## 4 Studies of the Molecular Profiling of EVs as Potential Biomarkers
### Lipidome Profiling Analysis of EVs in Multiple Cancers  
EVs represent an untapped source for the discovery of clinically relevant lipid biomarkers, which can be used in pre-clinical detection, as well as for following disease progression. Knowing the lipid profile of EVs from various tumor cell types is an important aspect, although a better understanding of how cancer cells evaluate their lipid resources to meet the metabolic demands of high proliferation rates could enhance novel anticancer therapeutic strategies. There is growing evidence about the transfer of biologically active lipids and lipid metabolites as one of the mechanisms used by cancer cells to alter the energy pathways within the tumor microenvironment [152]. It is now known that EVs transfer lipids and lipid-related proteins to influence target cell function [153, 154], making the study of the EV lipidome essential for the identification of biomarkers for diagnostic purposes. The lipidomic profiling of EVs isolated from cell cultures and biofluids includes differences in lipid composition. Based on published studies, EVs are mainly made up of membrane lipids, although small amounts of other lipids could be captured by the cytosol during ILV formation. Therefore, the lipid composition of EVs should therefore reflect the composition of a lipid bilayer. In fact, it has been shown that there is an asymmetrical distribution of lipid classes in the two leaflets of the plasma membrane, where sphingolipids and phosphatidylcholine (PC) are mainly present in the outer leaflet, while other lipid classes are mainly found in the inner leaflet [155]. Recent advances in lipid analysis by LC-MS/MS have identified lipid classes and lipid species that appear to be enriched in EVs. An important lipidomic profile study of EVs derived from single cell types was realized by [PERSON] and co-workers in ten exosome preparations, including cell-to-exosome enrichment factors in eight of them, showing an enrichment of 2-3 times in cholesterol (CHOL), sphingomyelin (SM), glycosphingolipids (GSLs), and phosphatidylserine (PS). Notably, the membranes of most EVs show lower phosphatidylcholine (PC) and phosphatidylinositol (PI) content than their cells of origin, but contain similar levels of phosphatidylcholine (PE) [156]. The synthesis of cholesterol, also known as the mevalonate pathway, is an important pathway of lipid biosynthesis, since cholesterol is a major component of membranes involved in controlling membrane fluidity and the formation of lipid rafts. Consequently, altered intracellular cholesterol levels can greatly modulate membrane architecture, promoting plasma membrane fluidity, and, therefore, contribute to cell migration and metastasis. Considering this, the active lowering of cholesterol in an advanced stage of the disease can have negative effects [157]. Conversely, the establishment of primary tumors is highly dependent on growth-stimulating signaling pathways, promoted by cholesterol concentrations on the membrane through the formation of lipid rafts. In fact, cholesterol-rich lipid rafts facilitate the accumulation of tyrosine kinase receptors, such as HER2. In this case, blocking cholesterol synthesis could inhibit the onset and proliferation of cancer in the early stages of the disease [158]. In order to use cholesterol metabolism as a therapeutic target in cancer, it is necessary first to understand why cancer cells depend on cholesterol and how this affects the progression of the disease. Glycopshingolipids (GSLs) are a subtype of glycolipids which mediate cell-cell interactions and modulate signal transduction pathways. Aberrant expression of specific GSLs and related enzymes is strongly associated with tumor formation and malignant transformation [159]. There are several studies in literature about the expression of various glycosphingolipids in specific tumors. For example, glycosphingolipids, such as GD3 and GD2, enhance the malignant properties of cancer cells, such as cell proliferation, cell invasion, and migration [160].