FLASH radiotherapy spares healthy astrocytes while stressing glioma cells

FLASH radiotherapy, which fires radiation at ultra-high dose rates, has long been reported to spare normal tissue while still controlling tumors, but the cellular reasons behind this so-called FLASH effect have remained murky. The researchers compared FLASH irradiation with conventional radiotherapy in murine glioma GL261 cells and in primary cultures of healthy astrocytes, the star-shaped support cells of the brain, tracking redox dynamics, mitochondrial function, bioenergetics and transcriptional stress programs in real time.

The team, led by Giulia Rosini and Beatrice D’Orsi with colleagues including Elisa De Santis, Riccardo Nieri, Matteo Paolini, Filippo Rossi, Silverio Sbrana, Esther Ciarrocchi, Andrea Cavalieri, Fabiola Paiar, Fabio Di Martino, Eleonora Vannini and Mario Costa, combined real-time kinetic assays, flow cytometry, Seahorse metabolic analysis and gene expression profiling. In healthy astrocytes, FLASH treatment reduced the acute burst of reactive oxygen species, limited cell death and was associated with better preservation of mitochondrial membrane potential and respiratory capacity than conventional irradiation. FLASH also triggered a sustained induction of cell-cycle checkpoint and senescence-associated transcripts in these normal cells.

Glioma cells told a strikingly different story. In GL261 cells, FLASH produced a lower initial ROS peak, but instead of settling down, the cells continued to accumulate reactive oxygen species over time. They showed enhanced accumulation in the G2/M phase of the cell cycle, persistent mitochondrial depolarization and suppressed respiration. At the 8 Gy dose, Annexin V analysis revealed comparable levels of Annexin V positivity between the two irradiation modalities, yet FLASH-treated cells displayed greater G2/M accumulation and metabolic dysfunction.

To probe which signaling pathways contribute to this radiation-induced metabolic dysfunction, the researchers applied four pharmacological modulators: DPQ, Calpeptin, Nec-1 and zVAD. These compounds differentially affected mitochondrial bioenergetic parameters depending on the irradiation modality and the cell type, providing functional evidence that distinct stress- and cell-death-associated signaling pathways are involved in how cells metabolically respond to ultra-high dose rate versus conventional irradiation.

The picture was not uniform across all tumor models. In human U87 glioblastoma cells, FLASH actually induced stronger ROS accumulation, greater Annexin V positivity and alterations in NAD⁺ levels associated with mitochondrial respiratory impairment compared with conventional treatment.

Taken together, the data indicate that FLASH radiotherapy elicits cell-type-specific redox-mitochondrial responses: relative preservation of mitochondrial function in healthy astrocytes, and prolonged metabolic stress with altered cell-cycle dynamics in glioma cells.

Several caveats matter for interpretation. This is a preclinical cell-culture study using murine GL261 glioma cells, primary healthy astrocytic cultures and human U87 glioblastoma cells, not living organisms or patients. The findings describe early cellular processes measured with kinetic assays, flow cytometry, Seahorse analysis and gene expression profiling over defined time windows, and the abstract notes that Annexin V comparisons were made at a single 8 Gy dose. Because the work examines isolated cells, it cannot by itself predict clinical outcomes in treated patients, and the authors emphasize that the mechanisms underlying the FLASH effect remain incompletely defined. The article is being shared early as a peer-reviewed, accepted, citable version with a permanent DOI, subject to further edits before the final Version of Record appears.

What makes the study notable is its methodological breadth: rather than measuring a single endpoint, the team simultaneously tracked ROS kinetics, mitochondrial membrane potential, respiratory capacity, NAD⁺ levels, cell-cycle distribution, Annexin V positivity and transcriptional stress programs across two irradiation modalities and three cell models. This systems-level view reveals that the FLASH effect is not a single phenomenon but a set of divergent, cell-type-dependent early responses, with healthy astrocytes mounting a protective, senescence-and-checkpoint-associated program while glioma cells slide into sustained redox and bioenergetic stress.

Subject of Research: Cell-type-specific redox and mitochondrial responses of astrocytes and glioma cells to ultra-high-dose-rate FLASH irradiation versus conventional radiotherapy.

Article Title: Differential redox-mitochondrial adaptation to ultra-high-dose-rate irradiation in astrocytes and glioma cells

Article References: Rosini, G., De Santis, E., Nieri, R., Paolini, M., Rossi, F., Sbrana, S., Ciarrocchi, E., Cavalieri, A., Paiar, F., Di Martino, F., Vannini, E., Costa, M., & D’Orsi, B. (2026). Differential redox-mitochondrial adaptation to ultra-high-dose-rate irradiation in astrocytes and glioma cells. Cell Death Discovery. https://doi.org/10.1038/s41420-026-03426-y

Image Credits: AI Generated

DOI: 10.1038/s41420-026-03426-y

Keywords: FLASH radiotherapy, ultra-high dose rate, astrocytes, glioma cells, mitochondrial dysfunction, reactive oxygen species, bioenergetics, cell cycle, senescence, glioblastoma, redox signaling, preclinical study