Collective exosome lipidomic analysis from various groups across various cell types has revealed selective enrichment of certain lipid species (Figures 2A,B). For instance, the abundance of phosphatidylcholine (PC) and diacylglycerol (DAG) in exosomes is reduced compared to the membranes of their cells of origin, but are enriched in sphingomylein (SM), cholesterol (Chol), gangliosides (GS), and disaturated lipids in general; PE remains constant.

Studies on exosomes released from reticulocytes and PC3 cells show highest enrichment for Chol, SM, hexosylceramide (HexCer), lactosylceramide (LacCer) in PC3 secreted exosomes and moderate enrichment of SM in reticulocyte (Llorente et al., 2013). Comparison of lipid species from Oli-Neu cells, HEPG2 and PC3 cells demonstrate a cell-type specific effect on exosomal lipidome (Figures 2A,B). For instance, while all cell lines showed a similar lipid enrichment profile, Oli-Neu cells have highest enrichment of Cer and lowest of SM (Trajkovic et al., 2008). Based on extensive reviewing by Skotland et al. (2017b), it can be stated that Chol and SM represent the two major lipid species highly enrichment in secreted exosomes compared to their parent cells. An interesting study by Pienimacki-Roemer et al. analyzed lipidome of vesicles with varying sizes released from platelets and showed that Chol, SM, PI, and PS re common entities in all released vesicles types, but are selectively enriched in exosomes (Pienimaeki-Roemer et al., 2015). A recent work by Zang et al. demonstrated high levels of TAGS, DAGs, MAGs and cardiolipid in exosomes from AcPC-1. MBA-MB-231, and B16-F10 cells, in addition to PC and SM (Zhang et al., 2018). Furthermore, PS36:1, PS39:1, and PS37:0 was most abundant in exosomes from AsPC-1, while PS35:0 was the dominant species in other cell types. Notably, the presence of odd chained lipids was surprising, given the fact that such phospholipids are present in very scare amounts in cells. Exosomic lipid analysis of LIM125 colorectal cells identified 500 lipid species with high enrichment of TAG and CE (Lydic et al., 2015). Similar enrichment has been reported in the exosomes in U87 glioblastoma, Huh7 heptacellular carcinoma and human-bone marrow derived mesenchymal cell (Haraszti et al., 2016). Intriguingly, these studies showed high abundance of CL and not of SM in Huh7 and mesenchymal exosomes, with opposite trend for exosomes from U87 cell. Higher amounts of PC, PE, and PS with unsaturated fatty acid chain, e.g., 38:4 have been found in exosomes from platelets compared with mesenchymal stromal cells (Valkonen et al., 2019). Sun et al. characterized 264 lipids isolated from circulating MVs from human plasma and serum and revealed remarkably high abundance of lysoglycerophospholipids such as lyso-PC, lyso-PE, lysp-PI followed by SM and Cer (Sun et al., 2019). Exosomes released from the apical side of polarized murine cortical collecting duct principal cells has been shown to differ substantially from those secreted from the basolateral side, however further investigations are warranted to delineate the specific lipid changes. Lipid content of exosomes also shows remarkable differences depending on the tumourigenicity of the parent cell lines. For example, the seminal work by Brzozowski et al. showed glycerolipids and prenol lipids to be most abundant in exosomes from non-tumorigenic prostrate cells, while sterol lipids, SM and glycerosphingolipids were highly enriched in exosomes from highly tumorigenic and metastatic prostate cancer cells (Brzozowski et al., 2018).

MVs within biological fluids contain a heterogeneous population originating from various cell-types. For instance, MVs in seminal fluid-prostasomes are highly abundant in SM and HexCer while urinary exosomes are enriched with Chol. Further PS18:0/18:1 was found be second most abundant lipid after Chol in urinary exosomes and all major PE lipid types were identified as ether lipids. Recently Singhto et al. using MALDI-TOF-MS and TLC compared lipidome profile of urinary microvesicles and exosomes and revealed that mannosyl-di-PI ceramides were detected only in exosomes, whereas PI-ceramides were found in microvesicles (Singhto et al., 2019). Similarly, Chen et al. analyzed the MVs present in human serum fluid, and demonstrated high levels of TAG, CE 30:3 and PC 12:0, 1:0 ad 14:1 (Chen et al., 2019). Exosome harbors more phosphatidylserine (PS) compared to the plasma membrane in general, however conflicting reports exists regarding the extracellular or intracellular location of PS in the exosomal lipid bilayer.

Compared to lipidome analysis of Eukaryotic MVs, there exist lesser number of reports on lipid analysis of bacterial MVs; though comparatively, Gram-positive bacteria are more investigated. Accumulation of phosphatidylglycerol (PG) and reduction of CL in MVs from Streptococcus pyogenes has been shown. Selective enrichment of cylindrical lipids has been proposed to foster MV formation in bacteria (Resch et al., 2016). MVs from Propionibacetrium acnes have reduced amount of TG compared to the parent cell membrane and may account for distinct membrane properties of MV membrane bilayers (Jeon et al., 2018). Compared to Gram-positive, Gram-negative bacteria have a distinct cell wall structure, and their MVs have been shown to be enriched in polar lipids such as PE, PG and diacylated phosphatidylinositol dimannoside (Ac2 PIM2); absence of mycolic acid ester implies the inner membrane to be the major site for MV origin (Prados-Rosales et al., 2011; Chowdhury and Jagannadham, 2013). MVs from Mtb under iron-deficient conditions are enriched with acylated glycerides and P, while under iron-sufficient conditions acyl trehalose was more abundant (Prados-Rosales et al., 2014b). Furthermore, higher abundance of saturated fatty acids in OMVs have been linked with low membrane fluidity of bacterial MVs.

