The Overlooked Vesicles with Great Diagnostic and Therapeutic Potential

2023-10-25

The scientific field now holds exosomes in high regard, acknowledging their profound potential in therapeutics and diagnostics. Exosomes were once labeled as 'cellular debris' and mere 'waste products' of the cell, even when they were first recognized as intraluminal vesicles, secreted from the cell through plasma membrane fusion in 1983. In time exosomes achieved their title of 'exosomes' in 1987, but it took until the early 21^st century – when researchers began to investigate what they might contain and why – for their true importance to be better appreciated and understood.

Exosomes are now respected as key intercellular communicators in cell biology and disease. They have 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. Despite the challenges associated with isolating exosomes, methods such as Biosynth's exosome isolation kits, based on the use of the pioneering Vn96 (ME-10) (US Patent # 8,956,878) peptide reagent, have provided an effective method to isolate exosomes, perfect for diagnostic and therapeutic applications. For more information about the different methods of exosome isolation, read our brochure on exosomes.

These tiny vesicles should not have been overlooked. Find out more about the diagnostic and therapeutic potential of exosomes in our blog below. Firstly what are exosomes and how are they made...

The Natural Manufacture of Exosomes

Exosomes are one of the two types of extracellular vesicles (EVs) released by the cells of prokaryotes and eukaryotes. 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.

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) go 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.

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.

NanoSight visualization of EVs isolated with Vn96 shows a greater number of exosomes with different morphology in disease state plasma when compared with a healthy control

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.

There is emerging evidence that extracellular vesicles like exosomes may contribute to neurodegenerative disease progression due to their role in transporting pathogenic protein aggregates between cells. Consequently exosomes could potentially be used as biomarkers for neurodegenerative diseases and even as a drug delivery method. The reasons that exosomes have such promising neurodegenerative disease diagnostic potential is that:

· Exosomes carry proteins and RNA that have become pathogenic during neurodegenerative diseases

· Exosomes have the ability to cross the blood-brain barrier and have a low level of immunogenicity

· Exosomes contain markers on their surface which is an indicator of their origin. Therefore exosomes from the central nervous systems can be captured.

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. Properties such as these can be harnessed by scientists to use exosomes as vectors in delivering drug molecules or other therapeutic agents.

Uptake of Vn96-isolated EVs (green) from breast cancer cell line MDA-MB-231 by normal breast cell line MCF10A

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 (IOX-3765-PI), a construct which 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 (FP10637).

Specific miRNA or small interfering RNA (siRNA) can also be used as payload molecules for the purpose of supressing gene expression. One instance of this is exosomes derived from dendritic cells modified with RVG (rabies virus glycoprotein) (FR110131) 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.

Biosynth see the value that exosomes bring to the field of medicine, and want to support vital research into their use as a diagnostic and therapeutic tools in a variety of disease areas. That is why we have available two exosome isolation kits, based on optimization for how different sample types behave: urine or media samples (ME-020) and plasma samples (ME-020P). Our kits are a novel technology which uses the peptides reagent, Vn96 (ME-10) (US Patent # 8,956,878), developed by New England Peptide (now Biosynth) in collaboration with the Atlantic Cancer Research Institute. Vn96 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. Binding of the peptide to exosomes produces a ‘snowball effect’ that allows easy precipitation into a pellet after spinning in a standard bench top centrifuge at 10,000g. The pellet is then suitable for use in many assays such as mass spectrometry, staining, blotting, PCR, EM and more. Although Vn96 isolation produces results comparable to ultracentrifugation, our method has a much greater efficiency as 1/30^th the sample size is required. Download our exosome kit flyer and read our publication (Ghosh, A. 2014) for more information on the effectiveness and flexibility of this Extracellular Vesicle isolation technology. Biosynth