• calcium signaling
• cell death
• CONV-RT
• FLASH-RT
• glioblastoma
• melanoma
• ROS

Conventional radiotherapy (CONV-RT) approach is widely used in clinic despite some limitations. A new approach called Flash radiotherapy (FLASH-RT), aims to spare the healthy tissue while leaving unchanged the therapeutic effect on tumor cells through transient hypoxia, reducing ROS and DNA damage. Also, calcium signaling, is important to proliferation, invasion and cell death in tumors and the role of Mitochondrial Ca2+, that regulates ATP synthesis, apoptosis, ROS generation and biosynthesis. We evaluate the role of radiobiological damage induced by FLASH- and CONV-RT in glioma GL-261 and melanoma B16-F10 murine cells. We performed analysis of Ca2+ homeostasis, ROS formations and cell death using combined population-based and single-cell imaging approaches at three different radiation dose (4,8,16 Gy) at various time points. Our data demonstrated that there are significant increases in cellular ROS in both treatments compared to control, with CONV-RT inducing greater ROS than FLASH-RT. Then we noticed a massive increase in intracellular Ca2+ with both modalities compared to control, at doses of 4 and 8 Gy for GL-261 and only at 8 Gy for B16-F10. Moreover, a greater cell damage occurred 72h after irradiation with both treatments, with FLASH inducing greater cell death at the lower dose (4 Gy). In FLASH-RT compared to CONV-RT was recognized 24h post-irradiation a greater transcriptional activation of genes involved in cell death, cell cycle arrest, senescence, and autophagy.