PO-0986: Downregulation of the oncoprotein SET enhances RT-induced apoptosis in hepatocellular carcinoma

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apoptosis (Annexin V and propidium iodin PI) 3/ Cell cycle modifications (PI) Results Our results showed that IR on RCCL not expressing HLA-G, ILT2 or ILT4 did not induce these molecules. However, in constitutively expressing HLA-G or ILT4 RCCL, IR decreased significantly HLA-G and ILT4 expression. Furthermore, we found that HLA-G, ILT2 and ILT4 transduction increased radioresistance. This effect was partially aborted by the use of antibodies directed against these molecules. Mechanisms of radio resistance are under investigations and will be presented at the meeting. Conclusion Ionizing radiation decreases the expression of HLA-G or its receptors in RCCL constitutively expressing these molecules. HLA-G and its ligands increase radioresistance. This finding could have some clinical implications for stereotactic radiotherapy of renal cancer or its metastasis. PO-0985 Tumor metabolic changes after neoadjuvant radiotherapy: consequences for surgery-related metastases N. Leroi1, F. Lallemand2, J. Leenders3, S. Blacher4, P. De Tullio3, P. Coucke5, A. Noel4, P. Martinive1 1 C.H.U. Liège - Université de Liège, Radiotherapy DeptLaboratoire de Biologie des Tumeurs et du Développement, Liège, Belgium 2 C.H.U. Liège - Université de Liège, Radiotherapy DeptResearch center of Cyclotron, Liège, Belgium 3 Université de Liège, CIRM- Chimie Pharmaceutique, Liège, Belgium 4 Université de Liège, Laboratoire de Biologie des Tumeurs et du Développement, Liège, Belgium 5 C.H.U. Liège - Université de Liège, Radiotherapy Dept., Liège, Belgium Purpose or Objective Neoadjuvant radiotherapy (NeoRT) aims at improving tumor local control and patient overall survival. In the case of locally advanced rectal cancer, NeoRT increases significantly local control compared to surgery alone, but patient overall survival is not improved. Currently, predicting tumor response and recurrences represent a major challenge for personalized medicine. Previously, we developed a pre-clinical model of NeoRT and showed that the timing of surgery and NeoRT schedules both influenced metastasis burden (Leroi et al., Oncotarget, 2015). Based on this model, we study the impact of RT schedule on the primary tumor metabolome at the time of surgery to predict local recurrence and metastatic profile. Material and Methods We locally irradiated primary tumors (MDA-MB231 cells and 4T1 cells), subcutaneously implanted to SCID and BalbC mice, with two NeoRT schedules (5x2Gy and 2x5Gy). We surgically removed tumors 4 or 11 days after the end of RT and kept the mice alive for the metastatic growth. Non-irradiated control tumors were also surgically collected at the same time. For metabolomic study, tumor samples were homogenized in deuterated phosphate buffer and supplemented with maleic acid and TMSP before Nuclear Magnetic Resonance (NMR) analyses. Data were analyzed with powerful statistical tool (supervised and multivariate analyses). Results Irradiated 4T1 and MDA-MB231 tumors displayed different metabolic profile than non-irradiated tumors, especially 4 days after the end of RT for 4T1 tumors and 11 days after NeoRT for MDA-MB231 tumors. Moreover, we observed a decrease in some metabolite levels (i.e. glutamate, taurine, glycine, myoinositol) in tumors following both NeoRT schedules. We also noticed an increase in general lipid signals in irradiated MDA-MB231 tumors. This was not related to adipocyte infiltration, as we observed, by immunostaining, decreased infiltration of perilipin and

FABP4+ cells in these tumors following NeoRT. Preliminary results with OPLS-DA analyses showed discrimination of primary tumor metabolome according to the propensity to induce loco-regional recurrence (significant for tumors collected 4 days after 5x2Gy). Furthermore, based on the metabolic profile of the primary MDA-MB231 tumors and OPLS linear regression, mathematical models were established in the different groups allowing to predict the metastatic burden (r²=0,80-0,90). Conclusion In preclinical models, we show profound modifications of the primary tumor metabolome following NeoRT through NMR analyses, offering new opportunities to understand tumor metabolism adaptation following NeoRT. Furthermore, others NMR results appear very relevant when transposed to clinic. Indeed, with mathematical models, local recurrence and metastatic profiles were predictable based on the metabolomic profile of the primary tumor at the time of surgery, which could be helpful to adapt adjuvant therapies in order to prevent relapse. PO-0986 Downregulation