The role of lipids in exosome biology and intercellular communication: Function, analytics and applications

Abstract Exosomes are extracellular vesicles that in recent years have received special attention for their regulatory functions in numerous biological processes. Recent evidence suggests a correlation between the composition of exosomes in body fluids and the progression of some disorders, such as cancer, diabetes and neurodegenerative diseases. In consequence, numerous studies have been performed to evaluate the composition of these vesicles, aiming to develop new biomarkers for diagnosis and to find novel therapeutic targets. On their part, lipids represent one of the most important components of exosomes, with important structural and regulatory functions during exosome biogenesis, release, targeting and cellular uptake. Therefore, exosome lipidomics has emerged as an innovative discipline for the discovery of novel lipid species with biomedical applications. This review summarizes the current knowledge about exosome lipids and their roles in exosome biology and intercellular communication. Furthermore, it presents the state‐of‐the‐art analytical procedures used in exosome lipidomics while emphasizing how this emerging discipline is providing new insights for future applications of exosome lipids in biomedicine.

The MVBs either fuse with the lysosome for the degradation of the ILVs or reach the cell membrane to release the ILVs as exosomes. 3 Exosomes, like other EVs, are limited by a lipidic membrane, which encapsulates the cargo molecules in an inner aqueous core. In the particular case of exosomes, these cargo molecules are mainly peptides, small proteins and nucleic acids, such as mRNA or miRNA, all of them used by the cell to transmit signals to other cell populations, coordinate biological functions and maintain homeostasis. 4 Despite its wide use in EVs reports, the application of the above-mentioned terminology is misleading in the practice due to the current limitations to isolate a particular type of EVs in a pure form. Therefore, the International Society for Extracellular vesicles on the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018) suggest the use of alternative terms such as "small EVs" (<200 nm) or "large EVs" (>200 nm). 5 Lately, exosomal proteins and nucleic acids have received particular attention in several studies exploring the biological processes in which they are involved with therapeutic purposes. [6][7][8] However, exosomal lipids represent other less-explored bioactive molecules abundantly present in exosomes, not only as part of their structure but also exerting regulatory functions in receptor cells. 9 Figure 1 shows  [9][10][11][12][13] In this sense, recent lipidomic studies over exosomes derived from different cell types describe the lipidic composition of these EVs and propose alterations under pathological conditions to contribute to the current knowledge about the physiology of exosomal lipids. [14][15][16] In this context and considering the increased interest in exosomal lipids as regulatory molecules and biomarkers observed during the last years, this review describes the most recent advances in exosome lipidomics and their applications, emphasizing the biological importance of exosomal lipids in producer cells and their regulatory function in receptor cells. We also analyze some critical challenges regarding the currently available methods for exosome lipidomic analysis, and some opportunities and future perspectives about the applications of this promising technology. It is important to note that according to the MISEV 2018, 5 the term "extracellular vesicles" is preferred over exosomes since it is difficult to ensure that a particular subtype of EVs (i.e., exosomes) is present in a sample without contamination with other EVs populations. Therefore, in this review, the term exosome is used only to refer to small EVs (50-150 nm) isolated by the commonly accepted methods (e.g., ultracentrifugation, ultrafiltration, precipitation, etc.), expressing cytosolic or transmembrane proteins specific for EVs (e.g., ALIX, syntenin, CD63, etc.), and reported as exosomes by the authors of the works cited in this review. Otherwise, the term EVs is used instead of exosomes.
F I G U R E 1 The number of publications between 2000 and 2019 in PubMed related to exosome genomics, proteomics, or lipidomics. The search terms were "exosome" and "proteomic", "proteomics" or "proteome" (green); "exosome" and "genomics", "genomic" or "genome" (red); "exosome" and "lipidomics", "lipidomic" or "lipidome" (blue) 2 | EXOSOME LIPID COMPOSITION Lipids are essential elements found in all cell types and abundantly distributed in EVs. Sphingomyelin, phospholipids, ganglioside GM3 and cholesterol are lipid classes commonly found in cell membranes and consequently in exosomes. 14 However, the relative abundance of these lipids in exosomal membranes may vary depending on the producer cell type, 17 the physiological stage of the producer cell, 16 and the fate and function of the exosome. 