Colon Cancer Stem Cells

Antonija Kreso1, Catherine Adell O'Brien2

1 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada, 2 Department of Surgery, Division of General Surgery, University Health Network, Toronto, Ontario, Canada
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 3.1
DOI:  10.1002/9780470151808.sc0301s7
Online Posting Date:  November, 2008
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Abstract

This unit describes protocols for working with colon cancer stem cells. To work with these cells one must start by generating single‐cell suspensions from human colon cancer tissue. These cell suspensions are sorted using flow cytometry–assisted cell sorting to fractionate the cells into tumor‐initiating and nontumor‐initiating subsets. Once the cells have been fractionated, they must be functionally tested to determine tumor‐forming capacity, the gold standard being the in vivo xenograft assay. Methods have also been developed to grow these cells in vitro in a sphere‐forming assay. This unit will describe how to isolate and functionally test colon cancer stem cells, as well as provide advice on the potential challenges of the research. Curr. Protoc. Stem Cell Biol. 7:3.1.1‐3.1.12. © 2008 by John Wiley & Sons, Inc.

Keywords: human colon cancer; cancer stem cells; in vivo xenograft assay; in vitro sphere assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generating Single‐Cell Suspensions from Human Colon Cancer Tissue
  • Support Protocol 1: Using a Ficoll Column to Remove Dead Cells
  • Basic Protocol 2: Flow Cytometry–Assisted Cell Sorting
  • Basic Protocol 3: In Vivo Xenograft Assay
  • Basic Protocol 4: Culturing Colon Cancer Cells as Spheres
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generating Single‐Cell Suspensions from Human Colon Cancer Tissue

  Materials
  • Colon tumor fragment
  • Colon cancer stem cell medium (SCM; see recipe)
  • Collagenase IV solution (200 U/ml SCM)
  • Ammonium chloride: 0.8% (w/v) NH 4Cl in 0.1 mM EDTA
  • Trypan blue
  • 35‐mm petri dishes
  • Razor blade and forceps
  • 5‐ml disposable pipets
  • 50‐ml conical tube
  • 45‐µm cell filter
  • Plunger from a 3‐ to 5‐ml syringe
  • Hemacytometer
  • Additional reagents and equipment for counting cells using a hemacytometer and trypan blue (unit 1.3)

Support Protocol 1: Using a Ficoll Column to Remove Dead Cells

  Materials
  • Ficoll
  • Colon cancer cell suspension ( protocol 1)
  • Colon cancer stem cell medium (SCM; see recipe)
  • 15‐ml conical tubes
  • 5‐ml pipet

Basic Protocol 2: Flow Cytometry–Assisted Cell Sorting

  Materials
  • Colon cancer cell suspension with <30% dead cells
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS) with 0.1% bovine serum albumin (BSA; CMF‐PBS/0.1% BSA)
  • ESA antibody conjugated to a fluorophore
  • Anti–mouse antibody conjugated to a fluorophore
  • CD133 APC or PE (Miltenyi Biotec)
  • Propidium iodide (final concentration: 1 µg/ml of PBS with 0.1% BSA)
  • 5‐ml polystyrene tubes
  • Additional reagents and equipment for flow cytometry–assisted cell sorting (Robinson et al., ) and counting cells using a hemacytometer (unit 1.3)

Basic Protocol 3: In Vivo Xenograft Assay

  Materials
  • Matrigel
  • ∼10 µl sorted cell suspension in SCM ( protocol 3)
  • Stem cell medium (SCM; see recipe)
  • NOD/SCID or SCID mice (8‐ to 10‐week‐old)
  • Iodine‐based solution (e.g., Betadine)
  • 70% ethanol
  • Normal saline, sterile
  • Pain medication (e.g., buprenorphine)
  • 1‐ml syringe without needle
  • 1‐ml insulin syringes with a 29‐G needle, ½‐in. long
  • Heating pad
  • Clippers
  • Sterile gauze
  • Scissors
  • Forceps
  • Sutures or surgical clips (Roboz)
  • Additional reagents and equipment for rodent anesthesia using isoflurane (unit 1.4)

Basic Protocol 4: Culturing Colon Cancer Cells as Spheres

  Materials
  • Colon cancer cell suspension, sorted ( protocol 3)
  • Stem cell medium (SCM; see recipe)
  • Trypsin/EDTA
  • Ultra‐low attachment surface dishes (Corning)
  • 5‐ml disposable pipet
  • 45‐µm filter
  • Additional reagents and equipment for counting cells using trypan blue (unit 1.3)
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Figures

Videos

Literature Cited

Literature Cited
   Al‐Hajj, M., Wicha, M.S., Benito‐Hernandez, A., Morrison, S.J., and Clarke, M.F. 2003. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 100: 3983‐3988.
   Dalerba, P., Dylla, S.J., Park, I.K., Liu, R., Wang, X., Cho, R.W., Hoey, T., Gurney, A., Huang, E.H., Simeone, D.M., Shelton, A.A., Parmiani, G., Castelli, C., and Clark, M.F. 2007. Phenotypic characterization of human colorectal cancer stem cells. Proc. Natl. Acad. Sci. U.S.A. 104: 10158‐10163.
   O'Brien, C.A., Pollett, A., Gallinger, S., and Dick, J.E. 2007. A human colon cancer cell capable of initiating growth in immunodeficient mice. Nature 445: 106‐110.
   Porter, E.H. and Berry, R.J. 1964. The efficient design of transplantable tumour assays. Br. J. Cancer 17: 583‐595.
   Ricci‐Vitiani, L., Lombardi, D.G., Pilozzi, E., Biffoni, M., Todaro, M., Peschle, C., and DeMaria, R. 2007. Identification and expression of human colon‐cancer‐initiating cells. Nature 445: 111‐115.
   Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Dressler, L.G., Rabinovitch, P.S., Stewart, C.C., Tanke, H.J., and Wheeless, L.L. (eds.) 2008. Current Protocols in Cytometry. John Wiley & Sons, Hoboken, N.J.
   Singh, S.K., Hawkins, C., Clarke, I.D., Squire, J.A., Bayani, J., Hide, T., Henkelman, R.M., Cusimano, M.D., and Dirks, P.B. 2004. Identification of human brain tumor initiating cells. Nature 432: 396‐401.
   Todaro, M., Alea, M.P., Di Stefano, A.B., Cammareri, P., Vermeulen, L., Iovino, F., Tripodo, C., Russo, A., Gulotta, G., Medema, J.P., and Stassi, G. 2007. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin‐4. Cell Stem Cell 1: 389‐402.
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