RBIO-08. FLASH radiotherapy demonstrates superior control over glioblastoma spheroid area when compared to conventional radiotherapy

Abstract BACKGROUND Glioblastomas (GBMs) are the most aggressive and prevalent primary brain tumour. Their high adaptability and resistance to therapy make them exceptionally challenging to treat. One key factor in GBM aggressiveness is a reliance on glycolysis for energy production, which facilitates rapid biomass generation and tumour growth. The glycolytic byproduct, lactate, acidifies the tumour microenvironment, promoting polarisation of macrophages into a tumour-supportive phenotype. These alterations contribute to radioresistance, leading to tumour recurrence. In this study, we investigated the effects of FLASH radiotherapy (FLASH-RT), which delivers ultra-fast, high-dose radiation. We monitored GBM spheroid area before and after FLASH-RT and conventional radiotherapy (CONV-RT). METHODS Patient-derived GBM cells from infiltrative tumour margins were cultured as spheroids in Ultra-Low Attachment plates. Over three days of culture, spheroid area was measured using a live-cell imager. On day four, spheroids were exposed to either CONV-RT (0.1Gy/s) or FLASH-RT (5Gy per 3.5µs pulse, 300Hz repetition rate) at total doses of 0, 5, 10, 15, or 20Gy (n=4-8 per condition). Following irradiation, spheroid area change was monitored for 70 hours post-treatment. RESULTS Spheroid area (µm²) varied with radiation dose (0-20 Gy) and demonstrated distinct patterns between CONV-RT and FLASH-RT. At 5Gy, both treatments showed high variability in the change in area of the spheroids; however, CONV-RT exhibited a wider range of values than FLASH-RT. Beyond 10Gy, spheroid areas under CONV-RT remained variable, with an average increase of 85,334µm². In contrast, FLASH-RT led to an average change in area of 29,663µm², with minimal variability and uniformly smaller spheroids. CONCLUSION The superior control of FLASH-RT on spheroid area suggests that FLASH-RT may enhance tumour response while potentially reducing variability in treatment outcomes. Future work will investigate the combined effects of FLASH-RT with metabolic inhibition and macrophage co-culture to better understand its impact on metabolic pathways and immune modulation in GBM.