Unraveling Heterogeneity in Tumor Evolution Induced by Diverse Radiation Modes: Insights from Systems Biology

The biomedical consequences of space radiation pose a significant concern for astronauts engaged in deep space. However, the effects of long-term low dose-rate exposures in space environments remain elusive. In this study, we simulated the space radiation environment by exposing human bronchial epithelial cells to low dose-rate (0.0067 Gy/day) a-particles, and continuously irradiated them multiple times to achieve cumulative total doses of 0.2 Gy, 0.4 Gy, and 0.5 Gy, respectively. At the same time, the cells were irradiated with the same total dose in a single exposure to investigate the potential of low dose-rate alpha particles to induce malignant transformation of human bronchial epithelial cells. A comprehensive suite of assays was employed to assess tumorigenic potential, including tumor formation in NOD/SCID mice, immunohistochemistry, CCK-8 proliferation assay, invasion assay, and the evaluation of multicellular spheroid formation during subsequent passages post-irradiation. Moreover, we dissected differential malignant mechanisms in tumor evolution ecosystem induced by the two distinct irradiation modes from systems biology views based on scRNA-seq technology. Our results showed that exposure to a-particles, whether through a single acute exposure or long-term low dose-rate exposures, induced the occurrence and development of tumors. Long-term low dose-rate exposures to a-particles increase the malignancy of induced tumors, but not the risk of carcinogenesis, compared to a single acute exposure with the same total dose. In addition, through scRNA-seq, we found that long-term low dose-rate exposures triggered more copy number variation (CNV) and epithelial-mesenchymal transition (EMT) events, and the activation of DNA damage repair pathways occurred significantly later than with a single acute exposure and involved more specific changes in cellular communication dynamics. In conclusion, our findings provide emerging yet convincing evidence that not only sheds light on why cells exposed to long-term low dose-rate exposures exhibit heightened malignancy, but also offers valuable insights into the genetic determinants driving tumor evolution and heterogeneity. Overall design: To elucidate the differential effects between single acute irradiation and long-term low dose-rate irradiation, we developed a series of a-particle exposure models to investigate the oncogenic effects of a-particles on human bronchial epithelial cells (BEAS-2B). Specifically, BEAS-2B cells cultured on cover slides were exposed to a-particles emitted by 241Am isotope at a dose rate of 0.14 Gy/min. The samples were divided into seven groups, Ctrl, sham control; R1_0.2, one acute exposure to 0.2 Gy; R1_0.4, one acute exposure to 0.4 Gy; R1_0.5, one acute exposure to 0.5 Gy; R10_0.2, low dose-rate exposure with a total dose of 0.2 Gy, i.e. one exposure with 0.02 Gy and subculture on the other day followed by another exposure two days later, so that totally 10 exposures in 30 days; R20_0.4, low dose-rate exposure to 0.4 Gy, 20 exposures in 60 days; R25_0.5, low dose-rate exposure to 0.5 Gy, 25 exposures in 75 days. So, the average dose rate of single acute exposures was 0.14 Gy/min while that of long-term low dose-rate exposures 0.0067 Gy/day. The seven groups of cells were treated and divided strictly following the same procedure. Once the exposure of the R25_0.5 group was completed, all the seven groups of cells were subcultured every three days, and cells were partially harvested at various passages for subsequent analysis (e.g., 10th, 30th, and 50th). To repeat the experiments independently, three independent sets of experiments were conducted from the very beginning by different individuals in parallel. To delve into the molecular processes and factors contributing to the disparities in malignancy between two irradiation modes, with a focus on genetic determinants, we performed single-cell RNA sequencing (scRNA-seq) on seven samples. These samples included sham control cells without irradiation (Ctrl), the cells after a single acute irradiation of 0.5 Gy and then subcultured for 10 (R1-10), 30 (R1-30), and 50 passages (R1-50), and the cells subjected to long-term low dose-rate exposures to 0.5 Gy and subcultured for 10 (R25-10), 30 (R25-30), and 50 passages (R25-50), respectively.