KEY POINTS
- The study compared conventional and ultra-high dose-rate electron irradiation in HN4 oral cavity squamous-cell carcinoma cells and non-malignant VH10 fibroblasts. Cells received 0, 2, 6, 10 or 20 Gy, with ultra-high dose-rate delivery at approximately 400 Gy/s versus conventional irradiation at approximately 0.1 Gy/s.
- High-resolution respirometry performed immediately and 48 hours after irradiation showed clear dose- and time-dependent increases in HN4 mitochondrial activity. Routine respiration, oxidative phosphorylation capacity and maximal electron-transfer capacity rose most strongly after 6–20 Gy at 48 hours.
- The response was tumor-cell specific within the tested models: VH10 fibroblasts showed little change in mitochondrial respiration across the same dose range, whereas HN4 cells demonstrated substantial post-irradiation metabolic adaptation.
- Despite numerically higher oxygen consumption after ultra-high dose-rate treatment in several conditions, no statistically significant differences between dose rates were detected for the principal respiratory endpoints. Both complex I- and complex II-supported pathways participated in the response.
- At 20 Gy, both irradiation modes also altered oxidative and metabolic stress markers. Conventional irradiation produced earlier increases in mitochondrial reactive oxygen species and membrane potential with reduced ATP-related signal, whereas ultra-high dose-rate irradiation showed more delayed mitochondrial reactive oxygen species elevation; these were descriptive temporal patterns rather than significant between-modality differences.
- The oxidative phosphorylation-to-electron-transfer ratio decreased between irradiation and 48 hours after 20 Gy with both dose rates, suggesting that ATP-production machinery became relatively more limiting despite the overall increase in respiratory capacity.
- RNA sequencing at 48 hours after 20 Gy showed substantial overlap between conventional and ultra-high dose-rate irradiation, including downregulation of gene sets involving mitochondrial membranes, matrix, ribosomes and oxidoreductase activity. The transcriptomic findings therefore reinforced remodeling after irradiation but not a distinct FLASH mitochondrial program.
CLINICAL TAKEAWAY
In this head and neck cancer model, mitochondrial adaptation was driven primarily by radiation itself rather than dose rate. The findings argue against a simple intrinsic mitochondrial explanation for FLASH under normoxic conditions, while leaving open effects dependent on hypoxia, tissue context or other biological systems.