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Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles.

Cell reports | 2019

Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.

Pubmed ID: 30943406 RIS Download

Associated grants

  • Agency: NCRR NIH HHS, United States
    Id: S10 RR028832
  • Agency: NCI NIH HHS, United States
    Id: P01 CA069246
  • Agency: NCI NIH HHS, United States
    Id: R35 CA232103
  • Agency: NINDS NIH HHS, United States
    Id: P30 NS045776
  • Agency: NCI NIH HHS, United States
    Id: R01 CA229777
  • Agency: NCRR NIH HHS, United States
    Id: S10 RR023440
  • Agency: NCRR NIH HHS, United States
    Id: S10 RR020936
  • Agency: NCI NIH HHS, United States
    Id: U19 CA179563
  • Agency: NIH HHS, United States
    Id: S10 OD016372
  • Agency: NCI NIH HHS, United States
    Id: U01 CA233360
  • Agency: NIH HHS, United States
    Id: S10 OD012027

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This is a list of tools and resources that we have found mentioned in this publication.


UniProt (tool)

RRID:SCR_002380

Collection of data of protein sequence and functional information. Resource for protein sequence and annotation data. Consortium for preservation of the UniProt databases: UniProt Knowledgebase (UniProtKB), UniProt Reference Clusters (UniRef), and UniProt Archive (UniParc), UniProt Proteomes. Collaboration between European Bioinformatics Institute (EMBL-EBI), SIB Swiss Institute of Bioinformatics and Protein Information Resource. Swiss-Prot is a curated subset of UniProtKB.

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Promega (tool)

RRID:SCR_006724

An Antibody supplier

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Abcam (tool)

RRID:SCR_012931

A commercial antibody supplier which supplies primary and secondary antibodies, biochemicals, proteins, peptides, lysates, immunoassays and other kits.

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BD Biosciences (tool)

RRID:SCR_013311

An Antibody supplier

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cBioPortal (tool)

RRID:SCR_014555

A portal that provides visualization, analysis and download of large-scale cancer genomics data sets.

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Crl:NU(NCr)-Foxn1nu (tool)

RRID:IMSR_CRL:490

Mus musculus with name Crl:NU(NCr)-Foxn1nu from IMSR.

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Anti-Gaussia Luciferase (antibody)

RRID:AB_2572413

This unknown targets Gaussia Luciferase

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Anti-β-Actin Antibody (antibody)

RRID:AB_476744

This monoclonal targets slightly modified β-cytoplasmic actin N-terminal peptide, Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys, conjugated to KLH

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Antisera-Gluc (antibody)

RRID:AB_2572411

This polyclonal targets Gaussia Luciferase

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Placental alkaline phosphatase (PLAP) antibody (antibody)

RRID:AB_10900125

This polyclonal targets Placental alkaline phosphatase (PLAP) antibody

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CD81 (B-11) (antibody)

RRID:AB_2275892

This monoclonal targets CD81 (B-11)

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Biotin anti-human CD47 (antibody)

RRID:AB_756134

This monoclonal targets CD47

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EpCAM antibody [VU-1D9] (Biotin) (antibody)

RRID:AB_1603294

This monoclonal targets Human EpCAM

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ImageJ (software resource)

RRID:SCR_003070

Open source Java based image processing software program designed for scientific multidimensional images. ImageJ has been transformed to ImageJ2 application to improve data engine to be sufficient to analyze modern datasets.

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FlowJo (software resource)

RRID:SCR_008520

Software for single-cell flow cytometry analysis. Its functions include management, display, manipulation, analysis and publication of the data stream produced by flow and mass cytometers.

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R Project for Statistical Computing (software resource)

RRID:SCR_001905

Software environment and programming language for statistical computing and graphics. R is integrated suite of software facilities for data manipulation, calculation and graphical display. Can be extended via packages. Some packages are supplied with the R distribution and more are available through CRAN family.It compiles and runs on wide variety of UNIX platforms, Windows and MacOS.

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GraphPad Prism (software resource)

RRID:SCR_002798

Statistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.

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OVSAHO (cell line)

RRID:CVCL_3114

Cell line OVSAHO is a Cancer cell line with a species of origin Homo sapiens (Human)

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A2780 (cell line)

RRID:CVCL_0134

Cell line A2780 is a Cancer cell line with a species of origin Homo sapiens (Human)

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Kuramochi (cell line)

RRID:CVCL_1345

Cell line Kuramochi is a Cancer cell line with a species of origin Homo sapiens (Human)

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OV-90 (cell line)

RRID:CVCL_3768

Cell line OV-90 is a Cancer cell line with a species of origin Homo sapiens (Human)

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Caov-3 (cell line)

RRID:CVCL_0201

Cell line Caov-3 is a Cancer cell line with a species of origin Homo sapiens (Human)

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OVCAR-5 (cell line)

RRID:CVCL_1628

Cell line OVCAR-5 is a Cancer cell line with a species of origin Homo sapiens (Human)

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GraphPad Prism (software resource)

RRID:SCR_002798

Statistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.

View all literature mentions

GraphPad Prism (software resource)

RRID:SCR_002798

Statistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.

View all literature mentions

R Project for Statistical Computing (software resource)

RRID:SCR_001905

Software environment and programming language for statistical computing and graphics. R is integrated suite of software facilities for data manipulation, calculation and graphical display. Can be extended via packages. Some packages are supplied with the R distribution and more are available through CRAN family.It compiles and runs on wide variety of UNIX platforms, Windows and MacOS.

View all literature mentions

ImageJ (software resource)

RRID:SCR_003070

Open source Java based image processing software program designed for scientific multidimensional images. ImageJ has been transformed to ImageJ2 application to improve data engine to be sufficient to analyze modern datasets.

View all literature mentions

FlowJo (software resource)

RRID:SCR_008520

Software for single-cell flow cytometry analysis. Its functions include management, display, manipulation, analysis and publication of the data stream produced by flow and mass cytometers.

View all literature mentions