Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

The scope of our research is to try to uncover mechanisms that promote glioblastoma cell spread in brain tissue. Specific questions revolve around differences in glioblastoma cell types, or states and molecular mechanisms that promote these cells to be highly invasive. For example, how does L1CAM expression promote invasiveness?

Both in vitro and in vivo models are insufficient for accurately analyzing glioblastoma cell behavior. In vitro models almost certainly modify and oversimplify in vivo cell behaviors. While in vivo models do not allow for easy observation of behaviors as they occur, instead allowing for analyses after the behaviors have occurred.

We have shown that the chick embryo brain is a good model for studying human brain cancer cell behavior, both in vivo and in ex vivo slice cultures. We also have established that L1CAM expression by glioblastoma cells can have profound effects on cell proliferation, invasion, and arrangement within the tumor. Besides several advantages of using chick embryos, our ex vivo spheroid protocol introduces cells onto live slices with no additional damage, whereas other methods of cell introduction involve piercing the tissue and implanting cells.

Additionally, the protocol allows for live time-lapse imaging that in vivo techniques do not. Our findings suggests that the chick embryo is a good model for cancer research, particularly brain cancer, and is extremely beneficial for the scientific community as it is much more readily available to those who may not have the funds, facilities, or expertise for rodent models. We will continue to focus on molecular mechanisms that control glioblastoma cell invasiveness in brain tissue, particularly along blood vessels.

Another crucial question is whether glioblastoma stem cells are a distinct and stable phenotype or a functional state that can be adopted when needed. That has huge implications for treatments. To begin, incubate the fertilized chicken eggs in a humidified egg incubator with the pointed end facing down at 37.5 degrees Celsius.

On the sixth day of incubation, or E5, sterilize the eggshell by spraying it with 70%ethanol. Using an egg candler, trace along the perimeter of the airspace above the embryo with a pencil and cover the outlined area with transparent tape. Gently cut around the traced area with curved scissors without cutting into the embryonic membranes or blood vessels.

Discard the top of the eggshell. Add a few drops of saline or cell culture media onto the airspace membrane to wet it so that it will detach easily. Using fine forceps, carefully pierce the airspace membrane over the top of the embryo.

Remove it. Grab the transparent amnion membrane that immediately surrounds the embryo to position the head and inject around 50, 000 cells in five microliters of suitable cell culture media into the optic tectum with a glass micro pipette and a pneumatic pico pump. Add a few drops of 50 milligrams per milliliter of ampicillin on top of the embryo.

And cover the hole in the top of the eggs with clear tape. Place them in a humidified egg incubator until E15 for dissection. The tumors that formed in the E5 optic tectum in vivo after the injection of glioblastoma or GBM stem cells or GSCs expressing green fluorescent protein at E15 are shown in this figure.

GSCs attach to the ventricular surface and form invasive tumors in the brain wall. Four colors were used to identify five features in this experiment:green GSCs, white nuclei, white blood vessels, blue integrin 6, and either red SOX2 or red Nestin. These figures show that the GSCs reside in your blood vessels and appear to be migrating along them.

Movies of rotating 3D volume renders to fixed and immuno stain slices of in vivo GSC tumors are shown here. Use an egg candler to trace along the perimeter of the air pocket above the E14 or E15 embryo with a pencil and cover the outlined area with transparent tape. Using curved or fine scissors, gently cut around the traced area.

Be careful not to cut into the embryo membrane or blood vessels. Then, discard the top piece of the shell. After decapitating the chick embryo, place the head in a 10 centimeter dish with a cold, sterile CMF solution.

Using sterile fine forceps, peel away the skin covering the brain to reveal the underlying dura mater. Remove the forming skull bones on the left and right sides of the brain. The forming skull bones do not yet cover the majority of the brain.

Gently use fine pointed forceps to tear through the meninges overlying the center of the brain and peel it to each side to uncover the brain. Using fine curved forceps, scoop the brain up from the bottom front and gently pull it out of its cavity. Dissect the brain into its three main parts:forebrain, midbrain or optic tecta and cerebellum.

Remove the overlying pia mater from the tecta using fine forceps and place the dissected brain regions in a small sterile dish on ice. For embedding and slicing the brain, gently pick up either the optic tectum or forebrain region with curved forceps as a scoop and blot slightly on sterile gauze to remove extra liquid. Prepare a simple mold by forming aluminum foil around an appropriately sized object.

Here, a small rectangular metal vibrating tissue slicer block is used as the object. Use a sterile transfer pipette and fill the mold with low melt agarose. Quickly place the brain region in agarose and let it solidify for approximately four to five minutes on ice.

Cut the sections on a vibrating tissue slicer using a sapphire knife. Use the sterile spatula to scoop up and remove the slice from the tray. Gently slide the section off the spatula and onto a membrane insert using another sterile spatula.

Place the six well plate of brain slices on membrane inserts in the cell culture incubator at 37 degrees Celsius and 5%carbon dioxide. On the day after plating, use a sterile pasteur or pipette to aspirate the media from below the insert and add one milliliter of fresh slice media to each well underneath the membrane insert. Next, to introduce the GBM cells onto the brain slices, remove one to several spheroids from their cultured dish using a 20 microliter micro pipette set to five microliters.

Gently expel the media with spheroids onto the desired brain slice and allow the spheroids to culture on the slice for two to five days. The viability results of ex vivo optic tectum slices after one week in culture are shown in this figure. The confocal images of a brain slice stained for nuclei with bisbenzamide and immunostained for laminin clearly shows normal and intact blood vessels optically sectioned in various configurations.

The maximum projection image of the confocal Z-stack of the brain slice stained for SOX2 transcription factor and total nuclei with bisbenzamide is shown here. These results suggest that chick embryo brain slices could be cultured on membrane inserts for approximately two weeks and remain viable with normal appearing blood vessels and transcription factor expression.