Painting Cell–Cell\nInteractions by Horseradish\nPeroxidase and Endogenously Generated Hydrogen Peroxide

Cell–cell\ninteractions are fundamental in biology for maintaining\nphysiological conditions with direct contact being the most straightforward\nmode of interaction. Recent advancements have led to the development\nof various chemical tools for detecting or identifying these interactions.\nHowever, the use of exogenous cues, such as toxic reagents, bulky\nprobes, and light irradiation, can disrupt normal cell physiology.\nFor example, the toxicity of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) limits the applications of peroxidases in the proximity\nlabeling field. In this study, we aimed to address this limitation\nby demonstrating that membrane-localized horseradish peroxidase (HRP-TM)\nefficiently utilizes endogenously generated extracellular H<sub>2</sub>O<sub>2</sub>. By harnessing endogenous H<sub>2</sub>O<sub>2</sub>, we observed that HRP-TM-expressing cells can effectively label\ncontacting cells without the need for exogenous H<sub>2</sub>O<sub>2</sub> treatment. Furthermore, we confirmed that HRP-TM labels proximal\ncells in an interaction-dependent manner. These findings offer a novel\napproach for studying cell–cell interactions under more physiological\nconditions without the confounding effects of exogenous stimuli. Our\nstudy contributes to elucidating cell–cell interaction networks\nin various model organisms, providing valuable insights into the dynamic\ninterplay between cells in their native network.