Abstract/Video opens by clicking at the talk title.
Abstract/Video opens by clicking at the talk title.
|12:00 p.m.||PS 20-01||
Introductory Lecture (#678)
1 Memorial Sloan Kettering Cancer Center, New York, United States of America
|12:18 p.m.||PS 20-02||
In vivo bioluminescence imaging of oncolytic virus infection using NanoLuc complementation and novel furimazine analogue (#125)
Natasa Gaspar1, 2, 3, Giorgia Zambito1, 4, Steve Cramer5, Wytske Weerden6, Clemens W. G. M. Lowik1, 2, Rob Hoeben5, Jeroen D. Vrij7, Thomas Kirkland9, Joel Walker9, Laura Mezzanotte1, 2
1 Erasmus Medical Center, Optical molecular Imaging, Department of Radiology and Nuclear Medicine, Rotterdam, Netherlands
Optical Molecular Imaging (OMI) plays an important role in the frontiers of preclinical medicine with discoveries leading to novel diagnostic and treatment tools. Our specific interest within this study is the establishment of a novel imaging platform to track in vitro and in vivo viral infection by applying a split-luciferase complementation. NanoLuc luciferase was split into 2 subunits referred as the Large BiT (LgBiT, 156AA) and the HiBiT peptide (11AA)  which demonstrated high affinity (0,7 nM) for LgBiT .
DELTA-24-RGD-HiBiT-GFP oncolytic adenovirus has been engineered to infect and lyse tumor cells selectively. Prostate cancer cell lines have been used to test the system. Cells were seeded in a 96-well plate, transfected for constitutive expression of LgBiT and later infected with the adenovirus. In case of infection, structural complementation occurs between NanoLuC subunits. After addition of furimazine, the photon flux (ph/s) was collected at the IVIS. To assess the feasibility of in vivo imaging, PC-3 cells expressing the LgBiT protein were injected subcutaneously in mice. 24 h and 7 days post intratumoral injection of virus, a furimazine analogue (PBI-6059, 4.2 µM) was intraperitoneally (IP) injected in nude mice enabling a longer imaging window and visualization of infection.
When using the oncolytic virus in vitro, signal was clearly visible already 24h post-infection, addition of pooled immunoglobulins (IVIG) resulted in 2 to 3 fold signal decrease, since it inhibits the infection. 24 h and 7 days after viral administration in vivo, a specific BLi signal, originating from the PC-3-LgBiT cells injected subcutaneously at the back left side of the mouse, indicating viral replication and persistence in the tumor. A constant signal from the tumor means that the virus is still replicating and infecting keeping the tumor growth arrested. The peak signal, when performing bioluminescence imaging with PBI-6059 in vivo, reaches its maximum 20 minutes post IP injection of the furimazine analogue.
With this study, for the first time in an in vivo model, we demonstrate that NanoLuC complementation can be used to track oncolytic virus infection and might become a preferred strategy to add an imaging marker to the genome of viral vectors that are already close to their maximum packaging capacity.
AcknowledgmentThis research was supported by the project grants; H2020-MSCA-ITN-2015-ISPIC GA number: 675743; H2020-MSCA-RISE-2017 GA number: 777682 and by the AMIE Core facility within the Erasmus MC.
 Dixon, A.S., et al., NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chemical Biology, 2016. 11(2): p. 400-408
 Stacer, A.C., et al., NanoLuc reporter for dual luciferase imaging in living animals. Molecular imaging, 2013. 12(7): p. 1-13.
 Taylor, A., et al., Multicolour In Vivo Bioluminescence Imaging Using a NanoLuc-Based BRET Reporter in Combination with Firefly Luciferase. Contrast media & molecular imaging, 2018. 2018: p. 2514796-2514796.