Membrane biophysical properties such as rigidity, elasticity, stiffness and order are crucial regulator of membrane fine structure, which in turn regulate various phenomena such as membrane curvature, lipid-protein interactions and their diffusion, cellular migration, adhesion and pathogen entry. Membrane properties in organelle specific fashion are critically controlled by the lipid compositional variation. For instance ER and Golgi have almost 10-fold lower amounts of SL and cholesterol with PM having maximum cholesterol content (Van Meer et al., 2008). This is the reason for high rigidity observed in plasma membrane compared to other intracellular membranes. Furthermore, lipid-specific structural variation impacts membrane curvature, which modulates membrane stiffness and elasticity. For example, phospholipid-like SM and phosphatidylcholine (PC) have cylindrical shapes due to their head and tail proportion. In contrast, lysophosphatidylcholine (LPC) and phosphoinositides (PI) have a higher head to tail ratio, leading to an inverted cone shape and a positive membrane curvature. On the other hand, PE, DAGs, TAGs, and PA have a cone-shaped structure due to their small head group, leading to negative membrane curvature. In line with these facts, a recently study on exosomes isolated from primary hepatocytes coupled with atomic force spectroscopy revealed these exosomes to be softer (less stiff) and less resistant to mechanical failure compared to those isolated from hepatocyte progenitor cell line MPL29 (Royo et al., 2019). This was attributed to the differential abundance of TAGs, PI, PE and LPC lipids between these exosomes. Study by Wubbolts et al. for the first time revealed high abundance of Chol in exosomal membranes with subtle similarities with detergent resistance fraction indicting that exosomes may have raft like membrane properties, namely, high order and rigidity (Wubbolts et al., 2003), Figure 2C. This fits well with recent reports showing exosomes to have high lipid order compared with microvesicles and apoptotic bodies released from same cells (Osteikoetxea et al., 2015). Moreover, exosome lipid bilayer due to the higher abundance of SM and di-saturated lipids has higher rigidity (Laulagnier et al., 2004), Figure 2C. This rigidity has shown to be pH dependent with low pH correlating to low rigidity and may be attributed to their site of origin, i.e., MVBs (Parolini et al., 2009). Moreover, the SM and Chol afforded high rigidity is also the reason behind degradation resistance behavior of MVs (Ridder et al., 2014). More work is needed to establish clear correlation with modulated lipidome and membrane biophysical properties and its impact on membrane-associated MV functions.

Cell to cell communication or quorum sensing (QS) in bacterial species is the ability of individual cells to communicate with one another by the use of extracellular signaling molecules (Waters and Bassler, 2005). The most common signaling molecule produced by a large number of Gram-negative bacteria is L-homoserine lactones (AHLs). The N-acyl side chains of these molecules typically range from 4 to 18 carbons in length. The chain length imparts varying degrees of hydrophobicity and free diffusion is only possible for short chain molecules, while long chain molecules require transporters (Schaefer et al., 2002; Ying et al., 2007; Huang et al., 2012). Recent studies have shown the importance of MVs in the transfer of such signaling molecules. For instance, Paracoccus denitrificans, releases the hydrophobic C16-homoserine lactone (HSL) by fusion of HSL containing MVs with different bacteria; albeit with varying propensities (Toyofuku et al., 2017). This seminal study by Toyofuku et al. demonstrated a novel MV-based binary trafficking mechanism for the targeted and specific delivery of hydrophobic signaling moieties to other bacteria. MVs bound with low affinity to the well-known P. aeruginosa strain, however the freely diffusing non-MV C16-HSL, showed no major difference in the binding affinity to E. coli and P. aeruginosa strains thus underscoring the ability of MVs to recognize various cell types. Clearly MV aided transport of hydrophobic signaling molecules in open aqueous bacterial environment enables efficient cellular communication, as non MV associated signals would be highly diluted impeding sufficient accumulation required for QS. Another study using the marine pathogen Vibrio harveyi, also showed OMV-aided trafficking of hydrophobic QS molecule, CAI-1, a long-chain amino ketone, in aqueous environments (Brameyer et al., 2018) fostering communication with CAI-1 non producing Vibrio cholerae and V. Harveyi. Apart from AHLs, P. aeruginosa also secretes another QS molecule, 2-heptyl-3-hydroxy-4quinolone (Pseudomonas Quinolone Signal, PQS), which is another highly hydrophobic molecule, thus hampering its free aqueous diffusion. MVs are therefore used to traffic this molecule within a population (Mashburn-Warren et al., 2008), and the removal of these MVs has been shown to halt communication and inhibit group behavior under the PQS control (Mashburn and Whiteley, 2005). Thus, MVs play an important role in cell-to-cell communication by aiding in the trafficking of signaling molecules in a targeted manner (Figure 3A).

