Early molecular changes induced by FLASH irradiation in MCF10A and MDA-MB-231 breast cell lines

Introduction: We used the non-tumorigenic MCF10A and triple-negative MDA-MB-231 breast cell lines to compare cell response to CONV and FLASH dose-rates under normoxic conditions. Beyond evaluating cell survival and DNA/microtubule damage, we assessed transcriptomic and immunological profiles to describe putative molecular changes. Oxidative stress induced by two different irradiation modalities was also investigated. Methods: Breast cell lines were irradiated with electron beams at increasing doses of 2, 4, 6, 9, 11, and 15 Gy delivered at either FLASH (230 Gy/s) or CONV (6 Gy/min) dose rates. Survival fractions were determined by clonogenic assay and dose-response curves. DNA damage was quantified by γ-H2AX and 53BP1 foci counting at 0.5, 1, and 24 hours after treatment with 2 and 5 Gy, while microtubule damage was evaluated by confocal microscopy. Transcriptomic profiling was performed by RNA sequencing 24 hours after RT with doses of 9 and 15 Gy. Immunological profiles were analyzed by using Luminex technique at 24, 48, and 72 hours post-RT. The GSH/GSSG ratio was also measured by mass spectrometry at 24 hours post-treatments to assess differences in cellular oxidative status. Results: Cell survival was comparable between FLASH and CONV regimens in both cell lines. However, a dose-rate effect was observed at the level of early DNA damage, with increased γ-H2AX foci 1 hour after FLASH-RT in both cell lines, and greater persistence of 53BP1 foci at 24 hours in MDA-MB-231 cells, suggesting a dose-dependent response. Immunological profiling showed no qualitative differences between dose rates; nevertheless, MDA-MB-231 cells produced higher levels of several factors after FLASH-RT, whereas MCF10A cells displayed minimal variation. Transcriptomic profiling revealed a broader gene modulation following FLASH-RT, with mitochondrial gene upregulation in MCF10A cells and induction of structural genes in MDA-MB-231 cells. No significant differences in glutathione balance were detected between FLASH and CONV irradiation in either breast cell line. Discussion: Our findings provide new insights into the early biological responses to ultra-high dose rates in vitro under normoxic conditions, suggesting that the dose-rate effect may influence early cellular processes at different levels, without directly affecting cell survival.