The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

This protocol allows the in vivo study of early interactions between tumor cells and nerves and the study of molecular pathways of nerve invasion in cancer. The main advantages of this technique are the short experimental time with the potential to study perineural invasion and other cancer phenotypes in the same experiment. Before trying this technique for the first time, we recommend practicing drilling commercial non-fertilized eggs and harvesting the dorsal root ganglia to optimize your technique for both procedures.

Demonstrating the procedure will be Min Liu, a research associate from my laboratory. Begin by incubating six commercial pathogen-free fertilized eggs per experimental group in an egg humidifying incubator at 38 degrees Celsius and 54%humidity on the first day post-fertilization for eight days with hourly rotation. On day eight, clean the dorsal skin of a euthanized rat with 70%ethanol and use scissors to remove the spine.

Separate the cervical, thoracic, and lumbar regions and place the spine sections in a 10 centimeter culture dish with PBS to keep the tissue samples hydrated. Using delicate bone scissors, open the thoracic and cervical vertebral bones in the dorsal and ventral aspects to separate the spine into two lateral halves and place the tissue sections into a clean 10 centimeter dish with fresh PBS. Using forceps, gently detach the spinal cord from the vertebral bones to visualize the dorsal root ganglions.

Placing the tips of a pair of fine forceps held under each ganglion, grasp the tissue and pull it from the bone cavity in which it is lodged. Immediately after harvesting, place each ganglion into DMEM culture medium supplemented with 2%penicillin and streptomycin or Pen-Strep and 10%fetal bovine serum or FBS to help prevent bacterial contamination of the nerve tissues. When all of the ganglia have been harvested, transfer the tissues into a new culture dish containing fresh DMEM culture medium supplemented with 2%Pen-Strep plus 10%FBS and 1.2 micrograms per milliliter of red fluorescent dye for a one-hour incubation in the cell culture incubator.

To prepare the eggs for the dorsal root ganglion graft, dim the light in a laminar flow cabinet and holding the egg with the naturally occurring air sac toward the light source, transilluminate each egg to check for chick viability and embryonic phase. Identify the attachment of the developing embryo to the chorioallantoic membrane as a dark moving vessel attached to the egg membrane and use a pencil to mark the attachment to avoid interventions in this region. Draw a 1.5 centimeter region in a well-vascularized area at least two centimeters from the embryo attachment to act as an operating window area and draw a 0.5 centimeter square in a less vascularized area approximately one centimeter from the operating window.

Then mark the center of the air sac region. Next, use a rotary tool and engraving drill with a three millimeter diameter bit to carefully drill the eggshell within the marked square. Use blunt forceps to remove the eggshell without removing the white outer eggshell membrane right under the shell.

Use the drill to carefully make a pinpoint drill perforation in the marked cross in the air sac area to allow airflow into the egg without breaking or damaging the shell and add 30 microliters of HBSS to the square opening over the intact outer eggshell membrane. Using a 30 gauge needle, make a pinpoint perforation in the outer membrane within the square area. Next, hold the egg up to the light source to visualize the air sac and apply pressure to an eyedropper rubber bulb.

Place the eyedropper bulb over the small perforation within the air sac and release the pressure on the bulb until a separation of the outer membrane and chorioallantoic membranes within the operating window area is observed repeating this step until a complete separation of the membranes is achieved. When all of the eggs have been modified in the same manner, drill the circular operating window in one egg taking care not to rupture the outer eggshell membrane and use the sticky side of a piece of adhesive tape to remove any loose particles from the shell. Using blunt forceps, remove the eggshell from the drilled area followed by the outer eggshell membrane taking care not to introduce small shell particles inside the egg to minimize contamination.

When the eggshell and outer membrane have been removed as demonstrated, cover the eggs with a paraffin wax membrane and place the eggs back into the egg incubator without rotation until the dorsal root ganglia are ready for grafting. For grafting of a dorsal root ganglion onto the chorioallantoic membrane, use fine sterile forceps to carefully grasp one ganglion in the culture dish and gently wash the tissue in a container of fresh HBSS. Place the ganglion onto the chorioallantoic membrane of one egg being careful not to puncture the membrane and cover all of the egg openings with sterile transparent film dressings.

When all of the eggs have been grafted, return them to the humidifying egg incubator for two days without rotation. At the end of the incubation, transfer the eggs to the laminar flow cabinet and use scissors and forceps to open the transparent film dressing. Next, add 0.5 to one million fluorescently labeled tumor cells in five microliters of HBSS onto the chorioallantoic membrane of each egg about two millimeters from each dorsal root ganglion keeping a uniform distance between the ganglia and the cells.

Then cover the eggs with a new film dressing and return the eggs to the egg incubator for seven days without rotation. At the end of the incubation, place the eggs onto the laboratory bench top. Use a syringe to perforate the film dressing of each egg and apply about 300 microliters of 4%paraformaldehyde over each chorioallantoic membrane to slightly stiffen the tissue to facilitate the harvesting process.

Using scissors, remove the upper half of the eggshell with the chorioallantoic membrane attached. Reduce the size of the shell to approximately three centimeters in diameter keeping the portion of the chorioallantoic membrane where dorsal root ganglion and tumor cells were grafted within the center of the shell fragment. Then grasp the chorioallantoic membrane with fine forceps and detach the tissue from the eggshell for transfer into one well of a six-well plate containing two milliliters of 4%paraformaldehyde per well dorsal root ganglion and tumor cell side up.

The integration of dorsal root ganglia into the chorioallantoic membrane is important as the chorioallantoic membrane provides nutrition to the dorsal root ganglion tissue during the experiment. Microscopically, the dorsal root ganglion is observed within the connective tissue of the chorioallantoic membrane and blood vessels are often seen inside the dorsal root ganglion tissue suggesting that the chorioallantoic membrane blood supply is nurturing the grafted tissue. Implanted tumors are also identified on the chorioallantoic membrane by hematoxylin and eosin staining.

Depending on how much invasion is present, tumors might present with no to numerous tumor islands invading the connective tissue. In this representative experiment, imaging with merge fluorescence analysis revealed an increased invasion of the dorsal root ganglion in chorioallantoic membranes grafted with tumor cells over expressing the galanin receptor 2 compared to control tumors. This useful and cost effective in vivo model of perineural invasion can be replicated even by laboratories with limited resources.