What are exosomes?
Exosomes are a class of cell-derived extracellular vesicles of endosomal origin, and are typically 30-150 nm in diameter – the smallest type of extracellular vesicle.1 Enveloped by a lipid bilayer, exosomes are released into the extracellular environment containing a complex cargo of contents derived from the original cell, including proteins, lipids, mRNA, miRNA and DNA.2 Exosomes are defined by how they are formed – through the fusion and exocytosis of multivesicular bodies into the extracellular space.
Multivesicular bodies* are unique organelles in the endocytic pathway that function as intermediates between early and late endosomes.3 The main function of multivesicular bodies is to separate components that will be recycled elsewhere from those that will be degraded by lysosomes.4 The vesicles that accumulate within multivesicular bodies are categorized as intraluminal vesicles while inside the cytoplasm – and exosomes when released from the cell.
*Confusingly, there is inconsistency in the literature; while some sources differentiate multivesicular bodies from late endosomes, others use the two interchangeably.
Why are they of interest and what roles do they have?
Exosomes are of general interest for their role in cell biology, and for their potential therapeutic and diagnostic applications. It was originally thought that exosomes were simply cellular waste products, however their function is now known to extend beyond waste removal. Exosomes represent a novel mode of cell communication and contribute to a spectrum of biological processes in health and disease.2
One of the main mechanisms by which exosomes are thought to exert their effects is via the transfer of exosome-associated RNA to recipient cells, where they influence protein machinery. There is growing evidence to support this, such as the identification of intact and functional exosomal RNA in recipient cells and certain RNA-binding proteins have been identified as likely players in the transfer of RNA to target cells.5,6 MicroRNAs and long noncoding RNAs are shuttled by exosomes and alter gene expression while proteins (e.g. heat shock proteins, cytoskeletal proteins, adhesion molecules, membrane transporter and fusion proteins) can directly affect target cells.7,8
Exosomes have been described as messengers of both health and disease. While they are essential for normal physiological conditions, they also act to potentiate cellular stress and damage under disease states.2
How are they generated?
Multivesicular bodies are a specialized subset of endosomes that contain membrane-bound intraluminal vesicles. Intraluminal vesicles are essentially the precursors of exosomes, and form by budding into the lumen of the multivesicular body. Most intraluminal vesicles fuse with lysosomes for subsequent degradation, while others are released into the extracellular space.9,10 The intraluminal vesicles that are secreted into the extracellular space become exosomes. This release occurs when the multivesicular body fuses with the plasma membrane.
The formation and degradation of exosomes.
This is an active area of research and it is not yet known how exosome release is regulated. However, recent advances in imaging protocols may allow exosome release events to be visualized at high spatiotemporal resolution.11
What role do they play in disease?
Exosomes have been implicated in a diverse range of conditions including neurodegenerative diseases, cancer, liver disease and heart failure. Like other microvesicles, the function of exosomes likely depends on the cargo they carry, which is dependent on the cell type in which they were produced.12 Researchers have studied exosomes in disease through a range of approaches, including:
- isolating exosomes from cultured cells and observing their effect in different cell culture studies
- comparing exosomes in various healthy and diseased biofluids
- blocking exosome secretion and observing changes
In cancer, exosomes have multiple roles in metastatic spread, drug resistance and angiogenesis. Specifically, exosomes can alter the extracellular matrix to create space for migrating tumor cells.13,14 Several studies also indicate that exosomes can increase the migration, invasion and secretion of cancer cells by influencing genes involved with tumor suppression and extracellular matrix degradation.15,16
Through general cell crosstalk, exosomal miRNA and lncRNA affect the progression of lung diseases including chronic obstructive pulmonary disease (COPD), asthma, tuberculosis and interstitial lung diseases. Stressors such as oxidant exposure can influence the secretion and cargo of exosomes, which in turn affect inflammatory reactions.17 Altered exosomal profiles in diseased states also imply a role for exosomes in many other conditions such as in neurodegenerative diseases and mental disorders.18,19
Cells exposed to bacteria release exosomes which act like decoys to toxins, suggesting a protective effect during infection.20 In neuronal circuit development, and in many other systems, exosomal signaling is likely to be a sum of overlapping and sometimes opposing signaling networks.21
How can they be used in diagnostics?
Exosomes can function as potential biomarkers, as their contents are molecular signatures of their originating cells. Due to the lipid bilayer, exosomal contents are relatively stable and protected against external proteases and other enzymes, making them attractive diagnostic tools. There are increasing reports that profiles of exosomal miRNA and lncRNA differ in patients with certain pathologies, compared with those of healthy people.17 Consequently, exosome-based diagnostic tests are being pursued for the early detection of cancer, diabetes and other diseases.22,23
Many exosomal proteins, nucleic acids and lipids are being explored as potential clinically relevant biomarkers.24 Phosphorylation proteins are promising biomarkers that can be separated from exosomal samples even after five years in the freezer25, while exosomal microRNA also appears to be highly stable.26 Exosomes are also highly accessible as they are present in a wide array of biofluids (including blood, urine, saliva, tears, ascites, semen, colostrum, breast milk, amniotic fluid and cerebrospinal fluid), creating many opportunities for liquid biopsies.
