Lipids in exosomes: Current knowledge and the way forward

Lipids are essential components of exosomal membranes, and it is well-known that specific lipids are enriched in exosomes compared to their parent cells. In this review we discuss current knowledge about the lipid composition of exosomes. We compare published data for different lipid classes in exosomes, and what is known about their lipid species, i.e. lipid molecules with different fatty acyl groups. Moreover, we elaborate on the hypothesis about hand-shaking between the very-long-chain sphingolipids in the outer leaflet and PS 18:0/18:1 in the inner leaflet, and we propose this to be an important mechanism in membrane biology, not only for exosomes. The similarity between the lipid composition of exosomes, HIV particles, and detergent resistant membranes, used as lipid rafts models, is also discussed. Furthermore, we summarize knowledge about the role of specific lipids and lipid metabolizing enzymes on the formation and release of exosomes. Finally, the use of exosomal lipids as biomarkers and how the lipid composition of exosomes may be of importance for researchers aiming to use exosomes as drug delivery vehicles is discussed. In conclusion, we have summarized what is presently known about lipids in exosomes and identified issues that should be taken into consideration in future studies.     

The Ether Lipid Precursor Hexadecylglycerol Stimulates the Release and Changes the Composition of Exosomes Derived from PC-3 Cells

Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes.     

Exosomes as new vesicular lipid transporters involved in cell–cell communication and various pathophysiologies

Exosomes are nanovesicles that have emerged as a new intercellular communication system between an intracellular compartment of a donor cell towards the periphery or an internal compartment of a recipient cell. The bioactivity of exosomes resides not only in their protein and RNA contents but also in their lipidic molecules. Exosomes display original lipids organized in a bilayer membrane and along with the lipid carriers such as fatty acid binding proteins that they contain, exosomes transport bioactive lipids. Exosomes can vectorize lipids such as eicosanoids, fatty acids, and cholesterol, and their lipid composition can be modified by in-vitro manipulation. They also contain lipid related enzymes so that they can constitute an autonomous unit of production of various bioactive lipids. Exosomes can circulate between proximal or distal cells and their fate can be regulated in part by lipidic molecules. Compared to their parental cells, exosomes are enriched in cholesterol and sphingomyelin and their accumulation in cells might modulate recipient cell homeostasis. Exosome release from cells appears to be a general biological process. They have been reported in all biological fluids from which they can be recovered and can be monitors of specific pathophysiological situations. Thus, the lipid content of circulating exosomes could be useful biomarkers of lipid related diseases. Since the first lipid analysis of exosomes ten years ago detailed knowledge of exosomal lipids has accumulated. The role of lipids in exosome fate and bioactivity and how they constitute an additional lipid transport system are considered in this review.     

Tumor cell-derived exosomes: A message in a bottle

Exosomes constitute the newest mode of intercellular communication, transmitting information between cells. This exchange of molecular information is facilitated by their unique composition which is enriched with enzymes, structural proteins, adhesion molecules, lipid rafts and RNAs. Following the discovery that cancer cells secrete excessive amounts of exosomes compared to normal cells, it became evident that i) these vesicles can be used as diagnostic markers; ii) their active secretion has functional implications, albeit unknown whether they are tumor promoting or suppressing. Notably, the interplay via the exchange of exosomes between cancer cells and between cancer cells and the tumor stroma may promote the transfer of oncogenes (e.g. β-catenin, CEA, HER2, Melan-A/Mart-1 and LMP-1) and onco-microRNAs (e.g. let7, miR1, miR15, miR16 and miR375) from one cell to another, leading to the reprogramming of the recipient cells. The molecular composition and functional role of tumor cell-derived exosomes in tumorigenesis, metastasis and response to therapy are slowly decrypted and the latest findings as well as potential therapeutic strategies are discussed in this review.     

