February 15, 2015 (Vol. 35, No. 4)

GEN Recently Interviewed a Number of Specialists Involved in Exosome Research

Exosomes are lipid nanovesicles secreted from cells and found in all bodily fluids such as plasma, urine, and cerebrospinal fluid. Originally viewed as garbage disposal systems for cellular debris and proteins, they are now seen as playing a potentially important role in the development of real-time molecular diagnostics, drug delivery vehicles, and tools for biotech research.

GEN recently interviewed a number of specialists involved in exosome research to learn about the promise and capabilities of these fascinating nanovesicles.

GEN: Why have exosomes become such a hot area of research activity?

Ms. Carnell: Exosomes appear to be ubiquitous in a broad range of prokaryotic and eukaryotic organisms, and for many years were regarded simply as cell debris. They are now known to be important in cellular communication and are believed to play a wide role in many physiological and pathological processes. Consequently they have the potential to be disease indicators, holding out the possibility of a role in early diagnostics, the focus of considerable R&D attention. They are also being investigated as drug delivery vehicles.

Dr. Clayton: The concept of exosomes challenges well-established dogma in cell biology, about cell-cell communication, and even perhaps the autonomy of a cell as the basic building blocks of living organisms. Countless studies now provide striking, highly compelling evidence for important functions of exosomes in driving disease processes, (cancer, neurodegeneration, cardiovascular disease, and many others).

Couple these with the idea of isolating exosomes from biofluids, as a source of multiple-molecular markers of disease, and we have a scenario where the field is now established as mainstream. Exosome research will be central to basic and translational biomedical science research in the future.

Dr. Henry: In addition to the potential for noninvasive diagnostics, exosomes and extracellular vesicles can also give us the timeliest information because they represent the state of the cell at that current moment. When we look at tissue, such as a tumor biopsy, we may actually be looking at years of history built up over time. With exosomes, we get a snapshot of what is happening in the body right now.

Exosomes also give us the ability to do liquid biopsies, which means that it’s easier for labs to get samples through blood, urine, saliva, various mucus membranes, etc. Easily obtained samples means faster processing times and potentially faster diagnoses.

Dr. Skog: The appeal of exosomes is their tremendous versatility. With the rise of targeted therapies, particularly for cancer, accurate and swift biomarker detection to match patients with the most tailored treatment is critical. Exosomes enable the detection of mutations, fusions, and splice variants, as well as RNA and protein profiling.

Additionally, exosome-based diagnostics have applications in many diseases beyond cancer, including neurological, endocrine, and cardiovascular disease, among others. In many cases, exosomes are part of the actual disease process, making them interesting from a biological standpoint, with clinical implications both for diagnostics as well as therapeutics.

Dr. Vlassov: Depending on the cell or tissue of origin, many different roles and functions have been attributed to exosomes: eradication of the obsolete molecules, facilitation of the immune response, antigen presentation, programmed cell death, angiogenesis, inflammation, coagulation, dissemination of oncogenes from tumor cells, and spread of pathogens such as prions and viruses between cells.

Importantly, exosomes deliver macromolecular messages (RNA and protein) that enable cell-to-cell communication and signaling. Interest in exosomes, from their function in the body to more practical applications, such as the use in diagnostics and therapeutics development, has grown exponentially in the last five years.

GEN: What are some of their most interesting applications?

Ms. Carnell: Mammalian cells shed exosomes that contain proteins and RNA fragments, which may be detected in various bodily fluids, such as plasma and urine. In the study of malignancies, analysis of exosome contents may help characterize a cancer’s molecular composition and level of progression.

Similarly exosomes are of interest as potential diagnostics indicators for other conditions including neuroinflammation, neurodegeneration, tissue repair, cardiovascular disease, and pre-eclampsia. Both natural and engineered exosomes also have potential application in macromolecular drug delivery, especially for drugs that have low solubility or are easily hydrolyzed. Exosomes produced by bacteria are also interesting from a clinical perspective.

Dr. Clayton: Stem cell exosomes—There are some exciting data showing that exosomes from stem cells hold remarkable tissue regenerative capacity, likely controlling endogenous tissue-resident stem cells and mobilizing their actions in situ.

Drug delivery—Because exosomes can naturally cross the plasma membrane of recipient cells, they may be open to manipulations, delivering therapeutic molecules (small molecules, macromolecules, siRNA, etc.), and this delivery may also be targetable in a selective manner.

Inhibiting exosomes—Many diseases involve a mechanistic contribution by exosomes, and selective inhibition of secretion or exosome action is an evolving area likely to bear fruit.

Dr. Henry: The more we learn about exosomes and extracellular vesicles, and the plethora of information they carry, such as proteins, DNA, RNAs, etc., the closer we move toward both personalizing medicine and improving our ability to provide point-of-care diagnostics and treatment or tailored and targeted therapeutics. We can easily and quickly get a clearer understanding of the events in the body, and this has the potential to open all sorts of novel applications.

