Culture and Isolation of Melanoma‐Initiating Cells

Barbara Stecca1, Roberta Santini1, Silvia Pandolfi1, Junia Y. Penachioni1

1 Tumor Cell Biology Unit, Core Research Laboratory, Istituto Toscano Tumori, Florence, Italy
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 3.6
DOI:  10.1002/9780470151808.sc0306s24
Online Posting Date:  February, 2013
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Abstract

Melanoma is the most aggressive skin cancer. This unit illustrates protocols for culture and isolation of human melanoma cancer stem cells/tumor‐initiating cells (CSC/TIC). We describe two complementary methods to enrich for melanoma CSC/TIC. The first approach exploits the ability of CSC/TIC to grow as tumor spheres in low‐adherent culture conditions, as previously shown for neural stem cells and human embryonic stem cells. As a second approach, melanoma CSC/TIC are enriched by fluorescence‐activated cell sorting for the aldehyde dehydrogenase (ALDH) enzyme activity. We previously showed that melanoma cells with high ALDH activity (ALDHhigh) are endowed with higher self‐renewal and tumorigenic abilities than the population with low activity (ALDHlow), suggesting that ALDH might be a good marker to select for melanoma CSC/TIC. This unit will also describe how to functionally test melanoma CSC/TIC by determining self‐renewal in vitro and tumor‐forming abilities in vivo using orthotopic xenograft assay. Curr. Protoc. Stem Cell Biol. 24:3.6.1‐3.6.12. © 2013 by John Wiley & Sons, Inc.

Keywords: melanoma; cancer stem cells; in vitro sphere assay; in vivo xenograft assay; Aldefluor assay; tumor spheres

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of Tumor Spheres from Human Melanomas
  • Support Protocol 1: Passaging of Tumor Spheres
  • Basic Protocol 2: Enrichment of Melanoma CSC/TIC by Aldefluor Assay
  • Basic Protocol 3: In Vitro Self‐Renewal Assay
  • Basic Protocol 4: In Vivo Orthotopic Xenograft Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culture of Tumor Spheres from Human Melanomas

  Materials
  • Human melanoma specimens on ice
  • 1× sterile phosphate‐buffered saline (PBS—both phosphate‐buffered saline with or without calcium and magnesium can be used to wash tissue) with 2% penicillin/streptomycin
  • Melanoma dissociation medium (see recipe)
  • DMEM/F12 serum‐free medium (see recipe)
  • Human embryonic stem cell (hESC) medium (see recipe)
  • Sterile forceps and razor blade
  • 100‐mm tissue culture dish
  • 15‐ml Falcon tubes
  • 5‐ml serological plastic pipet
  • 70‐µm cell strainer filter (Becton Dickinson, cat. no. 352350)
  • Centrifuge
  • 1000‐µl pipet and tips
  • Low‐adherence flasks
  • Additional equipment for counting cells using a hemacytometer and trypan blue (Phelan, )

Support Protocol 1: Passaging of Tumor Spheres

  Materials
  • Primary human melanoma cells growing in suspension as tumor spheres (see protocol 1)
  • 1× phosphate‐buffered saline (calcium‐ and magnesium‐free) containing 2 mM EDTA (made from a stock of 0.5M EDTA, pH 8.0)
  • 15‐ml Falcon tubes
  • Centrifuge
  • 200‐ to 1000‐µl pipets and tips
  • 5‐ml serological plastic pipets
  • Additional equipment for counting cells using a hemacytometer and trypan blue (Phelan, )

Basic Protocol 2: Enrichment of Melanoma CSC/TIC by Aldefluor Assay

  Materials
  • Dissociated primary melanoma cells or dissociated tumor spheres
  • Aldefluor kit (Stem Cell Technologies, cat. no. 1700), consisting of:
    • Dry ALDEFLUOR reagent, 50 µg
    • Diethylaminobenzaldehyde (DEAB)
    • 2 N HCl
    • Dimethyl sulfoxide (DMSO)
    • Aldefluor assay buffer
  • Activated Aldefluor reagent (see recipe)
  • 1× phosphate‐buffered saline (PBS; calcium‐ and magnesium‐free)
  • 200‐ to 1000‐µl pipets and tips
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge
  • 30‐µm filter (Cup Filcons; Becton Dickinson, cat. no. 340626)
  • Fluorescence‐activated cell sorter (FACS)
  • 5‐ml polystyrene FACS tubes
  • Additional reagents and equipment for counting cells using a hemacytometer (Phelan, ) and for fluorescence‐activated cell sorting (FACS; Robinson et al., )