Therapeutic applications of exosomes
Exosomes are being pursued for use in an array of potential therapeutic applications. While externally modified vesicles suffer from toxicity and rapid clearance, membranes of naturally occurring vesicles are better tolerated, offering low immunogenicity and a high resilience in extracellular fluid.27 These “naturally-equipped” nanovesicles could be therapeutically targeted or engineered as drug delivery systems.
Exosomes bear surface molecules that allow them to be targeted to recipient cells, where they deliver their payload. This could be used to target them to diseased tissues or organs.27 Exosomes may cross the blood-brain barrier, at least under certain conditions28 and could be used to deliver an array of therapies including small molecules, RNA therapies, proteins, viral gene therapy and CRISPR gene-editing.
Different approaches to creating drug-loaded exosomes include27:
- incorporating a drug into exosomes that have been purified from donor cells
- loading cells with a drug that is then contained within exosomes
- transfecting cells with DNA encoding therapeutically-active compounds that are then contained within exosomes
Exosomes hold huge potential as a way to complement chimeric antigen receptor T (CAR-T) cells in attacking cancer cells. CAR exosomes, which are released from CAR-T cells, carry CAR on their surface and express a high level of cytotoxic molecules and inhibit tumor growth.29 Cancer cell-derived exosomes carrying associated antigens have also been shown to recruit an antitumor immune response.30
Methods of isolation and detection
The purification of exosomes is a key challenge in the development of translational tools. Exosomes must be differentiated from other distinct populations of extracellular vesicles, such as microvesicles (which shed from the plasma membrane, also referred to as ectosomes or shedding vesicles) and apoptotic bodies.31 Although ultracentrifugation is regarded as the gold standard for exosome isolation, it has many disadvantages and alternative methods for exosome isolation are currently being sought. Exosome isolation is an active area of research (see Table 1) and many research groups are seeking ways to overcome the disadvantages listed below, while navigating the relevant regulatory hurdles along the way.
Table 1: An overview of exosome isolation methods
|Method||Overview of method||Advantages||Disadvantages|
|Differential centrifugation - including ultracentrifugation (centrifugation at very high speeds)32,33||Attempts to selectively sediment different components. Samples are centrifuged in successive rounds with increasing centrifugation forces and durations to remove cells, cellular debris and macromolecular proteins, followed by ultracentrifugation (at 100,000x g for 70 minutes) to obtain the exosomes in the supernatant.||Suitable for the extraction of exosomes from large samples, such as culture supernatant.|
Produces a low yield and low purity of the isolated exosomes as other types of extracellular vesicles have similar sedimentation properties.
Low efficiency as it is labor-intensive, time-consuming and requires a large amount of sample. specialized equipment. High centrifugal force can damage exosome integrity
|Charge neutralization-based precipitation (commercial kits)33.34||Based on the principle of compound polymerization precipitation. Samples are mixed with a reagent that reacts to form a mesh-like polymeric web that captures exosomes of a certain size.||Produced the highest yield in a comparative study against gel-filtration chromatography and differential ultracentrifugation. Fast, simple steps, requires only a basic centrifuge.||Impurity; certain contaminants that impair subsequent analysis may be co-extracted with the exosomes.|
|Gel-filtration/size-exclusion chromatography (commercial kits)35||Samples are passed through columns that exclude contaminants based on size.||Fast method.||Exosomal protein showed serum protein contamination, limiting proteomic analyses.|
|Affinity purification using immunomagnetic beads (commercial kits)33||Magnetic particles coated with antibodies against exosome-associated receptor molecules are incubated with the sample to form exosome-bead complexes. A magnetic field is applied to induce directional movement and separate the exosomes from the sample.||Target-specific, ensures the integrity of the extracted exosomes, relatively easy and does not require expensive instrumentation. Specific exosome subpopulations can be extracted, based on the expression of specific markers.||Eluting the exosomes from the magnetic beads is a challenge.|
|Stirred ultrafiltration33||An ultrafiltration membrane allows components of specific relative molecular mass to pass through or be intercepted. Externally supplied nitrogen causes the sample to pass through the membrane. ||Less time-consuming than ultracentrifugation and avoids the need for an ultracentrifuge. Suitable for the extraction of exosomes from large samples including culture supernatant. ||Lack of purity of the end product.|
