The Art of the Exosome

What we now know of as exosomes, were first observed decorating the outside of cells around 50 years ago, but at the time these were widely dismissed as no more than cellular debris. Even when it was recognized that intraluminal vesicles were secreted from the cell through plasma membrane fusion in 1983, their importance was still disregarded as being no more than the waste products of a cell. They were referred to as ‘exosomes’ for the first time in 1987, but it took until the early 21st century – when researchers began to investigate what they might contain and why – for their true importance to be better appreciated and understood.

Exosomes have since been found to play important roles in many biological processes, including communication, immune response, tissue regeneration, and tumor progression. They are also being studied for their potential use as biomarkers for various diseases, as well as for therapeutic potential in treating diseases such as cancer, neurodegenerative disorders, and inflammatory conditions. It is evident that these tiny vesicles should not have been overlooked.

What are Exosomes?

Exosomes are one of the two types of extracellular vesicles (EVs) released by the cells of prokaryotes and eukaryotes. [Ectosomes - the second type of EV - are produced by ‘pinching off’ the plasma membrane surface through outward budding.] Originally of endosomal origin, exosomes are secreted from cells when an intermediate compartment (sometimes referred to as a multivesicular body or MVB) fuses with the plasma membrane. As a result, the intraluminal vesicles (ILVs) contained within are released into the extracellular milieu and secreted as exosomes. Ranging in size from 30 to 160 nanometers in diameter, exosomes contain a wide assortment of biomolecules, including proteins, lipids, DNA, miRNA, and mRNA. Their primary function is intercellular communication by transferring their contents from the originating cell to other cells, both nearby and distant. Crucially, this transfer of information can affect the function and behavior of any recipient cells.

How are Exosomes Made?

First, an early sorting endosome (ESE) is produced after the first invagination of the plasma membrane occurs. ESEs may contain cell surface and extracellular milieu proteins or materials from the trans-Golgi network and endoplasmic reticulum. A matured form of ESEs (called late-sorting endosomes, or LSEs) goes on to develop into MVBs after the second inward invagination of the endosomal limiting membrane. These may be degraded when fusing to autophagosomes or lysosomes. Alternatively, through MVB docking and fusion to the plasma membrane, exocytosis occurs and the ILV contents are released as exosomes.

Proteins that are involved with the process of exocytosis are often found on the exosome surface, and thus are considered exosome markers: Alix (apoptosis-linked gene 2-interacting protein X), flotillin, CD63, CD9, CD81, TSG101, and canonical Heat Shock Proteins (HSPs).

The Importance of Exosomes

Exosomes and Diagnostics

As exosomes are present in a variety of biological fluids, are released from all cells, and contain important information regarding the state of their parent cells, they offer themselves as perfect candidates for non-invasive liquid biopsies to monitor diseases. This currently includes cancer and diseases targeting the central nervous and cardiovascular systems, and research into the use of exosomes in diagnosing liver, lung, and kidney diseases is also underway. Isolated exosomes have also proved themselves as a useful tool in a variety of assay formats such as immunohistochemistry, western blot, PCR, proteomic analysis by mass spectrometry, and structural or functional studies.

We have only begun to scratch the surface of the potential for this type of application. Exosomes carrying specific oncogenic and tumor suppressor miRNAs could offer insight into cancer prognosis because the expression of these miRNAs differs between normal and cancerous cells. In colon, colorectal, pancreatic, breast, oesophageal, and ovarian cancer, it has been observed that there are increased levels of circulating exosomal miR-21. Similarly, exosomes derived from urine have been found at elevated levels in prostate and bladder cancer. Tumor-suppressor miRNAs such as miR-146a and miR-34a have also been found in correlation with breast, colon, liver, and pancreatic malignancies. Exosomes have also been used to characterize the neuroinflammatory signaling cascade in stroke victims and their role in transporting pathogenic protein aggregates between cells may support their use as biomarkers for neurodegenerative diseases such as Alzheimer’s Disease.

Therapeutic Application of Exosomes

As a naturally occurring entity, exosomes can cross biological barriers and maintain their function with limited immune clearance and toxicity when introduced to new environments. Scientists can harness properties such as these to use exosomes as vectors in delivering drug molecules or other therapeutic agents.

