Abstract

--- - Background FLASH radiotherapy, characterized by ultra-high dose rates (> 40 Gy/s), potentially spares normal tissues while maintaining antitumor efficacy (the FLASH effect). While electron and proton FLASH are explored, pulsed X-ray sources like plasma focus devices offer unique possibilities. Previous work has reported hyper-radiosensitivity in colorectal cancer cells exposed to ultra-high-dose-rate pulsed X-rays from a kilojoule plasma focus (PF) device, without significant effects on non-cancerous cells. This study further investigates the biological effects of ultra-high-dose-rate (similar to 10(7) Gy/min), low-total-dose pulsed X-rays generated by a PF-2 kJ device on colorectal cancer cell lines, focusing on DNA damage, cell cycle progression, and gene expression. - Results Low-total-dose (similar to 0.25 Gy), ultra-high-dose-rate pulsed X-rays (0.025 Gy/pulse, a total of 10 pulses, pulses temporally separated by 15-20 s) generated by a PF-2 kJ device induced a significant increase in the SubG(1) population in HCT116 and DLD1 cells over 72 h, an effect indicative of apoptosis, which was not observed with conventional X-rays at similar total doses. In addition, pulsed X-rays induced apoptosis in radioresistant MCF-7 breast cancer cells. Whereas conventional X-rays did not cause a significant increase in double-strand breaks (DSBs), surrogate marker gamma-H2AX and phosphor-P53(Ser15) signal were detected 30 min following pulsed X-ray exposure and persisted for up to 24 h, and no evidence of G2/M cell cycle arrest was detected in exposed cells. Gene expression analysis and preliminary transcriptomic data further suggest a DNA damage response leading to cell death and global change in general biological processes related to regulation of gene expression. - Conclusion Low-total-dose (similar to 0.25 Gy), ultra-high-dose-rate pulsed X-rays generated by a PF-2 kJ device induce significant and sustained DNA damage (DSBs) leading to increased apoptosis in colorectal (HCT-116, DLD-1) and breast (MCF-7) cancer cells, compared to conventional X-rays. These effects, coupled with distinct changes in gene expression, suggest that ultra-high-dose-rate pulsed X-rays may overcomeradio-resistancee without eliciting a conventional DNA damage repair or cell cycle checkpoint response. These findings support the potential of PF-generated pulsed X-rays as a novel sourcof e radiotherapy modality and warrant further investigation, particularly in in vivo models, to assess clinical applicability and safety.