|Double filtration microfluidic device33||A microfluidic device contains two membranes (with pore sizes of 200 and 30 nm in diameter) that work following the principles of size-exclusion chromatography. Components larger than 200 nm are excluded by the membrane, and particles smaller than 30 nm pass into the waste chamber. Particles of a size between 30 and 200 nm remain in the sample chamber.||Inexpensive and more efficient than ultracentrifugation.||Exosomes may be squeezed and damaged during filtration.|
|Nanoplasmon-enhanced scattering36||Antibodies against exosomal membrane markers are conjugated to a silica surface on a sensor chip to capture exosomes in a sample. Antibody-coated gold nanoparticle probes (GNPs) are added and form complexes with the exosomes and are separated using a magnetic field. Under dark-field microscopy, GNPs scatter different colors to reflect the exosomes present.||Rapid, high-throughput, sensitive and specific.||High cost of antibodies. Complex statistical tools are required to detect radiation under dark-field microscopy.|
|Lab-on-a-chip devices||Approaches include acoustic nanofiltration, immunoaffinity, filtration, trapping on nanowires, viscoelastic flow sorting and lateral displacement.37||Scalable for high-throughput applications, low reagent consumption and potential for portability.||Sensitivity and detection limits vary.|
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- Isola, A., & Chen, S. (2016). Exosomes: The Messengers of Health and Disease. Current Neuropharmacology, 15(1), 157–165. https://doi.org/10.2174/1570159X14666160825160421
- Gruenberg, J., & Stenmark, H. (2004). The biogenesis of multivesicular endosomes.Nature Reviews Molecular Cell Biology, 5(4), 317–323. https://doi.org/10.1038/nrm1360
- Piper, R. C., & Katzmann, D. J. (2007). Biogenesis and Function of Multivesicular Bodies. Annual Review of Cell and Developmental Biology, 23(1), 519–547. https://doi.org/10.1146/annurev.cellbio.23.090506.123319
- Harding, C. V., Heuser, J. E., & Stahl, P. D. (2013). Exosomes: Looking back three decades and into the future. The Journal of Cell Biology, 200(4), 367–371. https://doi.org/10.1083/jcb.201212113
- Statello, L., Maugeri, M., Garre, E., Nawaz, M., Wahlgren, J., Papadimitriou, A., Lundqvist, C., Lindfors, L., Collén, A., Sunnerhagen, P., Ragusa, M., Purrello, M., Di Pietro, C., Tigue, N., & Valadi, H. (2018). Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes. PLOS ONE, 13(4), e0195969. https://doi.org/10.1371/journal.pone.0195969
- Behbahani, G. D., Khani, S., Hosseini, H. M., Abbaszadeh-Goudarzi, K., & Nazeri, S. (2016). The role of exosomes contents on genetic and epigenetic alterations of recipient cancer cells. Iranian Journal of Basic Medical Sciences, 19(10), 1031–1039.
- Di Leva, G., & Croce, C. M. (2013). MiRNA profiling of cancer. Current Opinion in Genetics & Development, 23(1), 3–11. https://doi.org/10.1016/j.gde.2013.01.004
- Huotari, J., & Helenius, A. (2011). Endosome maturation: Endosome maturation. The EMBO Journal, 30(17), 3481–3500. https://doi.org/10.1038/emboj.2011.286
- van Niel, G., D’Angelo, G., & Raposo, G. (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews Molecular Cell Biology, 19(4), 213–228. https://doi.org/10.1038/nrm.2017.125
- Bebelman, M. P., Bun, P., Huveneers, S., van Niel, G., Pegtel, D. M., & Verweij, F. J. (2020). Real-time imaging of multivesicular body–plasma membrane fusion to quantify exosome release from single cells. Nature Protocols, 15(1), 102–121. https://doi.org/10.1038/s41596-019-0245-4
- Muralidharan-Chari, V., Clancy, J. W., Sedgwick, A., & D’Souza-Schorey, C. (2010). Microvesicles: Mediators of extracellular communication during cancer progression. Journal of Cell Science, 123(10), 1603–1611. https://doi.org/10.1242/jcs.064386
- Weidle, U. H., Birzele, F., Kollmorgen, G., & Rüger, R. (2017). The Multiple Roles of Exosomes in Metastasis. Cancer Genomics & Proteomics, 14(1), 1–16. https://doi.org/10.21873/cgp.20015
- Mu, W., Rana, S., & Zöller, M. (2013). Host Matrix Modulation by Tumor Exosomes Promotes Motility and Invasiveness. Neoplasia, 15(8), 875-IN4. https://doi.org/10.1593/neo.13786
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- Wu, D., Deng, S., Liu, T., Han, R., Zhang, T., & Xu, Y. (2018). TGF-β-mediated exosomal lnc-MMP2-2 regulates migration and invasion of lung cancer cells to the vasculature by promoting MMP2 expression. Cancer Medicine, 7(10), 5118–5129. https://doi.org/10.1002/cam4.1758
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- Keller, M. D., Ching, K. L., Liang, F.-X., Dhabaria, A., Tam, K., Ueberheide, B. M., Unutmaz, D., Torres, V. J., & Cadwell, K. (2020). Decoy exosomes provide protection against bacterial toxins. Nature, 579(7798), 260–264. https://doi.org/10.1038/s41586-020-2066-6
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