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An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles

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Melanoma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2265))

Abstract

Most human cells release extracellular vesicles (EVs) of different sizes and composition, containing biomolecules characteristic from the originating tissue. In consequence, when EVs derive from a cancer cell, they also contain tumor antigens. Therefore, isolating and characterizing tumor-derived EVs has attracted great interest as an invaluable source of biomarkers, both for diagnosis and stratification of cancer. In this chapter, we describe a method for flow cytometry assessment of melanoma-derived EVs which are firstly captured onto antibody-coated beads recognizing either a common EV marker, namely, a tetraspanin, or a tumor antigen like the stress-related molecules MICA or PDL1. Then, after staining with a fluorophore-conjugated antibody directed against a different protein present on the EV surface, the EV-bead complex can be visualized in a conventional flow cytometer. The technique allows detection of proteins present on EVs isolated from tissue culture supernatants of melanoma cell lines and, more importantly, directly from plasma.

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Acknowledgments

We would like to thank Prof. J.L. Carrascosa, CNB-CSIC, for Electron Microscopy analysis of EVs immunocaptured on microspheres; Dr. V. Horejsi (Institute of Molecular Genetics of the ASCR, Czech Republic) for the gift of CD63, CD81, and CD9 antibodies; Prof A. Paschen (University Hospital Essen, Germany) for melanoma cell lines; H. Peinado (Spanish National Centre for Oncological Research (CNIO) for the use of Nanosight; C. Moreno and S. Escudero, from the Flow Cytometry Service, CNB-CSIC; and C. Patiño, from the Electron Microscopy Service, CNB-CSIC.

Conflict of Interest: R.J. is CEO of Immunostep, S.L.

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Author notes

  1. Sheila López-Cobo

    Present address: INSERM U932, Institut Curie, PSL Research University, Paris, France

Authors and Affiliations

  1. Department of Immunology and Oncology, Spanish National Centre for Biotechnology, CNB-CSIC, Madrid, Spain

    Carmen Campos-Silva, Yaiza Cáceres-Martell, Sheila López-Cobo & Mar Valés-Gómez

  2. Department of Molecular and Cell Biology, Spanish National Centre for Biotechnology, CNB-CSIC, Madrid, Spain

    María Josefa Rodriguez

  3. Immunostep, S.L., Centro Investigación del Cáncer (CIC), Salamanca, Spain

    Ricardo Jara

  4. Department of Molecular Biology, Universidad Autónoma de Madrid and Centro de Biología Molecular Severo Ochoa, IIS-IP, Madrid, Spain

    María Yáñez-Mó

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Correspondence to Mar Valés-Gómez .

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  1. Department of Biology, Hampden-Sydney College, Hampden-Sydney, VA, USA

    Kristian M. Hargadon

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Campos-Silva, C. et al. (2021). An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles. In: Hargadon, K.M. (eds) Melanoma. Methods in Molecular Biology, vol 2265. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1205-7_24

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  • DOI: https://doi.org/10.1007/978-1-0716-1205-7_24

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1204-0

  • Online ISBN: 978-1-0716-1205-7

  • eBook Packages: Springer Protocols

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