Tracking Dynamic Variations of Reactive Oxygen Species and Temperature during Ferroptosis-Induced Hepatic Stellate Cell Activation

Ferroptosis, driven by iron-dependent lipid peroxidation, represents a promising therapeutic strategy for hepatocellular carcinoma (HCC). However, reactive oxygen species (ROS) released from ferroptotic HCC cells may activate hepatic stellate cells (HSCs), potentially exacerbating liver fibrosis, an unexpected risk in HCC treatment. Resolving this rapid intercellular crosstalk requires methods that can track extracellular ROS release and key parameters of HSC activation ( i . e ., intracellular redox homeostasis and mitochondrial activity) with high spatiotemporal resolution. Here, we established an in vitro coculture model of HuH7 (HCC) and LX-2 (HSCs) cells and employed scanning electrochemical microscopy (SECM) to spatiotemporally resolve extracellular ROS fluxes from ferroptotic HuH7 cells, while subsequent intracellular ROS generation, mitochondrial respiratory activity, and intracellular temperature changes in LX-2 cells. We found that ROS released from ferroptotic HuH7 cells increased by ∼2.44 μM, and was temporally followed by NOX2-associated intracellular ROS bursts in LX-2 cells, with a maximum generation rate of approximately 5.44 × 10 –18 mol min –1 . This intracellular ROS burst was coupled to mitochondrial hypermetabolism in LX-2 cells, reflected by a ∼1.27-fold elevation in oxygen consumption and a ∼1.65 K rise in intracellular temperature. Finally, we also found that pirfenidone, an antifibrotic agent, could effectively suppress this ferroptosis-induced HSC activation. Our study introduces an SECM approach providing real-time single-cell evidence that ferroptotic HCC cells can trigger ROS-mediated activation of neighboring HSCs, and supports further evaluation of antifibrotic combination strategies for safer ferroptosis-based HCC therapy.