Basic Protocol 3: In Vitro Self‐Renewal Assay

  Materials
  • Primary (or secondary) melanoma spheres or FACS‐sorted ALDHhigh and ALDHlow cells
  • DMEM/F12 serum‐free medium (see recipe) or Human embryonic stem cell (hESC) medium (see recipe)
  • Low‐adherent 12‐well plates or 96‐well flat plates
  • Microscope
  • Additional equipment for counting cells using a hemacytometer and trypan blue (Phelan, )

Basic Protocol 4: In Vivo Orthotopic Xenograft Assay

  Materials
  • Primary (or subsequently passaged) melanoma spheres or FACS‐sorted ALDHhigh and ALDHlow cells
  • 1:1 Matrigel (Becton Dickinson, cat. no. 356234)/DMEM, freshly prepared (Matrigel must be stored undiluted at −20°C)
  • Athymic nude mice (8‐ to 10‐weeks‐old)
  • 1 ml syringe with a 29‐G needle, 1/ 2‐in. long
  • Ear punching or ear tags for identifying mice
  • Caliper
  • Additional equipment for counting cells using a hemacytometer and trypan blue (Phelan, )
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Figures

Videos

Literature Cited

Literature Cited
   Boiko, A.D., Razorenova, O.V., van de Rijn, M., Wetter, S.M., Johnson, D.L., Ly, D.P., Butler, P.D., Yang, G.P., Joshua, B., Kaplan, M.J., Longaker, M.T., and Weissman, I.L. 2010. Human melanoma‐initiating cells express neural crest nerve growth factor receptor CD271. Nature 466:133‐137.
   Boonyaratanakornkit, J.B., Yue, L., Strachan, L.R., Scalapino, K.J., LeBoit, P.E., Lu, Y., Leong, S.P., Smith, J.E., and Ghadially, R. 2010. Selection of tumorigenic melanoma cells using ALDH. J. Invest. Dermatol. 130:2799‐2808.
   Cheung, A.M., Wan, T.S., Leung, J.C., Chan, L.Y., Huang, H., Kwong, Y.L., Liang, R., and Leung, A.Y. 2007. Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential. Leukemia 21:1423‐1430.
   Civenni, G., Walter, A., Kobert, N., Mihic‐Probst, D., Zipser, M., Belloni, B., Seifert, B., Moch, H., Dummer, R., van den Broek, M., and Sommer, L. 2011. Human CD271‐positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long‐term growth. Cancer Res. 71:3098‐3109.
   Fang, D., Nguyen, T.K., Leishear, K., Finko, R., Kulp, A.N., Hotz, S., Van Belle, P.A., Xu, X., Elder, D.E., and Herlyn, M. 2005. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res. 65:9328‐9337.
   Ginestier, C., Hur, M.H., Charafe‐Jauffret, E., Monville, F., Dutcher, J., Brown, M., Jacquemier, J., Viens, P., Kleer, C.G., Liu, S., Schott, A., Hayes, D., Birnbaum, D., Wicha, M.S., and Dontu, G. 2007. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1:555‐567.
   Girouard, S.D. and Murphy, G.F. 2011. Melanoma stem cells: not rare, but well done. Lab. Invest. 91:647‐664.
   Huang, E.H., Hynes, M.J., Zhang, T., Ginestier, C., Dontu, G., Appelman, H., Fields, J.Z., Wicha, M.S., and Boman, B.M. 2009. Aldehyde dehydrogenase 1 is marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res. 69:3382‐3389.
   Kupas, V., Weishaupt, C., Siepmann, D., Kaserer, M.L., Eickelmann, M., Metze, D., Luger, T.A., Beissert, S., and Loser, K. 2011. RANK is expressed in metastatic melanoma and highly upregulated on melanoma‐initiating cells. J. Invest. Dermatol. 131:944‐955.
   Monzani, E., Facchetti, F., Galmozzi, E., Corsini, E., Benetti, A., Cavazzin, C., Gritti, A., Piccinini, A., Porro, D., Santinami, M., Invernici, G., Parati, E., Alessandri, G., and La Porta, C.A. 2007. Melanoma contains CD133 and ABCG2 positive cells with enhanced tumorigenic potential. Eur. J. Cancer 43:935‐946.
   Moreb, J., Schweder, M., Suresh, A., and Zucali, J.R. 1996. Overexpression of the human aldehyde dehydrogenase class I results in increased resistance to 4‐hydroperoxycyclophosphamide. Cancer Gene Ther. 3:24‐30.
