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

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.

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.

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.

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.