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Isolation of Melanoma Cell Subpopulations Using Negative Selection

  • Ana Slipicevic
  • Rajasekharan Somasundaram
  • Katrin Sproesser
  • Meenhard Herlyn
Part of the Methods in Molecular Biology book series (MIMB, volume 1102)

Abstract

Melanomas are phenotypically and functionally heterogeneous tumors comprising of distinct subpopulations that drive disease progression and are responsible for resistance to therapy. Identification and characterization of such subpopulations are highly important to develop novel targeted therapies. However, this can be a challenging task as there is a lack of clearly defined markers to distinguish the melanoma subpopulations from a general tumor cell population. Also, there is a lack of optimal isolation methods and functional assays that can fully recapitulate their phenotype. Here we describe a method for isolating tumor cells from fresh human tumor tissue specimens using an antibody coupled magnetic bead sorting technique that is well established in our laboratory. Thus, melanoma cells are enriched by negative cell sorting and elimination of non-tumor cell population such as erythrocytes, leukocytes, and endothelial cells. Enriched unmodified tumor cells can be further used for phenotypic and functional characterization of melanoma subpopulations.

Key words

Subpopulations Tumor cell isolation Magnetic beads Tumorigenic potential 

References

  1. 1.
    Nowell PC (1976) The clonal evolution of tumor cell populations. Science 194:23–28PubMedCrossRefGoogle Scholar
  2. 2.
    Fearon ER, Vogelstein B (1990) A genetic model for colorectal tumorigenesis. Cell 61:759–767PubMedCrossRefGoogle Scholar
  3. 3.
    Dick JE (2008) Stem cell concepts renew cancer research. Blood 112:4793–4807PubMedCrossRefGoogle Scholar
  4. 4.
    Lobo NA, Shimono Y, Qian D, Clarke MF (2007) The biology of cancer stem cells. Annu Rev Cell Dev Biol 23:675–699PubMedCrossRefGoogle Scholar
  5. 5.
    Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–111PubMedCrossRefGoogle Scholar
  6. 6.
    Gupta PB, Onder TT, Jiang G et al (2009) Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138:645–659PubMedCrossRefGoogle Scholar
  7. 7.
    Mani SA, Guo W, Liao MJ et al (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133:704–715PubMedCrossRefGoogle Scholar
  8. 8.
    Marusyk A, Polyak K (2010) Tumor heterogeneity: causes and consequences. Biochim Biophys Acta 1805:105–117PubMedGoogle Scholar
  9. 9.
    Roesch A, Fukunaga-Kalabis M, Schmidt EC et al (2010) A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth. Cell 141:583–594PubMedCrossRefGoogle Scholar
  10. 10.
    Jones PM, George AM (2004) The ABC transporter structure and mechanism: perspectives on recent research. Cell Mol Life Sci 61:682–699PubMedCrossRefGoogle Scholar
  11. 11.
    Dean M, Fojo T, Bates S (2005) Tumour stem cells and drug resistance. Nat Rev Cancer 5:275–284PubMedCrossRefGoogle Scholar
  12. 12.
    Szakacs G, Annereau JP, Lababidi S et al (2004) Predicting drug sensitivity and resistance: profiling ABC transporter genes in cancer cells. Cancer Cell 6:129–137PubMedCrossRefGoogle Scholar
  13. 13.
    Schatton T, Murphy GF, Frank NY et al (2008) Identification of cells initiating human melanomas. Nature 451:345–349PubMedCrossRefGoogle Scholar
  14. 14.
    Monzani E, Facchetti F, Galmozzi E et al (2007) Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential. Eur J Cancer 43:935–946PubMedCrossRefGoogle Scholar
  15. 15.
    Taghizadeh R, Noh M, Huh YH et al (2011) CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells. PLoS One 5:e15183CrossRefGoogle Scholar
  16. 16.
    Elliott AM, Al-Hajj MA (2009) ABCB8 mediates doxorubicin resistance in melanoma cells by protecting the mitochondrial genome. Mol Cancer Res 7:79–87PubMedCrossRefGoogle Scholar
  17. 17.
    Somasundaram R, Villanueva J, Herlyn M (2011) Will engineered T cells expressing CD20 scFv eradicate Melanoma? Mol Ther 19:638–640PubMedCrossRefGoogle Scholar
  18. 18.
    Tedder TF, Engel P (1994) CD20: a regulator of cell-cycle progression of B lymphocytes. Immunol Today 15:450–454PubMedCrossRefGoogle Scholar
  19. 19.
    Bittner M, Meltzer P, Chen Y et al (2000) Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 406:536–540PubMedCrossRefGoogle Scholar
  20. 20.
    Fang D, Nguyen TK, Leishear K et al (2005) A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65:9328–9337PubMedCrossRefGoogle Scholar
  21. 21.
    Schmidt P, Kopecky C, Hombach A, Zigrino P, Mauch C, Abken H (2011) Eradication of melanomas by targeted elimination of a minor subset of tumor cells. Proc Natl Acad Sci U S A 108:2474–2479PubMedCrossRefGoogle Scholar
