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Extracellular Vesicles as a New Source of Liquid Biopsy

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (15 November 2020) | Viewed by 29968

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Dr. Paola Lanuti

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Guest Editor

Department of Medicine and Aging Science, University “G. d’Annunzio” of Chieti-Pescara, Via dei Vestini, 31, 66100 Chieti, Italy
Interests: extracellular vesicles; flow cytometry; biomarkers
Special Issues, Collections and Topics in MDPI journals

Dr. Rodney J. Ouellette

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Co-Guest Editor

Atlantic Cancer Research Institute, 35 Providence Street, Moncton, NB EIC 8X3, Canada
Interests: liquid biopsy; extracellular vesicles; cancer; diagnostics; monitoring treatment response; artificial intelligence algorithms for multi-omic analysis in liquid biopsy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear colleagues,

Within the past decade, extracellular vesicles have progressively emerged as key regulators of organized communities of cells within multicellular organisms in health and disease. Extracellular vesicles have been described as shuttle vectors or signal transducers that can deliver specific biological information even at long distances. Due to their substantial stability, extracellular vesicles circulate through bodily fluids and have been identified in blood, urine, cerebrospinal fluid, saliva, milk, and tears. It is known that extracellular vesicles are a heterogeneous group of lipidic structures ranging from 30–50,000 nm in size, generically subclassified as small, if within 100 nm, and medium/large, if above 100–200 nm.

The extracellular vesicle cargo, consisting of genomic DNA, RNAs, miRNAs, proteins, and lipids dynamically reflecting the characteristics of the cells of origin, makes them an attractive source of biological information. For all of these reasons, extracellular vesicles have high potential as dynamic biomarkers of diseases and/or response to therapies. The deep characterization of extracellular vesicle general characteristics and cargoes related to their roles in tumorigenesis, immunity, and vascular biology may present interesting perspectives in the assessment of extracellular vesicles as a reliable liquid biopsy source.

Dr. Paola Lanuti
Dr. Rodney J. Ouellette
Guest Editors

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Keywords

Exosomes
Microvesicles
Apoptotic bodies
Large oncosomes
Extracellular vesicles
Biomarkers
Liquid biopsy
Intercellular communication
Cancer

Related Special Issue

Extracellular Vesicles as a New Source of Liquid Biopsy 2.0 in International Journal of Molecular Sciences (7 articles)

Published Papers (10 papers)

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Research

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15 pages, 14271 KiB

Open AccessArticle

Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits

by Sebastian Sjoqvist, Kentaro Otake and Yoshihiko Hirozane

Int. J. Mol. Sci. 2020, 21(24), 9425; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21249425 - 10 Dec 2020

Cited by 15 | Viewed by 3683

Abstract

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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13 pages, 1471 KiB

Open AccessArticle

A Novel Pool of Microparticle Cholesterol Is Elevated in Rheumatoid Arthritis but Not in Systemic Lupus Erythematosus Patients

by Shuaishuai Hu, Brenton L. Cavanagh, Robert Harrington, Muddassar Ahmad, Grainne Kearns, Steve Meaney and Claire Wynne

Int. J. Mol. Sci. 2020, 21(23), 9228; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21239228 - 03 Dec 2020

Cited by 1 | Viewed by 1951

Abstract

Microparticles are sub-micron, membrane-bound particles released from virtually all cells and which are present in the circulation. In several autoimmune disorders their amount and composition in the circulation is altered. Microparticle surface protein expression has been explored as a differentiating tool in autoimmune disorders where the clinical pictures can overlap. Here, we examine the utility of a novel lipid-based marker—microparticle cholesterol, present in all microparticles regardless of cellular origin—to distinguish between rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We first isolated a series of microparticle containing lipoprotein deficient fractions from patient and control plasma. There were no significant differences in the size, structure or protein content of microparticles isolated from each group. Compared to controls, both patient groups contained significantly greater amounts of platelet and endothelial cell-derived microparticles. The cholesterol content of microparticle fractions isolated from RA patients was significantly greater than those from either SLE patients or healthy controls. Our data indicate that circulating non-lipoprotein microparticle cholesterol, which may account for 1–2% of measured cholesterol in patient samples, may represent a novel differentiator of disease, which is independent of cellular origin. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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20 pages, 2161 KiB

Open AccessArticle

Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

by Catherine Taylor, Simi Chacko, Michelle Davey, Jacynthe Lacroix, Alexander MacPherson, Nicholas Finn, Gabriel Wajnberg, Anirban Ghosh, Nicolas Crapoulet, Stephen M. Lewis and Rodney J. Ouellette

