## 4 Studies of the Molecular Profiling of EVs as Potential Biomarkers
### Lipidome Profiling Analysis of EVs in Multiple Cancers
In contrast, mono-silyl gangliosides, such as GM1 and GM2 often suppress malignant properties of cancerous cells [161]. The analysis of GSLs does remain challenging due to their amphiphilic nature and inherent complexity. Sphingomyelin (SM) is also a key component of lipid rafts involved in the regulation of several signaling pathways [162]. It has been shown that low SM levels are associated with the tumorigenic transformation [163]. However, the role of SM in cancer is yet to be understood, since other studies have reported high levels of SM and different roles in cancer [164]. Considering the glycerophospholipids (GPLs) species, i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) are abundant in mammalian cell membranes, there is growing evidence that these GPLs (particularly PC and PS) might be potential biomarkers of cancers. Evidence supports that the overexpression of PCs observed in cancer cells is mainly due to upregulation of choline kinase and activation of phospholipase C specific to phosphatidylcholine, the latter activated in the cycles of phosphatidylcholine induced by mitogens and oncogenes, with effects on signaling pathways, regulation of the cell cycle, and cell proliferation [165]. The phosphatidylinositol (PI) resides on the cytosolic surface of cell membranes, and differential phosphorylation generates distinct phosphoinosititides, contributing to their signaling diversity, includingcell growth and proliferation. The phosphoinosititides have a distinct localization in the cell through which they carry out their specific function localization (e.g., PI (4,5)P2 is enriched at the plasma membrane, while PI(3)P in early endosomes). Aberrant phosphoinositide signaling has been observed in cancers, suggesting its potential role as a biomarker [166]. A pioneering work quantified 22 classes of EV lipids derived from metastatic prostate cancer cell lines, such as PC-3 cells, finding enrichment in CHOL, SM, glycosphingolipids, and phosphatidylserine, indicating a particular lipid sorting in the exosome membrane compared with the source cells by MS analysis [167] (Table 2). In contrast to healthy cells, tumor cells expose the phosphatidylserine (PS) at the cell surface. An early event in apoptosis is the appearance of PS on the cell surface, which reduces the inflammatory response by alerting phagocytic cells to engulf the cell. Macrophages recognize PS on the surface of apoptotic cells while viable cancer cells with high external PS inhibit phagocytosis by displaying CD47 [168]. In addition, EVs derived from tumor cells might expose PS, suggesting that the source of PS is mostly derived from them, thus constituting a diagnostic biomarker for cancer [169]. Research done on six different prostate cell lines observed differences in the relative abundance of the classes of glycerophospholipids between cells and their EVs [170] (Table 2). Glycerolipids comprise all glycerol-containing lipids; i.e., mono-, di- and tri-substituted glycerols (MAG, DAG, and TAG, respectively), and are important constituents of the EV membrane [171]. On the other hand, the EV lipidomes released from three prostate cell lines, i.e., RWPE1 (non-tumorigenic), NB26 (tumorigenic), and PC-3 (metastatic), have been recently published by [PERSON] et al. [74]. These authors have shown a relative enrichment of lipid species, fatty acids, glycerolipids, and prenolic lipids in EVs from RWPE1, while sterol lipids, sphingolipids, and glycerophospholipids were more abundant in EVs from NB26 and PC-3 cells. They also found that the average CHOL content of EVs derived from PC cells was three times higher than EVs derived from RWPE-1 cells [74]. Additionally, differences in the relative fraction of glycerophospholipids, sphingolipids, glycerolipids, and sterol lipids were identified in LIM1215 colorectal cancer cells, and in their secreted EVs, respectively. Besides, SM, CHOL, and PS were more common in EVs derived from the colorectal cancer cell line LIM1215 than in parental cells.