Along similar lines, MVs in eukaryotic cells also regulate cellular communication Particularly; their involvement in tempering with paracrine and autocrine communication in cancer is well studied (O’Brien et al., 2020), Figures 3A,B. Neoplastic cell release MVs, which modify the phenotype of recipient cells facilitating tumor growth (De Palma et al., 2017; O’Brien et al., 2020). For instance, ovarian cancer cells release microvesicles containing CD147 that promotes the expression of MMP-1,2- and nine- in endothelial cells (Yekula et al., 2020b; Lucchetti et al., 2020). Likewise, CD105 enriched microvesicles from renal cancer stem cells promote angiogenesis via stimulation of migration and tubule formation phenotype in endothelial cells (Parayath et al., 2020). Furthermore, angiogenic activity was stronger under hypoxic, oxygen deprived, conditions implying a regulatory role of tumor microenvironment on MV-mediated processes (Kumar and Deep, 2020). Similar to microvesicles, oncosomes also have been shown to participate in cell to cell communication and trigger migration of cancer associated fibroblasts (Minciacchi et al., 2017; Ciardiello et al., 2019). Notably, these fibroblasts in turn shed oncosomes with high content of microRNA-409 that contributes to EMT transition and cancer stem cell phenotype (Josson et al., 2015). Collectively, exchange of MVs, between cancer and normal cells regulate promotion of tumour growth, via not completely understood mechanisms.

Apart from being harbingers of communication signals, MVs also mediate transfer of cytotoxins, virulence factors and other biomolecules (Biller et al., 2014; Chattopadhyay and Jaganandham, 2015); many of these molecules lack identifiable signal sequences, Figure 3A. These MVs usually contain components of the bacterial outer membrane like proteins, lipopolysaccharides, phospholipids and peptidoglycans (Toyofuku et al., 2019; Nagakubo et al., 2020). For instance, P. aeruginosa use OMVs, to deliver an array of virulence factors such as ß-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, into the host airway epithelial cells via the fusion of OMVs with host cell lipid membrane. Particularly, host cell plasma membrane lipid rafts have been implicated as the most exploited site for fusion with bacterial OMVs carrying virulence cargo. N-WASP mediated actin pathway then traffics the vesicle cargo into the host cytoplasm (Bomberger et al., 2009) leading to systemic delivery to specific subcellular locations impact host processes. Moraxella catarrhalis, a pathogen responsible for the infection of the upper and lower respiratory tract (Augustyniak et al., 2018) use MVs to carry β-lactamase in high concentrations and subsequently helps protect susceptible strains of M. catarrhalis (Schaar et al., 2011b). Since M. catarrhalis is often found to be co-pathogens in infections with S. pneumoniae and H. influenzae, even these stains are protected against amoxicillin by the ß-lactamase rich OMVs by M. catarrhalis (Budhani and Struthers, 1998; Krishnamurthy et al., 2009). Interestingly, using bioinformatics and immunoproteomic approaches, Augustyniak et al. revealed various OMV associated proteins engaged in complement evasion and colonization strategies by M. catarrhalis, underlining their cross-reactive immunity (Augustyniak et al., 2018). Likewise, another proteomic study on the OMVs derived from clinically relevant E. faecium strains revealed the presence of proteins primarily associated with virulence factors, vaccine candidate and antimicrobial resistance, specifically vancomycin resistance (Wagner et al., 2018). E. coli and Salmonella enterica packs the cytotoxin ClyA with their OMVs that lyses the host cell membrane upon fusion. In fact, membrane lytic activity f ClyA was 8-fold higher when delivered via OMV pathway compared to its free form in solution. Similarly, targeted toxin delivery via OMV has been reported for Bacillus anthracis and enterotoxigenic E. coli as well. H. pylori also releases the vacuolating cytotoxin, VacA, associated with OMV for fusion with host cell membrane; VacA-containing MVs have less potent membrane lytic effect compared to ClyA. Mycobacterial MVs facilitate the release of various virulence-associated proteins and lipids during the course of infection. These include proteins such as LprG, LprA, LpqL, LppX, PBP-1, PSTS3 (Wang et al., 2019c) and lipids such as LAM, lipoarabinomannan, phosphatidylinositol, and acylated phosphatidylinositol-dimmannosides (Prados-Rosales et al., 2011) which have been isolated from intact bacteria or Mtb infected macrophage cells.

Similarly, eukaryotic microbes also release virulence factors packed with their MVs. Microvesicles ranging from 10 to 350 nm have been observed with various eukaryotic microbes such as Leishmania, Histoplasma, Candida, Sporothrix, and Saccharomyces. These are enriched in microbial lipids (including glucosylceramide and ergosterol), proteins and carbohydrates that are involved in virulence. These studies have collectively elucidated the functional involvement of MVs for release of virulence factors in close proximity of the host cell facilitating targeted delivery of pathogenic moieties not the target host cell. For example, proteomic analysis of the sectretome of Leshmania donovani revealed that 98% of the secreted proteins lack identifiable signal sequences required for secretion pathways, thus implicating non-classical MV (exosomes and microvesicles) mediated release during pathogenesis.

