This is relevant due to the role of EVs in the modulation of the cells in the tumor microenvironment (e.g. enrichment analysis was performed for a more general overview of the biological processes involved. More than 600 different Pipequaline hydrochloride proteins were recognized in EVs from each particular cell collection. Here, 14%, 10%, and 24% of the recognized proteins were unique in OSCC, PDAC, and melanoma vesicles, respectively. A specific protein profile was discovered for each cell collection, e.g., EGFR in OSCC, Muc5AC in PDAC, and FN1 in melanoma vesicles. Nevertheless, 25% of all the recognized proteins Pipequaline hydrochloride were common to all cell lines. Functional enrichment analysis linked the proteins in each data set to biological processes such as biological adhesion, cell motility, and cellular component biogenesis. EV proteomics discovered cancer-specific protein profiles, with proteins involved in processes promoting tumor progression. In addition, the biological processes associated to the melanoma-derived EVs were distinct from your ones linked to the EVs isolated from OSCC and PDAC. The malignancy specific biomolecular cues in EVs may have potential applications as diagnostic biomarkers and in therapy. 1. Introduction Extracellular vesicles (EVs) are released by cells into the extracellular space and are classified according to their size and biogenesis [1, 2]. Accordingly, EVs with diameters of 30C100 nm and of endosomal origin are defined as exosomes . The EVs which originate by direct outward budding of the cell membrane are named microvesicles (100C1000 nm) and apoptotic body ( 1000 nm) . EVs are important Pipequaline hydrochloride players in cell-cell communication in health and disease  due to their diverse content of biomolecules, such as lipids, nucleic acids, and proteins . EVs are quite abundant in biofluids as they are constantly released by cells . In some diseases, e.g., malignancy, the amount of EVs in the biofluids increases . The EVs in the blood of cancer patients are released both by normal and malignancy cells, and their number is usually estimated to be twice of that found in the blood of healthy individuals [6C8]. Oncogenes in cancer-derived EVs can modulate normal host cells, e.g. fibroblasts and macrophages, as well as local malignancy cells and metastatic cells [9C11]. In this manner, tumor-derived EVs can contribute to and maintain the Hallmarks of malignancy, a panel of acquired abilities of malignant tumors such as malignancy cell proliferation, evasion of growth suppressors, resistance to cell death, FLJ20285 migration, and invasion as well as modulating normal cells to favor tumor progression by transforming the microenvironment into a more permissive one [12C15]. Additionally, since EVs contain signaling molecules, they are considered be a potential source of diagnostic biomarkers for the prediction of disease, as well as in disease monitoring and treatment decision making [10, 16]. To explore the potential of cancer derived EVs as possible diagnostic and prognostic markers and to expand our understanding of their influence in cell signaling in disease progression, there is a need to isolate EVs from the other components in cell culture supernatant or biofluid of interest (e.g. blood, saliva, urine) . One method for EV separation is combining two size-based separation techniques (ultrafiltration (UF) and size exclusion chromatography (SEC)). These use the size of EVs to separate them from other components that are present in the biofluid or cell culture media. Once isolated, it is of great value to characterize the EV content. Mass spectrometry (MS)-analysis allows for the identification and characterization of proteins in EV samples. However, the enormous amount of data produced by this technique can be quite extensive . Therefore, to extract meaningful information from the extensive list of proteins, Gene Ontology (GO) has become a useful resource, by associating a GO term to each protein or group of proteins in the data set . Overall, GO is a standardized language, or ontology, that describes the function of a gene or a gene product (RNAs or proteins) in three key domains: biological processes, molecular functions, and cellular components [18C20]. In this study, we characterized the protein content of small EVs (diameters under 200 nm),.