In Vitro Enrichment of Tumor‐Initiating Cells from Human Established Cell Lines

Liat Benayoun1, Yuval Shaked1

1 Department of Molecular Pharmacology, Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 3.7
DOI:  10.1002/9780470151808.sc0307s24
Online Posting Date:  February, 2013
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Abstract

This unit describes protocols for culturing and subsequently enriching cancer stem cells (CSCs), also referred to as tumor‐initiating cells (TICs), from human established cell lines. TICs are thought to display the major cell population in the tumor with the proliferative capacity to seed tumors, implying that theyare critical in initiating and driving tumorigenesis. The protocols show the methods for enriching and subsequently characterizing TIC populations from a series of human tumors including glioblastoma, breast, and pancreatic tumors. Protocols evaluating the morphology, phenotypic, and functional properties of TICs are described. Long‐term cultures grown either as monolayers (“TIC‐low”) or as non‐adherent tumor spheres (“TIC‐high”) are generated. As a result, cells from the TIC‐high culture exhibit increased expression of stem cell surface markers, such as CD133, CD44, and CD24, high aldehyde dehydrogenase (ALDH) activity, and elevated expression levels of p21, in comparison to cells from the TIC‐low culture. Studying TICs by using such protocols is cost effective and is considered as a suitable and simple way for studying them. Curr. Protoc. Stem Cell Biol. 24:3.7.1‐3.7.15. © 2013 by John Wiley & Sons, Inc.

Keywords: tumor‐initiating cells (TICs); cancer stem cell (CSC); human cancer cell lines; ALDH (aldehyde dehydrogenase)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of TIC‐High and TIC‐Low from Human Established Cell Lines
  • Basic Protocol 2: Phenotypic Identification of TICs by Flow Cytometry
  • Basic Protocol 3: Phenotypic Identification of TICs by Immunoblotting
  • Basic Protocol 4: Functional Identification of TICs by ALDH Enzymatic Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generation of TIC‐High and TIC‐Low from Human Established Cell Lines

  Materials
  • Human established cell lines, such as U‐87MG glioma, MCF7 breast carcinoma, and PANC1 pancreatic adenocarcinoma frozen in 1.8‐ to 2‐ml cryovials
  • 1× sterile Dulbecco's phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • Complete Dulbecco's modified Eagle medium (DMEM; Sigma, cat. no. D5796) with 10% fetal bovine serum (FBS; Biological Industries, cat. no. 040071A), 1% (w/v) L‐glutamine, 1% (w/v) sodium‐pyruvate, and 1% (w/v) streptomycin
  • Trypsin‐0.25% EDTA (Biological Industries, cat. no. 030521A)
  • Tumor sphere medium DMEM/F‐12 (see recipe)
  • Growth factor cocktail mix for U‐87MG, MCF7, PANC1 (see recipe)
  • 37°C water bath
  • 15‐ml and 50‐ml centrifuge tubes, sterile
  • Centrifuge
  • 10‐cm2 dishes (Corning)
  • 37°C, 5% CO 2 incubator
  • 5‐, 10‐, and 25‐ml pipets
NOTE: All cell lines used in this protocol can be obtained from the American Type Culture Collection (ATCC).NOTE: Before thawing the tumor cells, ensure that the centrifuge is at room temperature.NOTE: Before thawing the tumor cells, ensure that the PBS and DMEM 10% FBS are prewarmed in a 37°C water bath.NOTE: When culturing TIC‐high cells, ensure that DMEM/F‐12, trypsin and growth factors cocktail mix are prewarmed in a 37°C water bath.

