Exosome Biomarkers for Disease

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Exosomes and other Extracellular Vesicles in Human Health and Disease

Due to their critical importance in intercellular communication, exosomes, and other extracellular vesicles (EVs), are emerging as important players in disease pathogenesis. EVs have been implicated in diseases such as cancer, Alzheimer’s Disease (AD), and Parkinson’s Disease (PD), as well as a host of other inflammatory pathologies. EVs can transfer cargo to cells in their immediate environment, as well as traverse the networks of blood, lymph, and cerebrospinal fluid (CSF) circulation to deliver messages to distal tissues.

In addition to their role in pathogenesis, exosomes and other EVs are of intense interest as a source for disease biomarkers. Acquiring samples from blood, urine, and CSF to diagnose and monitor disease progression is preferable to traditional invasive biopsy procedures for cancer. For diseases such as Alzheimer’s and Parkinson’s, clinical diagnosis relies heavily on behavioral symptoms, which means there has already been a substantial loss of neurons. Biomarkers to detect disease prior to symptom onset can allow for early interventions to protect neurons and delay symptoms.

Exosomes are Active Participants in Cancer Progression

Exosomes and other EVs are associated with many aspects of cancer disease progression.

Illustration depicting the role of Exosomes and Extracellular Vesicles (EV’s) in cancer

Role of EVs in Cancer. Tumor cells secrete exosomes and other EVs containing urgent messages related to tumor initiation, growth, progression, metastasis, and drug resistance. Figure adapted from Zhang, X., Yuan, X., Shi, H. et al. Exosomes in cancer: small particle, big player. J Hematol Oncol 8, 83 (2015). https://doi.org/10.1186/s13045-015-0181-x. License provided by CC License.

Broadly, the role of exosomes and other EVs in cancer can be divided into four categories:

Tumor Growth and Metastasis	Therapy Resistance
Immunosuppression	Biomarker

Exosomes Facilitate Tumor Growth and Metastasis

Exosomes and other EVs are implicated in each stage of tumor growth and metastasis. Cancer cells can secrete EVs containing growth factors to transform normal cells into malignant cells, directly increasing the number of malignant cells in the tumor. Additionally, oncogenic cells can secrete EVs containing anti-apoptotic factors, like Survivin, to prevent cell death, and growth factors, like VEGF, to increase angiogenesis in the tumor microenvironment (TME).

Exosome Cargo	Cancers Reported	Outcome
KRAS c-MET MIF EGFR/EGFRvIII	Colon, Melanoma, Pancreatic, Breast	Promotes tumor growth, metastasis
Survivin	Cervical, Breast, Lung	Inhibits apoptosis of tumor cells
VEGF Angiogenin	NSCLC, Renal, Colon, Glioblastoma	Enhances angiogenesis and thrombosis
Vimentin Rab3D TGF-β	NSCLC, Prostate, Bladder, Colorectal, Breast, Gastric	Involved in epithelial to mesenchymal transition (EMT) Induces myofibroblast differentiation
TGF-β HIF-1α c-KIT Wnt3a	NSCLC, NPC, GIST, Diffuse large B cell lymphoma	Enhancing tumor cell migration and invasion, establishing pre-metastatic niche, remodeling extra cellular matrix

Key: NSCLC-Non small-cell lung cancer; NPC- Nasopharyngeal carcinoma; GIST-Gastrointestinal stromal tumor

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Data image showing ELISA and flow cytometry data for exosome markers on intact and lysed EVs.

Growth factors are expressed on surface of small extracellular vesicles (sEVs). (Left) ELISA quantification of proteins from sEV lysate (grey) and intact sEVs (purple) from ovarian (ES2), colorectal (HCT116), and renal (786-0) cancer cell lines. Proteins CD63, TSG101, VEGF (Catalog # DVE00), GROa (Catalog # DY275-05) , IL-8 (Catalog # D8000C) , and FGF-2 (Catalog # DFB50). (Right) Detection of VEGF on surface of sEVs from VEGF+ cells (pink) and VEGF-/- cells (green). Microbeads were coated with Mouse Anti-Human VEGF (Catalog # MAB2391), anti-human CD63, or control IgG as indicated. EVs were labeled with fluorescent dye and detected by flow cytometry. Figures were adapted from Ko, S.Y., Lee, W., Kenny, H.A. et al. Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake. Commun Biol 2, 386 (2019). https://doi.org/10.1038/s42003-019-0609-x, licensed by CC License.

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Extracellular Vesicle-Mediated Immunosuppression

To be successful, tumors need to evade and suppress the immune response of the host. Some mechanisms of immunosuppression in the TME include cytotoxic inhibition of NK cells and effector T cells, expansion and activation of T regulatory (Treg) cells, and polarization of M2 macrophages. EVs play an important role in each of these immunosuppressive strategies.

