Why Exosomes Are Being Explored as Diagnostic and Therapeutic Tools

In 1983, two separate research groups reported novel observations regarding small, extracellular vesicles released by maturing red blood cells.^1,2 Five years later, the vesicles were named “exosomes” by Rose Johnstone, an author from one of the original studies.³ On reflection, Johnstone describes her group’s early discovery as somewhat “accidental”, explaining they stumbled upon exosomes when they were looking for an appropriate system to identify a specific transport protein.⁴ According to Johnstone, reports of the observation were initially met with disbelieving eyes.

Now, the initial skepticism has been overcome; instead of being considered an artifact, exosome formation is now accepted as a natural phenomenon. As biologists race to unlock the potential of exosomes, research in the field continues to gain momentum; enter “exosomes” in the PubMed search box and you will see over 4000 results from 2020 alone (up from 265 results in 2010). In this article, we provide an overview of the biology of exosomes and explore their potential uses as diagnostic and therapeutic tools – a market now valued at over $41 million and projected to reach $358.91 million by 2027.

The biology of exosomes

Biogenesis of exosomes

Exosomes are a subset of extracellular vesicles that are defined by how they are formed; through inward budding of the endosomal membrane during maturation of multivesicular bodies (MVBs). The inward budding creates intraluminal vesicles, which may follow one of two paths: lysosomal degradation (when the MVB fuses with a lysosome) or release into the extracellular space (when the MVB fuses with the plasma membrane and empties its contents). Upon release, each intraluminal vesicle earns its name as an “exosome”.⁵

Composition of exosomes

With a typical diameter of 30–150 nm, exosomes are the smallest class of extracellular vesicles. They have a double-layered lipid membrane and contain every basic cellular biomolecule, including proteins, lipids, DNA, mRNA and miRNA.^5,6

Biological function of exosomes

Once considered to be cellular debris, exosomes are now recognized as a fundamental mode of cell–cell communication and carriers that deliver bioactive contents (protein, nucleic acid and lipid cargoes) to recipient cells.⁷ Within the field of exosome research, many challenges have hindered the exploration of exosome dynamics; namely, the lack of established markers and the technicality and heterogeneity of isolation protocols.⁸

Nevertheless, there has been steady progress. Early studies suggested exosomes might target specific cells – for example, reports that purified B-cell-derived exosomes specifically bind to follicular dendritic cells but not to other cell types⁹ Collectively, studies indicate that exosomes are taken up by recipient cells via receptor-mediated endocytosis and/or fusion. Upon uptake, they appear to influence many processes such as cell proliferation, apoptosis and polarity.^10–12

Recently, developments in imaging and marker technologies have enabled exosome secretion and internal trafficking events to be captured in donor and recipient cells.^7,13 In a study published in Nature Communications, a novel dual-color reporter revealed the secretion of exosomes at the front of migrating cells, as well as strong pathfinding behavior of cells along the subsequent exosome trails.⁷ Strikingly, cancer cells not only followed the trail too – they actively endocytosed the exosomes. Alissa Weaver, professor of cell and developmental biology at Vanderbilt University and principal investigator of the study, outlines the value of live reporters:

“Live reporters like these can reveal unexpected cellular behaviors by allowing real-time visualization of the secretion and interaction of exosomes with cells. Although we know exosomes are important for directional migration, we were surprised by how closely the cells followed the exosome trails – as if the exosomes were releasing an attracting factor or pheromone.”

The findings support previous studies showing exosomes guide cell migration, adding to the long list of potential biological roles.^14,15 Ultimately, progress in marker technology represents a major step forward for the field of exosome research, explains Weaver: “Our reporter can be used to study both autocrine and paracrine interactions between cells, including in living animals. We expect it will be used in a variety of animal models to understand how exosomes mediate cell–cell communication.”

Exosomes as diagnostic tools

Considering exosomes contain unique cargoes, have a strong presence across almost all biofluids, offer a high biological stability and serve as intercellular messengers, the exploration of their utility as biomarkers in liquid biopsy was inevitable.^16,17 Furthermore, the implication of exosomes in certain pathologies, such as chronic obstructive pulmonary disease, provides further support for their utility as biomarkers.¹⁸

Characterizing the exosome proteome could be useful for identifying amplified signaling pathways indicative of cancer, and for observing the stromal response of tumors and response to cancer therapies.^19,20 Similarly, exosomal DNA (both intraluminal and extraluminal) and RNA could act as cancer-associated nucleic acid biomarkers.¹⁹ The diagnostic potential of exosomes is not limited to cancer; other therapeutic areas are also being explored, such as pain, autoimmune conditions and hypertensive disorders of pregnancy.^21-23

The path to biomarker verification, however, is not straightforward; exosomes are notoriously difficult to isolate and characterize. Ultracentrifugation, the most common method for exosome isolation, suffers from significant drawbacks; it is a multi-step, labor-intensive, and expensive process that can damage exosome integrity. Therefore, other exosome isolation methods are being pursued – and for good reason, says Weaver: “Exosome diagnostics is definitely an area worth pursuing, since exosomes are present in all body fluids and are involved in many aspects of cancer behavior. One avenue that will likely help is the development of purification-free approaches, such as single vesicle flow cytometry or microfluidic capture approaches. Indeed, many groups are working on this.”

