The LCGC Blog: Exosomes – A New Frontier in Cancer Biomarker Discovery and Quantitation

Exosomes are small lipid membrane-bound extracellular vesicles, on the order of 30–150 nm in diameter, which are shed by normal and tumor cells in the body. They are circulating within your body and can be isolated from virtually any biological fluid. Exosomes released from tumor cells have been shown to be enriched in certain proteins. These nanobodies hold significant promise for the discovery of cancer biomarkers, for cancer diagnosis and prognosis, and for biomarker quantitation.

I should qualify the term “new” in the title. Exosomes have been under investigation for some time. There are many good reviews on the nature of their existence and their isolation from biological samples (1–3). Exosomes are believed to be a means of cellular communication in the body. Their load of proteins, metabolites, and nucleic acids can be delivered to other cells upon fusion with their plasma membrane. However, at this time little appears to be known about specific mechanisms and specific consequences associated with this communication and transport of cellular material. It is clear that, for cancer, exosomes are involved in primary tumor growth and metastatic evolution (4).

Exosomes can be isolated from biological fluid, typically using ultracentrifugation (UC), ultrafiltration (UF), or size exclusion chromatography (SEC). Sometimes the combination of multiple isolation steps can be advantageous (5). Other means of isolating exosomes, including microfluidic and immunoaffinity-based methods, are also under development (3).

Exosomes are enriched in cellular membrane and cytosolic proteins. They do not generally contain proteins originating from other cellular machinery. Many transmembrane proteins are involved in disease pathogenesis. When originating from cancer cells, exosomes carry a protein signature of that cell. Thus, they are a source of cancer biomarkers specific to a given cancer. As such, exosomes isolated from circulating fluids have often been referred to as liquid biopsies. They are naturally enriched in these biomarkers relative to the biological fluid from which they are isolated; they are also cleaner than the biological fluid, as they have less background material.

Since exosomes are enriched in various signature proteins associated with the cells from which they are shed, the comparison of protein profiles from different cancer cell lines ought to lead to the discovery of new cancer biomarkers. Systematic evaluation of exosomes from various tumor versus normal cell lines (or cancer versus healthy patients) can easily be envisioned. Once the exosomes are isolated, the workflow associated with that discovery could encompass any number of proteomics-based analytical and informatics approaches.

Monitored changes in levels of validated (or, initially, candidate) biomarkers ought to be very useful for cancer diagnosis, prognosis, and treatment. For panels of multiple biomarkers, isolated exosomes may provide an indication not only of the presence of cancer, due to aberrant levels of various proteins relative to normal, but also the location of the cancer, due to a unique signature of the proteins. The extent of up- and down-regulation of the markers can be related to the severity of the disease at the time of monitoring. And of course, once treatment is undertaken, the levels of markers can be continually monitored to gauge the effectiveness of the treatment. Throughout this process, a key advantage of the use of exosomes or liquid biopsies could be that traditional tissue biopsies could be avoided.

Finally, the fact that proteins of interest are significantly enriched in exosomes, and that the background of potentially undesired proteins is reduced, provides a much improved sample matrix for biomarker quantitation relative to raw biological fluid. In our own efforts to demonstrate top-down quantitative analysis of intact proteins using liquid chromatography – tandem mass spectrometry (6,7), we have found that to achieve limits of detection more relevant for determination of the lowest level biomarkers, some sample enrichment will be necessary during sample preparation. In my view, as I have written recently (8), there are limited offerings commercially available for preparation of intact proteins. It may very well be that isolation of exosomes would provide the necessary enrichment needed in order to avoid extensive additional treatment of samples prior to analysis.

In my cursory discussions with scientists whom I encounter on a regular basis, I have often asked them what they know about exosomes. Many are still unfamiliar with the term and the promise held by these nanoscale biological particles. That said, research into the isolation and use of exosomes for cancer research is growing steadily. It is exciting to contemplate the possibilities they may provide for diagnosis, prognosis, and treatment of cancer, as well as for understanding and manipulating other interesting biological functions.

References:

1. G. Raposo and W. Stoorvogel, J. Cell Biol. 200, 373–383 (2013).
2. M. Record, K. Carayon, M. Poirot, and S. Silvente-Poirot, Biochim. Biophys. Acta1841, 108–120 (2014).
3. H. Shao, H. Im, C.M. Castro, X. Breakefield, R. Weissleder, and H. Lee, Chem. Rev.118, 1917–1950 (2018).
4. A. Becker, B.K. Thakur, J.M. Weiss, H.S. Kim, H. Peinado, and D. Lyden, Cancer Cell30, 836–848 (2016).
5. M. An, J. Wu, J. Zhu, and D.M. Lubman, J. Proteome Res.17, 3599–3605 (2018).
6. E.H. Wang, P.C. Combe, and K.A. Schug, J. Amer. Soc. Mass Spectrom.27, 886–896 (2016).
7. D.D. Khanal, Y.Z. Baghdady, B.J. Figard, K.A. Schug, Rapid Commun. Mass Spectrom.33, 821–830 (2019).
8. K.A. Schug, The LCGC Blog, May 1 (2019). http://www.chromatographyonline.com/lcgc-blog-commercial-sample-preparation-materials-isolation-intact-proteins-biological-samples-are-a

Kevin A. Schug is a Full Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington. He joined the faculty at UT Arlington in 2005 after completing a Ph.D. in Chemistry at Virginia Tech under the direction of Prof. Harold M. McNair and a post-doctoral fellowship at the University of Vienna under Prof. Wolfgang Lindner. Research in the Schug group spans fundamental and applied areas of separation science and mass spectrometry. Schug was named the LCGC Emerging Leader in Chromatography in 2009 and the 2012 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science. He is a fellow of both the U.T. Arlington and U.T. System-Wide Academies of Distinguished Teachers.