Int. J. Mol. Sci. 2020, 21(23), 9083; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21239083 - 29 Nov 2020

Cited by 14 | Viewed by 2933

Abstract

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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21 pages, 1735 KiB

Open AccessArticle

Affinity Captured Urinary Extracellular Vesicles Provide mRNA and miRNA Biomarkers for Improved Accuracy of Prostate Cancer Detection: A Pilot Study

by Michelle Davey, Sami Benzina, Marc Savoie, Guy Breault, Anirban Ghosh and Rodney J. Ouellette

Int. J. Mol. Sci. 2020, 21(21), 8330; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218330 - 06 Nov 2020

Cited by 23 | Viewed by 2627

Abstract

Serum prostate-specific antigen (sPSA) testing has helped to increase early detection of and decrease mortality from prostate cancer. However, since sPSA lacks specificity, an invasive prostate tissue biopsy is required to confirm cancer diagnosis. Using urinary extracellular vesicles (EVs) as a minimally invasive biomarker source, our goal was to develop a biomarker panel able to distinguish prostate cancer from benign conditions with high accuracy. We enrolled 56 patients in our study, 28 negative and 28 positive for cancer based on tissue biopsy results. Using our Vn96 peptide affinity method, we isolated EVs from post-digital rectal exam urines and used quantitative polymerase chain reaction to measure several mRNA and miRNA targets. We identified a panel of seven mRNA biomarkers whose expression ratio discriminated non-cancer from cancer with an area under the curve (AUC) of 0.825, sensitivity of 75% and specificity of 84%. We also identified two miRNAs whose combined score yielded an AUC of 0.744. A model pairing the seven mRNA and two miRNA panels yielded an AUC of 0.843, sensitivity of 79% and specificity of 89%. Addition of EV-derived PCA3 levels and clinical characteristics to the biomarker model further improved test accuracy. An AUC of 0.955, sensitivity of 86% and specificity of 93% were obtained. Hence, Vn96-isolated urinary EVs are a clinically applicable and minimally invasive source of mRNA and miRNA biomarkers with potential to improve on the accuracy of prostate cancer screening and diagnosis. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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16 pages, 4095 KiB

Open AccessArticle

Circulating MicroRNAs in Extracellular Vesicles as Potential Biomarkers of Alcohol-Induced Neuroinflammation in Adolescence: Gender Differences

by Francesc Ibáñez, Juan R. Ureña-Peralta, Pilar Costa-Alba, Jorge-Luis Torres, Francisco-Javier Laso, Miguel Marcos, Consuelo Guerri and María Pascual

Int. J. Mol. Sci. 2020, 21(18), 6730; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186730 - 14 Sep 2020

Cited by 25 | Viewed by 3024

Abstract

Current studies evidence the role of miRNAs in extracellular vesicles (EVs) as key regulators of pathological processes, including neuroinflammation and neurodegeneration. As EVs can cross the blood–brain barrier, and EV miRNAs are very stable in peripheral circulation, we evaluated the potential gender differences in inflammatory-regulated miRNAs levels in human and murine plasma EVs derived from alcohol-intoxicated female and male adolescents, and whether these miRNAs could be used as biomarkers of neuroinflammation. We demonstrated that while alcohol intoxication lowers anti-inflammatory miRNA (mir-146a-5p, mir-21-5p, mir-182-5p) levels in plasma EVs from human and mice female adolescents, these EV miRNAs increased in males. In mice brain cortices, ethanol treatment lowers mir-146a-5p and mir-21-5p levels, while triggering a higher expression of inflammatory target genes (Traf6, Stat3, and Camk2a) in adolescent female mice. These results indicate, for the first time, that female and male adolescents differ as regards the ethanol effects associated with the inflammatory-related plasma miRNAs EVs profile, and suggest that female adolescents are more vulnerable than males to the inflammatory effects of binge alcohol drinking. These findings also support the view that circulating miRNAs in EVs could be useful biomarkers for screening ethanol-induced neuroinflammation and brain damage in adolescence. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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10 pages, 3025 KiB

Open AccessArticle

miR-365 (microRNA): Potential Biomarker in Oral Squamous Cell Carcinoma Exosomes and Extracellular Vesicles

by Jeffery Coon, Karl Kingsley and Katherine M. Howard

Int. J. Mol. Sci. 2020, 21(15), 5317; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21155317 - 27 Jul 2020

