RNA-Protein Cargo Delineates Distinct Subpopulations of Cancer-Derived Extracellular Vesicles
Extracellular vesicles (EVs) are membrane-encapsulated particles that vary greatly in size and content. Although both normal and tumor cells produce an array of small EVs (S-EVs), only tumor cells have been found to release a population of atypically large EVs (L-EVs), also known as large oncosomes (LO). Little is known about the cargo of L-EVs, and how it differs from the S-EVs. Given the potential for tumor-derived L-EVs to provide insights into disease mechanisms and course, we performed proteomic analysis of L- and S-EVs derived from three cancer models followed by comparative proteomic and transcriptomic analysis of EVs from the prostate cancer model. 1) We identified both common and model-specific protein signatures for L- and S-EVs. 2) A subset of the proteins enriched in prostate cancer cell-derived L-EVs were also identified in L-EVs isolated from plasma of patients with metastatic prostate cancer. 3) Proteins enriched in L-EV that were also identified at the transcript level were mostly mitochondrial in origin, as confirmed by single vesicle RNA-Seq. Additionally, the L-EV mitochondrial signature was detected in plasma-derived L-EVs and distinguished patients with prostate cancer from cancer-free individuals as well as patients with metastatic prostate cancer from those with localized disease, corroborating the relevance of an integrative multi-analyte approach focused on L-EVs for liquid biopsy. Overall design: Large EVs (L-EVs) were enriched from the PC3 cell conditioned medium by differential centrifugation and purified by iodixanol density gradient; the number of particles was quantified by qNano using NP2000 and the L-EV  sample was diluted to contain 10,000 particles total to enable encapsulation of single L-EVs into individual droplets.