18 In this regard, several studies revealed that exosomes produced under different conditions modify their lipid and metabolite composition to modulate their biological function. For example, in vitro studies revealed that PC3 cells co-cultured with the ether lipid precursor hexadecylglycerol secret exosomes enriched in ether lipids and with different protein composition, demonstrating the impact of external stimuli to modify the lipidic and nonlipidic exosome composition. 19 Similar results were obtained with Huh7 cells co-cultured with palmitate and LPC, which resulted in an enhanced release of EVs with pro-inflammatory activity. 20 Furthermore, it has also been found that exosomes derived from mesenchymal stem cells (MSC) cultured under priming conditions are packaged with lipids and other metabolites associated with the immunomodulatory properties of MSC, including macrophage polarization. 21 The lipidic composition of exosomes derived from different sources and the enrichment of some lipid classes concerning producer cells has been extensively reported in several works. 17,22,23 In this sense, B-lymphocyte-derived exosomes are enriched in cholesterol (CHOL) up to 3 times more when compared to the cell membrane. 24 Apparently, CHOL starts to be accumulated in MVBs and this process appears to be essential for the formation of intraluminal vesicles, the precursors of exosomes. 22 Similarly, sphingomyelin (SM) enrichment in exosome membranes has caused these EVs to be considered as a new type of SM domain. This enrichment could originate from plasma membrane lipid rafts, and also at the expense of phosphatidylcholine (PC) through the activity of the sphingomyelin synthase. 22 Moreover, lipidomic studies in PC3 cells-derived exosomes confirmed that the exosomal membrane is a highly ordered structure enriched in glycosphingolipids, which confers the exosomes the demonstrated stability they present in extracellular environments. 17 This ordered distribution of lipids in the exosomal membrane could be responsible for several interactions during exosome formation, release and delivery to receptor cells, as discussed in the following subsections. It is important to note that the lipid distribution into exosomes and other EVs is a dynamic process that responds to several factors. For instance, significant variations in the lipidic composition of reticulocyte-derived exosomes were found in response to the physiological changes in the cell during the maturation to erythrocytes, demonstrating that the sorting of lipids for exosome biogenesis adapts to the cell requirements. 16 The distribution of lipids in the two leaflets of the lipid bilayer appears to be asymmetrical in the exosome membrane, with SM typically found in the outer leaflet and phosphatidylserine (PS) species in the inner leaflet. 25 However, it has been reported that PS is externalized in apoptotic and malignant cells, acting as an "eat me" signal for macrophages in the immune system. 26 Thus, exosomes and other EVs secreted by malignant cells also expose PS at the outer leaflet, opening novel perspectives for their potential use as exosomal biomarkers for cancer diagnosis. 27 In this context, a PS-targeted microfluidic device has been developed to isolate cancer-derived exosomes from plasma, achieving 90% capture efficiency for cancer cell exosomes, and resulting in a promising tool to explore the role of exosomes and exosomal lipids in cancer progression. 28 Conversely, other studies affirm that microvesicles and exosomes lack the membrane asymmetry found in producer cells because of the presence of a phospholipid scramblase in the exosome membrane, evidenced by the presence of PS and phosphatidylethanolamine (PE) in the outer leaflet. 29 Besides, recent lipidomic studies revealed that some lipids are exclusively or preferentially distributed to certain types of EVs, suggesting the existence of various highly controlled processes involved in the biogenesis of EVs and cargo packaging in which lipids play an indispensable role. 30 Some relevant studies regarding this differential distribution of lipids in EV subpopulations are presented in Table 1. It is also important to mention as well that the advances in purification methods have allowed the isolation of a novel and smaller vesicle that has been named "exomeres" ($35 nm). 15 Despite structural similarities with exosomes, exomeres seem to differ in lipidic composition, presenting higher content of triglyceride (TG), ceramide (Cer) and lysophosphatidylglycerol (LPG) when compared to exosomes, as shown in Table 1. Hence, this differential lipidic composition of EVs allows lipids to be considered important markers to assess the purity of exosome preparations. 31 Furthermore, recent studies reported that the lipid alterations in EVs isolated from pleural effusion of patients with pulmonary tuberculosis and lung cancer were different in small EVs regarding large EVs. 32 These findings suggest that the differential distribution of lipids in EVs subpopulations could be used to identify more sensitive biomarkers contained in a particular type of EVs.