Exosome Cargo	Cancers Reported	Outcome
NKG2D DNAM-1 Galectin-9 PD-L1 FasL CD39,CD73	Ovarian, NPC Melanoma, Bladder, Colorectal, Prostate, Breast	Inhibits cytotoxicity of NK cells and effector T cells. Promotes apoptosis of effector cells.
HSP70	NSCLC, Renal, Breast, Colon	Activates and expands myeloid-derived suppressor cells (MDSCs).
EGFR TGF-β CCL20	NSCLC, Melanoma, NPC	Promotes expansion and function of regulatory T cells (Tregs).
EGFR	NSCLC	Inhibits dendritic cell (DC) differentiation.

Key: NSCLC-Non small-cell lung cancer; NPC- Nasopharyngeal carcinoma

Breast cancer cells treated with paclitaxel (PTX) increase production of Survivin in treated exosomes. Membrane was stained with polyclonal Rabbit Anti-survivin (Catalog #NB500-201). Anti-Flotillin was used as loading control. Blot shows signal in PTX-treated exosomes. Use of siRNA targeting Survivin expression shows antibody is specific. This is evidence for knockdown (KD) genetic validation of antibody. Image adapted from Kreger, B. T., Johansen, E. R., Cerione, R. A., & Antonyak, M. A. (2016). The Enrichment of Survivin in Exosomes from Breast Cancer Cells Treated with Paclitaxel Promotes Cell Survival and Chemoresistance. Cancers, 8(12), 111. https://doi.org/10.3390/cancers8120111. License provided by CC-license.

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Exosome-Mediated Resistance to Drug Therapy

In addition to tumor metastasis and immunosuppression, exosomes and other EVs can contribute to tumor growth and disease progression by directly promoting resistance to anti-cancer therapy. EVs can transfer material, such as multi-drug resistant (MDR)-associated proteins and miRNAs to susceptible cells, transforming them into therapy-resistant cells. Additionally, drugs and drug metabolites can be encapsulated within EVs to facilitate transport out of the tumor cell, rendering the drug ineffective. Finally, due to their expression of cell surface markers that are also targets for therapy, such as CD20, Her-2, and PD-L1, EVs can serve as decoy targets, limiting therapeutic efficacy.

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Exosomes and Other Extracellular Vesicles as Biomarkers for Cancer

In general, a biomarker is a biological characteristic that can be measured and monitored. Biomarkers are an especially useful tool for diagnosis and monitoring cancer when the primary tumor site is in a hard-to-reach place, such as the brain or other solid organ.

EVs are under investigation as biomarkers to (1) diagnose disease, (2) determine disease burden, and (3) monitor disease progression and/or treatment efficacy.

Cancer	Exosome Marker(s)
Colon	VSIG3, GPA33, CD147
Ovarian	CD147, EpCAM, CA125/MUC16
Breast	EGFR
Pancreatic	Glypican-1, MIF
Lung	CD151, L1CAM/CD171, Tetraspanin 8, (NSCLC-EGFR, LRG-1)
Glioblastoma	Syndecan-1, EGFRvIII
Prostate	PSA, PSMA, PTEN
Melanoma	TRP2, VLA-4, HSP70, PD-L1

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Extracellular Vesicles in Neurodegenerative Diseases

Neurodegenerative diseases, like Alzheimer’s and Parkinson’s Disease, place a large burden on healthcare systems, with limited treatment options and no cure. Current methods of diagnosis rely on behavioral symptoms like memory loss and loss of motor functions. By the time an individual is symptomatic, there is irreparable damage to neurons and surrounding tissue. Given their accessibility in CSF and plasma, exosomes and other EVs are of great interest as a source of biomarkers for early detection of these diseases.

Exosomes and other EVs are involved in intercellular communication between all brain cells including neurons, astrocytes, microglia, and neural progenitor cells. EVs participate in many critical processes throughout the nervous system including neurogenesis, synaptic function, plasticity, and neuroinflammation.

Illustration of Extracellular Vesicles involved in intercellular communication between all cell types in the nervous system

EVs are involved in intercellular communication between all cell types in the nervous system. Arrows show flow of information from each cell type. Adapted from Riva, P.; Battaglia, C.; Venturin, M. Emerging Role of Genetic Alterations Affecting Exosome Biology in Neurodegenerative Diseases. Int. J. Mol. Sci. 2019, 20, 4113. https://doi.org/10.3390/ijms20174113. Licensed by CC License.

Role of Exosomes in Alzheimer’s Disease

Alzheimer’s Disease (AD) is the most common form of dementia, marked by a progressive cognitive decline including memory loss and behavioral changes. According to the WHO, AD accounts for 60-70% of dementia cases. AD is characterized by amyloid-beta (Aβ) accumulation in the extracellular space of neural tissues, as well as intracellular neurofibrillary tangles consisting of phosphorylated tau protein.