   Palma, V. and Ruiz i Altaba, A. 2004. Hedgehog‐GLI signaling regulates the behavior of cells with stem cell properties in the developing neocortex. Development 131:337‐345.
   Perego, M., Tortoreto, M., Tragni, G., Mariani, L., Deho, P., Carbone, A., Santinami, M., Patuzzo, R., Mina, P.D., Villa, A., Pratesi, G., Cossa, G., Perego, P., Daidone, M.G., Alison, M.R., Parmiani, G., Rivoltini, L., and Castelli, C. 2010. Heterogeneous phenotype of human cells with in vitro and in vivo features of tumor‐initiating cells. J. Invest. Dermatol. 130:1877‐1886.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Prasmickaite, L., Engesaeter, B.Ø., Skrbo, N., Hellenes, T., Kristian, A., Oliver, N.K., Suo, Z., and Maelandsmo, G.M. 2010. Aldehyde dehydrogenase (ALDH) activity does not select for cells with enhanced aggressive properties in malignant melanoma. PLoS One 5:e10731.
   Quintana, E., Shackleton, M., Sabel, M.S., Fullen, D.R, Johnson, T.M., and Morrison, S.J. 2008. Efficient tumour formation by single human melanoma cells. Nature 456:593‐598.
   Quintana, E., Shackleton, M., Foster, H.R., Fullen, D.R., Sabel, M.S., Johnson, T.M., and Morrison, S.J. 2010. Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized. Cancer Cell 18:510‐523.
   Reynolds, B.A. and Weiss, S. 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707‐1710.
   Reynolds, B.A. and Weiss, S. 1996. Clonal and population analyses demonstrate that an EGF‐responsive mammalian embryonic CNS precursor is a stem cell. Dev. Biol. 175:1‐13.
   Robinson, J.P., Darzynkiewicz, Z., Hoffman, R., Nolan, J.P., Orfao, A., Rabinovitch, P.S., and Watkins, S. (eds.) 2013. Current Protocols in Cytometry. John Wiley & Sons, Hoboken, N.J.
   Roesch, A., Fukunaga‐Kalabis, M., Schmidt, E.C., Zabierowski, S.E., Brafford, P.A., Vultur, A., Basu, D., Gimotty, P., Vogt, T., and Herlyn, M. 2010. A temporary distinct subpopulation of slow‐cycling melanoma cells is required for continuous tumor growth. Cell 141:583‐594.
   Santini, R., Vinci, M.C., Pandolfi, S., Penachioni, J.Y., Montagnani, V., Olivito, B., Gattai, R., Pimpinelli, N., Gerlini, G., Borgognoni, L., and Stecca, B. 2012. HEDGEHOG‐GLI signaling drives self‐renewal and tumorigenicity of human melanoma‐initiating cells. Stem Cells 30:1808‐1818.
   Schatton, T., Murphy, G.F., Frank, N.Y., Yamaura, K., Waaga‐Gasser, A.M., Gasser, M., Zhan, Q., Jordan, S., Duncan, L.M., Weishaupt, C., Fuhlbrigge, R.C., Kupper, T.S., Sayegh, M.H., and Frank, M.H. 2008. Identification of cells initiating human melanomas. Nature 451:345‐349.
   Stecca, B. and Ruiz i Altaba, A. 2009. A GLI1‐p53 inhibitory loop controls neural stem cell and tumour cell numbers. EMBO J. 28:663‐676.
   Sullivan, J.P., Spinola, M., Dodge, M., Raso, M.G., Behrens, C., Gao, B., Schuster, K., Shao, C., Larsen, J.E., Sullivan, L.A., Honorio, S., Xie, Y., Scaglioni, P.P., DiMaio, J.M., Gazdar, A.F., Shay, J.W., Wistuba, I.I., and Minna, J.D. 2010. Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling. Cancer Res. 70:9937‐9948.
   van den Hoogen, C., van der Horst, G., Cheung, H., Buijs, J.T., Lippitt, J.M., Guzmán‐Ramírez, N., Hamdy, F.C., Eaton, C.L., Thalmann, G.N., Cecchini, M.G., Pelger, R.C., and van der Pluijm, G. 2010. High aldehyde dehydrogenase activity identifies tumor‐initiating and metastasis‐initiating cells in human prostate cancer. Cancer Res. 70:5163‐5173.
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