Spatially fractionated mini-GRID radiotherapy differentially modulates radiation-induced senescence in murine glioma and normal cells.

BACKGROUND: Spatially fractionated radiotherapy (SFRT) is an emerging therapeutic strategy that enhances tumor control while reducing normal tissue toxicity. Although conventional radiotherapy is known to induce therapy-induced senescence (TIS), its induction by SFRT-particularly mini-GRID radiotherapy-remains poorly characterized. Addressing this question is essential to better understand SFRT-mediated biological responses and optimize its therapeutic potential. METHODS: Two rat glioma cell lines (F98, RG2), immortalized astrocytes (CTX-TNA2), and primary mouse embryonic fibroblasts (MEFs) were exposed to single radiation doses ranging from 5 to 20 Gy using conventional radiotherapy (Conv-RT) or mini-GRID RT. Seven days post-irradiation, senescence was evaluated by SA-β-galactosidase activity (X-Gal and FDG staining) and cell size analysis. Senescence markers (p53, p21, and p16) were analyzed by RT-qPCR and Western blotting, and SASP-related genes (Il1a, Il6, Serpine1, and Cxcl1) were quantified by RT-qPCR. RESULTS: In F98 and RG2 glioma cells, Conv-RT at 20 Gy induced a robust senescent phenotype, characterized by increased cell size, elevated SA-β-Gal activity, persistent DNA damage signaling, and upregulation of p53, p21, p16, and SASP-related genes. In contrast, mini-GRID RT achieved comparable cell number reduction while significantly limiting senescence, DNA damage signaling, and SASP activation, with expression levels approaching non-irradiated controls. In CTX-TNA2 astrocytes and MEFs, radiation induced senescence in a dose-dependent manner, with no significant differences between irradiation modalities. CONCLUSIONS: Mini-GRID RT maintains the cytotoxic effectiveness of conventional irradiation in glioma cells while reducing senescence and SASP induction. These findings highlight its potential to improve the therapeutic ratio and support further investigation.