Keywords: Optical Molecular Imaging, NanoLuc Complementation, onolytic virus, infection, furimazine
|12:30 p.m.||PS 20-03||
Multi-scale 3d virtual histology of human pancreatic tissue biopsies by propagation-based phase-contrast X-ray tomography (#229)
Jasper Frohn1, Jeannine Missbach-Guentner2, 3, Diana Pinkert-Leetsch2, 3, Frauke Alves2, 3, 4, Tim Salditt1
1 Georg-August-Universität, Institute for X-ray physics, Göttingen, Germany
Three dimensional (3d) analysis of the cancereous human pancreatic tissue can reveal important structural details underlying the disease mechanisms and progression with potential relevance for treatment. 3d data with sufficient resolution, contrast and field of view (FOV) can be provided by propagation-based phase-contrast X-ray tomography. Here we demonstrtate multi-scale 3d histology of human pancreas biopsies using an optimized setup of the synchrotron endstation „GINIX“ (Göttingen Intstrument for Nano-Imaging with X-rays)  at beamline P10 /PETRA III of DESY.
A biopsy (1 mm diameter) is taken from tumorous human pancreatic tissue embedded in paraffin and transferred to a polyimide tube. We have investigated unstained tissue and tissue stained with PTA (Phosphotungstic acid).The GINIX offers two complementary tomographic configurations. (1) For high resolution tomograms, the setup is used in a cone beam geometry. The effective pixelsize is 160 nm in 5 m distance. The FOV is 0.35 x 0.3 mm². The total acquisition time for a tomogram is ~ 40 min. This configuration showed excellent results for biological cells and tissues [2,3]. (2) Complementary to that configuration, we ran the endstation in a parallel beam geometry for overview tomograms. The FOV is ~ 1.6 x 1.4 mm² with a pixelsize of 650 nm. The total acquisition time for a tomogram is 75 s.
We have achieved large FOV‘s within fast acquisition times in the parallel beam geometry revealing the cytoarchitecture with cellular and sub-cellular details. Importantly, the 3d orientation of collagen fibers in the tumorous pancreas can be quantified. Furthermore, the geometry facilitates to locate and identify specific features such as islets of Langerhans. Resolution and contrast in the parallel geometry is already sufficient for 3d virtual histology. For further investigations on smaller length scales with higher resolution, we have switched to the cone beam geometry. With the combination of both geometries, the 3d structure of an islet of Langerhans was analysed with subcellular resolution. Different cell types can be distinguished. The workflow is compatible with correlative histological staining of sections prepared after the CT scans and different cell types can thus be validated.
With our propagation-based phase-contrast X-ray tomography setup at the synchrotron endstation GINIX we are able to analyse the 3d structure of tumorous human pancreatic tissue with single cells resolution. The endstation provides one configuration for fast overview tomograms with a pixelsize of 650 nm and one configuration for high resolution scans with a pixelsize of 160 nm. Further analysis is ongoing and will be published .
 Salditt, T., Osterhoff, Sprung, M. (2015). Compound focusing mirror and X-ray waveguide optics for coherent imaging and nano-diffraction. Journal of synchrotron radiation, 22(4), 867-878.
 Bartels, M., Krenkel, Salditt, T. (2015). X-ray holographic imaging of hydrated biological cells in solution. Physical review letters, 114(4), 048103.
 Töpperwien, M., van der Meer, F., Stadelmann, C., & Salditt, T. (2018). Three-dimensional virtual histology of human cerebellum by X-ray phase-contrast tomography. Proceedings of the National Academy of Sciences, 115(27), 6940-6945.
 Frohn, J., Missbach-Guentner, J., Pinkert-Leetsch, D., Alves, F., Salditt, T. , "Multi-scale 3d virtual histology of human pancreatic tissue biopsies by propagation-based phase-contrast X-ray tomography", unpublished.
Two GINIX configurations
X-ray beam path for parallel and cone beam illumination with the corresponding flat field images. a) Sketch of the parallel beam setup. b) Sketch of the cone beam setup. c) Flat field of the parallel beam setup with 35 ms. d) Flat field of the cone beam setup with 350 ms. Scalebars in the sample plane: (c) 200 µm, (d) 50 µm.
