Exosomes are membrane bound extracellular vesicles (EVs) that are produced in the endosomal compartment of most eukaryotic cells. The multivesicular body (MVB) is an endosome defined by intraluminal vesicles (ILVs) that bud inward into the endosomal lumen. If the MVB fuses with the cell surface (the plasma membrane), these ILVs are released as exosomes. In multicellular organisms, exosomes and other EVs are present in tissues and can also be found in biological fluids including blood, urine, and cerebrospinal fluid. They are also released in vitro by cultured cells into their growth medium. Since the size of exosomes is limited by that of the parent MVB, exosomes are generally thought to be smaller than most other EVs, from about 30 to 150 nanometres (nm) in diameter: around the same size as many lipoproteins but much smaller than cells. Compared with EVs in general, it is unclear whether exosomes have unique characteristics or functions or can be separated or distinguished effectively from other EVs. EVs including exosomes carry markers of cells of origin and have specialized functions in physiological processes, from coagulation and intercellular signalling to waste management. Consequently, there is a growing interest in clinical applications of EVs as biomarkers and therapies alike, prompting establishment of an International Society for Extracellular Vesicles (ISEV) and a scientific journal devoted to EVs, the Journal of Extracellular Vesicles.
Exosomes were first discovered in the maturing mammalian reticulocyte (immature red blood cell) by Stahl and group in 1983 and Johnstone and group in 1983 further termed 'exosomes' by Johnstone and group in 1987. Exosomes were shown to participate in selective removal of many plasma membrane proteins as the reticulocyte becomes a mature red blood cell (erythrocyte). In the reticulocyte, as in most mammalian cells, portions of the plasma membrane are regularly internalized as endosomes, with 50 to 180% of the plasma membrane being recycled every hour. In turn, parts of the membranes of some endosomes are subsequently internalized as smaller vesicles. Such endosomes are called multivesicular bodies because of their appearance, with many small vesicles, (ILVs or "intralumenal endosomal vesicles"), inside the larger body. The ILVs become exosomes if the MVB merges with the cell membrane, releasing the internal vesicles into the extracellular space.
Exosomes contain various molecular constituents of their cell of origin, including proteins and RNA. Although the exosomal protein composition varies with the cell and tissue of origin, most exosomes contain an evolutionarily-conserved common set of protein molecules. The protein content of a single exosome, given certain assumptions of protein size and configuration, and packing parameters, can be about 20,000 molecules. The cargo of mRNA and miRNA in exosomes was first discovered at the University of Gothenburg in Sweden. In that study, the differences in cellular and exosomal mRNA and miRNA content was described, as well as the functionality of the exosomal mRNA cargo. Exosomes have also been shown to carry double-stranded DNA.
Exosomes can transfer molecules from one cell to another via membrane vesicle trafficking, thereby influencing the immune system, such as dendritic cells and B cells, and may play a functional role in mediating adaptive immune responses to pathogens and tumors. Therefore, scientists that are actively researching the role that exosomes may play in cell-to-cell signaling, often hypothesize that delivery of their cargo RNA molecules can explain biological effects. For example, mRNA in exosomes has been suggested to affect protein production in the recipient cell. However, another study has suggested that miRNAs in exosomes secreted by mesenchymal stem cells (MSC) are predominantly pre- and not mature miRNAs. Because the authors of this study did not find RNA-induced silencing complex-associated proteins in these exosomes, they suggested that only the pre-miRNAs but not the mature miRNAs in MSC exosomes have the potential to be biologically active in the recipient cells. Multiple mechanisms have been reported to be involved in loading miRNAs into exosomes, including specific motifs in the miRNA sequences, interactions with lncRNAs localized to the exosomes, interactions with RBPs, and post-translational modifications of Ago.
Conversely, exosome production and content may be influenced by molecular signals received by the cell of origin. As evidence for this hypothesis, tumor cells exposed to hypoxia secrete exosomes with enhanced angiogenic and metastatic potential, suggesting that tumor cells adapt to a hypoxic microenvironment by secreting exosomes to stimulate angiogenesis or facilitate metastasis to more favorable environment.
Evolving consensus in the field is that the term "exosome" should be strictly applied to an EV of endosomal origin. Since it can be difficult to prove such an origin after an EV has left the cell, variations on the term "extracellular vesicle" are often appropriate instead.
Exosomes from red blood cells contain the transferrin receptor which is absent in mature erythrocytes. Dendritic cell-derived exosomes express MHC I, MHC II, and costimulatory molecules and have been proven to be able to induce and enhance antigen-specific T cell responses in vivo. In addition, the first exosome-based cancer vaccination platforms are being explored in early clinical trials. Exosomes can also be released into urine by the kidneys, and their detection might serve as a diagnostic tool. Urinary exosomes may be useful as treatment response markers in prostate cancer. Exosomes secreted from tumour cells can deliver signals to surrounding cells and have been shown to regulate myofibroblast differentiation. In melanoma, tumor-derived vesicles can enter lymphatics and interact with subcapsular sinus macrophages and B cells in lymph nodes. A recent investigation showed that exosome release positively correlates with the invasiveness of ovarian cancer. Exosomes released from tumors into the blood may also have diagnostic potential. Exosomes are remarkably stable in bodily fluids strengthening their utility as reservoirs for disease biomarkers. Patient blood samples stored in biorepositories can be used for biomarker analysis as colorectal cancer cell-derived exosomes spiked into blood plasma could be recovered after 90 days of storage at various temperatures.
