FLASH Radiotherapy selectively protects healthy glia while enhancing glioma cytotoxicity through redox - mitochondrial modulation

FLASH Radiotherapy selectively protects healthy glia while enhancing glioma cytotoxicity through redox - mitochondrial modulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article FLASH Radiotherapy selectively protects healthy glia while enhancing glioma cytotoxicity through redox - mitochondrial modulation Giulia Rosini, Elisa De Santis, Riccardo Nieri, Matteo Paolini, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8287444/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract FLASH radiotherapy (FLASH-RT) delivers radiation at ultra-high dose rates (UHDRs), achieving strong antitumor efficacy while minimizing normal tissue toxicity. However, the cellular and metabolic mechanisms underlying this ‘FLASH effect’ remain unclear. Here, we compared the impact of FLASH versus conventional (CONV) irradiation (8 Gy) in glioma GL261 cells and healthy primary glial cultures. Multiple parameters, including cytotoxicity, oxidative stress, mitochondrial function, and bioenergetics, were evaluated using real-time population-based assays, flow cytometry, Seahorse analysis, and gene expression profiling. In healthy glial cells, FLASH-RT markedly reduced ROS accumulation, preserved mitochondrial membrane potential and respiration, and maintained viability, while CONV-RT induced sustained oxidative stress and mitochondrial dysfunction. FLASH-treated glial cells showed preferential activation of cell-cycle arrest and senescence programs with minimal engagement of apoptotic pathways, indicating activation of repair and survival responses. In contrast, FLASH-RT in tumor cells produced a smaller initial ROS peak followed by a stronger and more sustained ROS accumulation over time, accompanied by mitochondrial depolarization, metabolic impairment, and early induction of autophagy-related genes. Both modalities reduced glioma cell viability by 72 h, but only FLASH preserved mitochondrial integrity in non-tumoral cells. Pharmacological inhibition of PARP, calpains, and necroptosis further revealed that CONV-RT–induced metabolic collapse is primarily PARP- and necroptosis-dependent, whereas FLASH-RT triggers a more distributed, modulable, and pathway-diverse stress response, with glial cells showing minimal inhibitor sensitivity and GL261 cells exhibiting multimodal vulnerability. Together, these results reveal that FLASH-RT orchestrates a cell-type-specific metabolic and redox reprogramming that selectively protects healthy glia while enhancing tumor susceptibility, providing mechanistic support for its development as a next-generation radiotherapy approach. FLASH-RT ROS Metabolic stress Mitochondrial Dysfunction Cell Metabolism bioenergetics cancer metabolism Full Text Supplementary Files SupplementaryinformationFiguresS.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8287444","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":557695421,"identity":"fb27e2d8-85f2-4ca8-885c-86ed5dc51c8c","order_by":0,"name":"Giulia Rosini","email":"","orcid":"","institution":"Consiglio Nazionale delle Ricerche","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Rosini","suffix":""},{"id":557695422,"identity":"d17b0639-33b2-4550-b12e-dbeedfec396e","order_by":1,"name":"Elisa De Santis","email":"","orcid":"","institution":"Consiglio Nazionale delle 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