Enhanced Radiation-Sparing Effects of Ultra-High Dose Rate Proton Radiation (FLASH-RT) in a Human Induced Pluripotent Stem Cell-Derived Cerebral Organoid Model

Abstract

<h3>Purpose/Objective(s)</h3> Superior normal tissue sparing with similar antitumoral effectiveness has been observed in preclinical animal models following delivery of radiation at ultra-high dose rates (FLASH-RT), relative to conventional dose rates (CONV-RT). Clinical application of FLASH-RT could reduce devastating radiation-related side effects for patients with aggressive brain tumors such as glioblastoma; however, the FLASH effect must be established in representative models of human systems before its implementation in the clinic. Our objective is to measure relevant response outcomes to proton FLASH-RT using advanced cerebral organoid (CO) models of normal and diseased human brain tissue, correspondingly generated with human induced pluripotent stem cells and patient-derived glioma stem-like cells (GSCs). <h3>Materials/Methods</h3> Mature COs were irradiated to 9 Gy using conventional (CONV, 0.2 Gy/s) and FLASH (100 Gy/s) dose rates using a proton beam and prepared for histological analysis at 30 days post-irradiation. Spatial distribution of hypoxia was visualized in COs by incubating for 2 hours with pimonidazole prior to preparation for histologic analysis. Image analysis was performed with QuPath. Lactate dehydrogenase (LDH) release, a marker of cell death, was sequentially quantified. COs implanted with GSCs expressing a luciferase reporter were co-cultured for 14 days and irradiated as above. Tumor cell proliferation was tracked via luciferase expression. <h3>Results</h3> At 2- and 4-days post-irradiation, there was an increase in LDH release for CONV versus FLASH-radiated COs (p<0.01, p<0.01 respectively) which normalized after 9 days. At 30 days, when specific cell populations in FLASH-radiated COs were histologically compared to CONV-radiated COs, there were higher numbers of proliferating neural progenitor cells (SOX2+/Ki67+, p<0.01), mature neurons (NeuN+, p=0.03), and deep cortical surface layer neurons (SATB2+, p=0.01). The spatial distribution of neural progenitor cells and mature neurons overlapped with hypoxic regions of the CO (pimonidazole+). A trend towards lower expression of activated microglia (IBA1+/CD68+) in FLASH-irradiated COs compared to CONV-radiated COs was noted (p=0.12). A 48.6% versus 51.2% reduction in tumor proliferation in FLASH versus CONV-irradiated GSC-laden-COs was observed at 4d post-irradiation (p=0.98). <h3>Conclusion</h3> We report preliminary findings of normal tissue toxicity reduction and iso-effective tumor response in a human-derived brain model following proton FLASH-RT. Potential protective effects following FLASH-RT to be further validated include preservation of specific cell populations within hypoxic niches including progenitor and differentiated neurons with a corresponding reduction in chronic neuroinflammation.