The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for CNS metastasis has been hindered by the lack of good preclinical models. Our patient-derived xenograft models recapitulate CNS metastasis better than historically used cell line models. By utilizing different routes of tumor inoculation, you can study different aspects of the metastatic cascade.

Each route has advantages that can be leveraged for your study. Demonstrating the procedure will be Ben Yi Tew, a postdoctoral fellow from my laboratory. For subcutaneous flank implantation of cryopreserved PDX tumors, quickly thaw the cryopreserved tumor tissue in a 37 degree Celsius water bath.

Then, rinse it in five milliliters of DPBS in a tissue culture dish. Next, after confirming anesthesia in a three to eight-week-old female NOG mouse, make a 0.5 to one centimeter incision on either the left or right flank and insert one 2x2x2 millimeter piece of the tumor tissue deeply into the pocket. After closing the incision, allow the mouse to recover from anesthesia with monitoring until it is ambulatory.

Once the tumor starts growing, measure the tumor three times per week with a caliper. At the appropriate experimental endpoint, identify metastases through a necropsy and confirm the presence of tumors within the target organ through a histological analysis. Place the resected PDX tumor in DMEM on ice.

Wash the tumor in five milliliters of DPBS in a tissue culture dish and remove the necrotic regions if there are any. Cut the tumor into small two to four-millimeter length pieces and transfer the pieces into a tube containing dissociation solution. Using the appropriate program on a Dissociator, mechanically dissociate the tissue and strain the resulting cell suspension through a 70-micrometer cell strainer.

Use 20 milliliters of DMEM to wash any remaining cells from the strainer and centrifuge the dissociated cell suspension. Then, resuspend the cells and DPBS for counting and adjust the cells to a concentration of five to 10 times 10 to the fourth cells per one to two microliters of DPBS. To prepare the surgical area, shave the fur on the mouse's head to expose the scalp.

Then, place the mouse into a stereotaxic frame, firmly securing the head of the mouse using ear bars. Make sure to also administer an appropriate analgesic. Disinfect the scalp with three alternating scrubs of povidone-iodine and 70%ethanol.

Make a five to seven-millimeter longitudinal incision to expose the skull and retract the scalp. Scrape off the periosteum with forceps to locate the bregma. Position the needle of the stereotactic frame on top of the bregma and reset the coordinates to zero.

Move the arm one millimeter posterior and one millimeter lateral to the right of the midline, and mark this location with a permanent marker. Then, drill a small burr hole in the skull at the marked location, taking care not to drill into the brain. Load to five microliter 26 gauge Hamilton syringe with one to two microliters of cells and attach the syringe to the stereotaxic arm.

Slowly insert the needle two millimeters into the brain and begin injecting cells at the desired rate. When all of the cells have been delivered, slowly retract the needle, fill the burr hole with bone wax, and close the incision. Place the mouse back into its cage with monitoring until recovery from anesthesia.

After euthanizing the animal at the appropriate experimental endpoint, confirm the presence of tumors in the brain through a histological analysis. To implant PDX tumors by intracardiac injection, prepare the tumor cells as demonstrated and place the anesthetized recipient mouse in the supine position. Shave the fur on the animal's chest and disinfect the exposed skin with povidone-iodine and 70%ethanol.

Draw 0.5 to 10 times 10 to the fifth tumor cells and up to 100 microliters of DPBS into a syringe equipped with a 28 gauge needle. Locate the injection site at slightly left of the sternum halfway between the sternal notch and the xiphoid process. Then, insert the needle vertically into the mouse at the injection site.

Once backflow is observed, indicating a successful entry of the needle into the left ventricle, slowly dispense the tumor suspension into the left ventricle without moving the needle. When all the cells have been delivered, slowly and vertically retract the needle and apply a piece of sterile gauze to the injection site for about one minute until the bleeding stops. Allow the mouse to recover on a heated pad with monitoring until it is ambulatory.

At the appropriate experimental endpoint, identify metastases through a necropsy and confirm the presence of tumors within the target organ through a histological analysis. Despite differences in the tumor microenvironment, the PDX tumors demonstrate similar morphologies containing cells with small nuclei and scant cytoplasm, regardless of the site of implantation. In this analysis, the intracardiac injection of human melanoma cells resulted in metastases of the tumor cells to the mouse brain, while the intracardiac injection of brain metastasizing human small cell lung cancer tumor cells resulted in metastasis to the mouse abdominal cavity and liver.

These protocols enable the setting up of preclinical studies for testing new treatments and treatment combinations, and can aid in studying biological processes and tumor metastasis.