Companies are already working on ways to use exosomes for drug delivery, vaccine development and, of course, diagnostics, specifically for various types of cancer. There are also implications for opening up other fields such as microfluidics and multiplexing technologies.

Dr. Skog: Exosome-based approaches will enhance disease detection, diagnosis, treatment, and monitoring. For instance, this year we will introduce several plasma-based tests that analyze exosomal RNA (exoRNA) or exoRNA plus cell-free DNA (cfDNA) to detect mutations in lung and other solid tumors. These diagnostics will enable real-time, serial, longitudinal monitoring that is difficult to accomplish via tissue biopsy.

We’ll also introduce a urine-based prostate cancer test analyzing exoRNA to identify high-grade prostate cancer and help determine whether a biopsy is necessary. There are many interesting potential applications beyond oncology, where exosomal RNA and proteins could be used to noninvasively monitor disease processes.

Dr. Vlassov: Exosomes, and in particular their RNA and protein cargo, may enable minimally or noninvasive next-generation highly specific and sensitive diagnostics and biomarkers. Exosomes definitely have potential as vesicles for the in vivo delivery of various therapeutic cargos—ranging from small molecules to protein and RNA (such as siRNA and miRNA mimics/inhibitors) and DNA. Exosomes/EVs can find applications in the fields completely unrelated to medicine, such as the food industry and environmental testing.

Molecular model of RNA exosome [Laguna Design/Media Bakery]

GEN: What are the major current challenges in exosome research?

Ms. Carnell: Researchers are still working toward a consensus in areas that include nomenclature—a descriptor for exosomes has not been fully defined or agreed—and techniques for exosome isolation, preparation and characterization.

Speciation of sub-populations of exosomes (by origin, markers or cargo for example) is not straightforward, nor is the removal (or understanding) of protein or systematic contaminants. The small size of exosomes demands super-sensitive characterization techniques to achieve the lower limits of detection required. Currently dynamic light scattering, SEM, flow cytometry, and Nanoparticle Tracking Analysis all offer different insights. Developing the methodologies to determine the levels of change in exosome number, size, cargo, or surface markers when moving from normal to disease states is essential for translating this knowledge into useful diagnostic tools.

Dr. Clayton: A bomb-proof definition for exosomes, and some consistency in terminology, preparative, and analytical methods will be helpful to the field. The International Society for Extracellular Vesicles (ISEV) will be central to issues of this nature.

Scientifically, we have only scratched the surface in relation to the dynamic nature of the vesicles, in terms of the quantity released, molecular cargo carried, and the impact of such changes in health and disease. We have little idea of how the vesicles are handled in simple cell systems and certainly not in complex physiological systems; and this will be key if we are to manipulate them therapeutically.

Biomarker studies face difficulties of isolating pure vesicles from fluids like plasma, and sorting through the molecular complexity of exosomes to find informative markers.

Dr. Henry: Major challenges are that our knowledge is still limited and that there are so many different types of extracellular vesicles. At this stage, our classification of extracellular vesicles is limited to size, organelle origin, and sometimes cell state.

As we continue profiling vesicles such as exosomes, we are discovering that not all vesicles have exactly the same composition and may not have the same purpose. Exosomes coming from all different cell types only compound this challenge. Looking at only a single or few markers may not be enough to determine actual cellular origin or the disease state.

Although the technology is coming, to unlock the true potential of exosomes, we must invest in basic research in vesicular trafficking and not focus solely on translational research.

Dr. Skog: The biggest challenge in exosome research is that not all isolation approaches are created equal, and pre-processing and isolation protocols must be tailored to fit the need of the particular application. Biofluid exosomes are a heterogeneous mix of vesicles, and for each application it is important to understand which isolation and vesicle types are needed for maximum sensitivity.

However, since the downstream analytics of exosome content is platform-agnostic, we are fortunate to benefit from the rapidly growing number of sensitive platforms such as NGS, ddPCR, allele-specific qPCR, qPCR, and even sensitive protein analytics platforms. We have been able to leverage this analytic versatility to advance the development of exosome-based diagnostics at a much quicker pace than most people originally envisioned.

Dr. Vlassov: The major challenges include specific isolation of exosomal subpopulations (e.g., based on cargo or function), isolation of other extracellular vesicles, and isolation of exosomes secreted by specific cells/organs/tissue. Analysis of formation of exosomes inside cells, their secretion, trafficking/pathways in the intracellular space, mechanism of uptake by recipient cells, specificity of uptake, analysis of exosomal effects in vitro/in vivo, and biological functions are other areas of increased scrutiny. 

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