Exosomes, ranging in size from 30 to 150 nanometers, are membrane-bound extracellular vesicles (EVs) that are produced in the endosomal compartment of most eukaryotic cells. In multicellular organisms, exosomes and other EVs are found in biological fluids including saliva, blood, urine and cerebrospinal fluid. EVs have specialized functions in physiological processes, from coagulation and waste management to intercellular communication.
Exosomes are formed through the inward budding of a late endosome, also known as a multivesicular body (MVB). The intraluminal vesicles (ILVs) of the multivesicular body (MVB) bud inward into the endosomal lumen. If the MVB fuses with the cell surface (the plasma membrane), these ILVs are released as exosomes. Exosomes were also identified within the tissue matrix, coined Matrix-Bound Nanovesicles (MBV). They are also released in vitro by cultured cells into their growth medium. Enriched with a diverse array of biological elements from their source cells, exosomes contain proteins (such as adhesion molecules, cytoskeletons, cytokines, ribosomal proteins, growth factors, and metabolic enzymes), lipids (including cholesterol, lipid rafts, and ceramides), and nucleic acids (such as DNA, mRNA, and miRNA). 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 in circulation carry genetic material and proteins from their cell of origin, proteo-transcriptomic signatures that act as biomarkers. In the case of cancer cells, exosomes may show differences in size, shape, morphology, and canonical markers from their donor cells. They may encapsulate relevant information that can be used for disease detection. 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.
Background 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.
The content of exosomes changes depending on the cells of origin, and they thereby reflect their originating cells. Analysis of the dynamic variation of exosomes may provide a valuable means of monitoring diseases. 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 who 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 long non-coding RNA (lncRNA) localized to the exosomes, interactions with RNA-binding proteins (RBP), and post-translational modifications of Argonaute (Ago) proteins.
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.
Critique of Extracellular Vesicle-Associated RNA Function
While the hypothesis that exosomal RNA can mediate biological effects in recipient cells is compelling, definitive, rigorous evidence demonstrating functional extracellular RNA (exRNA) transfer in mammals remains limited. Although functional nucleic acid exchange is well-documented in plants and nematodes, the mammalian context presents unique experimental challenges. The mere presence of RNAs in extracellular vesicles does not necessarily imply biological function, as RNA degradation, passive release, and contamination from cellular debris can confound interpretations.
Terminology Evolving consensus in the field is that the term "exosome" should be applied strictly 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.
Research Exosomes from red blood cells contain the transferrin receptor that 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.
In malignancies such as cancer, the regulatory circuit that guards exosome homeostasis is co-opted to promote cancer cell survival and metastasis. In breast cancers, neratinib, a novel pan-ErbB inhibitor, is able to downmodulate the amount of HER2 released by exosomes, thus potentially reducing tumor dissemination.
Urinary exosomes have also proven to be useful in the detection of many pathologies, such as genitourinary cancers and mineralocorticoid hypertension, through their protein and miRNA cargo."
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. The role played by exosomes in cell-cell or interorgan communication and metabolic regulation was reviewed by Samuelson and Vidal-Puig in 2018. 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. Exosomal export of miRNA molecules is also linked to the arrest of intercellular miRNA levels and affect their functionality by arresting them on heavy polysomes.
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.