of the oncoprotein SET enhances RT-induced apoptosis in hepatocellular carcinoma C.Y. Huang1, M.H. Hung2, C.W. Kuo3, C.T. Shih4, M.H. Chen4, K.F. Chen5 1 National Taiwan university hospital, Division of Radiation Oncology- Department of Oncology, Taipei, Taiwan 2 Taipei Veterans General Hospital, Division of Medical Oncology- Department of Oncology, Taipei, Taiwan 3 Yuanpei University of Medical Technology, Department of Medical Imaging and Radiological Technology, Hsinchu, Taiwan 4 National Yang-Ming University, Institute of Biopharmaceutical Sciences, Taipei, Taiwan 5 National Taiwan university hospital, Department of Medical Research, Taipei, Taiwan Purpose or Objective Hepatocellular carcinoma (HCC) is among the most lethal human malignancies worldwide. Radiotherapy (RT) is not commonly used to treat HCC with regard to both suboptimal treatment efficacy and toxicity. The current project aimed to characterize the role of a novel oncoprotein SET/ I2PP2A (Inhibitor-2 of protein phosphatase 2A) in mediating the radio-resistance of HCC cell and explore the potential on antagonizing SET to improve the anti-HCC effects of RT. Material and Methods The effects of RT in HCC cells with different expression of SET were assessed by colony formation and sphere formation assay. We generated a novel SET antagonist, EMQA (N4-(3-ethynylphenyl)-6,7-dimethoxy-N2-(4phenoxyphenyl) quinazoline-2,4-diamine), to validate the therapeutic potential of targeting SET. The combination effects of EMQA and RT were tested in vitro using four different HCC cell lines, Hep3B, PLC5, HA22T and HA59T, and a subcutaneous PLC5 xenografted model in vivo. HCC cells were exposed to 1 fraction of 4-Gy radiation using a cobalt 60 unit (at a dose rate of 0.5 Gy/min) with the source-axis-distance set at 80 cm to the bottom of the dish. After 48 hours, the cells were treated with or without EMQA. Results To explore the roles of SET in affecting the radiosensitivity in HCC, we first generated PLC5 and Hep3B cells with different SET activity, and assessed the effects of RT on these cells by colony formation and tumor sphere assay. Comparing to mock-treated cells, HCC cells transfected with shRNA against SET were shown with significant reduced viability under the same RT treatment. Oppositely, cells with ectopic expression of SET were more resistant to RT. Next, we used EMQA to test whether

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antagonizing SET could enhance the effects of RT against HCC. Using sub-G1 analysis, we showed that adding EMQA significantly increased RT-induced apoptosis of HCC cells. The number of tumor colony was also significantly decreased in HCC cells exposing to EMQA plus RT than either of the treatment alone. Lastly, using the PLC5 xenografted tumor model, the synergistic effects of SET antagonist combining RT were also observed. Conclusion SET is a novel oncoprotein that affects the radiosensitivity of HCC cells. A combination therapy with RT and the SET antagonist, such as EMQA, enhanced RTinduced apoptosis of HCC cells in vitro and in vivo. PO-0987 Gemcitabine-based chemoradiotherapy gets improved with PARP inhibitor in pancreatic cancer cells W. Waissi1, H. Burckel1, E. Magisson1, G. Larderet1, G. Noel1 1 CLCC Paul STRAUSS, EA3430- Laboratoire de Radiobiologie, Strasbourg, France Purpose or Objective Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a cumulative 5-year overall survival of less than 5% for all stages. Thirty percent of patients diagnosed with pancreatic adenocarcinoma present with a locally advanced disease and could benefit from chemoradiotherapy with gemcitabine, which is effective but toxic. Over the past few years, studies have focused on the development of targeted radiosensitizer such as poly(ADP-ribose) polymerase (PARP) inhibitor. We conducted this in vitro study to determine whether PARP inhibition enhances radiation-induced cytotoxicity of pancreatic adenocarcinoma. Material and Methods Pancreatic carcinoma cells, MIA PaCa-2 (BRCA1/2 wildtype) , were treated with olaparib and/or gemcitabine and/or irradiation (2,5 and 10 Gy). In vitro cell viability, clonogenic assay, cell cycle distribution, γ-H2AX quantification, apoptosis and autophagy were assessed. Results In vitro, treatment with olaparib alone at 1 µM was not cytotoxic but highly radiosensitized cells (standard enhancement ratio =1.23+/-0.02) and particularly at high dose per fraction (10 Gy). After 24 hours, the number of remaining γ-H2AX stained cells was higher when cells were treated with a combination of 10 Gy irradiation and olaparib compared to irradiation or olaparib alone. Furthermore, combination of olaparib and irradiation