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Melanoma pp 305–321Cite as

Immunoaffinity-Based Isolation of Melanoma Cell-Derived and T Cell-Derived Exosomes from Plasma of Melanoma Patients

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

Abstract

Tumor-derived exosomes (TEX), a subset of small extracellular vesicles (EVs) which originate from the endocytic compartment of tumor cells, are emerging as key players in cancer progression. TEX circulate freely in patients’ body fluids and transfer bioactive cargos from tumor to various recipient cells. The molecular cargo of melanoma cell-derived exosomes (MTEX) mimics that of the tumor, and MTEX serve as a liquid biopsy that provides potentially useful information for cancer diagnosis, prognosis, or responses to therapy. Plasma of melanoma patients contains a mix of MTEX and exosomes produced by nonmalignant cells (NMTEX). Isolation of these exosome subtypes from the bulk of plasma exosomes is necessary to evaluate contributions of each as potential biomarkers of melanoma progression and outcome. Here, methods for separation of MTEX from T cell-derived exosomes from a single small volume of plasma and their subsequent molecular and functional characterization are described. Following size exclusion chromatography (SEC) to isolate total plasma exosomes, immune affinity-based capture of MTEX with anti-CSPG4 antibody and then of exosomes produced by T cells with anti-CD3 antibody is used to sequentially isolate the two subsets. This immune capture method enables the recovery of MTEX and CD3+ exosomes in quantities sufficient both for molecular profiling by flow cytometry or western blotting and for functional analyses.

Key words

  • Melanoma cell-derived exosomes (MTEX)
  • Non–malignant cell-derived exosomes (NMTEX)
  • CD3+ exosomes
  • Immunoaffinity-based capture
  • Plasma-derived exosomes

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Acknowledgments

This work was, in part, supported by the NIH grant R01-CA 168628 to TLW.

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Authors and Affiliations

  1. Department of Pathology, University of Pittsburgh School of Medicine and UPMC Hillman Cancer Center, Pittsburgh, PA, USA

    Sujan Kumar Mondal & Theresa L. Whiteside

  2. Departments of Immunology and Otolaryngology, University of Pittsburgh School of Medicine and UPMC Hillman Cancer Center, Pittsburgh, PA, USA

    Theresa L. Whiteside

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  1. Sujan Kumar Mondal
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  2. Theresa L. Whiteside
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Correspondence to Theresa L. Whiteside .

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Editors and Affiliations

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

    Ph.D. Kristian M. Hargadon

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Mondal, S.K., Whiteside, T.L. (2021). Immunoaffinity-Based Isolation of Melanoma Cell-Derived and T Cell-Derived Exosomes from Plasma of Melanoma Patients. 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_23

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  • DOI: https://doi.org/10.1007/978-1-0716-1205-7_23

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