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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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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Basic Protocol 1: Culture of Tumor Spheres from Human Melanomas

  • 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

  • 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

  • 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

  • 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

  • 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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Literature Cited

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