Density-dependent regulation of ferroptosis depends on accumulation of extracellular metabolites

Ferroptosis is a type of cell death induced by the production of toxic iron-dependent lipid radicals. The use of ferroptosis in cancer therapy as a potential metastasis-suppressor mechanism has been gaining increasing attention in the last few years. Multiple cancer cell states, including those bearing a RAS mutation and those holding a mesenchymal phenotype, as well as dedifferentiated and drug-resistance cells, are sensitive to ferroptosis inducers. A critical tissue property which determines sensitivity to ferroptosis is cell density. For many years it has been known that cells seeded at high densities are more resistant, compared to those seeded at low densities, to what we know today as ferroptosis. However, whether and how the tissue microenvironment can regulate ferroptosis sensitivity in cancer cells remains poorly understood. This concept is important in light of the potential use of ferroptosis-inducing compounds as anti-cancer drugs, because it predicts if cancer cell arrangement could influence the response to therapy. Thus, shedding light on this mechanism could open new avenues to the precise utilisation or prevention of ferroptosis for therapeutic uses. In this study we show that ferroptosis can be regulated non-cell-autonomously. Cell crowding actively changes the composition of the extracellular environment by releasing important metabolites that in turn regulate cells susceptibility to ferroptosis. We found lactic acid, which provides ferroptosis resistance through the rewiring of the intracellular metabolism, to be the most enriched metabolite secreted by cells. Metabolomics analysis suggested that lactic acid empowers the glycerol shuttle in order to regenerate the mitochondrial ubiquinol pool, protecting cells from ferroptosis. This finding suggests the potential mechanism by which cell density provides protection from ferroptosis. Moreover, we demonstrated that this cell media conditioning also occurs when cancer cells are grown as spheroids, by co-culture of cancer cells with activated fibroblasts or even cell-autonomously by inhibition of lactate secretion. Finally, we found that such metabolic preconditioning can prime the ability of breast cancer cells to form lung metastasis. We think that this study could provide a new insight into how cancer cells can escape ferroptosis induction depending on their metabolic interactions and rewiring.