Apart from inducing an infection by targeted delivery of virulence factors, bacterial MVs also contribute to the complex host-pathogen interactions, specifically by tempering with the host immune response, Figure 3A. Typically, de-regulation of toll like receptor (TLR) and nucleotide-binding oligomerization domain containing protein (NOD) signaling has been extensively shown. For instance, P. aeruginosa OMVs activate TLR4 signaling pathway and contributes to the protection against pseudomonal lung disease in murine models (Zhao et al., 2013). Macrophage cells infected with M. tuberculosis or M. bovis release exosomes that contain pathogen-derived antigens including virulent lipid such as TDM, LAM etc. and activate host immune responses; both innate and acquired along with the production of pro-inflammatory cytokines (Schorey and Bhatnagar, 2008; De Toro et al., 2015). In fact lipoglycans such as LAM within Mtb MVs when released into infected macrophages provide a generic mechanism for these virulent lipoglycans to reach and inhibit T cells promoting immune evasion (Athman et al., 2017). Moraxella MVs induce pro-inflammatory response in epithelial cells with an upregulated IL-8 secretion resulting in the recruitment of TLR2 to the site of MV binding and ICAM-1 expression (Schaar et al., 2011a). MVs from Salmonella typhimurium potently stimulate APCs in vitro. They induce increased surface expression of MHC-II and CD86 and elevate the production of NO, TNF-α, and IL-12 to levels similar to that induced by whole bacterial cells (Alaniz et al., 2007). This shows the extent to which MVs can possess inflammatory properties and the ability to prime protective B and T cell responses in vivo against Salmonella. Similar observations on MVs from H. pylori leading to induction of IL-8 production in a MV dose dependent manner in a culture of gastric epithelial cells has been reported (Ismail et al., 2003). Typically, TLR and NOD ligands in bacterial MVs included capsular polysaccharide such as LPS, lipoproteins, peptidoglycan, and capsular proteins; supported by proteomic analysis. Arabidosis treated with MVs derived from Xanthomonas elicited an immune response in terms of defense gene activation, ROS burst and medium alkalinization (Bahar et al., 2016).

A variety of eukaryotic microbes, such as Trichomonas vaginalis, Trypanosoma cruzi, and helminths and intracellular parasites including Plasmodium falciparum, Toxoplasma gondii and Leishmania spp., have also been shown to employ their MVs for modulating host responses (Bhatnagar et al., 2007; Maxwell et al., 2008; Silverman et al., 2010; Martin-Jaular et al., 2011; Cestari et al., 2012; Marcilla et al., 2012; Mantel et al., 2013; Pope and Lässer, 2013; Regev-Rudzki et al., 2013; Twu et al., 2013; Marcilla et al., 2014; Arantes et al., 2016; Marti and Johnson, 2016; Schatz et al., 2017; Ribeiro et al., 2018). (Bhatnagar et al., 2007; Silverman et al., 2010; Martin-Jaular et al., 2011; Cestari et al., 2012; Marcilla et al., 2012; Mantel et al., 2013; Pope and Lässer, 2013; Regev-Rudzki et al., 2013; Twu et al., 2013; Marcilla et al., 2014; Arantes et al., 2016; Marti and Johnson 2016; Schatz et al., 2017; Ribeiro et al., 2018). For example, Leishmania spp. constitutively secrete exosomes within the lumen of the sand fly midgut and, following their ingestion into a new host these MVs play an important role in modulating host immunity promoting parasitic infection (Athman et al., 2015). In T. cruzi, a difference in MV production between strains has been shown to correlate with their infectivity and virulence affecting the host-parasite interactions, particularly invasion and proliferation (Ribeiro et al., 2018). Collectedly, MVs not only play a role in establishing an infection but also in the immune response associated with the disease.

Cancerous cells often encounter metabolic stresses due to their rapid growth which are characterized with deprivation of nutrients such as glucose, glutamine, lipids and oxygen within tumour microenvironment. However, the cancer cells adapt to such deficiencies by altering its metabolic pathways. For instance, under glucose deprivation, cancer cells upregulate the energetically and metabolically wasteful erobic glycolysis (Hay, 2016). In other cases, increased uptake of glutamine and amino acids is documented (Altman et al., 2016). Several genetic and epigenetic alterations endow cancer cells to manoeuvre alternate strategies for the production of all the necessary metabolites required for survival and proliferation. For instance, various receptor-mediated signalling pathways, such as Ras, RAF–mitogen-activated protein kinase (MAP kinase), phosphatidylinositol 3-kinases (PI3Ks) and mammalian target of rapamycin (mTOR) are exploited simultaneously. Alteration of these, rendered by activated oncogenes c-Myc (Myc), nuclear factor kB (NF-kB), AKT, and the tyrosine kinase receptors (epidermal growth factor, EGF; insulin-like growth factor 1, IGF-1; Her-2; etc.) foster glucose uptake by upregulating the expression of the glucose transporter GLUT1 and glycolysis (Levine and Puzio-Kuter, 2010). In addition to enhanced glycolysis, Myc also upregulates glutaminolysis by stimulating the transcription of glutaminase-1. This supports fatty acid (FA) synthesis, promotes mitochondrial gene expression and biogenesis (De Berardinis and Chandel, 2016). PI3K/AKT-mediated suppression of FA oxidation contributes to enhanced lipogenesis in proliferating cells. In addition, lipogenic genes including acetyl-CoA carboxylase (ACACA); FA synthase (FASN) and stearoyl-CoA desaturase (SCD) are induced by the mTORC1 activated sterol-regulatory element-binding protein family, which subsequently promote increased fatty acid biosynthesis (De Berardinis and Chandel, 2016). Another oncogene, KRas, under nutrient stressed condition, scavenges extracellular proteins to produce glutamine and other amino acids to fuel the tricarboxylic acid (TCA) cycle for survival and growth in pancreatic cancer (De Berardinis and Chandel, 2016). KRas also reprograms glucose metabolism by upregulating glycolytic flux via modulation of GLUT1, HK1 (hexokinase1), HK2 (hexokinase2), PFK1 (phosphofructokinase 1) and LDHA (lactate dehydrogenase A) and thus fulfill the increasing energy demands and biosynthesis (Bryant et al., 2014). Of note, some common oncogenes, such as chromatin binding protein polybromo 1 (PBRM1), which are otherwise not directly linked to above pathways, also play a profound role in cancer metabolism to support metabolic rewiring (Slaughter et al., 2018). Aside from oncogenes, tumour suppressor such as p53 transcription factor plays a pivotal role in metabolic stress management. p53 promotes a transcription-independent up-regulation of pentose phosphate pathway (PPP), which is essential for the production of lipid and nucleotide biosynthesis. p53 transcriptional target, TIGAR (TP53-induced glycolysis and apoptosis regulator), contributes to oxidative PPP up-regulation (Reid and Kong, 2013). Furthermore, p53 also maximizes mitochondrial oxidative phosphorylation (OXPHOS) by inducing the expression of ETC (electron transport chain) assembly factor SCO2 (Reid and Kong, 2013). In response to low glucose levels, another tumour suppressor AMPK (AMP – activated protein kinase), phosphorylates and activates p53. Activated p53 then enables adaptation to deprived glucose condition through metabolic check point arrest that provide rest to the cells along with a prolonged wait time in pro-proliferative conditions (Reid and Kong, 2013).

