Experimental Metastasis Assays in the Chick Embryo

Sylvia M. Wilson1, Ann F. Chambers1

1 London Regional Cancer Centre, London, Ontario
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.6
DOI:  10.1002/0471143030.cb1906s21
Online Posting Date:  February, 2004
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Abstract

Experimental metastasis assays are used to measure the ability of cancer cells to grow in secondary organs following injection of the cells into the circulation of an experimental animal. The chicken embryo provides an alternative to the more commonly used assays in mice. Details of the experimental metastasis assay in chick embryo are provided, including protocols for maintenance of fertilized chicken eggs, injection of cells into the circulation of 11‐day‐old chick embryos, recovery and quantification of cancer cells from chick liver using the ouabain plating assay, labeling of cells with fluorescent nanospheres, and monitoring of individual steps in the metastatic process in the chick chorioallantoic membrane using intravital videomicroscopy. These assays provide a cost‐effective, readily accessible, and rapid approach for studying the process of cancer metastasis.

Keywords: tumor metastasis; chick embryo; chorioallantoic membrane; intravital videomicroscopy; alternative assays

     
 
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Table of Contents

  • Basic Protocol 1: Experimental Metastasis Assay in the Chick Embryo
  • Support Protocol 1: Routine Maintenance of Eggs
  • Basic Protocol 2: Intravital Videomicroscopy of the Chick Embryo CAM
  • Support Protocol 2: Labeling Cells with Fluorescent Nanospheres
  • Support Protocol 3: Cell Accounting in Tissues
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Experimental Metastasis Assay in the Chick Embryo

  Materials
  • Eggs containing 11‐day‐old chick embryos (see protocol 2)
  • Adherent rodent tumor cells, optionally labeled with nanospheres (see protocol 4)
  • Medium and serum used to grow tumor cells
  • Paraplast wax
  • Paraffin oil
  • 70% ethanol
  • Sterile calcium‐ and magnesium‐free PBS, 4°C (see recipe)
  • Hank's balanced salt solution (HBSS), 4°C (see recipe)
  • 0.3% (w/v) collagenase/0.02% (w/v) trypsin (see recipe)
  • DNase I (see recipe)
  • 2 × 103 M ouabain (see recipe)
  • Detergent or other decontaminating solution
  • Methylene blue stain (see recipe)
  • Egg candler (local farm supply store)
  • Pencil
  • Enclosed still hood with glass front, interior light, and electrical outlet
  • Dremel Moto tool with polishing wheel, 6/8 × 1/8 –in. thick (available at local hobby shop)
  • 38.5°C (99° to 100°F) automatic rotating egg incubator with 80% to 82% humidity (e.g., March Farms Roll‐X incubators, Lyon Electric; available at farm supply stores)
  • Hemacytometer (unit 1.1) or Coulter counter
  • Adjustable gooseneck light source and fiber optic unit (e.g., Schott Glas Fiber Optics unit, Carl Zeiss unit) or focused intense light source
  • Egg support stand (i.e., three rubber corks glued on a plastic petri dish; Figs. and )
  • 1‐cc syringes
  • 30‐G ½‐in. needles
  • Cotton swab
  • Disposable underpad
  • 1‐liter plastic beaker lined with a 12‐lb. plastic bag
  • Small plastic beaker
  • Dissecting instruments, autoclaved:
    • Straight medium‐point dissecting forceps, 115 mm in length
    • Straight narrow‐blades dissecting scissors with fine points, 110 mm in length
    • Two straight fine‐point dissecting forceps, 110 mm in length
    • Two scalpel handles with no. 10 disposable blades
  • 150 × 20–mm plastic dishes
  • 100 × 15–mm nontissue and tissue culture dishes
  • 24‐well tissue culture plates
  • 100‐mm glass dishes (autoclave in a canister)
  • 5‐ml pipet
  • 17 × 100–mm polystyrene test tubes
  • 500‐ml wash bottle
  • Funnel
  • Plastic wash basin with drainage holes drilled half‐way up each end
  • Additional reagents and equipment for trypsinization (unit 1.1), adding accounting spheres (see protocol 5), and inspecting for gross surface tumors (Chambers et al., ; Chambers and Wilson, )
NOTE: During dissection, all instruments should be rinsed in 70% alcohol, wiped clean with a tissue, and flame sterilized between each step

Support Protocol 1: Routine Maintenance of Eggs

  Materials
  • Fresh fertilized eggs (standard outbred White Leghorn), 4°C
  • Pencil
  • 38.5°C (99° to 100°F) automatic rotating egg incubator with 80% to 82% humidity (e.g., March Farms Roll‐X incubators, Lyon Electric; available at farm supply stores)
  • Hygrometer to routinely monitor egg incubator humidity