Reconstruction islet of Langerhans
Reconstructed PTA stained pancreatic human tissue. a) Overview tomogram reveals islet of Langerhans inside the sample. b) Zoom red area in a). Single cells inside the islet of Langerhans are visible. c) Waveguide tomogram with higher contrast. Scalebars: (a) 300 µm and (b/c) 50 µm.
Keywords: Tomography, Pancreas, Phasecontrast, X-ray, Histology
|12:42 p.m.||PS 20-04||
In vivo image-based characterization of a pancreatic mouse model: effect of pro-oncogenic elastin-derived peptides (#419)
Lise Nannan1, 2, Bertrand Brassart2, Sarah Belderbos1, Jens Wouters1, Bella Manshian3, Stefaan Soenen3, Willy Gsell1, Uwe Himmelreich1
1 KU Leuven, Department of Imaging and Pathology / Biomedical MRI, Leuven, Belgium
Pancreatic cancer is currently the 4th leading cause of cancer death in Europe and is characterized by the presence of a transformed microenvironment with degradation of extracellular matrix, in particular elastin fibers1. Elastin is degraded by proteases during tumor progression and leads to the release of elastin-derived peptides (EDPs)2-3. In vivo preclinical multimodal imaging allows longitudinal assessment and can be used to follow efficacy of therapeutic treatments4. We aimed at characterizing the effect of EDPs in pancreatic cancer mouse model using multimodal, multiparametric imaging.
Xenografts were generated in Swiss nude mice after subcutaneous injection (inj.) of 3.5x106 human pancreatic cancer cell (MIA PaCa-2), which were transduced in-house with a lentiviral vector encoding for Firefly luciferase (Fluc) and Green Fluorescent Protein (GFP). Animals were monitored 3 times per week (w) for body weight and tumor size using a caliper. Mice were treated 3 times per week with AG9 and VG6 EDPs (10 mg/kg) or sterile PBS (control) (n=3 per group) and were scanned every two weeks using a 7T MRI (Bruker Biospin) and an IVIS Spectrum (BLI, Perkin Elmer). The following MR scans were acquired: 2D T2-weighted RARE, ADC and T2 parametric maps. After sacrificing the mice, tumors were removed, weighted, measured with a caliper and scanned using the IVIS Spectrum.
The treatment of EDPs didn’t impact the general health and weight of the animals. Mice treated with AG9 EDPs presented with higher total photon flux with BLI (Fig1A, Table I). Furthermore, 2D T2-weighted RARE anatomical images showed the highest effect on the tumor volume after the injection of the AG9 peptide when compared to the VG6 or control group (Fig1B, Table I). These results correlate to caliper measurements. Within the same group, T2 values and apparent diffusion coefficients (ADC) were lower at week 2, while they were comparably higher at week 4, indicative of a dense tumor mass at early time points and the presence of edema at the later time point (Fig1C-D, Table I). Moreover, tumor heterogeneity was seen in all groups, but was more prominent after injection of the AG9 EDPs. Last, ex vivo measurements correlate with the in vivo measurements and further imply AG9 to have the highest effect on tumor growth.
To conclude, multimodal imaging allowed the longitudinal follow-up of tumor development induced by EDPs. Our results indicate that AG9 has a larger effect compared to VG6 on tumor growth and tumor heterogeneity. Hereby, AG9 can be used as the reference peptide for further studies, contrary to previous work were VG6 was used5. Furthermore, we show that the EDPs receptor (RPSA) can be used as a marker for poor prognosis in pancreatic cancer.
