Exosomes as Disease Biomarkers

With rapid advancements in isolation and purification techniques, the potential role of exosomes as biomarkers in disease pathogenesis is becoming clearer

Nischal PM, PhD

Inherited genetic mutations are found in 5 to 10 percent of all cancers. When mutations in the BRCA1 gene were implicated in hereditary forms of breast cancer nearly 20 years ago, it sparked an interest in cancer genetic screening. Since then, several other genes have been identified as potential contributors to heritable forms of cancer. 

When exosomes were first discovered in the mid- 1980s, they were thought of as mere cellular waste disposal mechanisms. Scientists have since learned that they are involved in a variety of biological processes, including intracellular cell signaling, tissue regeneration, intercellular communications, and immune functions.1,2 

The macromolecular contents (cargo) present in exosomes are implicated in several diseases including cancers, neurodegenerative diseases, and respiratory diseases. Additionally, exposures to chemical pollutants and cellular stress mechanisms have the potential to change the various steps in biogenesis and export of exosomes.3 This exquisite sensitivity of the cargo is a boon for clinical toxicology studies. 

The fact that the exosome’s intracellular contents vary in patients with certain diseases when compared to healthy people, along with the ability to easily isolate exosomes from accessible body fluids such as blood and urine, have made them attractive as diagnostic targets.Overall, exosomes hold huge potential as diagnostic biomarkers.5 

Cancer 

Exosomes have a multi-layered role in the pathophysiology of cancers. They are involved in progression of the disease, metastases, angiogenesis, immune escape, and resistance to treatment.6,7 

Exosomes transfer several different miRNAs, mRNAs, lipids, and proteins that are thought to cause resistance to anticancer agents.8 For example, exosomes secrete miR-145 and miR-34a that increase resistance to 5-fluorouracil in colon cancer cells.

Additionally, exosomes are released from many different tumor types, including colorectal carcinoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, lung cancer, melanoma, and osteosarcoma.10 Once released by tumor cells, exosomes can alter recipient cells and play a crucial role in tumor development, angiogenesis, stromal fibroblast activation, development of drug-resistance, and stimulation of antiapoptotic proteins.10,11 

The analysis of the cargos from various tumor-associated exosomes has shown their potential as biomarkers for the following cancer types:10,12 

  • Ovarian cancer: Epithelial cell adhesion molecule, CD-24, CA-125, miR-21, miR-200b/c 
  • Melanoma: Caveolin-1, heat shock proteins 70 & 90, melanoma inhibitory activity (MIA) protein 
  • Breast cancer: Breast cancer resistance protein, glypican-1, miR-101, miR-372 
  • Prostate cancer: Carbonic anhydrase IX, survivin, phosphatase and tensin homolog (PTEN), miR-141, miR-1290 
  • Hepatocellular carcinoma: miR-18a, miR-222, miR-718 
  • Colorectal cancer: miR-1229, miR-150 
  • Gastric cancer: miR-423-5p

Furthermore, exosomes may represent a mechanistic link between exposure to certain environmental pollutants and cancers. Human bronchial epithelial cells, when exposed to arsenite, release exosomes containing miR-21 and PTEN.13 Exposure to ionizing radiation can result in the release of exosomes with integrins, tumor growth factor-β, and CXCL chemokines. These exosomes attach to naïve cells and cause cellular remodeling and enrich the microenvironment for tumor metastasis.4 

The difference in concentrations of the exosome contents isolated from cancer patients in the above examples, when compared to healthy individuals, makes them potentially important biomarkers. 

Neurodegenerative diseases 

Neurodegenerative diseases result from chronic and sustained degeneration of neural tissues. These diseases include Huntington’s, Alzheimer’s, Parkinson’s, prion disease, and amyotrophic lateral sclerosis.14 

Biomarkers for neurodegenerative diseases, such as α-synuclein, neurogranin, neurofilament light, and t/p-tau, are concentrated in cerebrospinal fluid. While lumbar puncture is considered safe and tolerable, it is contraindicated in several instances, and the concentration of the central nervous system biomarkers is usually very low in other body fluids.15 Thus, there is the urgent need for easily detectable and reliable biomarkers in neurodegenerative diseases. 