Cited by 22 | Viewed by 3253

Abstract

Introduction: miR-365 is a non-coding microRNA that regulates transcription and has been demonstrated to promote oncogenesis and metastasis in some cancers, while suppressing these effects in others. Many microRNAs are produced and then exported extracellularly in exosomes, which are small extracellular vesicles ranging from 30 to 100 nm that are found in eukaryotic fluids and facilitate many cellular functions. Exosomes and extracellular vesicles are produced by many cell types, including oral cancer cells—although no study to date has evaluated miR-365 and oral cancer exosomes or extracellular vesicles. Based on this information, our research question was to evaluate whether oral cancers produce exosomes or extracellular vesicles containing miR-365. Materials and Methods: Two commercially available oral cancer cell lines (SCC25 and CAL27) and a normal oral keratinocyte (OKF4) were grown in serum-free media, supplemented with exosome-depleted fetal bovine serum. Extracellular vesicles and exosomes were then isolated using the Invitrogen total exosome RNA and protein isolation kit for processing using the hsa-miR-365a-5p microRNA qPCR assay kit. Results: RNA was successfully isolated from the exosome-depleted supernatant from each cell line—SCC9, SCC15, SCC25, and CAL27 (oral squamous cell carcinomas) and OKF4 (oral epithelial cell line). Relative concentrations of RNA were similar among each cell line, which were not significantly different, p = 0.233. RNA quality was established by A260:A280 absorbance using a NanoDrop, revealing purity ranging 1.73–1.86. Expression of miR-16 was used to confirm the presence of microRNA from the extracted exosomes and extracellular vesicles. The presence of miR-365 was then confirmed and normalized to miR-16 expression, which demonstrated an increased level of miR-365 in both CAL27 and SCC25. In addition, the normalized relative quantity (RQ) for miR-365 exhibited greater variation among SCC25 (1.382–4.363) than CAL27 cells (1.248–1.536). Conclusions: These results confirm that miR-365 is not only expressed in oral cancer cell lines, but also is subsequently exported into exosomes and extracellular vesicles derived from these cultures. These data may help to contextualize the potential for this microRNA to contribute to the phenotypes and behaviors of oral cancers that express this microRNA. Future research will begin to investigate these potential mechanisms and pathways and to determine if miR-365 may be useful as an oral cancer biomarker for salivary or liquid biopsies. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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17 pages, 2784 KiB

Open AccessArticle

Plasma Extracellular Vesicle-Derived TIMP-1 mRNA as a Prognostic Biomarker in Clear Cell Renal Cell Carcinoma: A Pilot Study

by Francisca Dias, Ana Luísa Teixeira, Inês Nogueira, Mariana Morais, Joana Maia, Cristian Bodo, Marta Ferreira, Isabel Vieira, José Silva, João Lobo, José Pedro Sequeira, Joaquina Maurício, Jorge Oliveira, Carlos Palmeira, Gabriela Martins, Klaas Kok, Bruno Costa-Silva and Rui Medeiros

Int. J. Mol. Sci. 2020, 21(13), 4624; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21134624 - 29 Jun 2020

Cited by 11 | Viewed by 2348

Abstract

The tumor microenvironment has gained a lot of attention from the scientific community since it has a proven impact in the development of tumor progression and metastasis. Extracellular vesicles (EVs) are now considered one of the key players of tumor microenvironment modulation. Clear cell renal cell carcinoma (ccRCC) is the most lethal urological neoplasia and presents a high metastatic potential, which reinforces the need for the development of more effective predictive biomarkers. Our goal was to evaluate the applicability of EV-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) as prognostic biomarkers for ccRCC. To do so, we studied the plasma EV content of 32 patients with localized ccRCC and 29 patients with metastatic ccRCC. We observed that patients with localized disease and tumors larger than 7 cm presented higher levels of plasma EV-derived TIMP-1 mRNA when compared with patients presenting smaller tumors (p = 0.020). Moreover, patients with metastatic disease presented higher levels of EV-derived TIMP-1 mRNA when compared with patients with localized disease (p = 0.002) and when we stratified those patients in high and low levels of TIMP-1 EV-derived mRNA, the ones presenting higher levels had a lower overall survival (p = 0.030). EV-derived TIMP-1 mRNA may be a good prognostic biomarker candidate for ccRCC. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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Review

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20 pages, 1394 KiB

Open AccessReview

Extracellular Vesicles in Diagnosing Chronic Coronary Syndromes the Bumpy Road to Clinical Implementation

by Mirthe Dekker, Farahnaz Waissi, Nathalie Timmerman, Max J. M. Silvis, Leo Timmers and Dominique P. V. de Kleijn