Proteomics studies on eukaryotic MVs from cancer cells (i.e., exosomes) have elucidated them to be enriched with GLUT1 and glycolytic enzyme PKM2. For instance, Wan et al. showed enriched PKM2, and GLUT1 levels in secreted exosomes from hepatic stellate cells promoting metastasis, while another study demonstrated increased PKM2 exosomal content to be associated with pre-metastatic niche (Dai et al., 2019; Wan et al., 2019). Importantly, inhibition of exosome secretion by GW4869 inhibits glycolysis and reverse the phenotype restricting cancer progression, illustrating the potential of targeting exosomal pathways for anti-cancer therapy. Similarly, increased exosomal VEGF is associated with enhanced glycolysis in HUVECS (Wang et al., 2019a). Very recently, Sung et al. have demonstrated that triple negative breast cancer derived exosomes transport ITGB4 proteins that induce mitophagy, promote MCT4 expression in recpient cells that in turn increases glycolysis and lactate transport (Sung et al., 2020). In addition to proteins, exosomal cargo such as microRNA are also implicated in cancer cell metabolic reprgramming. For example, breast cancer cell derived exosomal miR-111 supresses glucosie uptake by niche cels by downregulating PKM2 and GLUT1; inhibition of miR-122 rescues glucose metabolism and restricts metastasis (Fong et al., 2015). Another breast cancer derived exosomal miR, miR-155 promotes lipolysis in adipocytes by decreasing PPARy expression (peroxisome proliferators-activated receptor (Wu et al., 2018). Recently, Shu et al. showed that human melanoma cells derived exosomes enriched with miR-155 and miR-210 remodel stromal cell metabolism by enahcing glycolysis and supressing OXPHOS expression (La Shu et al., 2018) which facilitates tumor migration and invasion. The same authors also interestingly showed that exosomes transferred with respective miRNA inhibitors can reverse the exosome-mediated metabolic reprogramming, and hence reduce the risk of tumor metastasis. FASN has been identified in prostate cancer cell derived exosomes hinting at their role in cancer cell lipogenesis (Lázaro-Ibáñez et al., 2017). Apart from the FA pathway, sugar related pathways are also modulated by MVs. For instance, proteomics of HCC showed a enhanced presence of enzymes associated with glycolysis (Zhang et al., 2017). In fact, glycolytic enzymes are some of the most commonly identified proteins in EV based proteomics and is accordance with the high levels of ATP in the tumor environment (Arab and Hadjati, 2019) which can help in the enhanced levels of adenosine and hence help with the immune escape of cancer cells (Vigano et al., 2019).

One of the most important treatment methods for cancer that is administered either on its own or along with chemotherapy is radiotherapy. As with any treatment for cancer, one of the major obstacles is the recurrence and relapse of the tumor and its resistance to radiotherapy (radioresistance) (Szatmári et al., 2019). Owing to the fact that majority of people do receive radiotherapy, it is a key issue that has been greatly studied to understand the underlying mechanism and cause of radioresistance. During radiation therapy, there is direct damage due to the energy of the radiation or indirect effects due to the reactive oxygen species on the target cells. However, not all cells are under the direct effect of the radiation and are prone to bystander effects. These bystander cells receive signals from damaged cells via the help of MVs (Ni et al., 2019).

Glioblastoma (GBM) based radiation therapy has been shown to possess limited efficacy due to the enrichment of cancer stem cells (CSCs). CSCs have the capacity to cause recurrent rumors and are resistant to therapies usually used against GBMs (Yekula et al., 2020a). In this regard, a study by Ramakrishna et al., showed that mass exporting of miR-603 in MVs led to the treatment resistant phenotype. The decreased levels of miR-603 drove the post radiation therapeutic resistance of GBM leading to the formation of IGF-1 mediated stem cell state in the GBMs. It was seen that ectopic expression of the miR-603 reversed this effect contributing to a positive effect of radiation and tumoricidal agents (Ramakrishnan et al., 2020). Studies of head and neck cancer cells have shown MVs to contain chromatin fragments having a functional significance toward signaling between irradiated and non-irradiated cells. Apart from this, altered levels of exosome markers were observed; while CD63 and CD81 levels were increased, CD9 and TSG101 levels remained unchanged. A similar upregulation of HSPs in exosomes released by the irradiated cells (Abramowicz et al., 2019) has also been documented. A study by Mrowczynski et al. demonstrated the effect of irradiated cells derived exosomes on recipient cells in vitro and in vivo models of nervous system cancer. The provided evidence that these exosomes contain upregulated oncogenic and downregulated tumor suppressive contents. There were downregulated levels of tumor suppressive miRNA like miR-365 and miR-516 which increased cancer cell proliferation and upregulated level of miR-889 that increased resistance, metastasis and decreased apoptosis. These collectively show that radiation exposure leads to the release of altered exosomes that contain signals which decrease tumor suppressive cargo and concomitantly increase oncogenic cargo to be taken up by the recipient cell leading to proliferation and enhanced ability to survive radiation treatment (Mrowczynski et al., 2018).

