The overall goal of this procedure is to examine the efficacy of tumor specific T-cell adoptive transfer immunotherapy in vivo. This is accomplished first by transplanting tumor cells via lateral tail vein injection to the mice in order to establish lung metastases. The second step of the procedure is to treat the tumor bearing mice with tumor specific cytotoxic T lymphocytes CTLs.
The final step is to use India ink as a means to visualize and quantify tumor nodules. Ultimately, results can be obtained that show the CIE of different treatments through measuring tumor size and quantifying the number of tumor nodules. Hi, my name is Mary Zimmerman and I'm a member of Dr.Kevin Lu's lab at the Medical College of Georgia.
Hello, my name is Shelene Hu.I'm second year student and I'm also working Dr.Lou's lab. The main advantage of this technique over traditional methods such as survival curves is that this procedure is both qualitative and Quantitative. The implication of the techniques can extend toward cancer therapy because it mimics T-cell adoptive immunotherapy in human cancer patients.
So let's get Started On the day before the tumor cell injections seed one T 75 flask with up to one times 10 to the seventh. CMS four met cells in 10 milliliters of RPMI medium containing 10%serum. To obtain fast-growing tumor cells incubate overnight at 37 degrees Celsius on the day of the injection.
Remove the medium and rinse the cells once with PBS, then harvest the tumor cells with 0.05%trypsin EDTA at 37 degrees Celsius for five minutes. Stop the reaction with 10 milliliters of RPMI medium containing 10%serum. Transfer the cells to a conical tube.
Spin down the cells at 1300 RPM in a val legend RT centrifuge for three minutes. At room temperature, remove the snat, resuspend the cells in 10 milliliters of fresh one XHB Ss to wash. Then spin down again and resuspend in the same manner.
Count the tumor cells on a hemo.Cytometer. Dilute the cells in HBSS so that the cells required for each injection are resuspended. In a total volume of 100 microliters warm the six to eight week old B SEA mice in a beaker, immersed in warm water.
To dilate the tail vein, place the mouse in a tail vein restrainer on the bench. Use a micros syringe to inject 100 microliters of tumor cells into the lateral tail vein. Use a septic technique to avoid infection after injection.
Use slight pressure to the injection site until bleeding has stopped. Return the mouse to the cage and allow the tumor to grow to the desired stage. On the day of the transfer, pipette up and down to resuspend purified cytotoxic T lymphocytes or CTLs prepared.
As described in the text, transfer all the cells from one plate to a 15 milliliter conical tube, not letting the volume exceed more than two thirds of the tube. Insert a sterile Pasteur pipette into the conical tube and layer lymphocyte separation. Medium or LSM under the cells until the total volume nears 14 milliliters.
Be careful not to disturb the layers. Centrifuge a 2000 RPM for 20 minutes at room temperature. Do not use the brake to slow the rotor after spinning, transfer the CTLs to a new 15 milliliter conical tube.
Add HBSS to a volume of approximately 10 milliliters. To wash. Count the cells spin down at 1300 RPM for five minutes At room temperature resuspend an HBSS to the required cell density for injections.
Keeping the total volume per injection at 100 microliters. Use the same tail injection technique as seen earlier in this video. To inject the CTLs into tumor bearing mice, return the mouse to the cage and allow the CTLs to interact with the tumor.
Mice are usually sacrificed for analysis 14 to 21 days after CTL treatment. To visualize lung metastases, place a sacrificed mouse on its back on a styrofoam board. Pin the legs to ensure unobstructed access to the trachea.
Spray the ventral side with 70%ethanol starting from the mid abdomen. Use scissors to cut along the midline through the rib cage and up toward the salivary glands. Expose the trachea.
Use forceps to remove tissues surrounding the trachea. Thread a 200 microliter pipette tip underneath the trachea holding the tip with one hand. Gently lift the trachea up and away from the body.
Rotate the platform 180 degrees. Use a 50 milliliter syringe to inject India ink into the lungs via the trachea. Completely inflate the lungs with ink until you feel a strong resistance.
Use scissors to cut the trachea. Slide a pair of forceps under the lungs and lift them out of the mouse. Rinse the lungs briefly in a one liter beaker of water in a chemical fume hood.
Transfer the lungs to a glass instalation bile containing five milliliters of FTI solution. The tumor tissue will emerge as white nodules on the black lungs. After a few minutes, the tumors can now be counted and start in FTI solution indefinitely.Here.
Lungs for mice transfected with mammary carcinoma. Cell line four T one show white spots indicating tumors. Mice transfected with an IRF eight dominant negative immune to K 79 E show the enhanced metastatic potential of tumor cells.
These images show a histological analysis of efficacy of CTL adoptive transfer immunotherapy. Tumor bearing mice injected with saline, showed multiple tumors six days later indicated by arrows. Mice injected with tumor specific T cells, on the other hand, showed a reduction of tumors in the same experiment.
India ink treatment gives a simpler way to measure the efficacy of CTL adoptive transfer. Here the white tumor nodules on tumor bearing lungs clearly distinguish them from lungs that have successfully undergone CTL adoptive transfer and counting the nodules allows for a degree of quantification not possible with histology. While attempting this procedure, it is important to remember to warm up the mice for easier and more accurate tail vein injections.
After watching this video, you should have a good understanding of how to assess the efficacy of the treatment through this simple procedure to quantify tumor size and numbers. Thanks for watching and good luck with your experiments.