Exosomal Lipids Impact Notch Signaling and Induce Death of Human Pancreatic Tumoral SOJ-6 Cells

Exosomes are of increasing interest as alternative mode of cell-to-cell communication. We previously reported that exosomes secreted by human SOJ-6 pancreatic tumor cells induce (glyco)protein ligand-independent cell death and inhibit Notch-1 pathway, this latter being particularly active during carcinogenesis and in cancer stem cells. Therefore, we asked whether exosomal lipids were key-elements for cell death and hypothesized that cholesterol-rich membrane microdomains were privileged sites of exosome interactions with tumor cells. To address these questions and based on the lipid composition of exosomes from SOJ-6 cells (Ristorcelli et al. (2008) FASEB J. 22; 3358–3369) enriched in cholesterol and sphingomyelin (lipids forming liquid-ordered phase, Lo) and depleted in phospholipids (lipids forming liquid-disordered phase, Ld), we designed Synthetic Exosome-Like Nanoparticles (SELN) with ratios Lo/Ld from 3.0 to 6.0 framing that of SOJ-6 cell exosomes. SELN decreased tumor cell survival, the higher the Lo/Ld ratio, the lower the cell survival. This decreased survival was due to activation of cell death with inhibition of Notch pathway. FRET analyses indicated fusions/exchanges of SELN with cell membranes. Fluorescent SELN co-localized with the ganglioside GM1 then with Rab5A, markers of lipid microdomains and of early endosomes, respectively. These interactions occurred at lipid microdomains of plasma and/or endosome membranes where the Notch-1 pathway matures. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death. To our knowledge this is the first report on such effects of lipidic nanoparticles on tumor cell behavior. This may have implications in tumor progression.     

Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.     

The Proteome of Pancreatic Cancer‐Derived Exosomes Reveals Signatures Rich in Key Signaling Pathways

Exosomes are membrane‐bound vesicles that traffic small molecular cargos. These cargos participate in cell–cell communication and contribute to the pathogenesis of many disease including cancer. How these mechanisms contribute to communication within the pancreatic adenocarcinoma (PDAC) microenvironment and how they contribute to PDAC biology are poorly understood. Performed in this study are comprehensive, quantitative comparisons of the proteomes of three PDAC cell lines to those of the exosomes they produce. Approximately 35% of whole cell proteins sort into exosomes. Analysis of composition of microbiomes (ANCOM) determined a cluster of 98 enriched pancreatic cancer exosome core proteins (ePC‐ECPs). Further, these proteins are predicted by ingenuity pathway analysis (IPA) as actively involved in signaling pathways regulating cell death and survival, cellular movement, and cell‐to‐cell signaling and interaction in particular (top three p‐value significant pathways). Significant enrichment of canonical pathways of acute phase response signaling (inflammatory response signaling pathways) and FXR and RXR activation in biosynthetic pathways are also predicted; 97 ePC‐ECPs are associated with cancer and among them, 34 are specifically associated with PDAC. In conclusion, exosomes from PDAC are enriched with cancer‐associated signaling proteins. Further assessment of these proteins as PDAC biomarkers or therapeutic targets is warranted.     

Exosome circ_0044516 promotes prostate cancer cell proliferation and metastasis as a potential biomarker

Circular RNAs (circRNAs) are important regulators in cancer growth and progression. Exosomes carry various molecules including RNA, protein, and lipid from one cell to another cell. But the role of circRNAs from the exosomes from prostate cancer patients are not elucidated. In this study, circ_0044516 was found upregulated in prostate cancer and the roles and molecular mechanism of Hsa_circ_0044516 (circ_0044516) was investigated. Firstly, the exosomes of prostate cancer patients were collected for human circRNAs microarray to screen the circRNA expression profile. There were 35 significantly expressed circRNAs with more than fivefolds from microarray analysis. Circ_0044516 was verified to be significantly upregulated in the exosomes from prostate cancer patients and the cell lines. Further investigation demonstrated that circ_0044516 downregulation inhibited the proliferation and metastasis of prostate cancer cells. By bioinformatics and luciferase reporter assays, circ_0044516 was verified to downregulate miR-29a-3p expression and negatively related to miR-29a-3p expression levels in prostate cancer. In a summary, the study indicated that circ_0044516 played an important role in prostate cancer cell survival and metastasis, which suggested that an oncogenic role of circ_0044516 in prostate cancer.     

Breast Cancer‐Derived Exosomes Reflect the Cell‐of‐Origin Phenotype

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell‐of‐origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway‐enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial‐mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell‐derived exosomes reflect the phenotype of the cells‐of‐origin.     

Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic

Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that approximately 10-20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1-2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic.     

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin‐1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non–small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin‐1 and flotillin‐2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin‐1 as well as its EV‐to‐cellular ratio vary drastically depending on cell type.     

