Novel Affinity Reagents for Analysis of Tumor Microvesicles and Their Metabolically Regulated Glycome
Additional Collaborators: Sachdev Sidhu (University of Toronto) and Wesley Levant (Janelia Research Campus)
The overall objective of our pilot proposal is to create a robust, integrated pipeline for the rapid discovery and characterization of selective, high-affinity Abs against defined glycan epitopes (glycotopes), in particular those associated with the tumor-specific MV glycome and the cellular glycocalyx (Aim 1). A parallel objective is to optimize the affinity, valency, and size of these reagents specifically for live-cell and super-resolution optical imaging (e.g., expansion microscopy (ExM)), and to develop and refine protocols for their application in imaging (Aim 2). In line with the Center’s vision, these reagents will enable physical sciences approaches (e.g., super-resolution microscopy; separation and classification of defined MV subpopulations) to analyze the biochemical and structural reprogramming of the glycocalyx in cancer, and its relationship to metabolism, MV biogenesis, and the role of the physical microenvironment in these processes. Notably, our reagents and approaches for ExM should make MV characterization accessible to all labs in the PSOC/PSON network and overcome the strict limitations imposed by our current reliance on nanoparticle tracking analysis, which is incapable of distinguishing MVs from other particles and aggregates in the media and also incapable of relating MV size with cargo and its sub-vesicular location. Moreover, the Abs discovered here could be developed for therapeutic purposes such as specific interference with MV formation by precision targeting and editing of the glycocalyx. To ensure the broadest impact possible, these reagents will be immediately available to all members in the PSOC/PSON network (and their outside collaborators) with protocols that include explicit recommendations for dye conjugation, reagent concentration, imaging parameters, cell preparation, as well as discussion of potential cross-reactivity of our reagents and other pitfalls.