Whole proteome and transcriptome analysis of cancer-derived extracellular vesicles overlap in specific pathways and distinct EV populations

Extracellular Vesicles (EVs) are particles of different sizes, covered by a lipid bilayer membrane and containing highly heterogeneous cargo. Cancer cell-derived EVs have been the main object of an extensive investigation in the field because they carry cancer-specific molecular cargo and can promote cancer progression. Cancer-derived EVs include populations of atypically large EVs (L-EVs), which have been referred to as tumor microvesicles, large oncosomes, or simply L-EVs. While small EVs (S-EVs), which include exosomes, have been investigated by a plethora of reports, little is known about L-EVs. The paucity of studies comparing protein cargo of L- and S-EVs, of studies focused on protein coding RNA, and the absence of integrative analyses to compare the protein and gene expression in different EV fractions, prompted us to perform mass spectrometry to profile three different, size-based EV fractions generated by three cancer cell models (glioma, prostate and breast cancer). We identified protein signatures for L- and S-EVs either common to all cell types or specific to each of them individually. The proteins enriched in prostate cancer cell-derived L-EVs were also identified in L-EVs from patients with metastatic prostate cancer by a SWATH proteomic assay. We also performed RNA-Seq on the prostate cancer model and integrated proteomic and transcriptomic datasets. GSEA revealed that mitochondrial function was enriched in L-EVs versus S-EVs at both the RNA and protein level. The mitochondrial signature at the transcriptome level was confirmed by single cell RNA-Seq of L- EVs in vitro. The integrated L-EV proteomic and transcriptomic signature enabled distinction between benign and localized prostate cancer, as well as between localized cancer and metastatic castration-resistant cancer.