 Kamisawa, T, Wood , LD, Itoi, T, Takaori, K 2016, ‘Pancreatic cancer’, Lancet Lond Engl, 388(10039), 73-85
 Maquart, FX, Siméon, A, Pasco, S, Monboisse, JC 1999, ‘Regulation of cell activity by the extracellular matrix: the concept of matrikines’, J Soc Biol, 193(4-5), 423-8
 Da Silva, J, Lameiras, P, Beljebbar, A, Berquand, A, Villemin, M, Ramont, L, Dukic, S, Nuzillard, JM, Molinari, M, Gautier, M, Brassart-Pasco, S, Brassart B 2018, ‘Structural characterization and in vivo pro-tumor properties of a highly conserved matrikine’, Oncotarget, 9(25), 17839-57
 Conway, JR, Carragher, NO, Timpson, P 2014, ‘Developments in preclinical cancer imaging: innovating the discovery of therapeutics’, Journal, 14(5), 314-28
 Robinet, A, Fahem, A, Cauchard, JH, Huet, E, Vincent, L, Lorimier, S, Antonicelli, F, Soria, C, Crepin, M, Hornebeck, W, Bellon, G 2005, ‘Elastin-derived peptides enhance angiogenesis by promoting endothelial cell migration and tubulogenesis through upregulation of MT1-MMP’, J Cell Sci, 118(Pt 2), 343-56
Table I: Quantitative imaging data_ evaluation of total photon flux, tumor size, T2 and ADC values.
Data are expressed as means ± SD. Statistical analyses were performed using an ordinary one-way ANOVA with Dunn’s post-hoc test for multiple correction. The results were considered statistically significant when we compared control to AG9 or VG6 EDPs * p < 0.01, ** p < 0.001, *** p < 0.0001 and when we compared AG9 to VG6 EDPs $ p < 0.01, $$$ p < 0.001.
Keywords: Pancreatic cancer, Elastin-derived peptides, PET/MRI, Optical Imaging
|12:54 p.m.||PS 20-05||
Tracing Nutrient Flux Following Monocarboxylate Transporter-1 Inhibition with AZD3965 (#474)
Marta Braga1, Maciej Kaliszczak2, Laurence Carroll1, Zachary Shug3, Gillian Mackay3, Francesco Mauri1, Chris Barnes1, Hector Keun1, Eric Aboagye1
1 Imperial College London, Surgery and Cancer, London, United Kingdom
The monocarboxylate transporter 1 (MCT1) is a key element in tumor cell metabolism, as it participates in the membrane’s transport of molecules such as lactate.1Selective inhibition of MCT1 with AZD3965 is undergoing clinical trials. Understanding the downstream effects of MCT1 inhibition is crucial for the development of biomarkers that can monitor therapeutic response.2 The purpose of this work was to investigate nutrient fluxes associated with MCT1 inhibition by AZD3965 in order to identify possible biomarkers of drug action.
We synthesized a 18F-labeled lactate analogue, 18F-fluorolactate (18F-FL), that was used alongside 13C-labeled glucose, lactate and 18F-FDG to determine the fate of lactate and its potential as a biomarker. Diffuse large B-cell lymphoma (DLBCL) was validated as a suitable model for investigation of AZD3965 treatment. Radioactive uptake of 18F-FL and 18F-FDG was used to investigate modulation of metabolism with AZD3965. DLBCL tumor-bearing mice were either imaged with 18F-FL-PET or infused with L-Lactate-1-13C and D-13C6-Glucose and tumors were analysed with GC-MS. To evaluate possible downstream effects on enzymes such as pyruvate dehydrogenase (PDH), enzyme-linked immunosorbent assays and genetic knockdown was also carried out.
Comparative radioactive incubation with 18F-FDG or 18F-FL show much higher intracellular accumulation of the former, showing that cultured cells commonly rely on glycolysis for metabolism. In vivo, however, lactate is also used as a metabolic fuel: metabolites extracted from DLBCL tumors after injection of D-Glucose-13C6 and L-lactate-1-13C show tumor uptake and intracellular accumulation of enriched lactate that is distant to glycolysis. AZD3965 treatment delayed tumor growth and caused a reduction in glycolytic intermediates, consistent with inhibition of glycolytic activity. Interestingly, intratumoral accumulation of both L-lactate-1-13C and 18F-FL was higher following treatment, which seems to indicate that AZD3965 causes differential inhibition of lactate import and export. The tricarboxylic acid cycle was shown to be inhibited following AZD3965 treatment, due to inactivation of PDH, the enzyme responsible for the conversion of pyruvate into acetyl-coA.