Exosomes as drug delivery vehicles

In addition to holding promise as diagnostic tools, exosomes also have many characteristics which make them attractive for drug delivery purposes; they are highly biocompatible, deliver payloads to recipient cells, and in theory, elicit low immunogenicity and cytotoxicity. Ultimately, if their homing abilities were harnessed, and hepatic clearance overcome, exosomes could serve as excellent drug nanocarriers.²⁴ This possibility is being taken seriously in the commercial world; in June 2020, over a billion dollars in exosome-related deals were announced by three large pharmaceutical companies alone.²⁵ Partnerships with gene therapy companies have come hand-in-hand, as the pharma industry seeks new means of delivering therapies to hard-to-reach tissues.²⁵

Improving the specificity of drug delivery reduces the likelihood of potentially toxic off-target interactions, creating new possibilities for previously untreatable diseases. Avoiding the unique toxicities of chimeric antigen receptor (CAR) T-cell therapy, for example, is a major challenge for the field of immunotherapy, with cytokine release syndrome and CAR T-related encephalopathy syndrome the two most commonly noticed toxic effects.²⁶ Furthermore, with their nanoscale size, exosomes may be more ideally suited to solid tumor therapies. With these toxicities and the promising characteristics of exosomes in mind, researchers have begun to explore the idea of exosomes as cancer-targeting agents.²⁷ In a preclinical in vivo model of cytokine release syndrome, exosomes derived from CAR T-cells showed promising antitumor efficacy, expressed a high level of cytotoxic molecules and promisingly, did not cause cytokine release syndrome – supporting the potential use of exosomes as therapeutic strategies against tumors.²⁶

Exosome-inspired therapeutics: Exosome mimetics

Therapeutic extracellular vesicles in clinical trials are typically separated by ultracentrifugation and tangential flow filtration, which may not be scalable for larger studies and commercial manufacturing.²⁸ Therefore, strategies have emerged to combat the challenge of time-consuming purification and low yield, with biotech companies channeling their energies toward improving the technology available for exosome purification. Meanwhile, there is a rising interest in exosome mimetics, i.e., the production of vesicles that retain the major characteristics of exosomes, but offer a greater yield.

For Wafa Al-Jamal, reader in nanomedicine and drug delivery at Queen’s University Belfast School of Pharmacy, Northern Ireland, UK, exosome mimetics represent a way forward in her mission to develop personalized, effective and safe nanomedicines targeting metastatic prostate cancer. “Our vision,” says Al-Jamal, “is to engineer targeted exosome mimetics from patients' blood cells, so the treatment is customized for each patient.” Al-Jamal notes how challenging it has been to deliver effective doses of chemotherapeutics to metastatic lesions – especially in bone – without causing toxicity to healthy tissues. The approach, described recently in the Journal of Controlled Release, aims to improve drug delivery by targeting drug-loaded exosome mimetics to advanced and metastatic prostate cancer lesions by combining the intrinsic affinity of exosome mimetics with active targeting via prostate-specific membrane antigen (PSMA).²⁹

“Our approach is based on filtering whole monocyte cells into smaller cell-mimetic vesicles using different pore size membranes, which generates vesicles similar to naturally-secreted extracellular vesicles (e.g., exosomes) – but accelerates production and increases yield,” explains Al-Jamal. “Moreover, our approach aims to prepare targeted vesicles expressing a prostate-targeting ligand on the cells' surface, eliminating the chemical procedures that would otherwise be required to attach the targeting ligand to the vesicles’ surface.”

Exosome mimetics also provide an opportunity for theranostics, i.e., the combination of diagnostic and therapeutic applications to predict therapy outcomes in animal models and patients. Al-Jamal explains how the co-delivery of diagnostics and therapeutics in a single nanocarrier could be achieved: “Due to the vesicular nature of exosome mimetics, drugs and imaging agents could be co-loaded into the same vesicles. Alternatively, imaging agents could be conjugated to the vesicles’ surface.”

The road to clinical application

The notion that cell-derived vesicles might be key players in intercellular communication has added new dimensions to our knowledge of cell biology. Exosomes are intriguing vesicles, and feature characteristics that make them attractive diagnostic biomarkers and therapeutic carriers. Going forward, understanding the basic biological functions of exosomes in health and disease will be instrumental to enabling clinical progress. Real-time imaging and reporters, such as those described by Weaver’s group, will be useful tools for understanding the autocrine and paracrine functions of exosomes and for tracking therapies such as those developed by Al-Jamal.^13,29 Ultimately, there is a need to understand how specific cargoes drive particular functions, says Weaver: “For cancer in particular, exosomes are known to be important players in driving and maintaining cancer metastasis. Identifying the cargoes that mediate that behavior is important to identifying new therapeutics and biomarkers of disease.”

References

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