Molecular Mechanisms of FLASH Radiotherapy in Alleviating Lung Normal Tissue Injury: Insights from Single-Cell Sequencing

FLASH radiotherapy (FLASH-RT), with its ultra-high dose rate (≥40 Gy/s) that causes the "FLASH effect," significantly reduces normal tissue toxicity while maintaining tumor cytotoxicity, but its molecular mechanisms have not been fully elucidated. Previous studies have identified a protective mitochondrial homeostasis mechanism in normal cells during proton FLASH-RT. To further investigate this mechanism, we used an orthotopic lung cancer mouse model to compare the effects of FLASH-RT with conventional radiotherapy and employed single-cell sequencing to comprehensively analyze differences in single-cell gene expression profiles between normal lung tissue and tumors after irradiation. The results indicate that FLASH-RT protects normal lung tissue by regulating the immune microenvironment, inhibiting oxidative stress, reducing epithelial cell apoptosis, and maintaining mitochondrial function. In tumors, a differential molecular response occurs, involving activation of autophagy/apoptosis pathways, enhancement of cellular stress responses, disruption of mitochondrial homeostasis to reduce ATP production, and regulation of the PI3K/Akt signaling pathway to induce cell cycle arrest and apoptosis. This work reveals the molecular mechanisms of FLASH-RT, providing a theoretical basis for its clinical application and advancing precision radiotherapy for cancer.