Similar to radioresistance, cancer cells also relay cargo and other components that are important for survival of neighboring cells in response to stress conditions brought about by chemotherapeutic drugs. For example, multiple myeloma (MM), a plasma cell malignancy has a range of immunomodulatory drugs, proteasome inhibitors and antibody drugs developed against it. MM cells are seen to acquire resistance on long term exposure to these drugs. MM derived exosomes have been shown to play a role in the transmission of this information from cell to cell and subsequently help in the cancer progression and drug resistance of surrounding cells (Colombo et al., 2019; Mogollón et al., 2019). Exosomes from breast cancer cells (MDAMB231) treated with paclitacel (PTX) has been shown to contain a cell survival protein and cancer marker, Survivin. These Survivin enriched exosomes promoted cell survival of fibroblasts and SKBR3 breast cancer cells when challenged with PTX treatment. Thus, these altered exosomes play an important role in mediating resistance to PTX (Kreger et al., 2016). Similar observations have been made with respect to resistance of breast cancer cells to Doxorubicin (DOX). For instance, the proteome profile of MCF-7/ADR cells show increased expression of CD44 in the DOX-resistant cells compared to that of the parental cell line. This leads to a increased exosome-mediated intercellular transfer of proteins in the cancer cells and its chemoresistance regulation. A siRNA based approach to target the CD44 could efficiently silence the expression of CD44 and in turn enhance the susceptibility of the cells to DOX and hence reduce cell proliferation (Wang et al., 2020b). A recent study by Faict et al. revealed that exosomes enriched with high acid SMase (ASM) content can transfer drug-resistant phenotype to drug-sensitive cells by altering lipid metabolism (Faict et al., 2019). Exosomes from patients with Kras chemoresistant lung cancer patients have been shown to remodel metabolism in a PKM2-dependent manner to maintain lung cancer cell metabolic chemoresistance (Petanidis et al., 2020). All these studies point to the MVs-mediated therapy resistance generation in cancer and consequently the potential of targeting exosomes in tumor drug resistant therapy.

Microvesicles and exosomes are the two major kinds of MVs involved in directional exchange of molecular messengers between cancer cells. Particularly, circulating exosomes found in various of samples, such as urine, blood, bile, etc. harbor tumor-specific molecular signatures including oncoproteins, nucleic acids, lipids and miRNAs and hence demonstrate features as next-generation non-invasive biomarkers for liquid biopsy in cancer diagnosis (Figure 3B) (Table 1). In addition, the ability to naturally surmount biological barriers makes them attractive modules for exploitation in cancer diagnosis (Morad et al., 2019).

The protein and nucleic acid contents of MVs have been thoroughly evaluated as biomarkers for disease diagnosis and prognosis (Fais et al., 2016). For instance, exosomal levels of glypican 1 (GPC-1), proteoglycan, in the blood was assessed for detection of pancreatic cancer. GPC-1 positive exosomes enabled distinguishing healthy individuals from those with early and late stage pancreatic cancer and from those with benign pancreatic disease (Melo et al., 2015). In fact, GPC-1 and its regulatory miRNAs have already been reported as specific biomarkers for the diagnosis of colorectal cancer as well (Melo et al., 2015). In another study protein expression of EGFR⁺ EVs were illustrated as effective diagnostic and prognostic markers of glioma, wherein EGFR expression in serum EVs were able to accurately differentiate high-grade and low-grade glioma patients (Wang et al., 2019b). The authors also showed the potential of NLGN3 and PTTG1 mRNA in EVs for detecting glioma patients. Interestingly, protein phosphorylation status is an emergent marker for early stage cancer. Chen et al. demonstrated that patients with breast cancer exhibit significantly higher levels of phosphoproteins in plasma MVs than healthy controls (Chen et al., 2017). Platelet factor 4 protein has surfaced as a new exosomal biomarker in the serum of patients with different liver diseases (Nguyen et al., 2019).

Exosomes containing mutants of Kras DNA have been used for predicting the status of pancreatic cancer and pancreatic ductal adenocarcinoma (Allenson et al., 2017; Yang et al., 2017b). Multiple exosomal miRNAs also have been used for distinguishing patients with lung adenocarcinoma and granulomas (Cazzoli et al., 2013); microRNAs (miRNAs) are a class of small, noncoding RNAs. Exosomal miR-375 expression level can accurately differentiate estrogen receptor-(ER) positive breast cancer at early stages (stages I, II) with a high accuracy (Zhao et al., 2020). Likewise, miR-21 has been used to differentiate breast cancer patients from healthy counterparts (Gao et al., 2019; Zhao et al., 2020). Furthermore, expression of exosomal miR-19a in serum has been identified as a prognostic biomarker for recurrence in CRC patients (Matsumura et al., 2015). In a recent study, Lee et al. employing a label free Raman MV fingerprinting approach identified diagnostic MV signatures such as CH₂ deformation stretching in lipids, proteins, and C=C stretching in lipids and were capable of distinguishing prostate cancer patients compared to healthy donors (Lee et al., 2018) adding to MV based diagnostic tool kit in prostate cancer.