  22. 22.
    Pinc A, Somasundaram R, Wagner C et al (2012) Targeting CD20 in melanoma patients at high risk of disease recurrence. Mol Ther 20(5):1056–1062PubMedCrossRefGoogle Scholar
  23. 23.
    Schlaak M, Schmidt P, Bangard C, Kurschat P, Mauch C, Abken H (2012) Regression of metastatic melanoma in a patient by antibody targeting of cancer stem cells. Oncotarget 3:22–30PubMedGoogle Scholar
  24. 24.
    Neuzil J, Stantic M, Zobalova R et al (2007) Tumour-initiating cells vs. cancer “stem” cells and CD133: what’s in the name? Biochem Biophys Res Commun 355:855–859PubMedCrossRefGoogle Scholar
  25. 25.
    Dembinski JL, Krauss S (2009) Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma. Clin Exp Metastasis 26(7):611–623PubMedCrossRefGoogle Scholar
  26. 26.
    Liu G, Yuan X, Zeng Z et al (2006) Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer 5:67PubMedCrossRefGoogle Scholar
  27. 27.
    Ricci-Vitiani L, Lombardi DG, Pilozzi E et al (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115PubMedCrossRefGoogle Scholar
  28. 28.
    Salmaggi A, Boiardi A, Gelati M et al (2006) Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype. Glia 54:850–860PubMedCrossRefGoogle Scholar
  29. 29.
    Shmelkov SV, Butler JM, Hooper AT et al (2008) CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors. J Clin Invest 118:2111–2120PubMedGoogle Scholar
  30. 30.
    Levina V, Marrangoni AM, DeMarco R, Gorelik E, Lokshin AE (2008) Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties. PLoS One 3:e3077PubMedCrossRefGoogle Scholar
  31. 31.
    Visvader JE, Lindeman GJ (2008) Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8:755–768PubMedCrossRefGoogle Scholar
  32. 32.
    Frank NY, Margaryan A, Huang Y et al (2005) ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res 65:4320–4333PubMedCrossRefGoogle Scholar
  33. 33.
    Klein WM, Wu BP, Zhao S, Wu H, Klein-Szanto AJ, Tahan SR (2007) Increased expression of stem cell markers in malignant melanoma. Mod Pathol 20:102–107PubMedCrossRefGoogle Scholar
  34. 34.
    Rappa G, Fodstad O, Lorico A (2008) The stem cell-associated antigen CD133 (Prominin-1) is a molecular therapeutic target for metastatic melanoma. Stem Cells 26:3008–3017PubMedCrossRefGoogle Scholar
  35. 35.
    Piras F, Perra MT, Murtas D et al (2010) The stem cell marker nestin predicts poor prognosis in human melanoma. Oncol Rep 23:17–24PubMedGoogle Scholar
  36. 36.
    Fusi A, Reichelt U, Busse A et al (2011) Expression of the stem cell markers nestin and CD133 on circulating melanoma cells. J Invest Dermatol 131:487–494PubMedCrossRefGoogle Scholar
  37. 37.
    Sharma BK, Manglik V, O’Connell M et al (2012) Clonal dominance of CD133+ subset population as risk factor in tumor progression and disease recurrence of human cutaneous melanoma. Int J Oncol 41(5):1570–1576PubMedGoogle Scholar
  38. 38.
    Lai CY, Schwartz BE, Hsu MY (2012) CD133+ Melanoma subpopulations contribute to perivascular niche morphogenesis and tumorigenicity through vasculogenic mimicry. Cancer Res 72(19):5111–5118PubMedCrossRefGoogle Scholar
  39. 39.
    Wang J, Sakariassen PO, Tsinkalovsky O et al (2008) CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells. Int J Cancer 122:761–768PubMedCrossRefGoogle Scholar
  40. 40.
    Quintana E, Shackleton M, Foster HR et al (2010) Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized. Cancer Cell 18:510–523PubMedCrossRefGoogle Scholar
  41. 41.
    Joo KM, Kim SY, Jin X et al (2008) Clinical and biological implications of CD133-positive and CD133-negative cells in glioblastomas. Lab Invest 88:808–815PubMedCrossRefGoogle Scholar
  42. 42.
    Boiko AD, Razorenova OV, van de Rijn M et al (2010) Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature 466:133–137PubMedCrossRefGoogle Scholar
  43. 43.
    Held MA, Curley DP, Dankort D, McMahon M, Muthusamy V, Bosenberg MW (2010) Characterization of melanoma cells capable of propagating tumors from a single cell. Cancer Res 70:388–397PubMedCrossRefGoogle Scholar
  44. 44.
    Sharma SV, Lee DY, Li B et al (2010) A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 141:69–80PubMedCrossRefGoogle Scholar
  45. 45.
    Magee JA, Piskounova E, Morrison SJ (2012) Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21:283–296PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Ana Slipicevic
    • 1
    • 2
  • Rajasekharan Somasundaram
    • 1
  • Katrin Sproesser
    • 1
  • Meenhard Herlyn
    • 3
  1. 1.The Wistar InstitutePhiladelphiaUSA
  2. 2.Department of PathologyThe Norwegian Radium Hospital, Oslo University HospitalOsloNorway
  3. 3.Wistar InstitutePhiladelphiaUSA

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