EV Fingerprinting: Resolving Extracellular Vesicle Heterogeneity using Multiparametric Flow Cytometry

Abstract

Cancer metastasis remains a major unsolved clinical challenge as over 90% or cancer-related deaths are attributed to metastasis. Dynamic regulation of cell adhesion is crucial for successful metastatic dissemination. Cell adhesion can be fine-tuned through direct modulation of cell adhesion molecules or the functional contributions of extracellular vesicle (EVs). We have previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a dynamic regulator of cell adhesion by expression of alternatively spliced isoform with differential proteolytic susceptibility that impacts metastasis. EVs are lipid bilayer enclosed particles secreted by cells that mediate of cell-cell communication in normal physiology as well as disease pathology. In cancer, EVs transfer bioactive cargoes (proteins, lipids, and nucleic acids) to facilitate directional migration, cell motility, and speed. Characterization of cargoes and biogenesis is therefore needed to identify populations of EVs with a distinctive function. ALCAM-mediated cell adhesion and EV biogenesis are linked through a common molecular factor, Syntenin-1. ALCAM is connected to the actin cytoskeleton through Syntenin-1 to form stable adhesion complexes. Syntenin-1 also participates in a distinctive class of EV biogenesis where it supports intraluminal vesicle budding through associations with Syndecan and ALIX on maturing endosomal compartments. This overlap in molecular machinery suggests that Syntenin-1 availability through ALCAM sequestration could affect EV biogenesis. Using a novel single-EV flow cytometry method called “EV Fingerprinting”, we showed that ALCAM shedding alters EV biogenesis by enhancing production of larger EVs. Additionally, cargo loading into EVs was affected by ACLAM expression. Examination of EV Fingerprints from bladder cancer cell lines cultured in a 3D organotypic model also showed similar profiles according to ALCAM shedding. In order to better understand the results of EV Fingerprinting, further validation of the method was required. Technical validation of EV Fingerprinting demonstrated that the lipophilic dye, di-8-ANEPPS, enables characterization of EVs based on relative fluorescence through size and liquid order by generating multiparametric data. Dimensional reduction and clustering of data revealed resolved populations of EVs. We used EV Fingerprinting to identify population-specific changes upon molecular perturbation of EV secretion through Rab27a knockdown and CD63 overexpression according to liquid order. Additionally, we determined that cargo partitioning is a non-random process by using multiplex analysis of CD63 and CD81 EV cargoes. In summary, this dissertation identifies a relationship between cell adhesion and EV biogenesis as well as establishes a tool to resolve heterogeneous EV populations.