The use of exosomal lipids as cancer biomarkers is not very widespread and currently emerging due to advances in lipidomics and effective methods for lipid isolation. In a seminal study by Skotland et al., analysis of exosomal lipids from the urine of prostate cancer patients revealed significant enrichment of LacCer 18:1/16:0 compared to control healthy controls. In contrast, PS 18:1/18:1 was highly abundant on control healthy samples. This method demonstrated a high sensitivity (93%) and specificity (100%), however reinforced the application of MS based lipidomic platforms as a routine method for clinical practice. This was due to the fact that various lipid classes displayed very little differences, which would go undetected using other semi-quantitative approaches (Skotland et al., 2017a). Specific to prostate cancer, several other Cer species have been found to be significantly different between stage 3 and 2 cancer patients (Clos-Garcia et al., 2018). Other work has furnished lipid species such as TAG and CE expressed in high amounts in urinary exosomes from prostate cancer patients (Yang et al., 2017a), however caution should be exercised when dealing with these lipid classes as these can be fall out of ineffective exosomes isolation causing contamination of TAG and CE enriched lipid droplets. Significantly deregulated lipid species, including LPC (22:0), PC (P-14:0/22:2), and PE (16:0/18:1), in serum exosomes, have been shown to be associated with tumor stage and tumor diameter in pancreatic cancer patients (Tao et al., 2019). In another very recent work, lipidomic analysis of urinary exosomes in α-tryptasemia patients elucidated 64 out of 521 lipid species from 19 lipid classes to be significantly different compared to healthy controls (Glover et al., 2019).

Apart from urine, other bio fluid MVs has also furnished lipid profiles amenable for diagnostic purposes. For example, serum EVs from 20 pancreatic cancer patients and healthy controls revealed PE 16:0/18:1 to be associated with the tumor stage, and significantly correlated with patient overall survival (Tao et al., 2019). Another study reported lipidomic analysis of MVs from plasma of multiple sclerosis patients and healthy volunteers and revealed presence of sulfatides, a class of glycosphingolipids; sulfated galactosylceramide. Particularly sulfatide C16:0 was elevated in the EVs isolated from patient samples (Moyano et al., 2016). Finally, Hough et al. isolated EVs from bronchoalveolar lavage fluid of asthmatics and healthy controls, and reported SM 34:to be highly abundant in EVs from second-hand smoke exposure asthmatics compared to healthy controls (Hough et al., 2018). Thus, lipid from various bio fluid MVs can serve as robust biomarkers for cancer diagnosis and prognosis, however this field has to reach its full potential in years to come.

Incorporation of mycobacterial proteins in exosomes released from infected macrophages has been exploited to elucidate robust and high fidelity biomarkers for tuberculosis (Kruh-Garcia et al., 2015). For example, an interesting study was able to differentiate between pulmonary and extra pulmonary tuberculosis based on the peptide markers (corresponding to Antigens 85b, BfrB, GlcB, Mpt64, and HspX) found in the serum exosomes of patients suffering from these, compared to healthy controls. Interestingly, some of the healthy patients (absence of active TB) showed evidence of latent disease dependent on the abundance of specific biomarker mycobacterial peptides belonging to (Kruh-Garcia et al., 2014). OMVs from BCG or Mtb have been demonstrated to elicit a cellular response while conferring protection against an Mtb aerosol (Prados-Rosales et al., 2014a). Moreover, Mtb MVs has also been shown to boost the effect of the BCG vaccine; surfacing as effective adjuvants. Probably, Mtb MVs may serve as vaccine even in the absence of adjuvants, due to the lipid immunogenic components such as TDM, LAM etc. However, the full diagnostic, prognostic and therapeutic value of MVs with respect to tuberculosis has yet to be fully realized.

The earliest report on the use of exosomes for drug delivery was reported by Zhang et al., wherein an enhanced anti-inflammatory effect by exosomal delivery of curcumin to target inflammatory cells was shown. Since then exosomes have been intensively investigated to encapsulate various therapeutic drugs for disease treatment (Sun et al., 2010). For instance, exosomes loaded with doxorubicin (DOX) and imperialine exhibit excellent potential for cancer treatments (Fan et al., 2019; Lin et al., 2019). Wu et al. developed engineered exosomes by coupling it with nanoparticles functionalized with platinum Pt (IV) prodrug (Pt (lau)), human serum albumin (HSA), and lecithin (Xiong et al., 2019). When compared with free cisplatin, the engineered exosomes facilitated breast-cancer-targeted drug delivery due to the tumour-homing ability of the macrophage-secreted exosomes. In another example of engineered MVs, Bi₂Se₃ nanodots and DOX co-embedded microparticles (Bi₂Se₃/DOX@MPs) were fabricated. Using ultraviolet light irradiation-induced budding of parent cells preloaded with Bi₂Se₃ nanodots and DOX, the drug-loaded microvesicles displayed dual-modal imaging capacity and excellent tumor suppression (Wang et al., 2020a). In a seminal work, using cancer-derived exosomes to deliver palladium catalyst in cells, a localized prodrug activation could be achieved; this underlines the development of new targeted therapy module-exosome-directed catalyst prodrug therapy (Sancho-Albero et al., 2019). In the last two years, indirect loading of drugs and targeting-ligands to produce functionalized MVs in vivo for drug delivery have surfaced (Tang et al., 2019; Yong et al., 2019; Zhang et al., 2019). Exosomes derived from immature dendritic cells lacking immunostimulatory markers like CD40, MHC-I, MHC-II were produced and engineered to target tumors by expressing lysosome associated membrane glycoprotein 2b (Lamp2b) which was fused with iRGD targeting peptide for αv integrin. These iRGD exosomes were then loaded with doxorubicin. These iRGD-exosomes showed high affinity to αv integrin positive breast cancer cells both in vitro and on BALC/c nude mice in vivo. This strategy helped in the administration of a low dose of doxorubicin that helped in the inhibiting of tumor growth compared to an equivalent dose of free doxorubicin which proved ineffective otherwise (Tian et al., 2014). Exosomes released by macrophages have been loaded with paclitaxel (a drug used to treat multi drug resistant cancer) and used to treat mouse 3LL-M27 lung carcinoma cells along with drug resistant and wild type strains of MDCK cells in vitro and C57Bl/6 mice bearing pulmonary metastases in vivo showing increased cytotoxicity against the lung cancer cell lines in vitro and reduced tumor size in vivo.

