A Melanoma Patient-Derived Xenograft Model

The PDX has revolutionized the cancer experimental pre-clinical studies. We can now mimic, in the laboratory, what is happening in the patients. And now we can work on how to overcome the resistance and how, on the longterm, we can increase survival of patients.

Demonstrating the procedure will be Min Xiao. To begin, transfer previously collected melanoma tissue to a sterile Petri dish. Working with surgical scissors, scalpel blade, and forceps, first remove normal tissue from the normal tissue as much as possible.

And then remove the necrotic tissue identified by pale white-ish tissue located centrally within the tumor. Then use a scalpel to subdivide an initial tumor chunk into approximately equal pieces for surgical mouse implantation. If the tumor tissue is too hard for mechanical dissociation, use a scalpel to mince the tumor chunks as finely as possible to form a slurry.

Transfer the slurry into a 50 milliliter tube with cold Hank's Balanced Salt Solution without calcium and magnesium ions. Centrifuge at 220 times g for four minutes at four degrees Celsius. After removing the supernatant, re-suspend the slurry in 10 milliliters of warmed, fresh digest media per one gram of tumor tissue.

Place the tube in a 37 degrees Celsius water bath for 20 minutes and mix vigorously every five minutes with a disposable pipette. Wash the slurry using up to 50 milliliters of HBSS, centrifuge at 220 times g for four minutes at four degrees Celsius, and then remove the supernatant. Add five milliliters of pre-warmed TEG buffer per one gram of tumor tissue, shake to gently re-suspend, and place the tube at 37 degrees Celsius for two minutes, without mixing.

To quench the trypsin, add at least one equal volume of cold staining media and centrifuge at 220 times g for four minutes at four degrees Celsius. After removing the supernatant, add 10 milliliters of staining media per one gram of tumor tissue, and re-suspend the pellet by pipetting up and down. Filter it through a 40 micrometer cell strainer to get a single cell suspension for mouse injection.

To implant surgical excision or surgical biopsy tissue, first shave hair from the lower back of NSG six to eight week old anesthetized mice, leaving an approximately 1.5 centimeters to three centimeters area without hair. Prepare tumor chunks, and divide tumor slurry in a Petri into individual mounds for surgical implantation. Using a scalpel blade, make an approximately five millimeter long incision on the center of the back of the mouse.

With one pair of forceps, life up the skin on the side of the incision opposite the operator. With scissors in the other hand, separate the skin from the muscle layer by gently cutting the fascial membrane with small scissor cuts, thereby creating a pocket for the tumor tissue. Use a scalpel blade to pick up one tumor chuck, as well as one individual mound of tumor slurry tissue, and gently place the tissue into the created pocket.

Then add 100 microliters of artificial extracellular matrix on the tumor tissue mound in the pocket. Using two pairs of forceps, pull up the incision on both ends so that the wound edges come close together. Then apply one or two wound clips to close the wound.

Subcutaneously inject one to five milligrams per kilogram of analgesic. When healing is completely, after approximately seven days, remove the wound clips. Monitor mice once weekly to check for palpable tumors.

Once tumors reach a desired volume, use surgical forceps to lift the skin adjacent to the tumor of the euthanized mouse and make a horizontal cut using curved scissors. Use a blunt separation technique to mobilize the skin on both sides of the tumor and over the tumor, exposing the tumor. Use scissors and a scalpel blade to separate the tumor from the fascia.

Cut out the tumor and transfer it to a sterile Petri dish. Cut the tumor into small pieces and remove necrotic tissue from the tumor. To bank tumor tissue for future implantation, take two to three small tumor pieces and mince them into pieces smaller than one by one millimeter.

Transfer all minced tissue to a two milliliter cryogenic vile. Add one milliliter of freezing media and mix well. Place the vials into a pre-cooled isopropanol-based cell freezing container on dry ice, store the container in a minus 80 degrees Celsius freezer overnight, and then transfer the vials to liquid nitrogen storage.

To snap freeze tissue for downstream assays, place the tumor tissue pieces into a cryogenic vial, put the cryogenic vile in liquid nitrogen immediately, and store the vials in a minus 80 degrees Celsius freezer. When three different methods of growing tumor tissue were compared, single cell suspension of tumor cells with the use of enzymatic digestion was most successful. Besides allowing for more rapid tumor growth, it was also sufficient for one initial tumor to be expanded into 10 to 20 mice, whereas the tumor chuck and slurry method can only be expanded into five to 10 mice.

Melanoma patient-derived xenograft models often reflect the drug sensitivity the patient displayed when on therapy. A xenograft derived from melanoma patient who initially responded to a BRAF inhibitor, but ultimately relapsed, also displayed initial sensitivity to BRAF protein inhibition, plus MEK kinase inhibitor, but the tumors ultimately relapsed. It's critical to take care when preparing PDX tissue for banking, as this will ensure success in future PDX expansions, therapy trials, as well as characterization.

With PDX material, single cell RNA sequencing, whole exome sequencing, and proteomic characterization, are among the many methods our lab leverages to dissect therapy resistance. The PDX technique allows researchers to investigate therapy resistance using tumor cells that better recapitulate the tumor biology in a human patient relative to traditional cancer models grown on plastic. This advantage allows for more predictive insights.

Researchers should always take care and use biosafety level two precautions when handling tumor tissue derived from a human patient.