induced a G2/M arrest. In contrast, a non-cytotoxic concentration of gemcitabine could also radiosensitize cells, but clearly less than olaparib (SER=1.11+/-0.04). Radiosenzitization by gemcitabine was associated with percentage of cells blocked in early S-phase just before irradiation. Finally, cell death quantification after 24 hours showed that none of the treatments induced apoptosis, whereas gemcitabine or 10 Gy irradiation alone induced autophagy. Conclusion Our results showed that MIA PaCa-2 cells could be radio sensitized with low dose of olaparib , through an increase of unrepaired double-strand breaks and a block in G2 phase. The radiosensitization was higher with high dose radiation. This may be translated into an enhancement of local control in vivo and better disease free survival. Investigations in three other pancreatic cells lines are in progress. PO-0988 Following tumour microenvironment after Neoadjuvant radiotherapy with IVIM perfusion analysis F. Lallemand1, N. Leroi2, M. Bahri3, E. Balteau3, A. Noël2, P. Coucke1, A. Plenevaux3, P. Martinive1 1 C.H.U. - Sart Tilman, Radiothérapie, Liège, Belgium 2 ULg, Laboratory of Tumor and Development Biology,

Liège, Belgium 3 ULg, Cyclotron Research Center, Liège, Belgium Purpose or Objective Neoadjuvant radiotherapy (NeoRT) improves tumor local control and facilitates tumor resection in many cancers. The timing between the end of the NeoRT and surgery is driven by the occurrence of side effects or the tumor downsizing. Some clinical studies demonstrated that the timing of surgery and the RT schedule influence tumor dissemination and subsequently patient overall survival. Previously, we developed a pre-clinical model demonstrating an impact of NeoRT schedule and the timing of surgery on metastatic spreading (Leroi et al. Oncotarget 2015). Here, we evaluate the impact of NeoRT on the tumor microenvironment by functional MRI (fMRI). We aim to identify non-invasive markers allowing to determine the best timing to perform surgery and avoiding tumor spreading. Material and Methods Based on our NeoRT model, MDA-MB 231 and 4T1 cells were implanted in the flank of SCID and BalbC mice, respectively. We locally irradiated tumors with 2x5Gy and then surgically removed at different time points after RT. Diffusion Weighted (DW) -MRI was performed every 2 days between RT and surgery. For each tumors we acquired 8 slices of 1 mm thickness and 0.5 mm gap with an 'in plane voxel resolution” of 0.5 mm. For DW-MRI, we performed FSEMS (Fast Spin Echo MultiSlice) sequences, with 9 different B-value (from 40 to 1000) and B0, in the 3 main directions. We performed IVIM (IntraVoxel Incoherent Motion) analysis to obtain information on intravascular diffusion, related to perfusion (F: perfusion factor) and subsequently tumor vessels perfusion. Results With the MDA-MB 231, we observed a significant peak of F at day 6 after irradiation, this increasing is about 60% of the basal value (n=6, p<0,05). Moreover, D* parameters (also related to perfusion) increase at the same time. The other parameters of the DW-MRI, ADC and D presented no modification. We observed similar results with 4T1 cells, where F increased at day 3 (about 55%, n=10, p<0,05) then returned to initial level. The difference in timing for the peak of F (day 6 vs day 3) could be related to the difference in tumor growth according to the cell line (four weeks for MDA-MB 231 cells vs one week for 4T1cells). We performed surgery at the time of the F parameter peak in the MDA-MB 231 and we observed a decrease of the metastasic burden compared to surgery performed at day 4 or day 11(absolute number of metastasis 23 VS 1 VS 8 with n=4). Conclusion For the first time, we demonstrate the feasibility of repetitive fMRI imaging in preclinical models after NeoRT. With these models, we show a significant difference in perfusion-related parameters (D* and F) at a specific time point depending of the tumor cells. These modifications are correlated to a decrease of metastasis spreading related to the surgery procedure. These results open new perspectives in the personalized medicine and MRI guided surgery timing after NeoRT. PO-0989 Sub-lethal radiation allows an efficient antitumor therapy with engineered T-cells in RIP-Tag2 mice F. Maione1, E. Garibaldi2, X. Zhuang3, J. Robinson3, R. Bicknell3, E. Delmastro2, A. Miranti4, S.P. Lee3, P. Gabriele2, E. Giraudo1 1 Candiolo Cancer Institute- Torino- Italy, Department of Science and Drug Technology, Candiolo TO, Italy 2 Candiolo Cancer Institute- Torino- Italy, Radiotherapy Unit, Candiolo TO, Italy 3 University of Birmingham, Institute of Immunology and Immunotherapy, Birmingham, United Kingdom