α-synuclein, amyloid-β, and tau are some of the native forms of proteins that are misfolded/aggregated and function as pathogenic mechanisms for the aforementioned neurodegenerative diseases. Exposure to pollutants, including pesticides, metals, and solvents further increase expression of these pathogenic proteins in neurons. Exosomes then transport these proteins to naïve cells.4 Exosomes originating from the CNS are able to cross the blood brain barrier and can be detected in peripheral fluids. 

In Alzheimer’s, a study found that astrocyte-derived and neuron-derived exosomes isolated from peripheral blood had significantly higher levels of soluble amyloid precursor protein α and β, γ-secretase, and phosphor-T181-tau when compared to healthy individuals. These properties make exosomes suitable biomarkers of brain plaque.15 

Other potential exosomal biomarkers of neurodegenerative diseases include: 

  • Parkinson’s disease: α-synuclein DJ1, apolipoprotein A1, clusterin 
  • Prion disease: cystatin c, H-FAB 
  • Amyotrophic lateral sclerosis: TAR DNA binding protein 43, phosphorylated neurofilament heavy chain, superoxide dismutase1

Respiratory diseases 

An array of cells in the lungs, including epithelial and endothelial cells, stem cells, macrophages, and tumor cells, release exosomes. These exosomes play key roles in the biology and function of the respiratory tract.16 

Airway epithelial cells release exosomes whose cargo includes α-2,6-linked sialic acid and mucins, which help neutralize human influenza viruses.17 In sarcoidosis of the lungs, inflammatory responses are enhanced by exosomes containing interleukin-13, interferon-¥, and C-X-C Motif Chemokine Ligand 8.18 Exosomes also serve as mediators of cell-to-cell communication in the lung microenvironment, and their cargo alters with the disease status. 

Their accessibility makes exosomes important biomarkers of respiratory disease. One study found that, when compared to healthy individuals, exosomes isolated from chronic obstructive pulmonary disease patients had elevated levels of several skeletal specific miRNAs, including miR-1, miR-133, and miR-206.18 

Other potential exosomal biomarkers of respiratory diseases include: 

  • Asthma: miR-658, miR-26a, miR-200c 
  • Idiopathic pulmonary fibrosis: miR-142-3p, miR-33a-5p 
  • Tuberculosis: Around 20 mycobacterial peptides including Mycobacterium tuberculosis antigens 85b, GlcB, and BfrB have been identified in serum exosomes obtained from these patients.21 

Summary

In addition to cancer, neurodegenerative diseases, and respiratory diseases, exosomes have also been implicated in other diseases including ocular diseases,22 bone disorders,23 infectious diseases,24 and metabolic and cardiovascular diseases.25 With rapid advancements in isolation and purification techniques, the potential role of exosomes as biomarkers in disease pathogenesis is becoming clearer. Moreover, their biocompatibility and ability to cross barriers, including the blood brain barrier, makes them especially interesting as next-generation therapeutic vehicles.24

References

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3. Harischandra, Dilshan S., et al. "Environmental neurotoxicant manganese regulates exosome-mediated extracellular miRNAs in cell culture model of Parkinson's disease: Relevance to α-synuclein misfolding in metal neurotoxicity." Neurotoxicology 64 (2018): 267-277. 

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13. Chen, Chao, et al. "NF-kB-regulated exosomal miR-155 promotes the inflammation associated with arsenite carcino genesis." Cancer Letters 388 (2017): 21-33. 

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20. Momen-Heravi, Fatemeh, et al. "Current methods for the isolation of extracellular vesicles." Biological Chemistry 394.10 (2013): 1253-1262. 

21. Kruh-Garcia, Nicole A., et al. "Detection of Mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS." PloS One 9.7 (2014): e103811. 

22. Yakimchuk, Konstantin. "Exosomes: isolation and character ization methods and specific markers." Mater Methods 5 (2015): 1450-3. 

23. Masaoutis, Christos, and Stamatios Theocharis. "The role of exosomes in bone remodeling: implications for bone physiol ogy and disease." Disease Markers (2019). 

24. Crenshaw, Brennetta J., Brian Sims, and Qiana L. Matthews. "Biological function of exosomes as diagnostic markers and therapeutic delivery vehicles in carcinogenesis and infectious diseases." Nanomedicines. IntechOpen, 2018. 

25. Guo, Dong, et al. "Roles and clinical applications of exosomes in cardiovascular disease." BioMed Research International (2020).