| THE ROLE OF THE LIPIDS IN EXOSOME BIOGENESIS
Exosome biogenesis is a high-regulated process in which the endosomal sorting complex required for the transport (ESCRT) plays an essential role, recruiting exosomal cargo components and inducing the formation of ILVs from the endosomal membrane. 30 However, more recently, novel ESCRT-independent mechanisms have received attention due to their capacity to induce EV formation in the absence of ESCRT machinery, one of them is the denominated lipid-driven mechanism. 38 Moreover, the enrichment of several lipid classes in exosomes and the differential lipidic composition of these EVs under different physiological conditions raises one question: what is the role of these lipids in the biology of exosomes? To answer this interrogation, this section focuses on the most relevant processes involved in exosome biogenesis in which lipids seem to play regulatory functions.  Some important findings in this field are presented in Table 2. Moreover, we present a discussion about some possible future applications of this biogenic role of lipids in exosome-related technologies.

| Cholesterol
The ESCRT machinery plays a fundamental role in the formation of MVBs and the packaging of cargo components into exosomes. 30 In vitro studies revealed that the ESCRT machinery induces the formation of ordered membrane microdomains in a CHOL-dependent manner, suggesting that CHOL content within endosomal membranes may provide adequate conditions for exosome formation, as shown in monocytes. 40 In this sense, the use of statins was reported to reduce the exosome release in BEAS-2B and THP-1 cells owing to its cholesterol-lowering effect, 41 opening novel perspectives regarding the use of statins as therapeutic agents to control exosome production in target cells.

| Sphingolipids
Ceramide is one of the most important lipids in exosome biogenesis because of its apparent capacity to trigger ESCRT-independent processes and induce spontaneous membrane invagination ( Figure 2). 42 Ceramide is synthesized from SM after removal of a phosphocholine moiety by sphingomyelinases, and the spontaneous budding of ceramide-containing membranes is attributed to its cone-shaped structure, which facilitates the negative curvature of the membrane. 38 In vitro experiments revealed the capacity of sphingomyelinases by themselves to induce membrane budding and vesicle formation in synthetic membranes containing SM after ceramide synthesis. 43 Therefore, the use of exogenous sphingomyelinases may represent an alternative to enhance the in vitro production of exosomes from cell lines of interest for scientific or therapeutic purposes.
Similarly, ESCRT-independent cargo sorting in exosomes occurs in the cells through the constitutive activation of inhibitory G proteincoupled sphingosine 1-phosphate (S1P) receptors by a constant supply of S1P, representing another lipid-regulated mechanism for the maturation of exosomal MVBs. 44 T A B L E 2 Role of some relevant lipids during exosome biogenesis

Lipid types Process involved Role in exosome biogenesis Reference
Cholesterol EVs formation, transport and release.
• Provide adequate membrane conditions for budding by maintaining the equilibrium between liquid-ordered and disordered domains. • Interact with ORP1L and control the movement of endosomes along microtubules. • Induce the fusion of MVBs with the cell membrane. 39,51 Ceramide EVs formation • Induce the negative curvature of the membrane. 38 Diacylglycerol EVs formation • Recruit soluble proteins in the cell membrane.
• Interact with cytoskeletal proteins. 52 Ether lipids EVs release • The fusion of MVBs with the cell membrane to release exosomes. 19 Phosphatidic acid EVs formation • Responsible for several protein-lipid interactions.
• Binding with ESCRT-0 to recruit ESCRT-I, -II and -III machinery in the membrane. • Interaction with Hrs protein to begin the cargo sorting into endosomes. 53,54 Bis(monoacyl-glycero) phosphate EVs formation and release.

| Phospholipids
Like ceramide, the phosphatidic acid (PA) is the simplest phospholipid with a small headgroup and cone-shaped structure that confers PA the capacity to induce spontaneous negative curvature in lipidic membranes ( Figure 2). 45,46 Moreover, the physicochemical properties of PA are given in part by its headgroup, allowing protein-lipid interactions between PA and the lysine and arginine residues of proteins. 47 Hence, PA is reported to interact with syntenin triggering the recruitment of syndecan, CD63 and ALIX in the membrane, stimulating the budding process of nascent ILVs. 48 Furthermore, it is proposed that sphingomyelinases interact with PA to enhance ceramide production and promote the ILVs budding in an ESCRT-independent way. 49 The synthesis of PA in the cells is regulated by the activity of phospholipases, 50 therefore the use of exogenous phospholipases could be explored to increase the production of exosomes in vitro and to support the development of exosome-related technologies. Other phospholipids that appear to play important regulatory functions during exosome biogenesis include phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-biphosphate, which seem to regulate the EVs formation, release and cargo sorting, as shown in Table 2.