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.     

Acyl chain-based molecular selectivity for HL60 cellular phosphatidylinositol and of phosphatidylcholine by phosphatidylinositol transfer protein alpha

Mammalian phosphatidylinositol transfer protein alpha (PITP) is an intracellular lipid transporter with a binding site that can accommodate a single molecule of phosphatidylinositol (PI) or phosphatidylcholine (PC). Phospholipids are a heterogeneous population of molecular species that can be distinguished by their characteristic headgroups as well as their acyl chains at the sn-1 and sn-2 position. In this study, we have defined the acyl chain preference for PITPalpha when presented with a total population of cellular lipids. Recombinant PITPalpha loaded with bacterial lipid, phosphatidylglycerol (PG), was incubated with permeabilised HL60 cells, followed by recovery of PITPalpha by affinity chromatography. Lipids extracted from the PITPalpha were analysed by tandem electrospray ionisation mass spectrometry (ESI-MS) and showed total exchange of acquired bacterial lipids for HL60 cellular PI and PC. Detailed comparison of the molecular species composition of bound phospholipids with those in whole cells permitted the assessment of selectivity of acyl chain binding. For both phospholipid classes, progressive fractional enrichments in bound species possessing shorter acyl chains were apparent with a preference order: 16:1>16:0>18:1>18:0>20:4. A recapitulation of this specificity order was also seen from a dramatically altered range of molecular species present in HL60 cells enriched with arachidonate over many weeks of culture. We speculate that short-chain, saturate-binding preferences under both conditions may reflect properties in vivo. This is consistent with target cell membranes actively remodelling newly delivered phospholipids after transport rather than relying on the transport of the specific molecular species conventionally found in mammalian membranes.     

Very-long-chain fatty acid sphingomyelin in nuclear lipid microdomains of hepatocytes and hepatoma cells: can the exchange from C24:0 to C16:0 affect signal proteins and vitamin D receptor?

Lipid microdomains localized in the inner nuclear membrane are considered platforms for active chromatin anchoring. Stimuli such as surgery, vitamin D, or glucocorticoid drugs influence their gene expression, DNA duplication, and RNA synthesis. In this study, we used ultrafast liquid chromatography–tandem mass spectrometry to identify sphingomyelin (SM) species coupled with immunoblot analysis to comprehensively map differences in nuclear lipid microdomains (NLMs) purified from hepatocytes and hepatoma cells. We showed that NLMs lost saturated very-long-chain fatty acid (FA; C24:0) SM in cancer cells and became enriched in long-chain FA (C16:0) SM. We also found that signaling proteins, such as STAT3, Raf1, and PKCζ, were increased and vitamin D receptor was reduced in cancer cells. Because recent researches showed a shift in sphingolipid composition from C24:0 to C16:0 in relation to cell life, we performed a comparative analysis of properties among C16:0 SM, C24:0 SM, and cholesterol. Our results led us to hypothesize that the enrichment of C16:0 SM could determine enhanced dynamic properties of NLMs in cancer cells with an increased shuttling of protein signaling molecules.     

Cobalt carbonyl complexes as probes for alkyne-tagged lipids

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a “tag” that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV_349nm. The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity “pull-down” strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection.     

Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations

Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer—the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.     

Role of exosomes in lung cancer: A comprehensive insight from immunomodulation to theragnostic applications

Exosomes are 30 to 150 nm-diameter lipid bilayer-enclosed extracellular vesicles that enable cell-to-cell communication through secretion and uptake. The exosomal cargoes contain RNA, lipids, proteins, and metabolites which can be delivered to recipient cells in vivo. In a healthy lung, exosomes facilitate interaction between adaptive and innate immunity and help maintain normal lung physiology. However, tumor-derived exosomes in lung cancer (LC) can, on the other hand, restrict immune cell proliferation, cause apoptosis in activated CD8+ T effector cells, reduce natural killer cell activity, obstruct monocyte differentiation, and promote proliferation of myeloid-derived suppressor and regulatory T cells. In addition, exosomes in the tumor microenvironment may also play a critical role in cancer progression and the development of drug resistance. In this review, we aim to comprehensively examine the current updates on the role of exosomes in lung carcinogenesis and their potential application as a diagnostic, prognostic, and therapeutic tool in lung cancer.