Intercellular communication is an essential function for multicellular organisms to maintain homeostasis. We have long known that direct cell-to-cell interactions and secretion of signaling molecules are key pathways for communication between cells. However, within the last decade there has been an increased interest in the role of extracellular vesicles (EVs), especially exosomes, in this process. While exosomes were initially discovered in 1983 (1,2), their role in intercellular communication and disease pathogenesis was initially overlooked. In fact, the term exosome has been incorrectly used to describe an array of extracellular vesicles without consideration to the differences in origin and size between exosomes and other EVs. Generally, there are three classes of EVs: microvesicles (formed by outward budding of the plasma membrane, size 50-1000 nm), apoptotic bodies (formed by membrane blebbing of an apoptotic cell, size 500-2000 nm), and exosomes.
Exosomes are distinct from microvesicles and apoptotic bodies in that they are of endocytic origin. Endocytosis from the plasma membrane leads to the formation of an early endosome in the cytosol. Inward budding of the endosomal membrane fills the lumen of the early endosome with appropriately named intraluminal vesicles. Sequestering of cargo to be included in intraluminal vesicles and the scission of exosomes from the endosomal membrane is mediated by two pathways: an endosomal sorting complex required for transport (ESCRT)-dependent pathway (3) and an ESCRT-independent pathway (4). The late endosome, now filled with intraluminal vesicles, is termed a multivesicular body (MVB). MVBs can then undergo one of three fates: fusion with a lysosome leading to the degradation of the MVB, contribute to the formation of specialized organelles (e.g. melanosomes), or it can fuse with the plasma membrane of the cell. Fusion with the plasma membrane allows the intraluminal vesicles to be released into the extracellular space. Only these intraluminal vesicles that are released in the extracellular space should be called exosomes.
Once exosomes are released into the extracellular space, they can interact with surrounding cells to evoke a variety of downstream effects. For instance, exosomes released from Epstein-Barr virus transformed B-lymphocytes bear MHC-II molecules that can interact with T cells and induce antigen-specific T cell response (5). Furthermore, exosomal cargo includes membrane proteins, cytosolic proteins, lipids, mRNA and miRNA that can alter gene expression. Their ability to alter gene expression in surrounding cells is of particular importance in cancer progression, metastasis, and resistance to cancer drugs (6). Additionally, exosomal proteins are being researched as prognostic and diagnostic biomarkers, which could allow for earlier detection of diseases (7).
Like microvesicles and apoptotic bodies, exosomes can contain surface markers from distinct cell types. For example, exosomes from colon cancer cell line LIM1215 express A33, a marker of colon epithelial cells (8). In addition to these cell type specific markers, there are several general markers common to exosomes, regardless of the cell type of origin The most widely used markers for the identification of exosomes are members of the tetraspanin protein family: CD9, CD63, and CD81, that are associated with exosomal cargo sorting (9,10). However, these tetraspanin proteins are not specific markers for exosomes as they are often associated with other extracellular vesicles as well.
In fact, isolating and characterizing exosomes has proven to be a difficult process. The traditional method of purification, differential ultracentrifugation, can separate extracellular vesicles based on size. However, this method cannot distinguish between EVs of the same size nor does it remove macromolecular aggregates from the sample. Therefore, differential ultracentrifugation alone can only provide exosome enriched samples, not purified exosome samples. Thus, differential ultracentrifugation must be used along with other isolation and characterization techniques to obtain purified exosome samples. The International Society of Extracellular Vesicles has provided a guideline for the minimal requirements of isolating and characterizing EVs (11).
To add to the tools available to researchers for exosome research, Chondrex, Inc. is currently developing a line of exosome isolation reagents. This includes a column-based system for the purification of intact exosomes from a variety of sources. Please subscribe to our mailing list to receive updates on this product line!
References
1. B.T. Pan, R.M. Johnstone, Fate of transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33, 967-978 (1983).
2. C. Harding, P. Stahl, Transferrin recycling in reticulocytes: pH and iron are important determinants of ligand binding and processing. Biochemical and Biophysical Research Communications 113, 650-658 (1983).
3. W. H. Henne, N.J. Buchkovich, S.D. Emr, The ESCRT Pathway. Developmental Cell 21, 77-91 (2011).
4. S. Stuffers, C. Sem Wegner, H. Stenmark, A. Brech, Multivesicular endosome biogenesis in the absence of ESCRTs. Traffic 10, 925-937 (2009).
5. G. Raposo et al., B lymphocytes secrete antigen-presenting vesicles. The Journal of Experimental Medicine 183, 1161-1172 (1996).
6. A. S. Azmi, B. Bao, F. H. Sarkar, Exosomes in cancer development, metastasis and drug resistance: a comprehensive review. Cancer and Metastasis Review 32, 9441-9449 (2013).
7. J. Lin et al., Exosomes: Novel Biomarkers for Clinical Diagnosis. The Scientific World Journal 2015, 8 (2015).
8. S. Mathivanan et al., Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue specific protein. Molecular & Cellular Proteomics 9, 197-208 (2010).
9. G. van Niel et al., The tetraspanin CD63 regulates ESCRT-independent and dependent endosomal sorting during melanogenesis. Developmental Cell 21, 708-721 (2011).
10. D. Perez-Hernandez et al., The intracellular interactome of tetraspanin-enriched microdomains reveals their function as sorting machineries toward exosomes. Journal of Biological Chemistry 288, 11649-11661 (2013).
11. J. Lotvall et al., Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. Journal of Extracellular Vesicles 2014, (2014).