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.

High-Specificity In Vivo Tumor Imaging Using Bioorthogonal NIR-IIb Nanoparticles.

Lanthanide-based NIR-IIb nanoprobes are ideal for in vivo imaging. However, existing NIR-IIb nanoprobes often suffer from low tumor-targeting specificity, limiting their widespread use. Here the application of bioorthogonal nanoprobes with high tumor-targeting specificity for in vivo NIR-IIb luminescence imaging and magnetic resonance imaging (MRI) is reported. These dual-modality nanoprobes can enhance NIR-IIb emission by 20-fold and MRI signal by twofold, compared with non-bioorthogonal nanoprobes in murine subcutaneous tumors. Moreover, these bioorthogonal probes enable orthotopic brain tumor imaging. Implementation of bio-orthogonal chemistry significantly reduces the nanoprobe dose and hence cytotoxicity, providing a paradigm for real-time in vivo visualization of tumors.

Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy.

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.

A Light-Up Probe with Aggregation-Induced Emission for Real-Time Bio-orthogonal Tumor Labeling and Image-Guided Photodynamic Therapy.

Bio-orthogonal tumor labeling is more effective in delivering imaging agents or drugs to a tumor site than active targeting strategy owing to covalent ligation. However, to date, tumor-specific imaging through bio-orthogonal labeling largely relies on body clearance to differentiate target from the intrinsic probe signal owing to the lack of light-up probes for in vivo bio-orthogonal labeling. Now the first light-up probe based on a fluorogen with aggregation-induced emission for in vivo bio-orthogonal fluorescence turn-on tumor labeling is presented. The probe has low background fluorescence in aqueous media, showing negligible non-specific interaction with normal tissues. Once it reacts with azide groups introduced to tumor cells through metabolic engineering, the probe fluorescence is lightened up very quickly, enabling rapid tumor-specific imaging. The photosensitizing ability was also used to realize effective image-guided photodynamic tumor therapy.