Basic Protocol 2: Phenotypic Identification of TICs by Flow Cytometry

  Materials
  • TIC‐low and TIC‐high cultured cells ( protocol 1)
  • Flow cytometry buffer (see recipe)
  • Ice
  • Mo‐Antibodies APC‐anti human CD133 (Macs Militenyi Biotec), PE anti‐human CD24 and APC anti‐human CD44 (BD Biosciences Pharmingen)
  • 15‐ml centrifuge tubes
  • Centrifuge
  • 70‐µm cell strainer (BD Falcon)
  • Flow cytometry microcentrifuge tubes (BD Falcon, cat. no. 352058)
  • Vortex mixer
  • Flow cytometry system: e.g., CyAn ADP (Beckman Coulter), equipped with 488‐nm, and 635‐nm lasers; a 575/25 bandpass (bp) filter for PE, and a 665/20 bp for APC
  • Summit Version 3.4 software (Beckman Coulter) or equivalent software for flow cytometry analysis
  • Additional reagents and equipment for counting cells using a hemacytometer and trypan blue (Phelan, )
NOTE: Before preparing the cells for flow cytometry acquisition, ensure that the centrifuge is at 4°C.NOTE: Work quickly and keep cells and flow cytometry buffer cold (at 4°C).NOTE: Master mix should be kept in the dark, and should not be stored longer than 1 week at 4°C.

Basic Protocol 3: Phenotypic Identification of TICs by Immunoblotting

  Materials
  • TIC‐low and TIC‐high cultured cells ( protocol 1)
  • Trypsin‐0.25% EDTA (Biological Industries, cat. no. 030521A)
  • Dulbecco's phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • DMEM/F‐12
  • Lysis buffer (see recipe) containing protease inhibitors cocktail tablets (Roche, cat. no. 11836145001)
  • Bradford reagent for protein assay (Bio Rad, cat. no. 50006)
  • PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Scientific, cat. no. 26619)
  • Mini‐protein precast gel 4% to 15%, 10‐well comb (Bio Rad, cat. no. 4561083)
  • Mini‐protein Tetra Cell for ready gel (Bio Rad, cat. no. 1658005)
  • Transfer buffer: 10 × Tris/Glycine buffer (Bio Rad, cat. no. 1610734)
  • 100% nonfat milk
  • TBST (see recipe)
  • Mo mouse anti‐p21 primary antibody (BD Biosciences Pharmingen, cat. no. 556430)
  • Peroxidase‐conjugated goat anti‐mouse IgG secondary antibody (Jackson, cat. no. 115035 146)
  • EZ‐ECL chemiluminescence detection kit for HRP (Biological industries, cat. no. 20500 120)
  • Antibody stripping buffer (Gene Bio Applications, cat. no. ST010)
  • Mo mouse anti‐actin (Milipore, cat. no. 691001)
  • 5‐ and 10‐ml pipets
  • 15‐ and 50‐ml centrifuge tubes, sterile
  • Centrifuge
  • 37°C, 5% CO 2 incubator
  • 1.5‐ml microcentrifuge tubes
  • PVDF membrane, 0.2 µm (Bio Rad, cat. no. 1620177)
  • Whatmann 3 MM filter paper
  • 4°C incubator
  • Shaker
  • Staining trays
  • Biomolecular digital imaging system (e.g., Image Quant LAS 4000)

Basic Protocol 4: Functional Identification of TICs by ALDH Enzymatic Activity

  Materials
  • TIC‐low and TIC‐high cultured cells ( protocol 1)
  • ALDEFLUOR kit (StemCell Technologies, cat. no. 01700) containing:
    • ALDEFLUOR assay buffer
  • 7‐Aminoactinomycin D (7AAD) solution (Calibiochem, cat. no. 129935)
  • 15‐ml centrifuge tubes
  • Centrifuge
  • 70‐µm cell strainer (BD Falcon)
  • Flow cytometry system, e.g., CyAn ADP (Beckman Coulter), equipped with 488‐nm laser (a 530/40 bandpass filter and a 680/30 bp filter for FITC and 7AAD detection, respectively)
  • Summit Version 3.4 software (Beckman Coulter) or equivalent software for flow cytometry analysis
  • Additional reagents and equipment for counting cells using a hemacytometer and trypan blue (Phelan, )
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Figures

Videos

Literature Cited

Literature Cited
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