The method visualizes and quantifies the ability of cancer cells to delaminate and invade, thereby being able to assess their metastatic aggressiveness before the action formation or metastasis. That the scoring is based on the delamination process as a prerequisite for metastasis formation, thereby achieving information regarding cells'metastatic aggressiveness in a relatively fast, easy, and cheap manner. Practice the different individual steps first before starting the actual experiment.
The CAM-Delam assay includes several technical steps which is difficult to explain in writing and much easier to appreciate and learn by visual demonstration. To begin place the desired number of eggs in egg trays and incubate the chick eggs horizontally in an egg incubator. Then sterilize 30 weighing boats and 30 small plastic boxes with transparent caps by spraying 70%ethanol.
Next, dry the weighing boats and boxes in a laminar hood overnight and store in a closed plastic box until further use on incubation day three. Sterilize two liters of distilled or deionized water per 30 incubated eggs, two pairs of scissors, and three pairs of forceps. First, add approximately 50 milliliters of sterilized water to the sterilized plastic boxes and close the lids.
On incubation day three, crack the egg shell using the sharp part of scissors and cut a straight opening in the shell. After opening the egg shell manually over a weighing boat, collect the egg white, the yolk, and it's attached healthy embryo in the weighing boat. Then look for an intact embryo with a beating heart, intact yolk, and developed blood vessels for the experiment.
Next, gently transfer the weighing boat into an internal humidified chamber and incubate the internal humidified chamber in the egg incubator. To prepare silicone rings, cut a silicone tube with an inner and outer diameter of four millimeters and five millimeters respectively in approximately one millimeter thickness, preferably using a paper cutter. Then transfer the silicone rings to small glass bottles.
Cover them with metal foil and sterilize using an autoclave or similar. Store the sterile silicone rings at room temperature. Culture the cancer cell lines of interest in the relevant culture medium in cell culture.
Then prepare one milliliter of collagen RPMI mix and keep it on ice. Next, trypsinize the cancer cells to isolate the cells by first removing the cell culture medium and wash. Then add three milliliters of trypsin solution per 15 centimeter diameter cell culture dish and incubate for two to three minutes in a cell culture incubator until the cells detach.
Using a tabletop inverted microscope, see the detached cells. Then inactivate trypsin by adding five milliliters of complete RPMI medium to each cell culture dish and collect the cell suspension from all cell culture dishes into a 50 milliliter tube. To count the cancer cells, add 10 microliters of the cell suspension to 10 microliters of 0.4%trypan blue stain.
After mixing the sample by pipetting up and down a few times, load 10 microliters of the cell mixture per chamber into the sample slide in the cell counter. Next, centrifuge the correct volume cell culture suspension in a 50 milliliter tube at 500 times G for five minutes. After discarding the supernatant, mix the cell pellet with one milliliter of the collagen RPMI mix.
Once the cells are mixed with a one milliliter pipette, keep the prepared cell suspension on ice. On incubation day 10, take out the internal humidified chambers with the incubated shell-less eggs from the incubator. After opening the internal humidified chamber, place up to six silicone rings on the CAM using sterilized forceps.
Once the cancer cell suspension is mixed by pipetting to get an even distribution of cancer cells, add 20 microliters of the prepared cancer cell suspension inside a silicone ring. After closing the internal humidified chamber, put it in the egg incubator. After 14 hours, 1.5 days, 2.5 days, and 3.5 days of incubation, take out approximately seven internal humidified chambers while opening their lids one at a time.
With a pair of scissors, dissect the cultured cancer cells attached to the CAM by cutting outside the silicone ring. Then immediately transfer the isolated CAM-Delam sample using forceps to 4%paraformaldehyde in 0.1 molar phosphate buffer in a Petri dish for fixation of the tissue and keep on ice or at four degrees Celsius for one hour. Next, remove the 4%paraformaldehyde solution and add 30%sucrose in 0.1 molar phosphate buffer to the CAM-Delam samples and equilibrate at four degree Celsius for one hour.
Under a dissection microscope, carefully remove the silicone ring using forceps. Next, cut the CAM-Delam sample in a rectangular shape with the cancer cells in the middle using a pair of scissors. Then transfer the CAM-Delam samples to frozen section medium in a Petri dish to remove excess sucrose and then to embedding molds in frozen section medium with a pair of forceps.
Under a dissection microscope, position the CAM-Delam sample in a U-shape in a vertical reaction in the embedding molds using any needle-like instrument and store the CAM-Delam samples at 80 degrees Celsius. Then section the frozen CAM-Delam samples at 10 micrometers on five to six consecutive slides using cryosectioning. First, make a line with a hydrophobic marker on the slides where the sections end and let them dry for a few minutes.
Then place the slides in a humidified chamber and cover the sections with approximately 200 to 500 microliters of blocking solution and incubate for 15 to 30 minutes. After pouring off the blocking solution, replace it with 100 to 150 microliters of the primary antibody of interest diluted in the blocking solution and incubate overnight at four degrees Celsius. Similarly, after pouring off the primary antibody solution, transfer the slides to the glass cuvettes and wash at least thrice for five minutes each in TBST.
Next, remove excess TBST from the slide and from the hydrophobic barrier area with a soft paper tissue and cover the slide with 100 to 150 microliters of a suitable secondary fluorescent antibody diluted in blocking solution combined with DAPI. Incubate the slide in the dark at room temperature for one hour. After pouring off the secondary antibody solution, transfer the slides to the glass cuvettes and wash at least thrice for five minutes each in TBST.
Then remove excess TBST from the slide and from the hydrophobic barrier area with a soft paper tissue. Next, mount the slides by putting one to two drops of fluorescent mounting medium on the slide and gently place a glass cover slip while avoiding air bubbles. Photograph the sections using an epifluorescence microscope equipped with a digital camera, preferably at 10 X magnification and analyze the sections using the following CAM-Delam scoring categories as described in the manuscript.
The use of internal humidified chambers significantly improved the survival rate of the chick embryos from less than 50%up to 90%at incubation day 10 and from approximately 15%up to 80%at incubation day 13. The results show that the capacity of cancer cells to degrade basal lamina and invade the mesenchyme can be scored into four categories including intact basal lamina without visible alterations, altered but undamaged basal lamina, damaged basal lamina without cell invasion, and damaged basal lamina with cell invasion. Antibody staining against von Willebrand's factor shows that the CAM was also thickened with an increase in blood vessel formation when cancer cells caused an altered or damaged basal lamina.
However, none of these two phenotypes were observed when the CAM was intact. The PC-3U cells induced damaged laminin after 1.5 days with clear invasion after 2.5 days. In contrast, U251 cells only induced minor alterations of laminin after 1.5 to 3.5 days, but never caused any visible damage to laminin.
After cobalt chloride treatment, U251 non-metastatic cancer cells acquired the ability to induce delamination and invasive cells, which were suppressed when combined with the MMP inhibitor, GM6001. Not to damage the CAM with the pipette tip when seeding the cancer cells since such damage of the membrane will destroy the readouts of the assay. A future possibility is to optimize the CAM-Delam method to determine metastatic properties in clinical tumor samples, which could future complement to today's used tumor node metastasis staging.
