Exosomes - novel tools in diagnostics and treatments

Written by: Vujic Vesna and Volnermanis Baraks

Supervisor: Dr. Gergely Jócsák

Physiology Department, University of Veterinary Medicine, Budapest

Introduction

Exosomes are a subtype of extracellular vesicles that are membrane-derived, they are formed as a result of a selective intracellular process in which plasma membrane lipoproteins are placed within endosomes, later to be released into the extra cellular space. They exist and are secreted from almost all cell types in the body, both prokaryotic and eukaryotic (Johnstone, 2006).

Théry et al (2002) stated that despite their similarity, exosomes differ from microvesicles in the fact that they are secreted only from living cells, whereas microvesicles can be produced from cells which have undergone apoptosis. In addition, contrary to microvesicles that bud from the plasma membrane, exosomes are released from the cell following a fusion between multi vesicular bodies with the cell plasma membrane (Skotland et al, 2019).

Being part of the cellular secretome, exosomes transfer cellular constituents (DNA, mRNA, lipids, proteins) extracellularly in a paracrine fashion to a nearby cell or, alternatively in a systemic fashion - travel through the blood stream to reach a more distant cell. Exosomes possess various additional attributes, such as the ability to penetrate the blood-brain barrier, avoid inducing any response of the immune system and withstand degradation of any enzymes (Sarko and McKinney, 2017). A recent study by Jeppesen et al (2019) has compared ‘classical exosomes’ (containing tetraspanins CD63, CD81, or CD9) and ‘non-classical exosomes’ (do not contain the mentioned tetraspanins). This study showed that ‘classical exosomes’ do not contain DNA and are not the mechanisms for active DNA release as previously believed. However, exosomes do contain RNA and RNA binding proteins.

Current research work is aimed at thoroughly understanding the way cells communicate with each other. Exosomal attributes mentioned beforehand are the reason why one of the main fields focuses at utilizing exosomes as a natural delivery vehicle system for therapeutic drugs to different cells (Sarko and McKinney, 2017).

Biogenesis

Exosomes are believed to originate from multivesicular bodies (MVB), a type of endosomes that contains membrane-bound intraluminal vesicles (ILV). ILVs form inside the MVBs by budding into their lumen. Once formed, MVBs can either undergo degradation by fusing with lysosomes or be released as exosomes to the extracellular environment by fusing with the cytoplasmic side of the plasma membrane of the cell (Sarko and McKinney, 2017).

Henne et al (2011) showed that endosomal sorting complex transport (ESCRT) mechanism has an important role in the formation process of ILVs, facilitation of MVB formation, vesicle budding, and exosomal cargo sorting into MVBs. However, a review by Zhang et al (2019) stated that the cargo sorting process can be ESCRT-independent as well, depending on the origin of the cell type.

Structure and chemical content

Structure

Exosomes are flattened ‘saucer-like’ spheres with a lipid bi-layer exterior, ranging approximately 30-150 nm in diameter with a density of 1.13 – 1.19 g ml^–1 (Théry et al, 2002).

Skotland et al (2019) showed that lipids have great functional importance in the biology of exosomes. Lipids are important for exosomal membrane structure and are essential for exosome formation and release to the extracellular environment as well. Exosomal lipid composition shows similarities with lipid rafts, it is rich and includes:

• Cholesterol
• Sphingolipids
• Sphingomyelin
• Glycosphingolipids
• Phospholipids
• Phosphatidylserine

Chemical content

Nucleic acid composition of exosomes includes both DNA and RNA. Lötvall and Valadi (2019) observed that exosomal RNA contains very low amounts of ribosomal RNA, which indicates presence of other RNA types, such as mRNA. Further profile analysis of the exosomal mRNA showed genetic difference between exosomes to their parental cell. These findings indicate selectivity of exosomal RNA and in the way in which parental cells allocate a certain number of genes for exosomal delivery. In addition to RNA, exosomal DNA has been found to be genomic, containing all chromosomes (Waldenström et al, 2012; Cai et al, 2016)

Théry et al (2002) stated that proteins currently detected in exosomes are of cytoplasmic origin, the membrane of endocytic compartments or the plasma membrane. These proteins vary based on the cell type from which the exosome has originated from. Protein composition includes:

Cytoskeletal proteins (tubulin, actin, keratin) Proteins involved in intracellular membrane fusion and transport (annexins, RAB proteins) Proteins involved in signal transduction (kinases) Metabolic enzymes (peroxidases, pyruvate kinases, lipid kinases) Heat-shock proteins Proteins involved in antigen presentation (MHC-I, MHC-II) Proteins involved in organization of large molecular complexes and membrane subdomains (tetraspanins) Cell specific transmembrane proteins (α- and β-chains of integrins, immunoglobulin family members, cell surface peptides)

Methods of exosome extraction

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Ultracentrifugation

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Ultrafiltration

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Size-exclusion liquid chromatography

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Precipitation

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Immunoaffinity techniques

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Microfluidics-based isolation techniques

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Exosomes as a diagnostic tool

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Biomarkers

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Therapeutics

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References

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Patents

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Figures

All the figures have been taken from open access articles

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