No longer thought to be just a cell’s way of getting rid of garbage, interest in exosomes and their role in cell-cell communication is at an all-time high. Their potential in research and clinical applications is thought to be huge. But the very basics of exosome biology are largely unknown. Here we review what is known and the status of exosomes for prognostic and therapeutic use, in addition to being biomarkers for health and disease.
What are they?
Exosomes are small extracellular vesicles (ECVs), consisting of a lipid bilayer surrounding a lumen, found in a variety of biofluids as well as in cell culture supernatants. Beyond that there is no standard, universally agreed-upon definition in terms of origin or defining characteristics, although the field seems to be headed toward at least a thumbnail set of practical criteria.
Exosomes are generally thought to be derived from multivesicular endosomal bodies that released their contents into the extracellular space upon fusion with the plasma membrane. The size range quoted by researchers varies, but typically falls between 30–150 nm in diameter. Exosomes are made up of all manner of biomolecule from the originating cell including lipids, membrane and cytosolic proteins, micro and messenger RNA, single- and double-stranded DNA, as well as metabolites.
Ultracentrifugation is the traditional way to isolate—or at least enrich for—exosomes. Size exclusion, hydrophobicity, density centrifugation, and other methods are employed in various kits and protocols as well, sometimes in combination.
Affinity capture methods allow for separation based on common exosomal markers such as certain tetraspanin proteins, rather than size and other physical parameters. Yet all markers to date have also been found on other vesicles, and no single marker has yet been demonstrated to be shared by all exosomes. James Edgar suggests in a review that perhaps exosomes can be defined biochemically by the relative levels of a combination of markers.
Each isolation method affords its own strengths and limitations, and each will yield a distinct but largely overlapping set of vesicles, notes Eva Surmann, product manager at Horizon Discovery. Without a reference standard it would be difficult (if not impossible) to assess the performance of the used extraction technique.
In the clinic
“If people are looking for biomarkers, or to use these vesicles for diagnosis, it doesn’t matter whether they are ‘exosomes’ or other vesicles: as long as they give the information needed to characterize the patient, that’s fine,” says Stefan Wild, senior scientist research and development at Miltenyi Biotec. “It must just be reproducible.”
There are about 200 trillion exosomes circulating in a healthy adult, and perhaps twice that number in the blood of a cancer patient, says Raghu Kalluri, chairman and professor of the department of cancer biology at the University of Texas MD Anderson Cancer Center, who uses the term loosely to mean vesicles from cells.
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“Pretty much anything in the cell has been found in the exosome at one point or the other, depending on how the experiment was done,” he points out. “They can give a window into the cells … to probe into the health or disease status of an individual.”
For example, exosomes from a pancreatic cancer patient will contain DNA with mutations associated with that cancer, along with differences in mRNA and miRNA as well. “I can sample the exosomes before therapy and after therapy to see if there has been any impact,” Kalluri says. “That’s called ‘liquid biopsy’.” Exosomes are also being used or explored in fields including neonatal screening, heart disease, and disorders of the central nervous system.
In contrast to circulating tumor cells, which are very rare and may thus represent only a single or perhaps two tumor clones in a blood draw, exosome’s abundance can give a perhaps more realistic representation of a tumor’s diversity, points out Ángel Picher, director of genomic research at Expedeon.
In the lab
From a basic research point of view, of course it makes a difference what an exosome is, says Wild. “Because then you want to know where these vesicles come from and for what purpose.”
It’s now widely accepted that exosomes can facilitate cell-to-cell communications.
It’s now widely accepted that exosomes can facilitate cell-to-cell communications. They are secreted by all cell types examined in culture. They can deliver contents by fusing with or becoming engulfed by the plasma membrane of the recipient cell or transmit signals by binding to surface receptors.
“It’s pretty clear that cancer cells interact with their surrounding cells via exosomes, and prepare their niche to survive within the body,” Wild notes. They can suppress the immune system, for example, promote angiogenesis, or enable metastasis.
Exosomes have been shown to carry cargos such as peptides implicated in the neurodegenerative Alzheimer’s and Parkinson’s diseases. And oncogenic horizontal transfer of mitochondrial and genomic DNA, mRNA, and miRNA, by exosomes has also been demonstrated. A database of molecules identified in exosomal experiments can be found at www.exocarta.org.
While exosomes’ contributions to a few pathologies are slowly being unraveled, their normal, physiological roles outside the immune system remain largely unknown, points out Kalluri. This is due in large part to the difficulties of isolating them from other small particles—even by size, since they are too small to be resolved by conventional light-scattering techniques such flow cytometry—and to “proving” their biological origin after the fact.
Image: Flow cytometry automation helps accelerate discovery of new exosome biomarkers. Image courtesy of Miltenyi Biotec.
The Future of exosomes
The biology of exosomes remains largely in its infancy, but their promise for medicine remains unabated. Companies have been established to explore their use as diagnostics, and there is hope that they can be used for screening in the not-too-distant future. Techniques such as electron microscopy allow them to be seen in high resolution, while imaging flow cytometry lets them be visualized in high throughput—with both allowing for staining of specific molecules. And exogenously produced exosomes are even being explored as delivery vehicles.