## 4 Studies of the Molecular Profiling of EVs as Potential Biomarkers
### Metabolome Profiling Analysis of EVs in Multiple Cancers  
The discovery of metabolic biomarkers in EVs is an important goal for diagnosing clinical relapses. A distinctive feature of cancer cells is their ability to perform metabolic reprogramming necessary for their high energy requirements [183]. To meet the metabolic needs associated with proliferation, a cancer cell must increase the import of nutrients from the environment. Classically, cancer metabolism has focused on carbon metabolism, including glycolysis and the tricarboxylic acid cycle (TCA cycle). Much is known about the role of glucose as a source of energy for cancer growth; however, amino acids are also important molecules in supporting cancer development. Glutamine is a non-essential amino acid abundant in circulation, which plays an important role in addition to its function as a constituent of proteins. It provides its two nitrogen atoms to synthesize hexosamines, nucleotides, and other amino acids; guides the uptake of essential amino acids; and is also a substrate for TCA, particularly under conditions of carbon diversion to the glycolytic pathways. The level of glutamine in tumor tissues _in vivo_ was found to be significantly lower than in healthy surrounding tissues or plasma. Cancer cells accumulate oncogenic alterations that convey a significant degree of independence to make up for this lacking of glutamine. For instance, _c-Myc_ hyperactivation results in altered levels of downstream transcriptional targets involved in glutamine uptake and metabolism. The lack of glutamine can induce apoptosis in a Myc-dependent manner [184]. On the other hand, glutamine, glycine, and aspartate serve as carbon and nitrogen donors for purine biosynthesis [185], whereas glycine, serine, and methionine provide one carbon unit through the methionine-folate cycle for nitrogenous bases [186]. Metabolites are small molecular analytes present inside the cell, which can provide real-time information on the biochemical events that occur at the time of sample collection,and, therefore, are indicative of the physiological state of the patient [187]. As a result, important clinical information on disease progression can be obtained by monitoring metabolic changes in the patient's bio-fluids, such as blood, urine, saliva, and others, as well as EVs derived from cell lines. Interestingly, during the formation of EVs, small metabolites can be packed inside the vesicle or they can be produced as a result of the activity of metabolic enzymes within the EVs [188]. It should be noted that there is a definitive crossover between EV metabolomics and lipidomics, as the size of the biologically relevant lipids make them classified as metabolites. Because their circulating levels are very low and difficult to detect, metabolites can only be distinguished through use of highly sensitive identification techniques, such as mass spectrometry (LC-MS/MS) and magnetic resonance spectroscopy. Analyses of urine-derived EVs (uEVs) have highlighted specific metabolites, including creatinine, glucuronate, pantothenic acid, 4-pyridyloxic acid, and others. Metabolites specific for plasma-derived EVs (pEVs) include lysine, kynure, threonine, tryptophan, cytidine. Metabolites were found to differ in abundance between uEVs and pEVs. Interestingly, the authors observed that EVs released in pre-prostatectomy present low levels of adenosine, glucuronate, isobutyryl-L-carnitine, and D-ribose 5-phosphate, compared to EVs secreted post-surgery, as well as in control and untreated samples. Specifically, they found greater differences in glucuronate between treated and untreated cancer groups when compared to a control group [5]. Another study observed a statistically significant difference in many of the key metabolites in PCa patients, including acyl carnitines, citrate, and kynure among benign prostate hyperplasia (BPH) samples. Importantly, they found significantly elevated levels of the steroid hormone dehydroenolrosterone sulphate (DHEAS) in uEVs from PCa patients compared to BHP patients.