Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

The engraftment of tumors onto the chicken chorioallantoic membrane, or CAM, can be used for studies of tumorigenicity, treatment efficacy or cellular crosstalk. This method permits cost effective rapid tumor growth in an immunotolerant in vivo setting. Using the CAM model, novel therapeutic approaches can be rapidly tested using cell lines or even patient tumor specimens.

The most crucial aspects of this protocol are ensuring proper CAM formation and not disrupting the CAM or vasculature while opening the eggs. On developmental day seven or eight when the CAM has fully developed, turn off the egg rotator and place approximately one to three eggs into the egg rack in the biosafety cabinet. With the lights turned off, place an egg candler against the egg shell to identify and mark the location of the air cell of one egg, then move the egg candler over the shell to find a large blood vessel network, rotating the egg as necessary.

An ideal vasculature will branch near the middle of the egg. Use a marker to trace the vasculature to be used for implantation. After turning back on the light in the hood, use a cordless rotary tool fitted with a silicon carbide grinding stone to drill a small hole in the shell directly over the center of the air cell.

When most of the shell has been removed but with white inner membrane intact, drill another small hole where the vascular window will be opened as just demonstrated. When the second hole is ready, use an 18 gauge needle to gently pierce the white inner membrane over the air cell and vasculature taking care that the white inner membrane, but not the CAM, is disrupted throughout the entirety of the drilled area. With the hood light off again, use the egg candler to verify that the air cell has been transferred from the end of the egg to the area over the vasculature.

If necessary, place vacuum tubing inserted into a pipette controller around the hole over the original air cell and gently apply suction in short bursts to move the air cell. Mark the outline of the new location of the air cell approximately 0.5 centimeters inside the air CAM boundary and fix a piece of packing tape just big enough to cover the hole over the new air cell. After preparing the other two eggs as just demonstrated, return the eggs to the incubator and open any additional eggs for the experiment in groups of one to three as demonstrated.

When all of the eggs have been opened, transfer one to three eggs with relocated air cells to the egg rack in a biosafety cabinet and use a cordless rotary tool fitted with a circular cutting wheel to cut a small line over the air cell boundary completely through the cell without disrupting the CAM or vasculature. Using curved scissors, cut around the remaining air cell to create a window in the shell and verify the viability of the embryo. Viable embryos will display an extensive vasculature, clear albumin, embryo movement, and/or a visible heartbeat.

In nonviable embryos, the CAM may appear opaque with few, if any, vessels present and the embryos may be small or absent without movement or a heartbeat. Using Semkin forceps, pull small pieces of cotton from a sterile cotton ball and gently blot the CAM surface of the viable embryos to remove any shell dust and debris, then cover the shell opening with a one-quarter piece of six by seven centimeter transparent film dressing without touching the CAM and return the eggs to the incubator with the opened window facing up. Use a piece of egg rack, the edge of the egg rotator, or another suitable item to prop any eggs that keep rolling.

To prepare cancer cell suspension for transplantation, harvest the cells from the cancer cell culture of interest and sediment the cells by centrifugation. Aspirate the supernatant and mechanically resuspend the cells in the residual medium. Place the cell suspension on ice and use a pipette to measure the volume of cells in medium.

Then resuspend the cells at a concentration of one to two million cells in 20 to 100 microliters of medium supplemented with 2.7 to 4 milligrams per milliliter extracellular matrix and any growth factors or other additives of interest. For cancer cell implantation, using a non-stick ring, place up to six eggs to be implanted onto the egg rack and roll the edges of the transparent film dressing toward the open window to remove the film from the shells. Check whether the eggs are viable and healthy.

Ideal eggs will have a large vessel with smaller branching vessels within the center of the opened area. Using curved iris forceps, place a sterile non-stick ring onto the CAM over the vessel ideally over a branch point. Use a sterile glass stir rod to gently abrade the CAM and pipette the cancer cell suspension into the center of the ring.

When all of the cells have been delivered, seal the opening with a one-quarter piece of six by seven centimeter transparent film dressing and label the egg with an appropriate implant designation. Then place the eggs securely in the incubator with the opening of the shell facing upright. To implant cancer cells without the use of a non-stick ring, aspirate the cell suspension into an appropriately sized pipette tip and holding the top portion of the tip carefully eject the pipette tip and place the tip horizontally into a sterile 10 centimeter tissue culture dish.

After the tips have been loaded, place the dish into a 37 degree Celsius incubator for 15 to 30 minutes, checking for polymerization after 15 minutes. A small amount of liquid will typically leak out as each tip is placed in the dish. Use this liquid to estimate the extent of the polymerization within the tip.

When the matrix is ready, place one tip onto an appropriately sized pipette and depress the plunger to force the partially polymerized cell suspension onto the CAM ideally over the branched point of a large well-developed vessel. Using these methods, both human and murine ovarian cancer cell lines and primary tumor pieces resulted in the successful engraftment of tumors with morphologies consistent with those observed in other in vivo models. Of the tested tumor types, ovarian cancer growth was much less pronounced and typically not visible without the assistance of bioluminescence imaging.

The effects of adding growth factors or hormones at the time of implantation can also be tested using these methods. For kidney cancer, the implantation of clear cell renal cell carcinoma cells from established cell lines or pieces from primary human renal tumors produces rapid, robust tumor formation. Further, multiple human and murine prostate cancer cell lines and human bladder cancer cell lines and primary tumor pieces had been used to establish tumors using this model.

It is crucial to avoid disrupting the CAM and the vasculature and to make sure that the CAM does not adhere to the shell while opening the window. After successful engraftment, the CAM model can be used to assess the therapeutic efficacy of treatments of interest or interactions between different cell types within a tumor.