With neurodegenerative disorders, exosomes appear to play a role in the spread of alpha-synuclein, and are being actively investigated as a tool to both monitor disease progression as well as a potential vehicle for delivery of drug and stem cell based therapy.
An online open access database containing genomic information for exosome content has been developed to catalyze research development within the field.
Exosomes and intercellular communication
Scientists are actively researching the role that exosomes may play in cell-to-cell signaling, hypothesizing that because exosomes can merge with and release their contents into cells that are distant from their cell of origin (see membrane vesicle trafficking), they may influence processes in the recipient cell. For example, RNA that is shuttled from one cell to another, known as "exosomal shuttle RNA," could potentially affect protein production in the recipient cell. By transferring molecules from one cell to another, exosomes from certain cells of the immune system, such as dendritic cells and B cells, may play a functional role in mediating adaptive immune responses to pathogens and tumors.
Conversely, exosome production and content may be influenced by molecular signals received by the cell of origin. As evidence for this hypothesis, tumor cells exposed to hypoxia secrete exosomes with enhanced angiogenic and metastatic potential, suggesting that tumor cells adapt to a hypoxic microenvironment by secreting exosomes to stimulate angiogenesis or facilitate metastasis to more favorable environment. It has recently been shown that exosomal protein content may change during the progression of chronic lymphocytic leukemia.
A study hypothesized that intercellular communication of tumor exosomes could mediate further regions of metastasis for cancer. Hypothetically, exosomes can plant tumor information, such as tainted RNA, into new cells to prepare for cancer to travel to that organ for metastasis. The study found that tumor exosomal communication has the ability to mediate metastasis to different organs. Furthermore, even when tumor cells have a disadvantage for replicating, the information planted at these new regions, organs, can aid in the expansion of organ specific metastasis.
Exosomes carry cargo, which can augment innate immune responses. For example, exosomes derived from Salmonella enterica-infected macrophages but not exosomes from uninfected cells stimulate naive macrophages and dendritic cells to secrete pro-inflammatory cytokines such as TNF-α, RANTES, IL-1ra, MIP-2, CXCL1, MCP-1, sICAM-1, GM-CSF, and G-CSF. Proinflammatory effects of exosomes are partially attributed to lipopolysaccharide, which is encapsulated within exosomes.
Exosome biogenesis, secretion, and uptake
Exosome formation starts with the invagination of the multi-vesicular bodies (MVBs) or late endosomes to generate intraluminal vesicles (ILVs). There are various proposed mechanisms for formation of MVBs, vesicle budding, and sorting. The most studied and well known is the endosomal sorting complex required for transport (ESCRT) dependent pathway. ESCRT machinery mediates the ubiquitinated pathway consisting of protein complexes; ESCRT-0, -I, -II, -III, and associated ATPase Vps4. ESCRT 0 recognizes and retains ubiquitinated proteins marked for packaging in the late endosomal membrane. ESCRT I/II recognizes the ESCRT 0 and starts creating involution of the membrane into the MVB. ESCRTIII forms a spiral shaped structure constricting the neck. ATPase VPS4 protein drives the membrane scission. Syndecan-syntenin-ALIX exosome biogenesis pathway are one of the ESCRT-independent or non-canonical pathways for exosome biogenesis.
The MVBs once formed are trafficked to the internal side of the plasma membrane. These MVBs are transported to the plasma membrane leading to fusion. Many studies have shown that MVBs having higher cholesterol content fuse with the plasma membrane thus releasing exosomes. The Rab proteins especially Rab 7 attached to the MVB recognizes its effector receptor. The SNARE complex (soluble N- ethylmaleimide- sensitive fusion attachment protein receptor) from the MVB and the plasma membrane interacts and mediates fusion.
Specific targeting by exosomes is an active area of research. The exact mechanisms of exosome targeting is limited to a few general mechanisms like docking of the exosomes with specific proteins, sugars, and lipid, or micropinocytosis. The internalized exosomes are targeted to the endosomes which release their content in the recipient cell.
