High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry. 2019

Carmen Campos-Silva, and Henar Suárez, and Ricardo Jara-Acevedo, and Estefanía Linares-Espinós, and Luis Martinez-Piñeiro, and María Yáñez-Mó, and Mar Valés-Gómez
Department of Immunology and Oncology, National Centre for Biotechnology, CNB-CSIC, Madrid, Spain.

Extracellular vesicles (EVs) provide an invaluable tool to analyse physiological processes because they transport, in biological fluids, biomolecules secreted from diverse tissues of an individual. EV biomarker detection requires highly sensitive techniques able to identify individual molecules. However, the lack of widespread, affordable methodologies for high-throughput EV analyses means that studies on biomarkers have not been done in large patient cohorts. To develop tools for EV analysis in biological samples, we evaluated here the critical parameters to optimise an assay based on immunocapture of EVs followed by flow cytometry. We describe a straightforward method for EV detection using general EV markers like the tetraspanins CD9, CD63 and CD81, that allowed highly sensitive detection of urinary EVs without prior enrichment. In proof-of-concept experiments, an epithelial marker enriched in carcinoma cells, EpCAM, was identified in EVs from cell lines and directly in urine samples. However, whereas EVs isolated from 5-10 ml of urine were required for western blot detection of EpCAM, only 500 μl of urine were sufficient to visualise EpCAM expression by flow cytometry. This method has the potential to allow any laboratory with access to conventional flow cytometry to identify surface markers on EVs, even non-abundant proteins, using minimally processed biological samples.

UI MeSH Term Description Entries
D001826 Body Fluids Liquid components of living organisms. Body Fluid,Fluid, Body,Fluids, Body
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000067128 Extracellular Vesicles Membrane limited structures derived from cell membranes and cytoplasmic material, and released into EXTRACELLULAR SPACE. They circulate through the EXTRACELLULAR FLUID and through the peripheral blood in the MICROVASCULATURE where cells, much larger, cannot, thereby affecting a variety of intercellular communication processes. Apoptotic Bodies,Exovesicles,Apoptotic Body,Bodies, Apoptotic,Body, Apoptotic,Exovesicle,Extracellular Vesicle,Vesicle, Extracellular,Vesicles, Extracellular
D000078722 PC-3 Cells An epithelial cell line initiated from a human bone metastasis of a grade IV prostatic ADENOCARCINOMA. Human Prostatic Carcinoma Cell Line PC3,PC-3 (Human Prostatic Carcinoma) Cell Line,PC-3 Cell Line,PC3 Cell Line,PC3 Cells,Cell Line, PC-3,Cell Line, PC3,Cells, PC-3,Cells, PC3,Line, PC-3 Cell,PC 3 Cell Line,PC 3 Cells,PC-3 Cell
D014556 Urine Liquid by-product of excretion produced in the kidneys, temporarily stored in the bladder until discharge through the URETHRA.
D015415 Biomarkers Measurable and quantifiable biological parameters (e.g., specific enzyme concentration, specific hormone concentration, specific gene phenotype distribution in a population, presence of biological substances) which serve as indices for health- and physiology-related assessments, such as disease risk, psychiatric disorders, ENVIRONMENTAL EXPOSURE and its effects, disease diagnosis; METABOLIC PROCESSES; SUBSTANCE ABUSE; PREGNANCY; cell line development; EPIDEMIOLOGIC STUDIES; etc. Biochemical Markers,Biological Markers,Biomarker,Clinical Markers,Immunologic Markers,Laboratory Markers,Markers, Biochemical,Markers, Biological,Markers, Clinical,Markers, Immunologic,Markers, Laboratory,Markers, Serum,Markers, Surrogate,Markers, Viral,Serum Markers,Surrogate Markers,Viral Markers,Biochemical Marker,Biologic Marker,Biologic Markers,Clinical Marker,Immune Marker,Immune Markers,Immunologic Marker,Laboratory Marker,Marker, Biochemical,Marker, Biological,Marker, Clinical,Marker, Immunologic,Marker, Laboratory,Marker, Serum,Marker, Surrogate,Serum Marker,Surrogate End Point,Surrogate End Points,Surrogate Endpoint,Surrogate Endpoints,Surrogate Marker,Viral Marker,Biological Marker,End Point, Surrogate,End Points, Surrogate,Endpoint, Surrogate,Endpoints, Surrogate,Marker, Biologic,Marker, Immune,Marker, Viral,Markers, Biologic,Markers, Immune
D060149 Tetraspanin 30 Ubiquitously-expressed tetraspanin protein that is found in late ENDOSOMES and LYSOSOMES. It functions in intracellular protein transport and signaling. Antigens, CD63,CD63 Antigen,Granulophysin,LIMP-CD63,Melanoma Antigen ME491,Platelet Glycoprotein 40 kDa,Pltgp40,Antigen ME491, Melanoma,Antigen, CD63,CD63 Antigens
D060245 Tetraspanin 29 A subtype of tetraspanin protein that plays a role in cell adhesion, cell motility, and tumor metastasis. It functions in platelet activation and aggregation, the formation of paranodal junctions in neuronal tissue, and the fusion of sperm with egg. Antigens, CD9,27 kDa Diphtheria Toxin Receptor-Associated Protein,CD9 Antigen,DRAP27 Protein,Leukemia-Associated Cell Surface Antigen p24,Motility-Related Protein-1,Tetraspanin-29,p24-CD9 Antigen,27 kDa Diphtheria Toxin Receptor Associated Protein,Antigen, CD9,CD9 Antigens,Leukemia Associated Cell Surface Antigen p24,Motility Related Protein 1,p24 CD9 Antigen

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