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

  • Unit Introduction
  • 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 Support Protocol 1)
  • Adherent rodent tumor cells, optionally labeled with nanospheres (see Support Protocol 2)
  • 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 × 10–3 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. 19.6.3 and 19.6.4)
  • 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 Support Protocol 3), and inspecting for gross surface tumors (Chambers et al., 1982; Chambers and Wilson, 1988)
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 Support Protocol 1)
  • Adherent tumor cells with or without nanospheres (see Support Protocol 2)
  • 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 Basic Protocol 1, steps to and step ) and injecting tumor cells into eggs (see Basic 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% CO2
  • 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 Support Protocol 2)
  • 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

  •  FigureFigure 19.6.1 Diagram of experimental metastasis assay in chick embryos. Cancer cells are injected via a chorioallantoic membrane (CAM) vein, through a window opened in the shell of 11-day-old chick embryos. Cells injected via this route travel via the circulation first to the embryonic liver. After incubation for up to 7 days, metastatic cells in an embryonic organ such as liver are quantified using the ouabain plating assay, or (for rapidly growing cell lines) macroscopic metastases can be quantified. Republished with permission of Anticancer Research from Chambers and Tuck (1988).
    View Image
  •  FigureFigure 19.6.2 The drilling of the marked square or window on the egg over the selected vein area with a Dremel Moto tool is done in an enclosed still hood. To minimize breathing of eggshell dust, hang a cloth from the bottom of the glass front (or alternatively wear a mask and protective eyewear). Drill all four sides of the window, with the egg at a slight angle and the air sac end (blunt end) up and the marked window facing toward the person drilling. The bit of the Dremel tool is brought at right angles to the surface of the shell. Gentle pressure is sufficient for cutting due to the high speed of the drill. Only the shell is cut while avoiding the underlying smoother papery-white membrane or the underlying CAM.
    View Image
  •  FigureFigure 19.6.3 The arrangement of equipment inside the tissue culture hood just before injections proceed. The front of the hood is opened for easier access during the procedure with adequate sterility still being maintained. The gooseneck arm of the fiber optics light is illuminating the air sac end of an egg positioned on the egg stand (made by gluing three rubber corks onto a plastic petri dish) ready for injection. The remaining eggs have had the shell of the drilled window carefully removed over the vein area and are placed in a tray with the windows facing upwards awaiting injection. The cell suspension has been prepared at a predetermined concentration ready to be drawn up into the 1-cc syringe following gentle vortexing. The other 1-cc syringe contains the paraffin oil of which a drop will be placed on the exposed window area prior to injection. The melted Paraplast in the glass beaker (left) is cooling in preparation for sealing of the entire window area following injection.
    View Image
  •  FigureFigure 19.6.4 Injection of cells into a chick embryo CAM vein. Position the egg on the egg stand, with the fiber optics light illuminating the air sac end for visualization of the large Y shaped vein. The addition of a small drop of paraffin oil onto the injection site enhances the clarity of the vein. The beveled tip of the needle is facing upwards for gentle insertion into the branch area of the Y (see inset) just until the beveled end is completely into the vein. The needle should be at the angle seen in the picture above to prevent damage to the vein. If a proper injection is performed, the vein will temporarily become clear as the 0.1-ml inoculum is smoothly injected, and there will be no blood seeping from the injection point either during or after the injection.
    View Image
  •  FigureFigure 19.6.5 Example of in vivo growth kinetics results that can be obtained with the chick embryo experimental metastasis assay and the ouabain plating assay to recover rodent (and thus, ouabain-resistant) cells at various times after injecting cells via a CAM vein. (A) B77-NRK (normal rat kidney) cells transformed with the src oncogene, or (B) LA23-NRK cells (NRK cells carrying a temperature-sensitive src oncogene, which is active at 36° and inactive at 38°C) were injected into CAM veins at 5 × 104 cells per embryo. Closed circles: embryos maintained at 36°C; Open circles: embryos transferred to 38°C at day 4. Each point represents the number of viable ouabain-resistant cells present in the liver of one embryo; lines connect median points. Republished with permission of American Society of Microbiologists from Chambers and Wilson (1985).
    View Image
  •  FigureFigure 19.6.6 Schematic of the intravital videomicroscopy technique, as used for real-time observations of the interactions of injected cancer cells with the CAM microcirculation. The CAM is exposed and positioned on a plastic platform with a coverslip window above the objective lens of inverted microscope. For visualization of fluorescently labeled cancer cells, oblique transillumination is provided by the fiber optic light and epifluorescence through the objective aids in positive identification of the cancer cells. Images are collected through the video camera and saved to SVHS tape or digitally on a computer. Diagram by S. Koop and I.C. MacDonald, republished with permission of Plenum Publishers from Chambers et al. (1995).
    View Image

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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