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

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.

Interception Proximity Labeling for Interrogating Cell Efflux Microenvironment.

The difficulty in elucidating the microenvironment of extracellular H2O2 efflux has led to the lack of a critical extracellular link in studies of the mechanisms of redox signaling pathways. Herein, we mounted horseradish peroxidase (HRP) to glycans expressed globally on the living cell surface and constructed an interception proximity labeling (IPL) platform for H2O2 efflux. The release of endogenous H2O2 is used as a "physiological switch" for HRP to enable proximity labeling. Using this platform, we visualize the oxidative stress state of tumor cells under the condition of nutrient withdrawal, as well as that of macrophages exposed to nonparticulate stimuli. Furthermore, in combination with a proteomics technique, we identify candidate proteins at the invasion interface between fungal mimics (zymosan) and macrophages by interception labeling of locally accumulated H2O2 and confirm that Toll-like receptor 2 binds zymosan in a glycan-dependent manner. The IPL platform has great potential to elucidate the mechanisms underlying biological processes involving redox pathways.

An inorganic prodrug, tellurium nanowires with enhanced ROS generation and GSH depletion for selective cancer therapy.

Organic prodrugs have been widely reported to avoid side effects and have been applied for precise tumor therapy in recent years. However, inorganic nano-prodrugs with localized generation of toxic products in the tumor have not been reported. Herein, we report an inorganic nano-prodrug, tellurium nanowires (TeNWs), that generate toxic TeO6 6- triggered by hydrogen peroxide (H2O2) for highly selective cancer chemotherapy. Bovine serum albumin and dextran conjugate coated TeNWs, with a length of ∼82 nm and a width of ∼7 nm, showed high stability in physiological medium. The interaction between TeNWs and intracellular H2O2 produces toxic TeO6 6- molecules greatly enhanced ROS generation, and the reaction product, verified as TeO6 6-, would react with glutathione (GSH) and thus decrease intracellular GSH levels, which greatly increases ROS levels in the tumor. Importantly, TeNWs selectively kill cancer cells by caspase-independent autophagic death and apoptosis, as well as exerting an immune response, while not affecting normal cells due to the high H2O2 levels in cancer cells. Moreover, after the sequential reaction with H2O2 and GSH, TeNWs were dissociated into small molecules and could be rapidly and completely removed from the body. Both in vitro and in vivo experiments indicate that TeNWs are a promising inorganic nano-prodrug that exerts good selective therapeutic effects on tumors.