We showed that DLBCL tumor model presents distinct metabolic phenotypes in vitro and in vivo, and that glucose (18F-FDG and D-Glucose-13C6) and lactate analogues (18F-FL and L-lactate-1-13C) can be successfully used as biomarkers for AZD3965 treatment. Furthermore, we showed that the TCA cycle is inhibited through downregulation of PDH activity.
 Halestrap, A. P. The SLC16 gene family – Structure, role and regulation in health and disease. Mol. Aspects Med. 34, 337–349 (2013).
 Hu, S. et al, 2013, 'MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program', Blood 121, 4021–4031.
Keywords: AZD3965, MCT1, 18F-Flurolactate, 18F-FDG
|1:06 p.m.||PS 20-06||
Design of a bimodal ligand of neurotensin receptor 1 for 68Ga-PET imaging and fluorescence-guided surgery of pancreatic cancer (#225)
Emma Renard1, Pierre-Alix Dancer2, Christophe Portal3, Franck Denat1, Aurélie Prignon4, Victor Goncalves1
1 Université Bourgogne Franche-Comté, Institut de Chimie Moléculaire de l’Université de Bourgogne, ICMUB UMR CNRS 6302, Dijon, France
Pancreatic cancer is the 4th leading cause of death by cancer in Europe. This cancer has a poor prognosis, which is due to late clinical presentation, rapid progression and limited effect of chemotherapy. Moreover, 30 to 50% of patients who have had surgery, relapse because of the difficulty to accurately delineate tumor margins. Therefore, there is real need to develop new methods that improve both the diagnosis and the accuracy of tumor resection.
Five bimodal imaging agents were synthesized, based on the structure of the NTSR1 peptide agonist NT20.3.1,2 The radiometal chelator, (R)-NODAGA, and three cyanine 5 derivatives were conjugated to the N-terminal lysine residue. Different spacers were introduced. The affinity of ligands for NTSR1 was determined in vitro. The compounds were radiolabeled with 68Ga and their biodistribution was evaluated on nude mice bearing a subcutaneous xenograft of AsPC1 (human pancreatic adenocarcinoma) cells. PET images were acquired 1 h post injection. Mice were sacrificed and the uptake of tracers into different organs was measured by gamma-counting. Fluorescence images were recorded at different times and tumors were removed through fluorescence-guided surgery.
Bimodal imaging agents were obtained with an overall yield of 10 to 33%. In vitro studies confirmed the high affinity of all compounds for NTSR1 with Ki values in the nanomolar range. Spacers did not have a significant impact on affinity for the receptor. In contrast, the addition of sulfonate groups resulted in a decrease in affinity from 0.76 nM to 12.1 nM. The radiolabeling with 68Ga was achieved with good radiochemical yields (51% to 90%) and purity (>99%). PET imaging studies allowed us to identify the compound [68Ga]-NODAGA-Lys(Cy5**)-PEG2-[Me-Arg8, Tle12]-NT(7-13), as the one showing the most promising pharmacokinetic profile. Biodistribution data showed a high tumor uptake (2.6%ID/g 90 min p.i.) with a high tumor-to-normal tissues ratio. Similar results were obtained by fluorescence imaging and confirmed during the fluorescence-guided resection of tumor masses.
A novel peptide-based bimodal imaging agent for NTSR1-positive tumors was developed. This compound showed high tumor uptake and fast clearance from non-targeted organs, leading to high contrast in PET and fluorescence imaging. These results highlight the potential of this agent for the diagnosis and the fluorescence-guided surgery of pancreatic cancer.
AcknowledgmentThe authors thank Christophe Piesse from the "Plateforme d'Ingénierie des Protéines de Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, IBPS” for help regarding the synthesis protocol of NT-20.3 and Aurélien Gibier for the synthesis of DOTA-NT-20.3. We also thank the "Plateforme d'Analyse Chimique et de Synthèse Moléculaire de l'Université de Bourgogne” (http://www.wpcm.fr) for access to analytical instrumentation and Pr. Anthony Romieu for providing Cy5 and the disulfonated cyanine 5.
