An In‐Situ‐Tag‐Generation Proximity Labeling Technology for Recording Cellular Interactions

Abstract Obtaining information about cellular interactions is fundamental to the elucidation of physiological and pathological processes. Proximity labeling technologies have been widely used to report cellular interactions in situ; however, the reliance on addition of tag molecules typically restricts their application to regions where tags can readily diffuse, while the application in, for example, solid tissues, is susceptible. Here, we propose an “in‐situ‐tag‐generation mechanism” and develop the GalTag technology based on galactose oxidase (GAO) for recording cellular interactions within three‐dimensional biological solid regions. GAO mounted on bait cells can in situ generate bio‐orthogonal aldehyde tags as interaction reporters on prey cells. Using GalTag , we monitored the dynamics of cellular interactions and assessed the targeting ability of engineered cells. In particular, we recorded, for the first time, the footprints of Bacillus Calmette‐Guérin (BCG) invasion into the bladder tissue of living mice, providing a valuable perspective to elucidate the anti‐tumor mechanism of BCG.