Listen in on Cellular Chatter with These Exosome-Analysis Tools

 Exosome-Based Biomarkers

When it comes to bodily fluids, we tend to think of them as homogeneous. A protein is either in the blood serum, say, or it isn’t. Ditto with urine, breast milk and cerebrospinal fluid.

But that isn’t precisely true. Yes, protein (and nucleic acids) do float free in serum, urine and other samples. But some biomolecules are encapsulated within lipid-bound compartments called exosomes or vesicles, and researchers increasingly are mining these as rich veins of molecular candidates for biomarker discovery.

Exosomes, says Andrew Hill, professor of biochemistry and molecular biology at the University of Melbourne, Australia, possess characteristics that make them particularly attractive for biomarker research. For one thing, macromolecules are stable inside a vesicle—nucleases and proteinases floating in circulation cannot harm the cargo inside a vesicle, meaning markers are likely to be stable for some time.

Exosomes also can be concentrated based on surface markers, and because those reflect the cells in which they were made, that provides a strategy for enriching, say, tumor-derived exosomes, says Xandra Breakefield, professor of neurology at Harvard Medical School and Massachusetts General Hospital.

Another obvious benefit, she says, is that exosomes reflect the contents only of live cells—that is, a cell has to actively make an exosome. Thus, researchers can use them to monitor live cells and ignore, for instance, those that are dying due to therapeutic treatments.

In 2013, the U.S. National Institutes of Health, recognizing the potential significance of exosomes, announced $17 million worth of funding for extracellular RNA research. Multiple companies are likewise ponying up dollars for internal research into biomarkers, diagnostics and therapeutics—companies like Exosome DiagnosticsExosome Sciences and Caris Life Sciences.

But to study exosomes, researchers need tools. Here, we review the small but growing exosome toolbox, and what researchers are doing with it.

What is an exosome?

Measuring between 40 and 100 nanometers in diameter, exosomes are extracellular vesicles excreted by most cells in the body. Some may be no better than membranous garbage bags, providing a mechanism for offloading specific macromolecules. Others appear to serve as a form of intracellular communication or manipulation, as exosome content is neither random nor uniform—while some molecules are underrepresented, others are highly enriched.

Researchers have cataloged many of the molecular components of these vesicles, including lipids, proteins and nucleic acids, at sites such as Vesiclepedia and ExoCarta, both developed by Suresh Mathivanan at La Trobe University in Australia.

Still, for a long time, the research community largely ignored exosomes. Then, several years ago, researchers realized exosomes actually provide a potential mechanism for communication of genetic information between eukaryotic cells. As Breakefield explains, nucleic acids are charged molecules that cannot cross cell membranes. “These vesicles provide a way to do that, for one cell to change the transcriptome of another cell through nucleic acid manipulation.”

“Exosomes are kind of like viruses,” says Alexander Vlassov, senior staff scientist at Thermo Fisher Scientific, which sells tools for exosome research through its recently acquired Life Technologies business. But instead of propagating pathogenic genomes, exosomes transmit selected endogenous genetic information from one cell to another.

Take microRNA miR-451, for instance. Tumors “release [miR-451] like crazy,” Breakefield says. “They stuff it in vesicles, possibly because it is inhibitory to growth.” When nearby cells pick up those vesicles, they receive a bolus of that transcript, which in normal cell types can induce a state more conducive to tumor growth and angiogenesis, thereby promoting tumor development.

And that’s just one molecule. According to Breakefield, cells chat all day long, as if over a molecular social network. “It’s like Twitter. They are swapping vesicles with one another, [producing] a huge exchange of information.”

According to Vlassov, exosomes have other functions, too, including elimination of obsolete molecules, antigen presentation and angiogenesis. Some 2,700 papers have been published on exosomes, according to PubMed, 1,700 of them since 2010.

Still, there’s much researchers don’t understand: How cells select exosome cargo, how exosomes are made, how they’re taken up and how many different types there are, for instance. “We haven’t even figured out a great nomenclature for the members of this clan yet,” Breakefield says.

Biomarker successes

While exosomal biomarker have shown promise in clinical studies (particularly with respect to tumors), thus far none have reached the clinic, Hill says. But one should be there soon.

Exosome Diagnostics has identified a three-transcript panel that uses urine exosomes to predict whether a patient has aggressive prostate cancer, thereby hopefully reducing unnecessary biopsies. “It’s not a detection test, it’s a rule-out test,” explains chief scientific officer Johan Skog. “It has a high negative predictive value. It’s very good at telling you are not likely to have an intermediate or high-grade prostate cancer.” According to Skog, the company will launch that test, called EXO106, some time in 2014.