| EXOSOMAL LIPIDS AND CELL-TO-CELL COMMUNICATION
Exosomes act as nanocarriers of bioactive lipids between cells to regulate specific biological processes. However, their activity is not limited to transport lipids from one cell to another, but also to produce bioactive lipids from other lipidic molecules through the activity of exosomal enzymes packaged into these EVs during their biogenesis. 58 In this sense, this section focuses both on the role of exosomes as lipidic particles as well as functional units for lipid transformation.

| Lipid transformations in exosomes
Exosomes contain all three A2 phospholipases classes (PLA2); the calcium-dependent PLA2 (cPLA2), the calcium-independent PLA2 (iPLA2), and the secreted PLA2 (sPLA2). These enzymes hydrolyze glycerophospholipids to produce arachidonic acid (AA) and other free fatty acids. 59 AA can be further processed by the 5-lipoxygenase to release a set of oxidized eicosanoids named leukotrienes such as LTB4, involved in the inflammation process, and the F I G U R E 2 Lipids in exosome biogenesis. Membrane domains enriched in cholesterol appear to provide adequate conditions for the recruitment of ESCRT machinery in MVBs. Ceramide and phosphatidic acid are cone-shaped lipids that seem to induce spontaneous curvature of the MVBs membrane in an ESCRT-independent manner. Ceramide is produced from sphingomyelin through the activity of the sphingomyelinases (SMase). On their part, phosphatidic acid is produced from phosphatidylcholine and diacylglycerol through the activity of the phospholipases (PLase) and diacylglycerol kinases (DGK), respectively. Furthermore, phosphatidic acid seems to interact with syndecan to enhance the recruitment of syntenin (Syn), ALIX and the ESCRT machinery angiogenesis-promoting LTC4 and LTD4. 60  Furthermore, the myoblast cells C2C12 exposed to palmitate produced palmitate-enriched exosomes with the capability to induce myoblast proliferation and to alter the expression of genes involved in cell cycle and muscle differentiation. Besides, these exosomes were able to be incorporated in various tissues in vivo, including the pancreas and liver, transferring by this way the deleterious effect of palm oil between muscle cells and other tissues. 68 In the brain, as an organ with one of the highest lipid concentrations, 69

| Advances in analytical procedures
Advances in mass spectrometry (MS) have made this method the dominating platform in the lipidomic analysis. 83 On its part, nuclear magnetic resonance (NMR) has become a less-used system limited by its lower sensitivity, the presence of overlapping signals, and the low natural abundance of 13 C for 13 CNMR. 84

| Data processing and bioinformatics
The growing interest in lipidomics as a tool for the evaluation of cell homeostasis demands the development of bioinformatic workflows to identify, quantify and study the influence of lipids on metabolism.
However, despite the existence of bioinformatic mechanisms for these purposes, some of them lack simplicity and interconnectivity and are not user-friendly. 103 Thus, the "Lipidomics Informatics for Life-Science" platform has recently provided its lipidomics software tools with integrative and user-friendly web interfaces. These tools include "LipidXplorer" for shotgun lipidomics, 104 "Skyline for Lipidomics" to assemble targeted mass spectrometry methods for complex lipids, 105 "LUX Score" for the quantification of systematic differences in the lipid composition of a lipidome, 106 and "LipidHome", to bridge the gap between theoretically identified lipid molecules and metadata. 107 All these tools have been recently used to identify exosome lipidic biomarkers in pancreatic cancer. 11 A similar bioinformatic platform extensively used to analyze 93% sensitivity and 100% specificity. 10 Similar research in the area includes the use of serum and blood plasma exosome lipids in pancreatic 11 and non-small cell lung cancer diagnosis, 13 as described in detail in Table 3. 93% sensitivity and 100% specificity by using the combination of the three lipids. 10 Non-small cell lung cancer  [121][122][123] Since this method for the fractionation of EVs is based on their lipidic composition, future research in this field should focus on the enhancement of the diagnosis properties of exosome lipids after a CTB, AV and STB fractionation (see Table 3).

