The overall goal of this procedure is to quantitatively evaluate the metastatic ability of cancer cells using the choal toic membrane or CAM as a xenograft model. This is accomplished by first incubating fertilized eggs until they have a mature cam. The second step of the procedure is to intravenously inject or topically xenograft tumor cells to the cam.
After an experimentally defined time interval tissue is collected for analysis, then metastasis is quantified using human specific PCR for the human allele sequence and the results are analyzed. My name is Trent Palmer. I'm a graduate student in the lab of Dr.Andre Zilstra.
I'll be demonstrating the xenograft and the spontaneous metastasis model where my mentor, Dr.Zilstra will be demonstrating the intravenous injection and the experimental metastasis model. The main advantage of this technique of existing models is the expedience and ease of which an experimentalist can quantitatively study. The metastasis of human tumor cells and experimentalists can set up approximately 120 animals and complete the protocol start to finish in a span of two weeks.
Because of the accessibility of the chick embryo and the ease of manipulation, the system is is readily extended to additional X ovo applications. This we have accomplished in the past years and is the subject of additional JoVE verticals. Freshly laid fertilized chicken eggs are incubated in a rotating incubator four times per hour for 10 days.
On the 10th day, turn the eggs onto their side on an egg rack and shine a bright direct light source into the egg shell at the blunt end of the egg where the air sock is located. Now locate the branch point of the choic vein. It is most easily identified by rotating the egg within 20 degrees of its long axis.
To visualize the point at which the vessel drops away from the shell, one centimeter away from the branch point of the cor toic vein, draw a one centimeter square box. Then clean the area around the box with a swab soaked in iodine. Using a dremel fitted with a silicone carbine grinding stone, first drill a hole through the blunt end of the egg into the air sack, and next drill a hole within the square stopping short of the eggshell membrane.
Now carefully use a 25 gauge needle with a fine burr on the end to pull back the eggshell membrane done correctly. This will not tear the underlying cam to detach the cam from the underlying membrane. Use an automatic pipette aid fitted with a piece of quarter inch tigon tubing.
Apply gentle suction to the air sack hole and check for an air pocket beneath the hole in the square, indicating success. If the cam becomes damaged during the disruption of the eggshell membrane, the can will not obviously drop. When the air sack is suctioned upon rotation of the egg, the air that is visible just underneath the shell is what is drawn into and under the damaged cam.
These eggs cannot be used after the cam is dropped. Tape both holes with a piece of laboratory tape and return the eggs to a stationary incubator. The eggs are now ready for xenograft.
Begin by preparing the tumor cells for xenograft. First, detach the cells from their culture dishes using trypsin EDTA. Finish preparing the cells by measuring their density with a hemo cytometer and resuspend them in PBS at 10 to 40 million cells per milliliter.
Now after sterilizing the hood and dremmel tool with ethanol, remove the eggs from the incubator. Open a window in the top of the egg shell and using a cotton tipped applicator, a braid the cam five times a little blood should be evident on the cotton onto the damaged area. Immediately inject 25 microliters of the cell suspension between one half and 1 million cells.
Now seal up the window with laboratory tape and allow the cells to settle for 10 to 15 minutes. After 15 minutes, return the eggs to the stationary incubator where the tumors are allowed to grow for five to eight days. Using the same incubation demonstrated in section two, collect 12 day old eggs.
Position an egg in front of the light source to locate and mark the corry and toic vein directly above the vein. Make a one centimeter by 0.5 centimeter box and wipe the area clean with a swab soaked in iodine. Now using a drmo fitted with a drill bit, make a small window in the egg shell at the rectangle.
Carefully remove the eggshell window to expose the underlying membrane. Add a drop of mineral oil to the exposed membrane, rendering it transparent. Retrieve the eggs from the incubator and using a 30 gauge insulin syringe.
Inject 100 microliters of cell suspension into the auic vein of each embryo. Then place a piece of laboratory tape over the exposed membranes and return the eggs to a stationary incubator over the next one to seven days. The lung or lower cam is harvested for the detection of tumor cells as demonstrated in the following section.
It is vital to execute this step of the procedure in an area dedicated to dissections and with an absolute minimum chance of contact with DNA. To prevent cross-contamination of samples, use three separate sets of tools for cutting the egg, removing the primary tumor, and removing the internal organs before beginning and between dissections. Wash each set of tools once with bleach, once with 70%ethanol and twice with double distilled water.
The eggs are removed from the stationary incubator and placed on ice for 15 minutes. To anesthetize and euthanize the animals, the windows are opened such that the tumors become visible. It may be necessary to enlarge the windows to fully expose the tumors.
Now trim away the primary tumors from the cam. Using a clean set of tools, weigh the tumors and process them for histology. Next, proceed with harvesting the organs.
Begin by removing the chick from the eggshell. To do this, cut the shell radially into equal halves. Then decant the chick embryo from the cut.
Shell also harvest two pieces of cam from each side of the egg shell using a punch tool and place them in an eend tube. Transfer the chick to a whey boat breast side up. Using a clean set of dissection tools, open the embryo by cutting through the abdomen and sternum and opening up the abdominal space.
First, collect a piece of the liver from either the right or left lobe. Now the organs can be carefully pulled out while cutting the viscera that connects the organs. Select a lung and cut around its four sides to free it from the rib cage.
For consistency. Collect the same lung from each animal. If any of the organs are not processed immediately store them at minus 20 degrees celsius.
After removal of a xenograft hep three tumor histological analysis was applied. This is a tri RME stain of the Hep three tumor. The stromal component of the tumor is clearly evident among the distinct lobes of the tumor.
Here intravenously injected hep three cells expressing GFP are visualized with fluorescent microscopy. One day after IV injection only scattered individual tumor cells are visible three days after injection, it is clear that the cell number is increasing and small colonies of metastatic cells are forming here. The level of metastasis over the course of seven days post grafting has been quantified using real-time PCR for the human A LU sequence.
The technique we have demonstrated here today is a highly quantitative high end value model. Once mastered, an experimentalist can set up approximately 30 animals per hour, and additionally, in two weeks span, metastasis can be quantitatively analyzed using the avian embryo model after is development. This technique allowed researchers in the field of cancer biology to quantitatively analyze metastasis using a naturally immunodeficient model.
