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Effects of Malignant Melanoma Initiating Cells on T-Cell Activation

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Methods in Molecular Biology

Part of the book series: Methods in Molecular Biology

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Abstract

Although human malignant melanoma is a highly immunogenic cancer, both the endogenous antitumor immune response and melanoma immunotherapy often fail to control neoplastic progression. Accordingly, characterizing melanoma cell subsets capable of evading antitumor immunity could unravel optimized treatment strategies that might reduce morbidity and mortality from melanoma. By virtue of their preferential capacity to modulate antitumor immune responses and drive inexorable tumor growth and progression, malignant melanoma-initiating cells (MMICs) warrant closer investigation to further elucidate the cellular and molecular mechanisms underlying melanoma immune evasion and immunotherapy resistance. Here we describe methodologies that enable the characterization of immunoregulatory effects of purified MMICs versus melanoma bulk populations in coculture with syngeneic or allogeneic lymphocytes, using [3H]thymidine incorporation, enzyme-linked immunosorbent spot (ELISPOT), or ELISA assays. These assays were traditionally developed to analyze alloimmune processes and we successfully adapted them for the study of tumor-mediated immunomodulatory functions.

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References

  1. Henry C, Kimura J, Wofsy L (1972) Cell separation on affinity columns: the isolation of immunospecific precursor cells from unimmunized mice (lactoside hapten-lymphocyte receptors-immunology). Proc Natl Acad Sci U S A 69:34–36

    Article  PubMed  CAS  ADS  PubMed Central  Google Scholar 

  2. Julius MH, Masuda T, Herzenberg LA (1972) Demonstration that antigen-binding cells are precursors of antibody-producing cells after purification with a fluorescence-activated cell sorter. Proc Natl Acad Sci U S A 69:1934–1938

    Article  PubMed  CAS  ADS  PubMed Central  Google Scholar 

  3. Watt SM, Gilmore DJ, Davis JM, Clark MR, Waldmann H (1987) Cell-surface markers on haemopoietic precursors. Reagents for the isolation and analysis of progenitor cell subpopulations. Mol Cell Probes 1:297–326

    Article  PubMed  CAS  Google Scholar 

  4. Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730–737

    Article  PubMed  CAS  Google Scholar 

  5. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367:645–648

    Article  PubMed  CAS  ADS  Google Scholar 

  6. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM (2006) Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66:9339–9344

    Article  PubMed  CAS  Google Scholar 

  7. Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, Zhan Q, Jordan S, Duncan LM, Weishaupt C, Fuhlbrigge RC, Kupper TS, Sayegh MH, Frank MH (2008) Identification of cells initiating human melanomas. Nature 451:345–349

    Article  PubMed  CAS  ADS  PubMed Central  Google Scholar 

  8. Boiko AD, Razorenova OV, van de Rijn M, Swetter SM, Johnson DL, Ly DP, Butler PD, Yang GP, Joshua B, Kaplan MJ, Longaker MT, Weissman IL (2010) Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature 466:133–137

    Article  PubMed  CAS  ADS  PubMed Central  Google Scholar 

  9. Civenni G, Walter A, Kobert N, Mihic-Probst D, Zipser M, Belloni B, Seifert B, Moch H, Dummer R, van den Broek M, Sommer L (2011) Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth. Cancer Res 71:3098–3109

    Article  PubMed  CAS  Google Scholar 

  10. Frank NY, Schatton T, Kim S, Zhan Q, Wilson BJ, Ma J, Saab KR, Osherov V, Widlund HR, Gasser M, Waaga-Gasser AM, Kupper TS, Murphy GF, Frank MH (2011) VEGFR-1 expressed by malignant melanoma-initiating cells is required for tumor growth. Cancer Res 71:1474–1485

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Manson LA, Simmons T (1969) Induction of the alloimmune response in mouse lymphocytes by cell-free transplantation antigens in vitro: enhancement of DNA synthesis and specific sensitization. Transplant Proc 1:498–501

    PubMed  CAS  Google Scholar 

  12. Czerkinsky CC, Nilsson LA, Nygren H, Ouchterlony O, Tarkowski A (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65:109–121

    Article  PubMed  CAS  Google Scholar 

  13. Schatton T, Schutte U, Frank NY, Zhan Q, Hoerning A, Robles SC, Zhou J, Hodi FS, Spagnoli GC, Murphy GF, Frank MH (2010) Modulation of T-cell activation by malignant melanoma initiating cells. Cancer Res 70:697–708

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  14. Wei J, Barr J, Kong LY, Wang Y, Wu A, Sharma AK, Gumin J, Henry V, Colman H, Sawaya R, Lang FF, Heimberger AB (2010) Glioma-associated cancer-initiating cells induce immunosuppression. Clin Cancer Res 16:461–473

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  15. Di Tomaso T, Mazzoleni S, Wang E, Sovena G, Clavenna D, Franzin A, Mortini P, Ferrone S, Doglioni C, Marincola FM, Galli R, Parmiani G, Maccalli C (2010) Immunobiological characterization of cancer stem cells isolated from glioblastoma patients. Clin Cancer Res 16:800–813

    Article  PubMed  PubMed Central  Google Scholar 

  16. Frank NY, Pendse SS, Lapchak PH, Margaryan A, Shlain D, Doeing C, Sayegh MH, Frank MH (2003) Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassette transporter. J Biol Chem 278:47156–47165

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported by NIH/National Cancer Institute grants 1RO1CA113796-01A1, 1R01CA138231-01, and 2P50CA093683-06A20006 (to M.H. Frank). T. Schatton is the recipient of a Research Career Development Award from the Dermatology Foundation and an Innovative Research Grant from the Melanoma International Foundation.

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Authors and Affiliations

  1. Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Tobias Schatton & Markus H. Frank

  2. Transplantation Research Program, Division of Nephrology, Children’s Hospital Boston, Harvard Medical School, 300 Longwood Avenue, Boston, MA, 02115, USA

    Tobias Schatton, Ute Schütte & Markus H. Frank

  3. Center of Integrated Oncology (CIO) Cologne-Bonn, Department of Internal Medicine III, University Hospital of Bonn, Bonn, Germany

    Ute Schütte

Authors
  1. Tobias Schatton
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  2. Ute Schütte
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  3. Markus H. Frank
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Correspondence to Markus H. Frank .

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Schatton, T., Schütte, U., Frank, M.H. (2015). Effects of Malignant Melanoma Initiating Cells on T-Cell Activation. In: Methods in Molecular Biology. Humana Press. https://doi.org/10.1007/7651_2015_299

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  • DOI: https://doi.org/10.1007/7651_2015_299

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  • Publisher Name: Humana Press

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