Isolation and Characterization of Potential Cancer Stem Cells from Solid Human Tumors—Potential Applications

Zachary C. Dobbin1, Charles N. Landen1

1 Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.28
DOI:  10.1002/0471141755.ph1428s63
Online Posting Date:  December, 2013
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Abstract

Cancer stem cells (CSCs) are a subpopulation of cells within a heterogeneous tumor that have enhanced biologic properties, e.g., increased capacity for self‐renewal, increased tumorigenicity, enhanced differentiation capacity, and resistance to chemo‐ and radiotherapies. This unit describes protocols to isolate and characterize potential cancer stem cells from a solid tumor. These involve creating a single‐cell suspension from tumor tissue, tagging the cell subpopulations of interest, and sorting them into different populations. The sorted subpopulations can be evaluated for their ability to meet the functional requirements of a CSC, which primarily include increased tumorigenicity in an in vivo xenograft assay. Use of the protocols described in this unit makes it possible to study populations of cells that may have properties of CSCs. Curr. Protoc. Pharmacol. 63:14.28.1‐14.28.19. © 2013 by John Wiley & Sons, Inc.

Keywords: cancer stem cells; xenograft assay; cell separation and sorting

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Mechanical Dissociation of Primary Tumor or Mouse Xenografts
  • Alternate Protocol 1: Chemical Dissociation of Primary Tumor or Mouse Xenograft
  • Basic Protocol 2: Identification of Cells Based on Surface Marker Expression and Separation by Flow Cytometry
  • Alternate Protocol 2: Isolating Cancer Stem Cells Using Magnetic Bead Separation
  • Support Protocol 1: Using the EasySep “Do‐It‐Yourself”
  • Basic Protocol 3: Isolating Cancer Stem Cells Based on Hoechst Dye Exclusion: The Side Population (SP)
  • Basic Protocol 4: Determining Tumorigenicity of Putative Cancer Stem Cells
  • Basic Protocol 5: Spheroid Assay of Putative Cancer Stem Cells
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mechanical Dissociation of Primary Tumor or Mouse Xenografts

  Materials
  • At least 1 cm3 of viable tumor tissue with minimal necrotic component
  • Serum‐free RPMI cell culture medium or medium preferred for the tumor type under analysis
  • Trypan blue
  • PBS (optional)
  • 10‐cm Petri dish
  • Tissue forceps
  • Scalpel with #22 blade
  • 10‐, 5‐, and 1‐ml pipets
  • 16‐gauge needles and 3‐ to 5‐ml syringes
  • 70‐μm sterile mesh filter (Sefar Filtration)
  • 50‐ml conical tube
  • 200‐ and 100‐μm sterile mesh filters (optional; Sefar Filtration)
  • Additional reagents and equipment for assay of viability by trypan blue exclusion (Strober, )

Alternate Protocol 1: Chemical Dissociation of Primary Tumor or Mouse Xenograft

  Materials
  • Tumor fragment
  • 1× PBS
  • Enzymatic digestion solution (0.25% trypsin/EDTA) with or without hyaluronidase and collagenase (see step 2 note)
  • RPMI‐1640 medium with 10% fetal bovine serum (FBS)
  • Trypan blue
  • 50‐ml, 10‐ml (optional), and 15‐ml (optional) conical tubes
  • 10‐cm Petri dish
  • #22 scalpel blade and blade handle
  • Tissue forceps
  • 70‐μm sterile mesh filter
  • 10‐ml, 5‐ml, and 1‐ml serological pipets
  • Additional reagents and equipment for assay of viability by trypan blue exclusion (Strober, )

Basic Protocol 2: Identification of Cells Based on Surface Marker Expression and Separation by Flow Cytometry

  Materials
  • Single‐cell suspension of cancer cells ( protocol 1)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free, with 0.1% bovine serum albumin (CMF‐PBS/0.1% BSA)
  • Antibodies for desired surface marker(s) conjugated to a fluorophore (antibodies validated for use in flow cytometry work best; BD Biosciences)
  • RPMI or cell culture medium of choice containing 0.1% BSA (optional)
  • 5‐ml conical polystyrene tubes
  • Flow cytometer with appropriate channels for the fluorochromes being employed

Alternate Protocol 2: Isolating Cancer Stem Cells Using Magnetic Bead Separation

  Materials
  • Single‐cell suspension of cancer cells ( protocol 1)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free with 2% fetal bovine serum and 1 mM EDTA (CMF‐PBS/2% FBS/1 mM EDTA)
  • Species‐specific FcR blocking antibody (same species as the FITC‐ or biotin‐conjugated antibody)
  • FITC‐conjugated (or biotin‐conjugated) antibody of marker of interest
  • EasySep FITC (or biotin) selection cocktail (STEMCELL Technologies)
  • EasySep (or equivalent) magnetic nanoparticles (STEMCELL Technologies)
  • Medium of choice
  • 12 × 75‐mm polystyrene tubes (e.g., Falcon 5‐ml round‐bottom tubes)
  • EasySep (or equivalent) magnet (STEMCELL Technologies)

Support Protocol 1: Using the EasySep “Do‐It‐Yourself”

  Additional Materials
  • Mouse IgG 1 monoclonal antibody for surface marker of interest
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
  • EasySep “Do‐It‐Yourself” Selection Kit (STEMCELL Technologies)

Basic Protocol 3: Isolating Cancer Stem Cells Based on Hoechst Dye Exclusion: The Side Population (SP)

  Materials
  • Preferred culture medium
  • Single‐cell suspension of cancer cells ( protocol 1)
  • Hoechst 33342 dye (Thermo Scientific, cat. no. 62249)
  • Verapamil (Sigma)
  • Sterile phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
  • 5‐ml polystyrene tubes
  • Additional reagents and equipment for flow cytometry of side population (Goodell, ; Petriz, )

Basic Protocol 4: Determining Tumorigenicity of Putative Cancer Stem Cells

  Materials
  • Putative cancer stem cell and non‐CSC populations sorted via protocol 3, protocol 4, protocol 5, or protocol 6
  • Preferred cell culture medium (e.g., DMEM), serum‐free
  • Matrigel, preferably without added growth factors (optional; BD Biosciences)
  • 6‐ to 8‐week old SCID mice (e.g., NCI Frederick, Taconic, Charles River Labs, Jackson Labs) of desired gender kept in approved IACUC and ARP conditions
  • Isoflurane
  • 1‐ml syringe
  • 25‐gauge needles
  • Vaporizing anesthesia device (e.g., Drager 19.1, Anesthesia Service and Equipment) with access to clinical‐grade oxygen
  • Betadine scrub
  • Hair removal equipment (shaver or Nair) if using non‐nude mice
  • Warming equipment, e.g., heat lamp, isothermal pad (Deltaphase Isothermal Pad, Braintree Scientific)

Basic Protocol 5: Spheroid Assay of Putative Cancer Stem Cells

  Materials
  • Heterogeneous cell population, or subpopulation
  • B‐27 supplement, 50× (Gibco #12587‐010)
  • Serum‐free medium of choice
  • Recombinant FGF‐basic (Gibco #PHG0021L)
  • Recombinant EGF (Gibco #PHG0311)
  • Costar ultra‐low‐attachment multiple‐well plates (Corning #3473)
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Figures

Videos

Literature Cited

Literature Cited
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