Labile iron pool dynamics do not drive ferroptosis in colorectal cancer cells.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality. CRC tumors exhibit aberrant iron accumulation, which supports tumor cell proliferation through multiple metabolic pathways. However, the elevated iron must be counterbalanced given its potential to generate damaging reactive oxygen species. Ferroptosis is a regulated, non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation. Selenoenzyme glutathione peroxidase 4 controls this process by reducing lipid peroxides and can be pharmacologically inhibited by agents such as RSL3 and JKE1674. A key source of redox-active iron is the labile iron pool (LIP), yet its role in regulating ferroptosis remains incompletely defined and whether ferroptosis is accompanied by dynamic changes in the LIP is unknown. To examine this, we treated CRC cells with exogenous iron and pharmacologic ferroptosis inducers. Iron supplementation significantly reduced cell viability, suggesting that expansion of the LIP potentiates ferroptotic cell death. However, by assessing expression of iron regulatory genes as well as employing two orthogonal approaches to measure labile iron, we found that the LIP did not measurably increase during ferroptosis induction with glutathione peroxidase 4 or SLC7A11 inhibition. These findings suggest that the LIP does not expand upon pharmacologically initiated ferroptosis, despite the potentiating effect of exogenous iron supplementation.