This work was funded by the French National Research Agency (ANR) under the French Investissements d’Avenir programs Equipex (IMAPPI, ANR-10-EQPX-05-01), France Life Imaging (FluoNTEP, ANR-11-INBS-0006), and AAP Générique 2017 (ZINELABEL, ANR-17-CE18-0016-01). This work is also part of the project Pharmaco-imagerie et agents théranostiques funded by the Conseil Régional de Bourgogne Franche-Comté through the Plan d’Action Régional pour l'Innovation (PARI) and by the European Union through the PO FEDER-FSE 2014/2020 Bourgogne program.
 Alshoukr, F, Prignon, A, Brans, L, Jallane, A, Mendes, S, Talbot, J.-N, Tourwé, D, Barbet, J, Gruaz-Guyon A. 2011, Novel DOTA-Neurotensin Analogues for 111In Scintigraphy and 68Ga PET Imaging of Neurotensin Receptor-Positive Tumors, Bioconjugate Chem., 22, 1374-1385.
 Prignon, A, Provost, C, Alshoukr, F, Wendum, D, Couvelard, A, Barbet, J, Forgez, P, Talbot, J.-N, Gruaz-Guyon, A. 2019, Preclinical Evaluation of 68Ga-DOTA-NT-20.3: A Promising PET Imaging Probe To Discriminate Human Pancreatic Ductal Adenocarcinoma from Pancreatitis, Mol. Pharmaceutics, 16, 2776-2784.
Keywords: neurotensin, PET, Gallium-68, fluorescence-guided surgery, bimodal
|1:18 p.m.||PS 20-07||
INDUSTRY TALK (PerkinElmer)Combined tomographic optical and morphological imaging in cancer- and drug research (#722)
Manfred Ogris1, Fatih Alioglu1, Magdalena Billerhart1, Simon Decker1, Haider Sami1
1 University of Vienna, Department of Pharmaceutical Chemistry, Vienna, Austria
Optical imaging has evolved as a key imaging modality in preclinical research, and with near infrared imaging it is also in clinical use. Near infrared emitting fluorophores allow tracking of biomolecules and nanoparticles in vivo [1,2], while luciferase reporter genes are versatile tools to study intracellular signaling, gene delivery efficiency and therapy response both in vitro and in vivo [3,4]. To overcome limitations of 2-D imaging, we apply optical tomographic imaging combined with contrast agent enhanced computed tomography [1,2,4-6]. Applying these technologies, we could follow the biodistribution of intratracheally administered nucleic acids and nanoparticles labelled with the near infrared emitting fluorophore AF750 [1,2]. The growth of tumor lesions could be tracked in a disseminated model of syngeneic triple negative breast cancer (Fig. 1 and ref ). Combining bioluminescence imaging and MRI, we could demonstrate efficient transfection of tumor lesions in lung via plasmid loaded and peptide targeted nanoparticles .
 Geyer et al Int J Pharm. 2017 Jun 20;525(2):359-366.
 Sami et al ACS Appl Mater Interfaces. 2020 Jul 8;12(27):30095-30111.
 Maier et al PLoS One. 2019 Dec 20;14(12):e0226570.
 Groza et al Oncoimmunology. 2018 Feb 16;7(5):e1424676.
  Taschauer et al Mol Ther Nucleic Acids. 2019 Dec 6;18:774-786.
Geyer et al Hum Gene Ther. 2017 Dec;28(12):1202-1213.
Fig. 1: near infrared emitting reporter genes: FLIT/CT
Balb/c mice carrying metastatic triple negative 4T1 breast cancer tumors were analyzed by Fluorescence Imaging Tomography (FLIT) in combination with contrast enhanced CT (iopamidol). 4T1 tumors were lentivirally transduced prior to implantation to express the near infrared emitting reportergene iRFP720 .
Keywords: tomographic optical and morphological imaging