Abstract

Versatile and Multiplexed Extracellular Vesicle and Single Extracellular Vesicle Analysis

David Juncker, Professor and Chair, McGill University

Extracellular vesicles (EVs) have emerged as a fundamental cell signaling and cargo system. EV proteomic analysis face challenges such difficulty to detect internal EV proteins, difficult to normalize concentration, co-expression, and lack of reproducibility. Single EV analysis, while highly desirable, still suffers from imprecision owing to lack of a protein markers universally expressed in EVs, low fluorescence of single molecules, and low scattering signals and the ensuing difficulty to detect small EVs. Here our efforts to address these challenges will be presented including multiplexed (i) inner and outer protein profiling in EVs; (ii) co-enrichment analysis which builds on co-expression analysis and quantifies protein co-enrichment, either negative or positive, and is illustrated with >100 protein pairs in EVs from metastatic, organotropic breast cancer cell lines. Next, (iii) a single EV analysis platform for label-free interference scattering (iSCAT) on a common (fluorescence) microscope will be introduced, and sizing of EVs from ~35 nm to ~200 nm in diameter shown, outperforming existing methods such as nanoflow cytometry; (iv) the co-expression and co-enrichment profiles of ~20 proteins on single EVs will be demonstrated by combining iSCAT, fluorescence imaging, and microfluidic exchange of DNA-barcoded antibodies. These new EV analysis methods will be useful to detect, profile and analyse EVs in health and disease, and deepen our understanding of their function.