Assessment of ferroptosis inducers and Nrf2 inhibitors as radiosensitisers in 2D and 3D breast cancer cell cultures

Ferroptosis is a form of programmed cell death that is modulated in some cancer cells as a pro-survival mechanism. Induction of ferroptosis is a potential anti-cancer strategy, and enhancement of ferroptosis using ferroptosis inducers has the potential to enhance current anti-tumour mechanisms. In this study, we assessed the effect of the ferroptosis inducers Erastin, RSL-3 and FIN-56 on radiosensitivity in 2D cell culture, and in 3D alginate tumour spheroids from breast cancer cell lines. Since some tumours modulate ferroptosis via increased Nrf2 production, and MCF-7 and MDA-MB-231 both produce Nrf2 protein, we also assessed the effects of the Nrf2 inhibitor ML385 on radiosensitivity. MDA-MB-231 was highly sensitive to all ferroptosis inducers, and ferroptosis was reversed by the ferroptosis inhibitors Ferrostatin-1, Liproxstatin-1 and Deferoxamine. MCF-7 was resistant to all ferroptosis inducers. MDA-MB-231 and MCF-7 cells were sensitive to irradiation in 2D cell culture but resistant to irradiation in 3D alginate spheroids. Ferroptosis inducers did not synergistically enhance irradiation-induced cell death in 2D cell cultures. There was also no robust enhancement to irradiation effects with ferroptosis inducers in 2D or 3D cell culture. Ferroptosis inducers did, however, show a heterogeneous response in 3D cell culture, in that isogenic spheroids responded differently within the same spheroid. The Nrf2 inhibitor ML385 showed no synergistic enhancement of ferroptotic cell death when combined with irradiation. These studies suggest targeting ferroptosis does not induce short-term enhancement of ferroptotic cell death.