| EXOSOME LIPIDS AS THERAPEUTIC TARGETS
Given the previously mentioned role of exosomes in the pathogenesis of some diseases, several strategies have been proposed to highlight their potential as novel therapeutic targets by inhibiting key aspects in their biology, such as biogenesis, release and cell uptake. 124 These novel methods could be applied in disorders in which exosomes induce a pathological effect (Figure 4). For example, in cancer, it has been demonstrated that the amount of circulating EVs is correlated with cancer progression, and with the survival of patients with melanoma. 125 In this case, a therapeutic intervention could be aimed at reducing the load of exosomes in blood by inhibiting their biogenesis or release.
One of the strategies proposed with this aim is the reduction of the endosomal sorting and exosome biogenesis through the inhibition of the sphingomyelinase, the enzyme that synthesizes ceramide from SM. This inhibition can be achieved with the blood-pressure-lowering drug amiloride, which has demonstrated an efficient in vivo reduction of the circulating tumor-derived EVs with the subsequent reduction in tumor growth. 38,126 Similarly, the biosynthesis of ceramide has also been inhibited using GW4869 and some specific small interfering RNA, reducing exosome release. 127,128 However, a study in PC3 cells revealed that this interference in exosome biogenesis attained by inhibiting the synthesis of ceramide could be cell-type specific, with lower or no effect in certain cell types. 129 Similar lipid-related molecules acting as therapeutic targets in exosomes include the diacylglycerol kinase α (DAGK α) and PS. In this sense, the inhibition of the DAGKα by the DAGK inhibitor II resulted in a decreased secretion of exosomes in J-HM1-2.2 cells. 130 On their part, PS is a lipid molecule exposed on exosome surface important for cell adhesion. 131 The evidence suggests that blocking PS with diannexin reduces the cellular uptake of exosomes, resulting also in a decreased growth of tumor xenografts in mice. 132,133 Furthermore, to prevent the exosome-mediated cholesterol accumulation in atheroma-associated cells, anti-PS receptor antibodies were evaluated, resulting in a diminished internalization of exosomes in cells with favorable outcomes. 9 However, therapeutic strategies based on blocking PS should be carefully designed to avoid interference with other physiological functions regulated by PS, such as the clearing of apoptotic cells. 124 In brain astrocytes, it has been found that the amyloid-β (Aβ) peptide stimulates the secretion of exosomes enriched in both ceramide and the ceramide-sensitizer protein PAR-4, with apoptotic effects.
This deleterious effect was suppressed by inhibiting the activity of sphingomyelinase 2. 134 Furthermore, exosomes can also act as Aβ scavengers by sequestering Aβ through the glycosphingolipids on the exosome surface. 135 The inhibition of the sphingomyelinase 2 activity to avoid exosome-induced apoptosis in astrocytes and the Aβ clearance effect of exogenous exosomes in the brain provides novel insights for therapeutic intervention in Alzheimer's disease. [136][137][138] The cellular internalization of exosomes derived from glioblastoma cells involves the non-classical, lipid raft-dependent endocytosis negatively regulated by the lipid raft-associated protein caveolin-1 (CAV-1). 139 This study revealed that exosome internalization depends First, standardized methods for sample preparation and storage need to be developed, especially considering the instability of lipids under certain temperatures, 141 pH, 142 or freezing conditions. 143 Besides, conventional exosome isolation methods such as ultracentrifugation, polymer-based precipitation, size exclusion, density gradient centrifugation and immunoaffinity capture, may induce loss of exosome integrity and co-isolation of other non-exosome EVs, disturbing the results of lipidomic analysis. 81 Therefore, the optimization of these traditional methods for lipidomic studies or new exosome isolation systems is required. As an alternative, flow field-flow fractionation has been proposed as a new size-based isolation method with favorable outcomes in exosome lipidomics that needs to be further studied. 144 Improvements in analytical methods to achieve full coverage of lipidomes are also required. Many isomeric/isobaric lipid species precludes the use of the shotgun approach in future lipidomic research.
Therefore, innovative chromatographic separations need to be developed, like those previously proposed using mobile-phase modifier systems. 101 Similarly, migration from HPLC to 2.1 mm UHPLC or microflow LC systems may improve the sample throughput and the quality of the results. 83 The absolute quantification of all lipid species in a lipidome remains a major challenge due to the limited number of commercially available lipid standards. Consequently, it is also necessary to establish a consensus in the field of lipidomics about how much accuracy is required to quantitate individual molecular lipid species. Moreover, structural validation of lipids is essential, especially in those proposed as biomarkers.
In this sense, tandem MS analysis should be needed, as well as sample derivatization methods to validate functional groups. 145 In summary, despite the recent achievements in the field of exosome lipidomics, research in this field is still incipient. Therefore, future comprehensive studies are indispensable to increase the cur-