A feedback loop between cell proliferation and ROS regulates ferroptosis sensitivity

Background: Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation and membrane rupture. While cellular populations reaching confluence are known to have limited sensitivity to ferroptosis, an understanding of the interplay between growth dynamics, reactive oxygen species (ROS) levels, metabolism and ferroptosis is currently lacking. This study aimed to establish a regulatory framework for the systemic interplay of these biological processes. Results: Here we use live-cell imaging coupled to ROS tracing to reveal a feedback loop between population growth and ferroptotic cell death. Starting out from the observation that the cellular proliferation rate declines with increased cellular density, we find that ROS levels also decline with increasing cellular density. In turn, low ROS levels make cells insensitive to ferroptosis, which enables population growth. Conversely, keeping cell numbers and drug concentration/cell constant while restricting growth space led to reduced proliferation, reduced ROS and decreased ferroptotic cell death. We find that this feedback between population growth and ferroptotic cell death leads to two steady states: (i) a ferroptosis-insensitive state characterized by slow growth, low levels of ROS and low rates of cell death and (ii) a ferroptosis-sensitive state characterized by rapid growth, ROS accumulation, and high rates of ferroptosis. A mathematical model of the feedback mechanism predicts the long-term fate of populations as well as their ferroptosis sensitivity when external conditions impacting cell proliferation rates, ROS, or both are changed. We tested the proposed feedback mechanism experimentally by interfering with lipid hydroperoxide clearance and by increasing cellular and lipid ROS production through a galactose-promoted OXPHOS switch. Conclusion: We find a feedback loop between population growth and ferroptotic cell death that dictates cellular fate (growth or cell death via ferroptosis) and is mechanistically determined by the levels of metabolic ROS. These results provide a unifying framework that dynamically links population growth and metabolic ROS regulation with ferroptosis sensitivity.