Int. J. Mol. Sci. 2020, 21(23), 9128; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21239128 - 30 Nov 2020

Cited by 7 | Viewed by 2241

Abstract

Coronary artery disease (CAD), comprising both acute coronary syndromes (ACS) and chronic coronary syndromes (CCS), remains one of the most important killers throughout the entire world. ACS is often quickly diagnosed by either deviation on an electrocardiogram or elevated levels of troponin, but CCS appears to be more complicated. The most used noninvasive strategies to diagnose CCS are coronary computed tomography and perfusion imaging. Although both show reasonable accuracy (80–90%), these modalities are becoming more and more subject of debate due to costs, radiation and increasing inappropriate use in low-risk patients. A reliable, blood-based biomarker is not available for CCS but would be of great clinical importance. Extracellular vesicles (EVs) are lipid-bilayer membrane vesicles containing bioactive contents e.g., proteins, lipids and nucleic acids. EVs are often referred to as the “liquid biopsy” since their contents reflect changes in the condition of the cell they originate from. Although EVs are studied extensively for their role as biomarkers in the cardiovascular field during the last decade, they are still not incorporated into clinical practice in this field. This review provides an overview on EV biomarkers in CCS and discusses the clinical and technological aspects important for successful clinical application of EVs. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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14 pages, 994 KiB

Open AccessReview

Circulating 16S RNA in Biofluids: Extracellular Vesicles as Mirrors of Human Microbiome?

by Veronica Ricci, Davide Carcione, Simone Messina, Gualtiero I. Colombo and Yuri D’Alessandra

Int. J. Mol. Sci. 2020, 21(23), 8959; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21238959 - 25 Nov 2020

Cited by 26 | Viewed by 4488

Abstract

The human body is inhabited by around 10¹³ microbes composing a multicomplex system, termed microbiota, which is strongly involved in the regulation and maintenance of homeostasis. Perturbations in microbiota composition can lead to dysbiosis, which has been associated with several human pathologies. The gold-standard method to explore microbial composition is next-generation sequencing, which involves the analysis of 16S rRNA, an indicator of the presence of specific microorganisms and the principal tool used in bacterial taxonomic classification. Indeed, the development of 16S RNA sequencing allows us to explore microbial composition in several environments and human body districts and fluids, since it has been detected in “germ-free” environments such as blood, plasma, and urine of diseased and healthy subjects. Recently, prokaryotes showed to generate extracellular vesicles, which are known to be responsible for shuttling different intracellular components such as proteins and nucleic acids (including 16S molecules) by protecting their cargo from degradation. These vesicles can be found in several human biofluids and can be exploited as tools for bacterial detection and identification. In this review, we examine the complex link between circulating 16S RNA molecules and bacteria-derived vesicles. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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16 pages, 719 KiB

Open AccessReview

Role of Extracellular Vesicles in Epithelial Ovarian Cancer: A Systematic Review

by Alessandro Lucidi, Danilo Buca, Carlo Ronsini, Sara Tinari, Giuseppina Bologna, Davide Buca, Martina Leombroni, Marco Liberati, Francesco D’Antonio, Giovanni Scambia, Paola Lanuti and Marco Petrillo

Int. J. Mol. Sci. 2020, 21(22), 8762; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21228762 - 19 Nov 2020

Cited by 32 | Viewed by 2615

Abstract

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived submicron vesicles released under physiological or pathological conditions. EVs mediate the cellular crosstalk, thus contributing to defining the tumor microenvironment, including in epithelial ovarian cancer (EOC). The available literature investigating the role of EVs in EOC has been reviewed following PRISMA guidelines, focusing on the role of EVs in early disease diagnosis, metastatic spread, and the development of chemoresistance in EOC. Data were identified from searches of Medline, Current Contents, PubMed, and from references in relevant articles from 2010 to 1 April 2020. The research yielded 194 results. Of these, a total of 36 papers, 9 reviews, and 27 original types of research were retained and analyzed. The literature findings demonstrate that a panel of EV-derived circulating miRNAs may be useful for early diagnosis of EOC. Furthermore, it appears clear that EVs are involved in mediating two crucial processes for metastatic and chemoresistance development: the epithelial–mesenchymal transition, and tumor escape from the immune system response. Further studies, more focused on in vivo evidence, are urgently needed to clarify the role of EV assessment in the clinical management of EOC patients. Full article

(This article belongs to the Special Issue Extracellular Vesicles as a New Source of Liquid Biopsy)

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