Early effects of ionizing radiation on molecular modifications and hallmarks of Immunogenic cell death in mouse tumor cells

BACKGROUND: Ionizing radiation (IR) is known to activate the immune system by releasing damage-associated molecular pattern (DAMP) molecules. To combine the IR with novel immunotherapies it is important to know how different irradiation doses influence the release of DAMPs and induce other molecular changes within the initial 48 h post-irradiation, a period preceding extensive cell death. Therefore, our aim was to determine the activation of most common markers of immunogenic cell death, calreticulin (CRT), HMGB1 and ATP and the changes in the expression of MHC I, MHC II and PD-L1 molecules to evaluate early post-IR events. METHODS: We used three immunologically different mouse tumor cell lines, B16-F10, 4T1 and CT26, which form differently immunogenic tumor models in vivo. They were irradiated in vitro with doses at which 30 (IC30), 50 (IC50) or 70% (IC70) of the cells died. We determined the type of cell death, membrane exposure of CRT, the release of HMGB1 and ATP, as well as the expression of MHC I, MHC II and PD-L1 molecules at different IC doses up to 48 h after irradiation. RESULTS: Most of the cells were still alive at 24 to 48 h after IR, regardless of dose and cell type. The percentage of apoptotic and necrotic cells slightly increased with increasing radiation dose and with increasing time after IR. The release of HMGB1 and ATP and membrane localization of CRT after IR were increasing with radiation dose and time after IR. Only the CT26 cell line exhibited significant elevation in all three hallmarks of immunogenic cell death (HMGB1, ATP, and CRT). MHC I and PD-L1 expressions increased in all three cell lines and increased with IR dose. There was no significant change in MHC II expression following IR. CONCLUSIONS: Our results indicate that IR induced the most potent immunological changes in CT26 cell line, which forms the most immunogenic tumor model. In general, the effect increased with the IR dose and time after IR, with the most significant changes observed 48 h post-IR. Our results highlight the onset of immunological changes in initial 48 h post-irradiation, a period preceding extensive cell death, which were cell type and IR dose dependent, indicating the importance of tumor type and its associated microenvironment in the potential systemic activation of the anti-tumor immune response after IR in vivo and in patients.