CNSC-49. COMPARATIVE ASSESSMENT OF RADIOTOXICITY INDUCED BY PROTON FLASH AND PROTON CONVENTIONAL RADIOTHERAPY IN A NEONATAL ORGANOTYPIC BRAIN SLICE MODEL

Abstract Brain cancer is a complex, heterogeneous disease with a poor prognosis, reflected in a 5-year survival rate of approximately 33%. Current treatment includes surgery, radiotherapy, and chemotherapy. However, conventional whole-brain photon radiotherapy causes cognitive impairment due to collateral damage to non-tumoral neural tissue. Proton Conventional radiotherapy (pCONV-RT) has emerged as a more targeted alternative delivering precise tumor irradiation with reduced off-target toxicity. A novel form of this therapy is Proton FLASH radiotherapy (pFLASH-RT), which delivers an ultra-high dose rate within milliseconds. Preclinical studies suggest that pFLASH-RT may offer neuroprotective effects; nonetheless, its impact on healthy, particularly developing brain tissue, remains poorly understood. To address this knowledge gap, we developed a preclinical organotypic brain slice model from neonatal mice, which preserves 3D tissue architecture, reduces animal use, and supports diverse cellular and molecular analyses. Using this system, we hypothesized that pFLASH-RT induces less DNA damage and inflammation in healthy tissue compared to pCONV-RT. Coronal brain slices (250μm thick) from 7-day-old C57BL/6 mice (both sexes) were cultured for one week before irradiation with 10Gy pFLASH-RT (~50Gy/min) or pCONV-RT (0.1Gy/s). Tissues were harvested 24h post-irradiation for immunofluorescence and gene expression analyses. Our results showed that both radiation modalities increased nuclear γH2AX foci, indicating DNA damage. Cell proliferation decreased significantly in irradiated samples compared to unirradiated controls (SHAM), as assessed by Ki67 gene and protein expression. Inflammatory genes Il-1β and Il-6 were upregulated by both radiotherapies. Importantly, this inflammatory response was sex-dependent, with a significantly higher effect in females exposed to pCONV-RT, underscoring potential age- and sex-specific susceptibilities in the developing brain. Overall, our organotypic model represents a robust platform to investigate the mechanisms and safety profiles of emerging radiotherapy strategies. These findings support the potential of pFLASH-RT for pediatric brain cancer and emphasize the need for further research into sex- and age-dependent radiotoxicity.