## 4 Studies of the Molecular Profiling of EVs as Potential Biomarkers
### Proteome Profiling Analysis of EVs in Multiple Cancers  
Proteins are an important class of molecules that are transported by EVs, and some of them are integral constituents of EV structures. Analysis of EV protein composition is crucial to understanding the mechanisms of their biogenesis and function under physiological and pathological conditions. It is well-known that EVs are highly enriched in membrane proteins, such as tetraspanins, including CD9, CD63, CD81, CD82, CD151, and Tspan8 [73; 74]; proteins involved in EVs biogenesis as well as ESCRT-related proteins, such as ALIX, TSG101 (the stereotypical biomarkers for EVs characterization), and syntenin [75]. Other typical EV proteins include cell adhesion-related proteins (integrins, LFA-1, and ICAM -1) [76], and those participating in cytoskeletal construction (actin and tubulin) and vesicle trafficking (e.g., Rab family proteins). MHC class I and II complexes, involved in antigen presentation, have been also reported [77], as well as heat shock proteins, which facilitate protein folding and balance of proteostasis (i.e., Hsp60, Hsp70, and Hsp90) [78]. In addition to self-proteins, EV content reflects the physiological state of the cell from which they originated. Since EVs are normally isolated in small amounts, highly sensitive analyses are needed. In recent years, technology have been widely used to allow the massive identification and relative quantification of proteins present in EVs from different biological samples [79; 80]. Moreover, many public databases have been created to share data among the scientific community, such as EVpedia, ExoCarta, and Vesiclepedia [81, 82, 83]. Current proteomic technology based on mass spectrometry (MS) is the basis for the study and discovery of specific non-invasive biomarkers in the oncology field and beyond. Its high sensitivity makes it capable of identifying low abundance proteins over wide dynamic ranges, including post-translational changes. Additionally, ELISA and Western blotting can be used to identify potential biomarkers. Although many analytical methods have been standardized, unique challenges are associated with different applications of proteomics. On this note, we have considered some studies on cancer-derived-EVs to assess the recent research progress in the field, and at the same time highlight the potential biomarkers reported. In the literature there are many examples of potential biomarkers found in EVs derived from breast cancer (BC) from both cell lines and biological fluid. An early study on serum-derived EVs of BC patients revealed the presence of CD24, with low epithelial cell adhesion molecules (EpCAM) expression, providing evidence that EpCAM could be cleaved from EVs via serum metalloproteinases [84] (Table 1). CD24 is a glycosylphosphatidylinositol-anchored membrane protein considered to be a negative cancer stem cell marker, specifically in BC [85]. EpCAM is an epithelial cell marker which modulates biological processes, such as cell proliferation, migration, and invasion. Human epidermal growth factor receptor-2 (HER2) positive breast cancer represents 15-20% of all breast cancers. The primary mechanism of HER2 activation in breast cancer is its gene amplification, which causes its overexpression, ultimately activating several signaling pathways [86]. It has been revealed that HER2 is overexpressed in EVs derived from plasma of BC patients [87] (Table 1).