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

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

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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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 IgG₁ 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

Figure 14.28.1 Spheroid growth in a heterogeneous sample. Unsorted A2780 ovarian cancer cells were plated in serum‐free differentiation‐inhibiting medium on ultra‐low‐attachment plates. A spheroid of fused cells is noted to have a different phenotype than the majority of dividing cells, which remain attached as aggregates but not spheroids (A ). Cells plated as a single cell per well can sometimes form spheroids, which can be maintained for several weeks (B ).

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Figure 14.28.2 CD555 expression in a heterogeneous population. Dissociated cells from a freshly collected solid tumor were subjected to flow cytometric analysis after exposure to anti‐CD555‐FITC antibody. Gating based on the negative control shows that 1.8% of cells are CD555‐positive. Side scatter plotted on y axis against intensity of FITC signal on x axis.

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Figure 14.28.3 Platinum resistance in the CD555 population. Dissociated cells were sorted by flow cytometry based on CD555 expression, and plated into 96‐well plates, 2000 cells per well. After 6 hr, the medium was replaced with medium with increasing concentrations of carboplatin or vehicle. After 5 days, cells were subjected to MTT. CD555‐positive cells were much more resistant, demonstrating a carboplatin IC₅₀ value of 82 nM, while CD555‐negative cells had an IC₅₀ value of 4.4 nM.

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Literature Cited

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