Non-coding RNAs as biomarkers in liquid biopsies with a special emphasis on extracellular vesicles in urological malignancies

Brain tumors are the most common malignant primary intracranial tumors of the central nervous system. They are often recognized too late for successful therapy. Minimally invasive methods are needed to establish a diagnosis or monitor the response to treatment of CNS tumors. Brain tumors release molecular information into the circulation. Liquid biopsies collect and analyze tumor components in body fluids, and there is an increasing interest in the investigation of liquid biopsies as a substitute for tumor tissue. Tumor-derived biomarkers include nucleic acids, proteins, and tumor-derived extracellular vesicles that accumulate in blood or cerebrospinal fluid. In recent years, circulating tumor cells have also been identified in the blood of glioblastoma patients. In this review of the literature, the authors highlight the significance, regulation, and prevalence of molecular biomarkers such as O6-methylguanine-DNA methyltransferase, epidermal growth factor receptor, and isocitrate dehydrogenase. Herein, we critically review the available literature on plasma circulating tumor cells (CTCs), cell-free tumors (ctDNAs), circulating cell-free microRNAs (cfmiRNAs), and circulating extracellular vesicles (EVs) for the diagnosis and monitoring of brain tumor. Currently available markers have significant limitations.While much research has been conductedon these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature.

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Extracellular Vesicles: Novel Opportunities to Understand and Detect Neoplastic Diseases

Veterinary Pathology ◽

10.1177/0300985821999328 ◽

2021 ◽

pp. 030098582199932

Author(s):

Laura Bongiovanni ◽

Anneloes Andriessen ◽

Marca H. M. Wauben ◽

Esther N. M. Nolte-’t Hoen ◽

Alain de Bruin

Keyword(s):

Extracellular Vesicles ◽

Biologically Active ◽

Liquid Biopsies ◽

Donor Cells ◽

Recent Developments ◽

Veterinary Pathology ◽

Cell Components ◽

Potential Applications ◽

Intercellular Communications

With a size range from 30 to 1000 nm, extracellular vesicles (EVs) are one of the smallest cell components able to transport biologically active molecules. They mediate intercellular communications and play a fundamental role in the maintenance of tissue homeostasis and pathogenesis in several types of diseases. In particular, EVs actively contribute to cancer initiation and progression, and there is emerging understanding of their role in creation of the metastatic niche. This fact underlies the recent exponential growth in EV research, which has improved our understanding of their specific roles in disease and their potential applications in diagnosis and therapy. EVs and their biomolecular cargo reflect the state of the diseased donor cells, and can be detected in body fluids and exploited as biomarkers in cancer and other diseases. Relatively few studies have been published on EVs in the veterinary field. This review provides an overview of the features and biology of EVs as well as recent developments in EV research including techniques for isolation and analysis, and will address the way in which the EVs released by diseased tissues can be studied and exploited in the field of veterinary pathology. Uniquely, this review emphasizes the important contribution that pathologists can make to the field of EV research: pathologists can help EV scientists in studying and confirming the role of EVs and their molecular cargo in diseased tissues and as biomarkers in liquid biopsies.

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Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids

Scientific Reports ◽

10.1038/s41598-021-86910-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jingjing Zhang ◽

Luong T. H. Nguyen ◽

Richard Hickey ◽

Nicole Walters ◽

Xinyu Wang ◽

...

Keyword(s):

Extracellular Vesicles ◽

Culture Media ◽

Metastatic Breast ◽

Clinical Samples ◽

Clinical Use ◽

Immunomagnetic Beads ◽

Liquid Biopsies ◽

Cell Culture Media ◽

Non Invasive ◽

Healthy Donors

AbstractExtracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

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Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽

10.3390/diagnostics11081320 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1320

Author(s):

Kristýna Pekárková ◽

Jakub Soukup ◽

Marie Kostelanská ◽

Jan Širc ◽

Zbyněk Straňák ◽

...

Keyword(s):

Flow Cytometry ◽

Cord Blood ◽

Extracellular Vesicles ◽

Correlation Coefficients ◽

Flow Cytometer ◽

Liquid Biopsies ◽

Physiological Conditions ◽

Flow Cytometric ◽

Term Births ◽

And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

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Nanoparticle-based biosensors for detection of extracellular vesicles in liquid biopsies

Journal of Materials Chemistry B ◽

10.1039/d0tb00861c ◽

2020 ◽

Vol 8 (31) ◽

pp. 6710-6738

Author(s):

Beatriz Martín-Gracia ◽

Alba Martín-Barreiro ◽

Carlos Cuestas-Ayllón ◽

Valeria Grazú ◽

Aija Line ◽

...

Keyword(s):

Extracellular Vesicles ◽

Liquid Biopsies ◽

Speed Up

Selecting the appropriate nanoparticle, functionalization chemistry and sensing methodology can speed up the translation of liquid biopsies into the clinic.

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Protein biomarkers in breast cancer-derived extracellular vesicles for use in liquid biopsies

AJP Cell Physiology ◽

10.1152/ajpcell.00048.2021 ◽

2021 ◽

Author(s):

Dan Li ◽

Wenjia Lai ◽

Di Fan ◽

Qiaojun Fang

Keyword(s):

Breast Cancer ◽

Early Diagnosis ◽

Extracellular Vesicles ◽

Liquid Biopsy ◽

Breast Cancer Diagnosis ◽

Extracellular Vesicle ◽

Detection Methods ◽

Protein Markers ◽

Liquid Biopsies ◽

Diagnosis And Prognosis

Breast cancer is the most common malignant disease in women worldwide. Early diagnosis and treatment can greatly improve the management of breast cancer. Liquid biopsies are becoming convenient detection methods for diagnosing and monitoring breast cancer due to their non-invasiveness and ability to provide real-time feedback. A range of liquid biopsy markers, including circulating tumor proteins, circulating tumor cells, and circulating tumor nucleic acids, have been implemented for breast cancer diagnosis and prognosis, with each having its own advantages and limitations. Circulating extracellular vesicles are messengers of intercellular communication that are packed with information from mother cells and are found in a wide variety of bodily fluids; thus, they are emerging as ideal candidates for liquid biopsy biomarkers. In this review, we summarize extracellular vesicle protein markers that can be potentially used for the early diagnosis and prognosis of breast cancer or determining its specific subtypes.