Basic Protocol 2: Intravital Videomicroscopy of the Chick Embryo CAM

  Materials
  • Eggs containing 11‐day‐old chick embryos (see protocol 2)
  • Adherent tumor cells with or without nanospheres (see protocol 4)
  • Medium and serum used to grow tumor cells
  • Citrate saline (see recipe)
  • Paraffin oil
  • Plasticine
  • Egg candler (local farm supply store)
  • Pencil
  • Enclosed still hood with glass front, interior light, and electrical outlet
  • Dremel Moto tool with polishing wheel, 6/8 × 1/8 –in. thick (available at local hobby shop)
  • 38.5°C (99° to 100°F) automatic rotating egg incubator with 80% to 82% humidity (e.g., March Farms Roll‐X incubators, Lyon Electric; available at farm supply stores)
  • 30‐G ½ –in. needle attached to a PE‐10 cannula and 1‐cc syringe
  • Hemacytometer (unit 1.1) or Coulter counter
  • Vinyl tape
  • No. 1 coverslip, 45 × 50–mm
  • 180 × 130 × 3–mm acrylic viewing center with 40 × 40–mm hole cut in the center
  • Masking tape
  • Inverted microscope (e.g., Nikon Diaphot TMD) with 10× to 60× or 100× objectives and mercury arc lamp with B2‐A filter block (570‐nm dichroic mirror and 520‐nm barrier filter; 450‐ to 490‐nm excitation wavelength; Nikon)
  • Infrared heat lamp
  • Fiber optic light source with 150 W halogen bulb
  • Newvicon tube video camera with extended red sensitivity (Panasonic WV1550 or Hamamatsu C2400)
  • Additional reagents and equipment for preparing windows in eggs (see protocol 1, steps to and step ) and injecting tumor cells into eggs (see protocol 1, steps to and steps to )

Support Protocol 2: Labeling Cells with Fluorescent Nanospheres

  Materials
  • 0.05‐µm‐diameter, fluorescent, carboxylated P(S/V‐COOH), dragon‐green (480/520 nm) nanospheres (Bangs Laboratories) for cell labeling; store at 4°C in the dark
  • OptiMEM serum‐reduced medium (InVitrogen)
  • Cells to be labeled
  • Sonicator
  • 50‐ml conical polystyrene centrifuge tubes
  • 0.2‐µm syringe filter and 10‐ml syringe
  • Aluminum foil
  • 75‐cm2 tissue culture flask
  • 150 × 15–mm tissue culture dishes
  • Tissue culture incubator, standard, 37°C, 5% CO 2
  • Additional reagents and solutions for culturing, trypsinizing, and counting cells (unit 1.1)

Support Protocol 3: Cell Accounting in Tissues

  Materials
  • Tumor cell suspension, with or without fluorescent nanospheres (see protocol 4)
  • 10‐µm‐diameter, yellow/green (505/515 nm) fluorescent, plastic microspheres (fluorospheres; Molecular Probes)
  • 60‐mm tissue culture dishes
  • Additional reagents and equipment for fluorescent microscopy (unit 4.2)
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Figures