Others are focusing on exosome protein content. Lance Liotta, codirector of the Center for Applied Proteomics and Molecular Medicine at George Mason University, for instance, with colleague Virginia Espina, has used exosomes isolated from cultured retinal pigment epithelia and reverse-phase protein microarrays to identify phosphoproteins associated with the development of macular degeneration [1].

The study, Liotta says, identified hundreds of proteins related to signaling pathways that could play a role in disease etiology—proteins that represent both potential therapeutic targets and diagnostic biomarkers. “We are now planning a larger scale analysis of human vitreous samples this summer though an ongoing collaboration with Francesco Facchiano of the L'Istituto Superiore di Sanità.” But that will not be a treatment trial, he says. “We will gather biomarker data and correlate this with the disease state of the retina.”

Hakho Lee and Ralph Weissleder of Massachusetts General Hospital focus on the proteins found on exosomes themselves. Working with Breakefield, the pair in 2012 demonstrated that antibodies to three proteins—the cell-surface protein CD63 (a pan-exosomal marker), the receptor EGFR and a tumor-specific mutant of that protein called EGFRvIII—can be used to assess tumor burden and treatment efficacy in glioblastoma multiforme. That study used a bespoke “miniaturized nuclear magnetic resonance” microfluidics system, labeling exosomes first with antibodies to select markers, and then with magnetic nanoparticles that could be detected by measuring the resulting 1H NMR signal [2].

More recently, Lee and Weissleder showed they could use antibodies to CD63, EpCAM and CD24 to monitor ovarian cancer in blood serum via a microfluidics-based surface plasmon resonance approach [3].

According to Lee, the team’s findings, though preliminary, suggest exosomes can be used as a relatively noninvasive biomarker for tumor burden and treatment efficacy. “It confirms that exosomes do reflect the signature of the proteins on the primary tumor, and we can use those properties to use exosomes as tumor diagnostics … instead of a more invasive tissue biopsy.”

Working with exosomes

The traditional method of exosome isolation is differential ultracentrifugation, the method Lee and Weissleder used in their studies. “It’s the gold standard, really,” says Hill. In this approach, the sample first is pelleted at relatively low speed to remove cellular debris, and then spun again at progressively higher speeds until a final 100,000 x g for an hour and a half or so pellets the exosomes. The result is a crude exosome preparation suitable for functional assays or biomarker discovery.

But ultracentrifugation is neither fast nor high-throughput. It also is difficult to translate into a clinical setting, says Skog, as it requires standardizing such variables as rotor selection, centrifugation conditions and how long the rotor sits following the spin. As a result, companies have developed alternative approaches.

Hill’s lab has tested several alternatives. In one paper, his team compared RNA from exosomes prepared by ultracentrifugation to RNA prepared directly using a spin-column-based Norgen Biotek exosomal RNA isolation kit. The team’s analysis detected some apparent RNA contamination with the commercial kit when applied to plasma, but not serum [4].

Exosome Diagnostics also sells kits for RNA enrichment based on spin-column concentration. Thermo Fisher Scientific’s Total Exosome Isolation Reagent line uses a proprietary polymer that causes the vesicles to precipitate, enabling collection with a rapid spin. Or, researchers can collect specific exosomal subsets using Thermo’s antibody-conjugated Dynabeads®, with antibodies targeting CD63 or other user-specified antigens. (HansaBioMed’s ExoTEST™ assays serve the same purpose.)

Other reagents are available to fluorescently label exosomes, isolate their cargo, and more. The point is, the exosome toolbox is filling rapidly, and those tools are powering exosome research at a furious pace. With exosome-based diagnostics just over the horizon, Vlassov says, “I’m pretty sure that after one success, this field will explode.”

References

[1] Biasutto, L., et al., “Retinal pigment epithelium (RPE) exosomes contain signaling phosphoproteins affected by oxidative stress,” Exp Cell Res, 319:2113–23, 2013. [PubMed ID: 23669273]

[2] Shao, H., et al., “Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy,” Nat Med, 18:1835–40, 2012. [PubMed ID: 23142818]

[3] Im, H., et al., “Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor,” Nat Biotechnol, published online April 20, 2014. [Nature Biotechnology]

[4] Cheng, L., et al., “Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood,” J Extracell Vesicles, 3:23743, 2014. [PubMed ID: 24683445]

Image: Extracellular RNAs, or ExRNAs, travel in body fluids including fluid surrounding the brain and spinal cord, urine, and blood. Credit: NIH Common Fund.

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