Ferroptosis is a Pivotal Player in Radiation-induced Cell Death of Colorectal Cancer Cells

Ferroptosis, an iron-dependent type of regulated cell death (RCD), has recently been associated with radiation efficacy. However, the impact of Ferroptosis inducers (FINs) on colorectal Cancer (CRC) cell lines varies tremendously. This study aims to elucidate the importance of Ferroptosis in radiation-induced RCD, comparing it with Apoptosis and Necroptosis. Human CRC cell lines (DLD-1, HT29, HCT116), and a murine CRC cell line (CT26) were included in this study. radiation-induced RCD was assessed by flow cytometric analysis. To determine the precise percentage of cells undergoing RCD, a colony formation assay (CFA) was employed following treatment with the cell death inhibitors ferrostatin-1, Z-VAD-FMK or necrostatin-1. The impact of hypoxia (1% O2) and fractionation on RCD percentages was analysed using a CFA. In vitro results were confirmed in 3D spheroid models and validated in a CT26 tumor model. Radiation significantly elevated the levels of Apoptosis, Necroptosis and Ferroptosis, irrespective of oxygen concentration. Inhibition of Ferroptosis reduced cell death similarly to the inhibition of Apoptosis and Necroptosis. These findings were confirmed in 3D models. Hypoxia and fractionation decreased overall RCD. In vivo experiments confirmed the pivotal role of Ferroptosis, showing it to be similarly involved as Necroptosis and greater than Apoptosis. Ferroptosis is equally involved in radiation-induced RCD in CRC cells compared to Apoptosis and Necroptosis. However, its importance decreases under hypoxic conditions and after fractionation. Nonetheless, the reduction was less pronounced than for Necroptosis, suggesting that Ferroptosis is an ideal type of RCD to trigger in a clinical setting. Overall, this study highlights the potential of FINs as effective clinical radiosensitizers.