## 4 Studies of the Molecular Profiling of EVs as Potential Biomarkers
### Proteome Profiling Analysis of EVs in Multiple Cancers
Since PSC is a major risk factor of CCA development, serum EV proteins were compared between CCA and PSC, and a selection of 10 overexpressed proteins in CCA were identified, including fibrinogen gamma chain (FIBG), alpha-1-acid glycoprotein 1 (A1 AG1), and S100A8 proteins [98]. A1 AG1 is a protein mainly expressed during the acute phase of the inflammatory process, that can play an important role in the tumor microenvironment, affecting immune modulation, drug resistance, and cancer progression [123], whereas S100A8 protein (calgranulin A) belongs to the S100 multigenic family of calcium-modulated proteins with roles in inflammation [124]. Discrimination between early-stage CCA (I-II) and PSC is possible through detection of ficolin-2 (FCN-2) proteins, which are involved in cancer immunity, suppression of EMT, and metastasis of HCC [125]. On this note, comparison of early-stage CCA (I-II) with PSC showed that ficolin-2, inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), and FIBG were most abundant in early-stage CCA. Further, MUC1, EGFR, EpCAM, among others, were abundant in EVs released from CCA human cell lines compared to normal human cholangiocarcinomaytes (NHC) [98] (Table 1). Other enriched proteins after tumor resection found in serum EVs were EpCAM, asialoglycoprotein 1 receptor (ASGPR1), annexin V, and tumor-associated microparticles (taMPs), showing sensitivity/specificity scores and positive/negative predictive values (>78%), indicating their potential in diagnosis [99] (Table 1). Hepatic ASGPR1 is a transmembrane molecule specifically expressed on the sinusoidal and basolateral hepatocellular membranes, and not in other human tissue [126]. Furthermore, taMPs have recently emerged as novel vehicles for a horizontal crosstalk between different cells, especially in the setting of inflammatory conditions [127]. Differential exosomal phosphoproteome analysis of invasive M213 and M213D5 CCA cells has shown its potential as a biomarker in cancer. Reduced exosomal fetuin-A phosphorylation and high HSP90B phosphorylation has been detected in tissues from CCA patients with a low TNM stage, compared to those with a single high-stage TNM [100] (Table 1). The analysis of EVs obtained from high- and low-grade metastatic HCC cell lines discriminated CCA from other tumors, highlighting adenylate cyclase associated protein 1 (CAP1) as enriched in metastatic tumor cells when compared to non-tumor/primarycell lines [101] (Table 1). CAP1 is an actin monomer binding protein involved in the reorganization of the actin filament essential for cell migration. Its overexpression in HCC is closely related to tumor metastases [128], although its role needs to be further investigated. EVs secreted in gastric cancer (GC) are also involved in tumorigenesis. A recent study suggests that proteasome subunits PSMA3 and PSMA6 levels in patients with metastatic GC (stage III/IV) were significantly higher in serum EVs than those in healthy controls and patients with early-stage GC [102] (Table 1). PSMAs are major components of the 20S proteasome core complex with potential diagnostic utility [129]. In prostate cancer (PC), there is a need to identify new markers, due to the fact that prostate specific antigen (PSA), both in free form and in EVs derived from plasma of PC lacks specificity and sensitivity [130], and therefore could lead to overdiagnosis [131]. In the search for new biomarkers, it has been shown that levels of fatty acid binding protein 5 (FABP5) in urine-derived EVs were higher in pathological groups, as well as the levels of granulin, AMBER, CHMP4A, and others [103] (Table 1). FABP5 belongs to the family of intracellular lipid-binding proteins, and is responsible for uptake and transport of fatty acid [132]. In PC cell lines, FABP5 regulates energy metabolism via ERR\(\alpha\) activation, suggesting that a new FABP5-ERR\(\alpha\) signaling axis plays an important role in the regulation of AMPK activity, which is a cellular energy state sensor that directs metabolic adaptation to support cell proliferation and survival [133].