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Development of extracellular vesicles-based classifier for detection of early-stage bladder, ovarian, and pancreatic cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.3048 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 3048-3048

Author(s):

Juan Pablo Hinestrosa ◽

Razelle Kurzrock ◽

Jean Lewis ◽

Nick Schork ◽

Ashish M. Kamat ◽

...

Keyword(s):

Pancreatic Cancer ◽

Confidence Interval ◽

Extracellular Vesicles ◽

Metastatic Disease ◽

Cancer Detection ◽

Early Stage ◽

Expression Patterns ◽

Stepwise Logistic Regression ◽

Liquid Biopsies ◽

Cancer Early Detection

3048 Background: Many cancers are lethal because they present with metastatic disease. Because localized/resectable tumors produce vague symptoms, diagnosis is delayed. In pancreatic cancer, only ̃10% of patients survive five years, and it will soon become the second leading cause of cancer-related deaths in the USA. For patients with metastatic disease, the 2- and 5-year survival is < 10% and ̃3%, respectively. For the few patients with local disease, 5-year survival is ̃40%. Many other cancers have comparable differences between early- and late-stage disease. It is apparent a diagnostic assay for early-stage cancers would transform the field by minimizing the need for aggressive surgeries and other harsh interventions, and by its potential to increase survival. Identifying cancer-specific aberrations in blood-based “liquid” biopsies offers a prospect for a non-invasive cancer detection tool. In the bloodstream, there are extracellular vesicles (EVs) with cargoes including membrane and cytosolic proteins, as well as RNA and lipids derived from their parent cells. Methods: We used an alternating current electrokinetics (ACE) microarray to isolate EVs from the plasma of stage I and II bladder (N = 48), ovarian (N = 42), and pancreatic cancer patients (N = 44), and healthy volunteers (N = 110). EVs were analyzed using multiplex protein immunoassays for 54 cancer-related proteins. EV protein expression patterns were analyzed using stepwise logistic regression followed by a split between training and test sets (67%/33% respectively). This process enabled biomarker selection and generation of a classifier to discriminate between cancer and healthy donors. Results: The EV protein-based classifier had an overall area under curve (AUC) of 0.95 with a sensitivity of 71.2% (69.4% – 73.0%, at 95% confidence interval) at > 99% specificity. The classifier’s performance for the pancreatic cancer cohort was very strong, with overall sensitivity of 95.7% (94.6% – 96.9%, at 95% confidence interval) at > 99% specificity. Conclusions: EV-associated proteins may enable early cancer detection where surgical resection is most likely to improve outcomes. The classifier’s performance for the initial three cancers studied showed encouraging results. Future efforts will include examining additional cancer types and evaluating the classifier performance using samples from donors with related benign conditions with the aim of a pan-cancer early detection assay.

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Extracellular Vesicles and Matrix Remodeling Enzymes: The Emerging Roles in Extracellular Matrix Remodeling, Progression of Diseases and Tissue Repair

Cells ◽

10.3390/cells7100167 ◽

2018 ◽

Vol 7 (10) ◽

pp. 167 ◽

Cited By ~ 52

Author(s):

Muhammad Nawaz ◽

Neelam Shah ◽

Bruna Zanetti ◽

Marco Maugeri ◽

Renata Silvestre ◽

...

Keyword(s):

Extracellular Matrix ◽

Extracellular Vesicles ◽

Tissue Repair ◽

Metabolic Diseases ◽

Bioactive Molecules ◽

Tissue Inhibitors Of Metalloproteinases ◽

Matrix Remodeling ◽

Pathological Conditions ◽

Potential Applications ◽

Non Coding Rnas

Extracellular vesicles (EVs) are membrane enclosed micro- and nano-sized vesicles that are secreted from almost every species, ranging from prokaryotes to eukaryotes, and from almost every cell type studied so far. EVs contain repertoire of bioactive molecules such as proteins (including enzymes and transcriptional factors), lipids, carbohydrates and nucleic acids including DNA, coding and non-coding RNAs. The secreted EVs are taken up by neighboring cells where they release their content in recipient cells, or can sail through body fluids to reach distant organs. Since EVs transport bioactive cargo between cells, they have emerged as novel mediators of extra- and intercellular activities in local microenvironment and inter-organ communications distantly. Herein, we review the activities of EV-associated matrix-remodeling enzymes such as matrix metalloproteinases, heparanases, hyaluronidases, aggrecanases, and their regulators such as extracellular matrix metalloproteinase inducers and tissue inhibitors of metalloproteinases as novel means of matrix remodeling in physiological and pathological conditions. We discuss how such EVs act as novel mediators of extracellular matrix degradation to prepare a permissive environment for various pathological conditions such as cancer, cardiovascular diseases, arthritis and metabolic diseases. Additionally, the roles of EV-mediated matrix remodeling in tissue repair and their potential applications as organ therapies have been reviewed. Collectively, this knowledge could benefit the development of new approaches for tissue engineering.

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