Videos

Literature Cited

Literature Cited
   Bondy, G.P., Wilson, S., and Chambers, A.F. 1985. Experimental metastatic ability of H‐ras‐transformed NIH 3T3 cells. Cancer Res. 45:6005‐6009.
   Chambers, A.F. and Ling, V. 1984. Selection of experimental metastatic ability of heterologous tumor cells in the chick embryo after DNA‐mediated transfer. Cancer Res. 44:3970‐3975.
   Chambers, A.F. and Tuck, A.B. 1988. Oncogene transformation and the metastatic phenotype. Anticancer Res. 8:861‐872.
   Chambers, A.F. and Wilson, S. 1985. Cells transformed with ts viral src mutant are temperature‐sensitive for in vivo growth. Mol. Cell. Biol. 5:728‐733.
   Chambers, A.F. and Wilson, S. 1988. Use of NeoR B16F1 murine melanoma cells to assess clonality of experimental metastases in the immune‐deficient chick embryo. Clin. Exper. Metast. 6:171‐182.
   Chambers, A.F., Shafir, R., and Ling, V. 1982. A model system for studying metastasis using the embryonic chick. Cancer Res. 42:4018‐4025.
   Chambers, A.F., Wilson, S.M., Tuck, A.B., Denhardt, G.H., and Cairncross, J.G. 1990. Comparison of metastatic properties of a variety of mouse, rat, and human cells in assays in nude mice and chick embryos. In Vivo 4:215‐219
   Chambers, A.F., Schmidt, E.E., MacDonald, I.C., Morris, V.L., and Groom, A.C. 1992. Early steps in hematogenous metastasis of B16F1 melanoma cells in chick embryos studied by high‐resolution intravital videomicroscopy. J. Natl. Cancer Inst. 84:797‐803.
   Chambers, A.F., MacDonald, I.C., Schmidt, E.E., Koop, S., Morris, V.L., Khokha, R., and Groom, A.C. 1995. Steps in tumor metastasis: New concepts from intravital videomicroscopy. Cancer Metast. Rev. 14:279‐301.
   Chambers, A.F., Groom, A.C., and MacDonald, I.C. 2002. Dissemination and growth of cancer cells in metastatic sites. Nat. Rev. Cancer 2:563‐572.
   Hill, S.A., Wilson, S., and Chambers, A.F. 1988. Clonal heterogeneity, experimental metastatic ability, and p21 expression in H‐ras‐transformed NIH 3T3 cells. J. Natl. Cancer Inst. 80:484‐490.
   Koop, S., Khokha, R., Schmidt, E.E., MacDonald, I.C., Morris, V.L., Chambers, A.F., and Groom, A.C. 1994. Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth. Cancer Res. 54:4791‐4797.
   Koop, S., MacDonald, I.C., Luzzi, K., Schmidt, E.E., Morris, V.L., Gratten, M., Khokha, R., Chambers, A.F., and Groom, A.C. 1995. Fate of melanoma cells entering the microcirculation: Over 80% survive and extravasate. Cancer Res. 55:2520‐2523.
   Koop, S., Schmidt, E.E., MacDonald, I.C., Morris, V.L., Khokha, R., Grattan, M., Leone, J., Chambers, A.F., and Groom, A.F. 1996. Independence of metastatic ability and extravasation: Metastatic ras‐transformed and control fibroblasts extravasate equally well. Proc. Natl. Acad. Sci. U.S.A. 93:11080‐11084.
   MacDonald, I.C., Schmidt, E.E., Morris, V.L., Chambers, A.F., and Groom, A.C. 1992. Intravital videomicroscopy of the chorioallantoic microcirculation: A model system for studying metastasis. Microvas. Res. 44:185‐199.
   MacDonald, I.C., Schmidt, E.E., Morris, V.L., Groom, A.C., and Chambers, A.F. 1998. In vivo videomicroscopy of experimental hematogenous metastasis: Cancer cell arrest, extravasation, and migration. In Motion Analysis of Living Cells, Chapter 12 (D.R. Soll, and, D. Wessels, eds.), pp. 263‐285. John Wiley & Sons, New York.
   MacDonald, I.C., Groom, A.C., and Chambers, A.F. 2002. Cancer spread and micrometastasis development: Quantitative approaches for in vivo models. BioEssays 24:885‐893.
   Morris, V.L., Koop, S., MacDonald, I.C., Schmidt, E.E., Grattan, M., Percy, D., Chambers, A.F., and Groom, A.C. 1994. Mammary carcinoma cell lines of high and low metastatic potential differ not in extravasation but in subsequent migration and growth. Clin. Exp. Metast. 12:357‐367.
   Morris, V.L., Schmidt, E.E., MacDonald, I.C., Groom, A.C., and Chambers, A.F. 1997. Sequential steps in hematogenous metastatasis of cancer cells studied by in vivo videomicroscopy. Inv. Metast. 17:281‐296
   Naumov, G.N., Wilson, S.M., MacDonald, I.C., Schmidt, E.E., Morris, V.L., Groom, A.C., Hoffman, R.M., and Chambers, R.M. 1999. Cellular expression of green fluorescent protein, coupled with high‐resolution in vivo videomicroscopy, to monitor steps in tumor metastasis. J. Cell Sci. 112:1835‐1842.
   Naumov, G.N., MacDonald, I.C., Weinmeister, P.M., Kerkvliet, N., Nadkarni, K.V., Wilson, S.M., Morris, V.L., Groom, A.C., and Chambers, A.F. 2002. Persistence of solitary mammary carcinoma cells in a secondary site: A possible contributor to dormancy. Cancer Res. 62:2162‐2168.
   New, D.A.T. 1966. The Culture of Vertebrate Embryos. Chapter 3 (The Chick) pp. 47‐98. Logos‐Academic Press, London.
   Rizzo, V. and Defouw, D.O. 1993. Macromolecular selectivity of chick chorioallantoic membrane microvessels during normal angiogenesis and endothelial cell differentiation. Tissue Cell 25:847‐856.
   Sethi, N. and Brookes, M. 1971. Ultrastructure of the blood vessels in the chick allantois and chorioallantois. J. Anat. 109:1‐15.
   Sturkie, P.D. 1976. Avian Physiology (Third Edition). Springer‐Verlag, New York.
   Welch, D.R. 1997. Technical considerations for studying cancer metastasis in vivo. Clin. Exper. Metast. 15:272‐306.
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