## 4 Studies of the Molecular Profiling of EVs as Potential Biomarkers
### Proteome Profiling Analysis of EVs in Multiple Cancers
Granulins, also known as granulin-epithelial precursors, are a growth factor which regulates inflammation and tumorigenesis. They can promote migration, invasion, and proliferation in PCa [134]. CHMP4A is a subunit of the charged multivesicular body, ESCRT-III complex, which is involved in multivesicular body (MVBs) formation and sorting of endosomal cargo proteins into MVBs [135]. Although urine is an excellent source of protein biomarkers for bladder cancers, there is a high degree of variability in these samples. In this regard, the enrichment of urine EVs could reduce the variability between samples, allowing us to identify functionally relevant proteins. For instance, 1222 total proteins were detected with high confidence in EVs derived from bladder cancer, validating some of them by Western blotting, such as mucin-1 (MUC1), carcinoembryonic antigen (CEA), epidermal substrate of growth factor receptor kinase 8-protein 2 (EPS8L2), and moesin, as proteins directly associated with cancer [104] (Table 1). MUC1s are membrane glycoproteins that play important roles in cell physiology, such as mediating anti-adhesive properties between cells and the extracellular matrix (ECM). Its deregulated expression is associated with cancer progression [136]. CEA, also known as CD66, is expected to have potential value in the early diagnosis of invasive urinary bladder cancer [137]. Eps8L2 belongs to the family of epidermal growth factor receptor kinase substrate 8 (EPS8)-related proteins, which are involved in actin remodeling in response to EGF [138]. Moesin is known to be associated with an aggressive phenotype in several malignant tumors, showing predictive ability for early detection of bladder urothelial carcinoma (BUC) invasion [139]. EGFR is a transmembrane receptor whose function is to regulate both cell proliferation and apoptosis via signal transduction pathways [140], which are highly related to lung cancer [141]. A large number of biomarkers is currently being investigated in lung cancer. Consequently, it has been shown that EVs released by non-small-cell lung cancers (NSCLC) patients' plasma are also particularly enriched in MUC1 [105] (Table 1). Furthermore, EVs isolated from lung cancer cells, lung biopsies, and plasma are enriched in EGFR, making them the most powerful prognostic biomarker [106] (Table 1). [PERSON] and co-workers also reported an enrichment in NY-ESO-1, phospholipase A-2-activating protein (PLAP), and EpCam proteins. NY-ESO-1, also known as cancer-testis antigen (CTAs), is regularly limited in its expression to germ and placental cells, but is re-expressed in tumor cells. Similarly, NY-ESO-1 expression was found in NSCLC and has been associated with a higher risk of relapse, poorer response to treatment, and shorter survival [142]. PLAP is a member of the WD-repeat protein, G-protein-transducin superfamily, which mediates eicosanoid generation and participates in inflammatory responses [143]. On the other hand, EpCAM is a transmembrane glycoprotein that affects intercellular adhesion, and is oveexpressed in various human epithelial carcinomas. It is involved in many important functions relevant to tumor progression, including cell proliferation [17]. Other works have shown that serum EVs can help distinguish patients with metastatic NSCLC from nonmetastatic NSCLC by monitoring lipopolysaccharide-binding protein (LBP) levels [107] (Table 1). BPIFA1, CRNN, MUC5B, and IQGAP1 were also found highly abundant in salivary exosomes of lung cancer patients [108] (Table 1). BPIFA1 is a protein specifically expressed in the upper airways and nasopharyngeal regions. It was identified as a potential marker for the micro-metastasis of non-small cell lung cancer (NSCLC) [144]. MUC5B is a gel-forming mucin secreted from airway epithelial cells in the lung, associated with longer survival in primary EGFR mutant NSCLC [145], and IQGAP is a scaffold protein that may promote the regulation of cancer cell migration and metastasis [146].