By Ryan Wallis, PhD student, QMUL, Blizard Institute
The microscopic world of cell biology was first explored by Robert Hooke in the 17th century and published in his work, Micrographia, in 1665. He marvelled at the revelation that an organism such as a cork plant was actually comprised of tiny “Cellula” resembling the small chambers occupied by monks. His insight into this invisible domain was the first in a line of discoveries which have transformed our understanding of biology, health and disease. As technology has progressed, scientific discovery has flourished, and the invention of electron microscopy has allowed us to observe the world on an even smaller scale. Exosomes are tiny structures ̶ a thousand times smaller than a typical cell – which were discovered in 1983 by Rose Johnstone (Pan and Johnstone, 1983). These nanoscale, membrane-bound vesicles are secreted by all cell types and were initially thought to function as a waste disposal system, a kind of “cellular trash can” (H. Rashed et al., 2017). However, more recently, it has been established that exosomes contain and deliver a range of biologically active molecules, contributing to the signalling which underlies both normal biological functions, such as the immune system, as well as pathological processes, such as cancer and neurodegeneration (Zitvogel et al., 1998; Wada et al., 2010; Vella, Hill and Cheng, 2016; Soria et al., 2017). Research into these novel mediators of cellular communication has grown exponentially over the past decade and many hope it will present a new route for therapeutic innovation. So, let us delve into a world a thousand times smaller than that observed by Hooke – the world of exosomes.
The Discovery of Exosomes
The circumstances which led to the discovery of exosomes may be best described as serendipitous. In her original study, Rose Johnstone was investigating the loss of the transferrin receptor from the surface of reticulocytes as they matured. By labelling transferrin with gold particles, she was able to observe the internalisation of the receptors into endosomes, but, more specifically, into internal vesicles approximately 50 nm in diameter. These “intraluminal vesicles” were then released by the cells via exocytosis and so termed “exosomes”(Pan and Johnstone, 1983; Johnstone et al., 1987). This discovery sits alongside many others in science which occurred when perceptive investigators observed a phenomenon which was, up to that point, unanticipated. Perhaps the most famous example of this is Alexander Fleming’s discovery of penicillin, serving as a reminder that, as scientists, we should always be on the lookout for the unexpected. However, it would be over 20 years before exosomes began to be seen as anything more than a waste disposal system, when it was reported that the exosomes produced by B-lymphocytes could invoke responses in T-cells via the display of MHCII molecules on their surface (Raposo et al., 1997). Since these early days, our understanding of exosomes and their functions has increased exponentially and we now have a far greater understanding of where these vesicles come from and the role they play in health and disease.
In simple terms, exosomes can be thought of as miniature versions of cells. Firstly, formation of an endosome occurs with inward budding of the plasma membrane leading to an internal structure with an identical membrane composition, only inside-out. Inward budding of this endosomal membrane leads to the formation of tiny “intraluminal vesicles” which, again, have an identical composition to the plasma membrane but, this time, with the correct topology. During the formation of these vesicles cytoplasmic content is taken up, resulting in a cargo which is representative of their parental cell. In this way, the vesicles exist as a “snapshot” of the parental cell, at the time of their production. Endosomes containing these vesicles have been termed “multivesicular bodies” and must undergo one of two fates. Either they may traffic to lysosomes for enzymatic degradation, or they may traffic to the plasma membrane, where the vesicles are released as exosomes (Trajkovic et al., 2008). This complex biogenesis pathway distinguishes exosomes from other extracellular vesicles, such as microvesicles (which bud directly from the plasma membrane) or apoptotic bodies (which randomly form as cells are undergoing apoptosis) (Willms et al., 2016).
Exosomal Cargo and Functions
As we have seen, exosomes derive their contents from their parental cell; however, enrichment in certain proteins has also been reported. Tetraspanins – including CD63, CD81 and CD9 – are found abundantly in exosomes and have been implicated in their formation (Andreu and Yáñez-Mó, 2014). The machinery which allows the vesicular scission from the endosomal membrane is the “endosomal sorting complex required for transport” (ESCRT). This machinery contains components such as ALIX and TSG101, both of which are demonstrated to be enriched in exosomes (van Niel, D’Angelo and Raposo, 2018). To date, these are the canonical proteins which are often used to identify exosomes; however, the lack of specific markers is a major hurdle within the field (Lotvall et al., 2014). It has also been demonstrated that functional proteins are packaged inside, or displayed on the membrane of, exosomes, propagating responses often associated with the parental cells. This includes the display of antigen specific markers on exosomes from dendritic cells or the release of TGF-Beta positive exosomes from cancer cells, aiding immune evasion (Raposo et al., 1997; Wada et al., 2010). It is important to note that, as exosomes are produced by almost all cell types, their contents and functions are likely to be equally diverse (Edgar et al., 2016).
Another area which has garnered considerable attention is the inclusion of nucleic acids within exosomes. This was first described by Valadi and colleagues in 2007 but generated an explosion of interest in 2010, when four groups independently demonstrated the transfer of functional miRNA between cells, via exosomes (Kosaka et al., 2010; Pegtel et al., 2010; Zhang et al., 2010). This entirely novel means of cellular communication has been the most exciting revelation in a field still in its infancy. We have only begun to scratch the surface in understanding the role of these vesicles, but it is clear that they have the potential to propagate an enormously varied array of responses – both physiological and pathological.
What does the future hold for Exosomes?
As with any burgeoning field, there are many challenges facing researchers wishing to study exosomes. Until recent efforts by the “International society for extracellular vesicles” (ISEV), even the nomenclature was inconsistent, with many different terms being used for the same vesicles (Gould and Raposo, 2013). The nanoscale size of exosomes means that all but the most expensive and technically challenging microscopy techniques are appropriate and the lack of specific markers means flow-cytometry is far more challenging than with cells. Furthermore, whilst nanoparticle tracking has made the characterisation of exosome preparations more straightforward, the lack of standardisation with regards to their isolation remains a challenge for the field (Ramirez et al., 2018). Recent attempts to encourage consistency in reporting, such as the EV-Track platform, are commendable; yet, the technical challenges of studying these key mediators of cellular communication is likely to always be limiting (Van Deun, Hendrix and EV-TRACK consortium, 2017). Currently, one major area of interest is the use of exosomes as clinical biomarkers of disease, providing an opportunity to detect conditions early on or to differentiate between those which present similar symptoms (Kawikova and Askenase, 2015). The use of exosomes as natural drug delivery vectors is also being explored, most notably in the idea of loading exosomes with a therapeutic agent – a potentially revolutionary concept (Johnsen et al., 2014). Clearly, the potential of these vesicles is massive and their study is a crucial aspect of modern biomedicine. As technologies advance, it is hoped we will be able to gain a greater insight into the nanoscale world of exosomes and perhaps reveal novel treatment strategies to conditions which currently elude us.
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