Immunohistochemistry (IHC) staining of beta-amyloid in brain of wild-type and 5xFAD mouse using DAB with hematoxylin counterstain

Immunohistochemistry (IHC) staining of beta-amyloid in brain of wild-type (left) and 5xFAD (right) mouse using DAB with hematoxylin counterstain. Mouse Anti-Human beta Amyloid (MOAB-2) (Catalog # NBP2-13075) was used at 1:20 dilution in wild-type brain tissue and 1:400 dilution in 5xFAD mouse brain tissue. Beta amyloid plaques indicated by blue arrows.

Learn more about Alzheimers’ Disease

Extracellular Vesicles in AD Pathogenesis: Pathogenic or Protective?

The role of EVs in AD pathogenesis is not well defined, though there is consensus that exosomes are able to carry Aβ. However, there are conflicting reports about whether this association is protective or pathogenic. Some research shows a reduction in synaptic inflammation and increased disease pathology associated with an increase in EV-associated Aβ. Yet, other research shows exosomes protecting neurons transporting excess Aβ from neurons to microglia for degradation.

Exosome Markers	Role in AD
Alix	Associated with Aβ plaques of AD brain, but not healthy controls.
TSG101, VPS4A	Inhibition of transcription led to decreased spread of Aβ.
Amyloid Precursor Protein (APP)	Precursor protein that is processed to Aβ.
Aβ	Accumulation of plaques is associated with progressive cognitive decline.
Tau, pTau (T181)	Major component of neurofibrillary tangles.

Exosome Biomarkers for Alzheimer’s Disease

Early detection of AD could prove critical for intervention efforts to prevent or delay disease progression. Thus, intensive research is underway to identify biomarkers of AD prior to diagnosis. In addition to Aβ, proteins related to function of the neuronal synapse are a high priority for biomarker research.

Exosome Cargo	Expression compared to healthy controls	Present in preclinical Associated with disease state
Aβ42	Increased in plasma	EV-associated levels can predict risk of disease progression.
TAR DNA-binding protein 43 (TDP43)	Increased in plasma	No correlation with cognitive function or other neurological symptoms.
Synaptophysin	Decreased in plasma	Already decreased in pre-clinical state. Correlated with cognitive decline.
Synaptotagmin	Decreased in plasma	Already decreased in pre-clinical state. Correlated with cognitive decline.
25-kDa synaptosomal-associated protein (SNAP-25)	Decreased in plasma	Can be detected several years before clinical onset of AD.
GAP43	Decreased in Plasma	Can be detected several years before clinical onset of AD.
Neurogranin	Increased in CSF, decreased in plasma	Present in preclinical state. Potentially predictive of disease progression.

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Exosomes and Other Extracellular Vesicles in Parkinson’s Disease

Parkinson’s Disease (PD) is another common neurodegenerative disease, characterized by the loss of dopaminergic neurons, primarily in the substantia nigra.

PD is defined by the presence of alpha-synuclein aggregates (Lewy bodies) and accumulation of defective mitochondria, leading to neuronal death. Exosomes, specifically L1CAM-expressing neuronal derived exosomes (NDEs) have been implicated in the pathological transport of alpha-synuclein in the brain. Diagnosis of PD relies on neurological examinations and presentation of symptoms. The most consistent exosome biomarker in PD patients is increased levels of NDE-associated alpha-synuclein in plasma. Other targets associated with plasma NDEs in PD include Park7/DJ1 and clusterin.

Explore more targets for Parkinson’s Disease

Currently, there is no cure for PD, but motor symptoms can be temporarily alleviated with supplemental dopamine. New therapies concentrate on neuroprotective and disease-modifying strategies, making early detection a critical research focus.

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Expanding Role of Exosomes in Inflammatory Diseases

Exosomes and other EVs are implicated in the pathogenesis of many inflammatory diseases including cardiovascular disease, kidney disease, autoimmune disorders, and viral infections. Thus far, much of this work has investigated the role of various microRNAs (miRs), with an increasing emphasis on protein cargo.

Disease	Exosome/EV Cargo
Cardiovascular Disease	Cystatin C, Serpin F2, CD14, PolyIg Receptor, C5a, Tissue Factor, CD31, CD62e
Renal Disease	AQP1, AQP2, Aminopeptidase N,
Systemic Lupus Erythematosus (SLE)	IFN-α, TNF-α, C1q, CD54
Type 1 Diabetes	GAD65, Proinsulin
HIV	HMGB1, NF-L, HIV gp120

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Select References

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Liu H, Qiao S, Fan X, Gu Y, Zhang Y, Huang S. Role of exosomes in pancreatic cancer (Review). Oncol Lett 2021;21:1–13. https://doi.org/10.3892/OL.2021.12559.

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