In addition to drug, MVs have also been tapped for therapeutic delivery of proteins. MV contain a significant number of binding sites for specific targeting/homing ligands for targeted protein (Yang et al., 2019). As such, therapeutic proteins can be delivered by engineering the MV surface using the fusion of the moiety of interest adding to the ever increasing repertoire of smart engineered membrane vesicles for precision medicine (Tran et al., 2020) and have been extensively reviewed by Xing et al. (2020). Similarly, MVs harbor excellent potential for targeted delivery of nucleic acids while obviating issue of degradation and have been reported extensively over the years (Alvarez-Erviti et al., 2011). Injection of Exosomes derived from marrow stromal cell (MSC) after the transfection with miR-146b miRNA, into the primary brain tumor of rats showed significant reduction in the glioma xenograft growth suggesting a new treatment strategy for malignant glioma (Katakowski et al., 2013). Similarly, stellate cell derived EVs loaded with miRNA-335-5p when delivered to hepatocellular carcinoma cells inhibited cancer growth and invasion both in vitro and in vivo. It also led to the shrinking of the HCC and also the identification of mRNA down regulated due to the miRNA such as CDC42, CDK2, EIF2C2, CSNK1G2, ZMYND8 and others that have been implicated in hepatocellular carcinoma (Wang et al., 2018). In recent years, Levenberg et al. using mesenchymal stem cell derived exosomes loaded with phosphatase and tensin homolog small interfering RNA (ExoPTEN) showed robust axonal outgrowth of neurons in vitro, and improved neovascularization and therapeutic efficacy in rats with spinal cord injury (Guo et al., 2019). In an excellent study, reprogramming of native MVs for RNA delivery was elucidated. An arrow-shaped RNA to control ligand display on MV membranes for specific cell targeting was installed. Placement of membrane anchoring cholesterol at the tail of the arrow resulted in the display of RNA aptamer or folate on the outer surface of MVs; the resulting MVs were able to specifically deliver siRNA to cells, and halt tumor growth in prostate, breast and colorectal cancer models (Pi et al., 2018).

Stem cells have been shown to hold great promise for treating several diseases, and hence stem cell derived MVs are emerging as attractive modality for tissue generation. For example, perivascular stem cells (PSCs) derived MVs facilitate issue repair of perivascular stem cells while providing an ‘off-the-shelf' substitute for bone tissue regeneration (Xu et al., 2019). Interestingly MVs derived from human dermal fibroblasts have been shown to exhibit anti-skin-aging abilities (Hu et al., 2019). Similarly, lung spheroid cell secreted exosomes have been shown to be effective in different models of lung injury and fibrosis. Attenuation of lung fibrosis by re-establishing normal alveolar structure and decreasing collagen accumulation and myofibroblast proliferation indicate their great innate therapeutic potential (Dinh et al., 2020).

MVs are advantageous due to their low immunogenicity and innate ability to interact with target cells owing to their biocompatibility which is an important pre-requisite for clinical applications. Other factors that propel MVs based clinical use are high stability in biological fluids, cell-cell communication, active targeting by modification/functionalization, suitability for multidrug delivery and a wide range ofdrug encapsulation methods. However, MV-based therapeutics have their own challenges (Ayala-Mar et al., 2019) such as sourcing of an optimal donor cell type, preserving the structural integrity of the MV, large-scale production, storage and high purity (Akuma et al., 2019) are some roadblocks for attaining a more practical means of exploiting MVs for theranostic use. In addition, poorly defined compositions and uptake mechanisms, lack of good manufacturing practice standards, in ability to translocate through renal barriers and in-depth lack of pre-clinical evaluation are some impediments that hamper heightened clinical transition of MVs based therapy. Further, engineered MVs require the precise evaluation of the interaction of the MVs with cells in order to determine their uptake routes. Finally, the route of exosome delivery is another key factor that affects the therapeutic application of exosomes as intravenous admiration has shown to lead to the faster clearance of the exosomes and lead to their accumulation in either the liver, spleen or kidney (Grange et al., 2014; Wiklander et al., 2015). This leads to the limited systemic admiration. To circumvent this issue reports have shown the alteration in the ligand of exosomes lead to enhanced target accumulation with certain limitations pertaining to the overall quantity of exosomes in the target organ (Ohno et al., 2013). Nonetheless MVs present new paradigms for diagnosis and prognosis against various diseases.