ESTRO 2021 Abstract Book

S592

ESTRO 2021

upregulation of profibrotic signaling was markedly enhanced after coculturing the HCF with macrophages, after direct and indirect cell contact, and additionally enhanced after radiation. This profibrotic macrophage effect was associated with predominantly M2 marker polarization after HCF coculture which was in turn enhanced after radiation, and accompanied by IL10 and IL1b induction. Collectively, inhibition of PDGF and TGF-ß signaling reduced the profibrotic effects in the HCF/macrophage system. Conclusion In conclusion our results suggest that macrophage - human cardiac fibroblast interactions prompted by radiation play an important role in the initiation of cardiac fibrosis, that TGF-β, CTGF and PDGF have a role in this process, and that inhibitors of these pathways may be able to attenuate RIHD. PD-0761 Radiation-induced anti-tumor immunogenicity in pancreatic cancer cells – protons vs. X-rays S. Kerschbaum-Gruber 1,2 , A. Falkner 3 , K. Stasny 3 , L. Komornik 3 , K. Schwarz 3 , S. Demaria 4 , D. Georg 3,5 1 Medical University Vienna, Department of Radiation Oncology, Vienna, Austria; 2 EBG MedAustron, Ion Therapy and Research Center, Wr. Neustadt, Austria; 3 Medical University of Vienna, Department of Radiation Oncology, Vienna, Austria; 4 Weill Cornell Medicine, Department of Radiation Oncology, New York, USA; 5 EBG MedAustron , Ion Therapy and Research Center, Wr. Neustadt, Austria Purpose or Objective Pancreatic cancer remains a disease with dismal outcome and limited treatment possibilities. The difficult anatomical location results in significant dose burden for several organs at risk (OAR) in curative photon-based radiotherapy (RT). Particles, including protons, yield in favorable dose distributions with improved OAR. Thus, particle therapy has potential to improve radiotherapy of pancreatic cancer. Although successful for other malignancies, Immune checkpoint blockade (ICB) has failed to improve outcome for pancreatic cancer patients in clinical studies. RT has been shown to synergize with ICB and improve ICB efficacy in previously unresponsive preclinical tumors. This has been observed in several malignancies but only limited data exists for pancreatic cancer. Therefore, this study aims at investigating photon-, as well as proton-mediated response patterns. We investigate immunogenic cell death and key regulators of anti-tumor immunity and tumor immune escape. These include the anti-tumor immunity triggering IFN-I pathway, immune evasion key player Galectin-1 and the immune checkpoint proteins PD-L1 and CD73 in 3 human cancer cell lines. Materials and Methods Radiation was delivered as single dose (0Gy, 2Gy, 8Gy, 24Gy) or with 3 fractions of 8 Gy on 3 consecutive days. Single dose treatment response was investigated for X-rays as well as protons. Fractionation was only performed with X-rays. For proton treatment, cell cultures (Panc-1, Mia-Paca-2 and BxPc3) were positioned at a central SOBP position at 6cm depth in a water phantom. Soluble HMGB1 (ELISA) and ecto-Calreticulin (immunofluorescence) were assessed as markers of immunogenic cell death. Cytosolic DNA was quantified fluorescence-based after subcellular fractionation. IFN-I pathway and the nucleic acid sensing response (STAT- 1, cGAS, STING, IFN-I, CXCL10), as well as PD-L1, CD73 and Galectin-1 expression were assessed using qRT- PCR, Western Blotting and ELISA for soluble IFN-I, CD73 and Galectin-1. Results All 3 cell lines yielded similar results. Both radiation types induced immunogenic cell death markers with 3x8 Gy yielding most pronounced effects. Cellular Galectin-1 expression as well as soluble Galectin-1 were found to be upregulated in response to radiation, particularly after 3x8Gy. cGAS protein expression was upregulated, most pronounced after 3x8Gy, based on robust accumulation of cytosolic dsDNA. However, neither STAT1 nor STING were phosphorylated at any dose level and no INF-I could be detected using ELISA, indicating that both, X-rays and protons, failed to activate an IFN-I response. Furthermore, no changes of PD-L1 or CD73 expression were observed at any dose level of either X-rays or protons. Conclusion Our findings suggest only a moderate effect of radiation on modulating pancreatic cancer cell immunogenicity. Protons and X-rays yielded similar effects for all endpoints investigated. PD-0762 Abscopal effect by ablative RT on lung metastases in an oligoprogressive tumor model E. Firat 1 , G. Niedermann 2 1 University Clinics Freiburg, Dept. of Radiation Oncology, Freiburg, Germany; 2 University Clinics Freiburg, Dept. of Radiation Oncology, Freiburg, Germany Purpose or Objective To tackle the controversial question about the degree of T cell-mediated abscopal effects induced by high- dose ablative RT. Materials and Methods In BALB/c mice bearing two 4T1 mammary carcinoma tumors in opposite flanks, the primary tumor was irradiated with two fractions of 20 Gy (ablative RT); in addition, anti-PD-1 antibodies were given once per week. Mice of the combined treatment group and of the control groups were monitored for growth of the two subcutaneous tumors and for survival with and without antibody-mediated depletion of CD8+ T cells ( n = 5–8 mice per group). Spontaneously arising lung metastases were determined by ex vivo clonogenic assay. In single-cell suspensions of the subcutaneous tumors, lung, and spleen, tumor-specific CD8+ T cells (and their expression of exhaustion markers) and immunosuppressive myeloid-derived suppressor cells (MDSCs) were determined by FACS using MHC tetramers and various antibodies. Systemic immunity was also assessed by peptide restimulation assays and in an ex vivo kill of syngeneic tumor cells by tumor-specific T cells found in the spleen. Results Anti-PD-1 monotherapy was not effective at all. In mice treated with ablative RT or RT + aPD-1, the non- irradiated subcutaneous metastasis progressed as rapidly as in untreated mice and as in aPD-1-treated mice. In