Characterizing the DNA damage response in fibrosarcoma stem cells by in-situ cell tracking

Purpose: Radiotherapy works by generating large amounts of DNA double-strand breaks (DSBs). Cancer stem-like cells (CSCs) are the principal cause of tumor radioresistance. Therefore, investigating the dynamics and mechanisms of DNA damage response (DDR) in CSCs is of great importance.

Materials and methods: Human fibrosarcoma cell line HT1080 stably transfected with 53BP1-GFP was used to investigate the real-time cellular response to DSBs induced by γ-rays. HT1080 CSCs were sorted based on aldehyde dehydrogenase (ALDH1) levels by flow cytometry and verified by mammosphere formation assay. We set the number, area and intensity of ionizing radiation-induced foci (IRIF) as endpoints. Using live-cell imaging to track single IRIF in-situ, we compared the IRIF induction and dispersal in HT1080 cells and CSCs.

Results: ALDH1⁺ cells showed much stronger mammosphere-forming capability, indicating the property of CSCs and could be considered as HT1080 CSCs. After γ-irradiation, CSCs had fewer IRIF number and smaller IRIF size than HT1080 cells. Different repair kinetics (with plateau and without plateau) were observed both in CSCs and HT1080 cells. Similar to HT1080 cells, IRIF with a plateau in CSCs showed higher intensity, larger area and slower decay rate of intensity than IRIF without plateau. Additionally, the level of IRIF merging in HT1080 cells was significantly higher than that in CSCs.

Conclusions: CSCs have fewer and smaller IRIF, indicating the reduced complexity of DNA damage. This may contribute to tumor radioresistance. Heterogeneous repair kinetics (with and without plateau) were observed although the dynamics of IRIF with or without plateau in CSCs resemble the dynamics in HT1080 cells based on single IRIF analysis.