Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

Overview

In this video, we describe the implantation of cancer cells onto a chicken chorioallantoic membrane to generate an in ovo model. The model successfully engrafts the ovarian cancer cells to study the progression of gynecological cancer.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing the cancer cell suspension for transplantation (option 1)

NOTE: This is to be completed just prior to the implantation, which should ideally take place between days 7 and 10. Please see notes at the beginning of step 3 or 4 for further information concerning the implantation date. This approach was used for all the cell lines and cultured kidney cancer tumor digests.

1. Thaw the extracellular matrix solution on ice.
2. Using mechanical and/or enzymatic digestion, obtain a single cell suspension using a method appropriate for the cell type being implanted.
3. Resuspend the total number of cells to be implanted in an appropriate medium for implantation. Implants of 1-2 x 10⁶ cells per egg are typical.
   NOTE: The medium used for the implantation of the cell lines is typically the complete medium used for culturing the cells, containing FBS or serum replacement. The implantation of tumor digests typically uses the complete medium used for culturing cell lines of the same cancer type. However, adjustments to the medium formulation may be made if needed experimentally. The effects on tumor development and growth would need to be empirically determined.
4. Pellet the cells using a centrifuge speed and time appropriate for the cells being used. Typical speeds are 250-300 x g, and times are 5-10 min.
5. Remove the supernatant from the pelleted cells by pipetting. Mechanically resuspend the cells in the residual medium via flicking or pipetting. Place on ice to cool. Measure the volume of cells and medium using an appropriately sized pipette.
6. Calculate implantation volumes such that 1-2 x 10⁶ cells are implanted in a volume of 20-100 μL per egg with a final extracellular matrix concentration of 2.7-4 mg/mL protein. Add medium, any desired growth factors or additives, and extracellular matrix solution according to this calculation. Keep on ice until ready to implant.
   NOTE: For the calculations in steps 1.3 and 1.6, an extra volume of at least one-half egg implant should be incorporated to ensure an adequate volume for all eggs in the group. For implantation of the ovarian cancer cell lines (i.e., SKOV3 and ID8), 10⁶ cells per egg were implanted. For implantation of the renal cell carcinoma cell line RENCA, cultured cells derived from digested primary human renal cell carcinoma, prostate cancer cell lines (i.e., CWR, C4-2, and MyC-CaP), and bladder cancer cell lines (i.e., HT-1376 and T24), 2 x 10⁶ cells were implanted.

2. Preparing tumor pieces for implantation (option 2)

NOTE: This is to be completed just prior to implantation, which should ideally take place between days 7 and 10. Please see notes at the beginning of step 3 or 4 for further information concerning the implantation date. Primary ovarian and bladder cancers were implanted as tumor pieces.

1. Thaw extracellular matrix solution on ice. Dilute to a final protein concentration of 2.7-4 mg/mL in an appropriate medium containing any desired growth factors or additives. Keep on ice.
   NOTE: Implantation of tumor pieces typically uses the complete medium used for culturing cell lines of the same cancer type. However, adjustments to the medium formulation may be done if needed experimentally. The effects on tumor engraftment and growth would need to be empirically determined.
2. Using a scalpel or scissors, excise pieces from the fresh tumor. Ideal sizes range from 2-5 mm on each side. Keep tissues immersed in the medium until ready to implant.

3. Implantation using a nonstick ring (option 1)

NOTE: Cells may be implanted beginning on development day 7 if the CAM is fully developed. Implantation can occur any time prior to hatching that permits adequate time for tumor development and the desired experiment but note that the embryo's immune cells begin to be present around day 10 postfertilization. Tumor growth rate varies considerably by cell type and needs to be empirically determined for the cell type of interest. The ovarian cancer and the prostate cancer cells were implanted using the nonstick ring method. Note that when a nonstick ring is not available, a pipette tip may be cut to a similar size and used.

1. Use 70% ethanol to disinfect a biosafety cabinet and all the required tools: an egg rack, curved iris forceps, nonstick rings (1/4 inch inner diameter), glass stir rod, appropriate volume pipettes and tips, 6 x 7 cm transparent film dressing, office scissors, and marker or pencil. Whenever possible, sterile, disposable or autoclaved tools should be used.
2. Place eggs to be implanted on an egg rack in a biosafety cabinet. Select a manageable number of eggs. Up to six is typical. Avoid allowing the eggs to cool substantially while working with them.
3. Remove transparent film dressing from the shell by rolling the edges towards the open window to avoid pulling away pieces of shell. Check that the eggs are viable and healthy. Ideal eggs will have a large vessel in the center of the opened area with smaller vessels branching from it.
4. Using curved iris forceps, place a sterile, nonstick ring onto the CAM over the vessel, ideally over a branch point. Use a sterile glass stir rod to gently abrade the CAM.
5. Pipet the cell suspension from step 1.6 into the center of the nonstick ring. Alternatively, use forceps to place a tumor piece from step 2.2 into the center of the nonstick ring and cover with 20-50 μL of the extracellular matrix solution generated in step 2.1.
6. Seal the opening with a one-quarter piece of 6 x 7 cm transparent film dressing. Label the eggs with an appropriate implant designation.
   NOTE: Numbering the eggs within a group facilitates longitudinal observations.
7. Return the egg to the incubator. Ensure that the opening of the shell sits upright and that the egg is secure.
   NOTE: Eggs can be returned to an egg incubator without rotation. Alternatively, high postimplant viability can be obtained using a 37-38 °C cell incubator with the CO₂ deactivated and a hygrometer to monitor the humidity, which should be 50%-80%.

Materials

Name	Company	Catalog Number	Comments
010 Teflon (PTFE) White 55 Duro Shore D O-Rings	The O-Ring Store	TEF010	Nonstick ring for cell seeding. 1/4"ID X 3/8"OD X 1/16"CS Polytetrafluoroethylene (PTFE).
Delicate Operating Scissors; Curved; Sharp-Sharp; 30mm Blade Length; 4-3/4 in. Overall Length	Roboz Surgical	RS6703	This is provided as an example. Any similar curved scissors would work as well.
Dremel 8050-N/18 Micro 8V Max Tool Kit	Dremel	8050-N/18	This kit contains all necessary tools
Fertilized chicken eggs (Rhode Island Red - Brown, Lab Grade)	AA Lab Eggs Inc.	N/A	A local egg supplier would need to be identified, as this supplier only delivers regionally.
Hovabator Genesis 1588 Deluxe Egg Incubator Combo Kit	Incubator Warehouse	HB1588D-NONE-1102-1588-1357	Other egg incubators may be used, but their reliability would need to be verified. After implantation, a cell incubator with the CO2 disabled may also be used.
Incu-Bright Cool Light Egg Candler	Incubator Warehouse	1102	Other candlers may be used; however, this is preferred among those that we have tested. This candler is included in the aforementioned incubator kit.
Matrigel Membrane Matrix HC; LDEV-Free	Corning	354248	Extracellular matrix solution
SKOV3	ATCC	HTB-77	Human ovarian cancer cell line.
Stirring Rods with Rubber Policeman; 5mm diameter, 6 in. length	United Scientific Supplies	GRPL06	This is provided as an example. Any similar glass stir rods would work as well.
Specimen forceps	Electron Microscopy Sciences	72914	This is provided as an example. The forceps used for pulling away the shell for bioluminescence imaging are approximately 12.8 cm long with 3 mm-wide tips.