAM model for an initial evaluation of the biodistribution of radiolabeled compounds by combining the highly sensitive PET and high-resolution MRI techniques was demonstrated. These results prove the potential of the HETCAM model as an attractive alternative to established small animal models for the assessment on the target specificity.

AcknowledgmentThe authors received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 667192, and by the Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Center 1279 – The exploration of the Human Peptidome. Further the general support of the MoMAN imaging center of Ulm University is acknowledged.
References
[1] Zuo, Z. et al. Scientific reports 2017, 7, 46690.

[2] Zuo, Z. et al. NMR in biomedicine 2015, 28 (4), 440-7.

[3] Fedorov, A. et al. Magn Reson Imaging. 2012;30:1323-41
PET/MRI of radiolabelled of [68Ga]PSMA-11

LNCaP C4-2 and PC-3 xenotransplanted tumors on the CAM (a), respective t2-weighted MRI (b), and fused PET-MRI as 3D (c) and 2D (d) visualization.

Quantitative dose analysis
Gamma-counting results (a) and dose-time curves (b) of the investigated tumors.
Keywords: HET-CAM, MRI, PET, xenograft
BO-07-08

Metabolic imaging-based subtype prediction in orthotopically transplanted murine pancreatic ductal adenocarcinoma

Moritz Mayer1, Irina Heid1, Lukas Kritzner1, Geoffrey Topping2, Katja Peschke3, Martin Grashei2, Christian Hundshammer2, Katja Steiger4, Franz Schilling2, Max Reichert3, Rickmer Braren1

1 TUM/MRI, Department of diagnostic and interventional Radiology, Munich, Germany
2 TUM/MRI, Nuclear Medicine Department, Muncih, Germany
3 TUM/MRI, Innere Medizin II, Munich, Germany
4 TUM/MRI, Institute of Pathology, Munich, Germany

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease. Two molecular subtypes recently have been identified: classical epithelial (CE) and quasi-mesenchymal (QM)1 PDAC. Metabolic imaging may enable differentiation of these subtypes allowing non-invasive stratification in lipogenic CE and glycolytic QM PDAC subtypes. In this study, diffusion-weighted imaging (DWI), chemical-shift imaging (CSI) with 1-13C-pyruvate and 18F-FDG positron-emission tomography (PET) were used to test multiparametric imaging for the differentiation of orthotopically transplanted murine CE and QM cell lines.

Methods

CE and QM mPDAC cell lines were transplanted orthotopically into CrTac:NCr-Foxn1numice (N=8). Animals was subjected to endpoint static 18F-FDG PET (Siemens Inveon) and subsequently T2-weighted, DWI (0.25x0.25x1mm; 12 b-values 12-1500) and multi-frame single-slice axial 2D phase encoded CSI (2x2x3mm, TR=5s) with hyperpolarized 1-13C-Pyruvate (80mM) in a 7T MRI (Bruker BioSpec) using a dual-tuned 1H/13C volume coil (31mm ID). Data was reconstructed and analyzed either in MATLAB (MRI) or IRW (Siemens, Erlangen). Tumors were removed, fixated and embedded according to the axial imaging plane. Tissues were formalin-fixed, H&E and immunostained (CD31, GLUT1, MCT4) and co-registered with respective image regions.

Results/Discussion

The transplanted tumors imitated the growth patterns of the primary PDAC (F. 1 A-B, G-H). A multimodal imaging protocol was performed (F. 1 . C-F, I-L). DWI showed no significant differences in calculated ADC values (F. 1 M), indicating comparable tumor cellularity2 (F. 2 I). In contrast, 18F-FDG PET and CSI showed significant differences in SUV and AUClac/AUCpyrvalues between CE and QM tumors, enabling good subgroup differentiation (F. 1 N, O). SUV and AUClac/AUCpy correlated significantly (Fig. 1 P). Histological analyses revealed comparable tumor cellularity (F. 2 A and I) and a good correlation with the ADC parameter (F. 2 H). CD31 staining (F. 2 B and E) showed no difference between subtypes2. Differences were detected in GLUT1 (F. 2 C and F) and MCT 4 (F. 2 D and G) and a good correlation between GLUT1 and MCT 4 immunostaining was noted (F. 2 J).

Conclusions

We have established a comprehensive multimodal multiparametric imaging platform for orthotopically transplanted mPDAC at preclinical 7T MRI and PET. CE and QM PDAC showed differences in the activation of glycolytic pathways, which could be observed by metabolic imaging (CSI and PET) and confirmed by immunohistochemical analysis. Metabolic imaging may enable tumor stratification for the testing of personalized therapeutic interventions.

Acknowledgment

Thank you to Sybille Reder and Markus Mittelhäuser for performing the PET measurements.
Additionally we acknowledge support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – 391523415, SFB 824).

References
[1] Collison, EA, Sadanandam, A 2011, ‘Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy’, NatMed, p.500-503
[2] Heid, I, Steiger, K, Trajkovic-Arsic, M 2017, ‘Co-clinical assessment of tumor cellularity in pancreatic cancer’, Clin Can Res, p.1461-1470
Fig. (F.) 1: Multiparametric imaging of orthotopically transplanted CE and QM cell lines

A,G) Primary tumor histology (H&E) of CE and QM cell lines B,H) Histology (H&E) of transplanted and orthotopically grown lesions C-F,I-L) Co-registered imaging (T2w, DWI, PET, CSI) of epithelial (C-F) and mesenchymal (I-L) lesions M) Mean regional ADC values grouped by tumor subtype N) SUVmean values gruped by tumor subtype O) AUClactate/AUCpyruvatevalues gruped by tumor subtype P) Linear regression of respective SUVmeanand AUClactate/AUCpyruvatevalues

Fig. 2: Immunohistochemistry of CE and QM tumors

A) H&E stained histology B) Cd31 immunostaining as a marker for vasculature C,D) GLUT1 and MCT4 immunostaining as a marker for activation of glycolytic pathways E-G) Percentage of stained area of the respective immunohistochemistry grouped by tumor subtype H,I) ADC correlated with cellularity determined by histology J) Cellularity determined by histology grouped by tumor subtype

 

Keywords: PDAC, MRSI, PET, Biomarker