Abstract

Colorectal cancer (CRC), a widespread malignancy, is closely associated with tumor microenvironmental hydrogen peroxide (H2O2) levels. Some clinical trials targeting H2O2 for cancer treatment have revealed its paradoxical role as a promoter of cancer progression. Investigating the dynamics of cancer cell H2O2 eustress at the single-cell level is crucial. In this study, non-contact hopping probe mode scanning ion conductance microscopy (HPICM) with high-sensitive Pt-functionalized nanoelectrodes was employed to measure dynamic extracellular to intracellular H2O2 gradients in individual colorectal cancer Caco-2 cells. We explored the relationship between cellular mechanical properties and H2O2 gradients. Exposure to 0.1 or 1 mmol/L H2O2 eustress increased the extracellular to intracellular H2O2 gradient from 0.3 to 1.91 or 3.04, respectively. Notably, cellular F-actin-dependent stiffness increased at 0.1 mmol/L but decreased at 1 mmol/L H2O2 eustress. This H2O2-induced stiffness modulated AKT activation positively and glutathione peroxidase 2 (GPX2) expression negatively. Our findings unveil the failure of some H2O2-targeted therapies due to their ineffectiveness in generating H2O2, which instead acts eustress to promote cancer cell survival. This research also reveals the complex interplay between physical properties and biochemical signaling in cancer cells' antioxidant defense, illuminating the exploitation of H2O2 eustress for survival at the single-cell level. Inhibiting GPX and/or catalase (CAT) enhances the cytotoxic activity of H2O2 eustress against CRC cells, which holds significant promise for developing innovative therapies targeting cancer and other H2O2-related inflammatory diseases.