Analysis of Cell Death and Proliferative Activity of Cell Cultures under Proton Irradiation in Flash Mode

The study of the biological effects of accelerated protons with ultrahigh dose rates in and outside the Bragg peak on tumour and normal cell lines is essential for understanding the consequences of such exposure and for selecting optimal doses and modes for further application in tumour radiotherapy. In this study, human colorectal adenocarcinoma cells (HT-29) and normal human adipose-derived mesenchymal stem cells (ADSC)—fibroblasts—were irradiated. The irradiation was performed using the high-current linear proton accelerator at the Institute for Nuclear Research of the Russian Academy of Sciences, which allows for energy variation in the range of 70–230 MeV. The dose was delivered in three modes: conventional mode (dose rate $$\dot{D}<1$$ Gy/s), Splash mode ( $$\dot{D}\sim 100$$ Gy/s), and single-pulse Flash mode ( $$\dot{D}>10^{4}$$ Gy/s, denoted as Splash from single-pulse Flash) in the region of the spread out Bragg peak. To analyze cell death, staining with propidium iodide and annexin was performed. The proliferative potential was assessed using the EdU assay. After 24 h, an increase in the number of apoptotic HT-29 cells was observed in all irradiation modes, while for fibroblasts, a relative increase in the number of necrotic cells was noted under conventional irradiation with a higher dose compared to other modes. After 48 h, a tendency toward a dose-dependent decrease in the number of necrotic HT-29 cells was observed in Flash/Splash modes, with consistently low levels of necrotic fibroblasts. The number of DNA-synthesizing cells decreased significantly by 120 h across all doses and irradiation modes. The curves for Flash and Splash irradiation modes were nearly parallel. Further studies involving a broader range of cell lines and doses are required.