Sorting and packaging of cargoes in exosomes
Exosomes contain different cargoes; proteins, lipids, and nucleic acids. These cargoes are specifically sorted and packaged into exosomes. The contents packaged into exosomes are cell type specific and also influenced by cellular conditions. Exosomal microRNAs (exomiRs) and proteins are sorted and packaged in exosomes. Villarroya-Beltri and colleagues identified a conserved GGAG specific motif, EXOmotif, in the miRNA packaged in the exosomes which was absent in the cytosolic miRNA (CLmiRNA), which binds to sumoylated heterogeneous nuclear riboprotein (hnRNP) A2B1 for exosome specific miRNA packaging Proteins are packaged in ESCRT, tertraspanins, lipid- dependent mechanisms. Exosomes are enriched in cholesterol, spingomyelin, saturated phosphatidylcholine and phosphatyletanolamine as compared to the plasma membrane of the cell.
The isolation and detection of exosomes has proven to be complicated. Due to the complexity of body fluids, physical separation of exosomes from cells and similar-sized particles is challenging. Isolation of exosomes using differential ultracentrifugation results in co-isolation of protein and other contaminants and incomplete separation of vesicles from lipoproteins. Combining ultracentrifugation with micro-filtration or a gradient can improve purity. Single step isolation of extracellular vesicles by size-exclusion chromatography has been demonstrated to provide greater efficiency for recovering intact vesicles over centrifugation, although a size-based technique alone will not be able to distinguish exosomes from other vesicle types. To isolate a pure population of exosomes a combination of techniques is necessary, based on both physical (e.g. size, density) and biochemical parameters (e.g. presence/absence of certain proteins involved in their biogenesis). The use of reference materials such as trackable recombinant EV will assist in mitigating technical variation introduced during sample preparation and analysis.
Often, functional as well as antigenic assays are applied to derive useful information from multiple exosomes. Well-known examples of assays to detect proteins in total populations of exosomes are mass spectrometry and Western blot. However, a limitation of these methods is that contaminants may be present that affect the information obtained from such assays. Preferably, information is derived from single exosomes. Relevant properties of exosomes to detect include size, density, morphology, composition, and zeta potential.
Since the diameter of exosomes is typically below 100 nm and because they have a low refractive index, exosomes are below the detection range of many currently used techniques. A number of miniaturized systems, exploiting nanotechnology and microfluidics, have been developed to expedite exosome analyses. These new systems include a microNMR device, a nanoplasmonic chip, and an magneto-electrochemical sensor for protein profiling; and an integrated fluidic cartridge for RNA detection. Flow cytometry is an optical method to detect exosomes in suspension. Nevertheless, the applicability of flow cytometry to detect single exosomes is still inadequate due to limited sensitivity and potential measurement artifacts such as swarm detection. Other methods to detect single exosomes are atomic force microscopy, nanoparticle tracking analysis, Raman microspectroscopy, tunable resistive pulse sensing, and transmission electron microscopy.
Exosomes contain RNA, proteins, lipids and metabolites that is reflective of the cell type of origin. As exosomes contain numerous proteins, RNA and lipids, large scale analysis including proteomics and transcriptomics is often performed. Currently, to analyse these data, non-commercial tools such as FunRich can be used to identify over-represented groups of molecules. With the advent of Next generation sequencing technologies, the research on exosomes have been accelerated in not only cancer but various diseases. Recently, bioinformatics based analysis of RNA-Seq data of exosomes extracted from Trypanosoma cruzi has showed the association of these extracellular vesicles with various important gene products that strengthens the probability of finding biomarkers for Chagas disease.
Therapeutics and carriers of drugs
Increasingly, exosomes are being recognized as potential therapeutics as they have the ability to elicit potent cellular responses in vitro and in vivo. Exosomes mediate regenerative outcomes in injury and disease that recapitulate observed bioactivity of stem cell populations. Mesenchymal stem cell exosomes were found to activate several signaling pathways important in wound healing (Akt, ERK, and STAT3) and bone fracture repair. They induce the expression of a number of growth factors (hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), nerve growth factor (NGF), and stromal-derived growth factor-1 (SDF1)). Exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling, exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo. Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-126, miR-130a, miR-132) and anti-inflammatory (miR124a, miR-125b) microRNAs, and a microRNA regulating collagen deposition (miR-21). Researchers have also found that exosomes released from oral keratinocytes can accelerate wound healing, even when human exosomes were applied to rat wounds. Exosomes can be considered a promising carrier for effective delivery of small interfering RNA due to their existence in body's endogenous system and high tolerance. Patient-derived exosomes have been employed as a novel cancer immunotherapy in several clinical trials.
Exosomes offer distinct advantages that uniquely position them as highly effective drug carriers. Composed of cellular membranes with multiple adhesive proteins on their surface, exosomes are known to specialize in cell–cell communications and provide an exclusive approach for the delivery of various therapeutic agents to target cells. For example, researchers used exosomes as a vehicle for the delivery of cancer drug paclitaxel. They placed the drug inside exosomes derived from white blood cells, which were then injected into mice with drug-resistant lung cancer. Importantly, incorporation of paclitaxel into exosomes increased cytotoxicity more than 50 times as a result of nearly complete co-localization of airway-delivered exosomes with lung cancer cells.
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