Exosomes derived from fibroblasts, mesenchymal and dendritic cells can initiate immunomodulation and neoantigen presentation, or the delivery of a drug to targets such as immune, cancer, or parenchymal cells. The ligands expressed by exosomes on their surface can also be engineered to customize their specificity towards a target cell. A prime example of this is exosomes derived from dendritic cells and enriched with the addition of an αv integrin-specific RGD-modified peptide and doxorubicin, a construct that has demonstrated a therapeutic response in mice with mammary tumors. Similar results were seen in mice with lung tumors in response to exosomes derived from macrophages loaded with paclitaxel.

Specific miRNA or small interfering RNA (siRNA) can also be used as payload molecules to suppress gene expression. One instance of this is exosomes derived from dendritic cells modified with RVG (rabies virus glycoprotein) and Bace1-targeting siRNA were shown to inhibit the expression of BACE1 in the brains of mice. Methods such as this could have ground-breaking consequences for diseases of the central nervous system and cancer.

Challenges of Exosome Isolation

Regardless of the application intended for use, there can be obstacles to overcome when attempting to isolate exosomes from biological fluids. Exosomes have a high heterogeneity, with each differing in their origin, size, surface markers, and cargo type. It can also be difficult to separate them from nanoscale contaminants such as retroviruses and lipoproteins.

Ultracentrifugation (>100,000g) has been considered the gold standard for isolation for years, yet this requires working with expensive specialized instrumentation and necessitates the use of large volumes of material that may not be easily attained, especially with the smaller patient sample volumes used in diagnostics. The high levels of force involved can also mechanically damage the integrity of exosomes, which could present issues if bioactivity is to be maintained.

Polymer and size-based precipitation methods are also available. Although adaptable to multiple formats and relatively easy to use, these methods also tend to produce material with more contaminants.

Utilizing immunomagnetic beads coated with exosome marker antibodies is another method that allows isolation with greater yield and undisturbed bioactivity. This limits the captured material to exosomes expressing that particular marker, however, which may only be a subset of what is available. The use of beads also may have adverse chelator effects on the captured exosomes.

Still, other methods capture exosomes by covalent chemistry or lipid microarrays, but these too have limitations in terms of yield or suitability for clinical applications.

A Novel and Specific Exosomes Capture Method

Vn96 (ME-10) (US Patent # 8,956,878) is a peptide reagent developed by New England Peptide (now Biosynth) in collaboration with the Atlantic Cancer Research Institute that binds to canonical HSPs found on the surface of exosomes. As HSPs are cellular chaperones that are upregulated when cells are under conditions of stress, exosomes containing these on their surface are more likely to be derived from those in a disease state. The binding of the peptide to exosomes produces a ‘snowball effect’ that allows easy precipitation into a pellet after spinning in a standard benchtop centrifuge at 10,000g. The pellet is then suitable for use in many assays such as mass spectrometry, staining, blotting, PCR, EM, and more.

Vn96 isolation produces results comparable to ultracentrifugation, but at much greater efficiency as 1/30th the sample size is required. Two kits are available for purchase online based on optimization for how different sample types behave: urine or media samples (ME-020) and plasma samples (ME-020P).

Download our exosome kit flyer and read our publication (Ghosh, 2014) for more information on the effectiveness and flexibility of this EV isolation technology.

References

Edgar, J. R. (2016) What are exosomes, exactly? BMC Biology, 14(46).

Gao, J., Li, A., Hu, J., Feng, L., Liu, L., Shen, Z. (2023) Recent developments in isolating methods for exosomes. Frontiers in Bioengineering and Biotechnology, 10 (2022), 1100892.

Kalluri, R., LeBleu, V. S. (2020) The biology, function, and biomedical applications of exosomes. Science, 367(6478): eaau6977.

Younas, N., Flores, L. C. F., Hopfner, F., Höglinger, G. U., Zerr, I. (2022) A new paradigm for diagnosis of neurodegenerative diseases: peripheral exosomes of brain origin. Translational NeurodegenerationTranslational Neurodegeneration, 11(28).