FLASH Radiotherapy selectively protects healthy glia while enhancing glioma cytotoxicity through redox - mitochondrial modulation

FLASH radiotherapy (FLASH-RT) delivers radiation at ultra-high dose rates (UHDRs), achieving strong antitumor efficacy while minimizing normal tissue toxicity. However, the cellular and metabolic mechanisms underlying this ‘FLASH effect’ remain unclear. Here, we compared the impact of FLASH versus conventional (CONV) irradiation (8 Gy) in glioma GL261 cells and healthy primary glial cultures. Multiple parameters, including cytotoxicity, oxidative stress, mitochondrial function, and bioenergetics, were evaluated using real-time population-based assays, flow cytometry, Seahorse analysis, and gene expression profiling. In healthy glial cells, FLASH-RT markedly reduced ROS accumulation, preserved mitochondrial membrane potential and respiration, and maintained viability, while CONV-RT induced sustained oxidative stress and mitochondrial dysfunction. FLASH-treated glial cells showed preferential activation of cell-cycle arrest and senescence programs with minimal engagement of apoptotic pathways, indicating activation of repair and survival responses. In contrast, FLASH-RT in tumor cells produced a smaller initial ROS peak followed by a stronger and more sustained ROS accumulation over time, accompanied by mitochondrial depolarization, metabolic impairment, and early induction of autophagy-related genes. Both modalities reduced glioma cell viability by 72 h, but only FLASH preserved mitochondrial integrity in non-tumoral cells. Pharmacological inhibition of PARP, calpains, and necroptosis further revealed that CONV-RT–induced metabolic collapse is primarily PARP- and necroptosis-dependent, whereas FLASH-RT triggers a more distributed, modulable, and pathway-diverse stress response, with glial cells showing minimal inhibitor sensitivity and GL261 cells exhibiting multimodal vulnerability. Together, these results reveal that FLASH-RT orchestrates a cell-type-specific metabolic and redox reprogramming that selectively protects healthy glia while enhancing tumor susceptibility, providing mechanistic support for its development as a next-generation radiotherapy approach.