Subcutaneous Transplantation Assay: A Method to Transplant Melanoma Cells from a Donor to a Recipient Zebrafish Model

- During melanoma transplantation assay, cancerous cells from tumor-bearing zebrafish are transplanted into an immunocompromised recipient model to study tumor development. Begin by taking a euthanized tumor-bearing donor zebrafish and surgically resect the tumor from its body. Transfer the tumor into a Petri dish containing a suitable buffer and mince it into small pieces.

Next, triturate the tumor fragments using a pipette. This dissociates the tumor pieces into individual cells. Now, load the tumor cell suspension into a syringe. Subsequently, prep an immunocompromised recipient Casper zebrafish on a filter paper. This pigmentation-deficient, mutant variant of zebrafish lacks melanocytes and iridophores, making it transparent and versatile for imaging and transplantation experiments.

Next, inject the preloaded melanoma cell suspension subcutaneously into the region above the peritoneal cavity, midway between the hindbrain and the dorsal fin of the Casper zebrafish. Now, transfer the recipient fish into the water and monitor it to observe tumor development. The transparent body of zebrafish helps in the easy visualization of the developed tumors.

In the following protocol, we will show the transplantation of melanoma cells from a transgenic tumor-bearing zebrafish donor to an immunocompromised recipient Casper fish model.

- After irradiating recipient 2 to 3 month old Casper fish with 25 Gy of gamma irradiation one day prior to transplantation, allow the fish to recover in fish water. Use tricaine to euthanize a tumor-bearing fish. Cut off the tumor and put it in a Petri dish with approximately 5 milliliters of filter sterilized 0.9x PBS with 5% FPS. With a razor, dice the tumor while in solution.

Working at room temperature and with a P1000 pipette, triturate to produce single cells. Bring up the volume to 25 milliliters. Filter the solution through a 40 micrometer mesh filter and spin in a tabletop centrifuge at 453 rcf for 10 minutes. Resuspend the pellet in 0.9% PBS 5% FBS to the desired concentration.

Using a hemocytometer, calculate the exact cell number and dilute the cell suspension so that the final injection volume is approximately 5 microliters. For example, if 50,000 cells are to be injected, the cell suspension should be diluted to a final concentration of 10,000 cells per microliter. Flick the tube containing the cell suspension every few minutes to prevent clumping of the cells.

With 100% ethanol and 0.9x PBS, wash a 26s gauge (bevel tip) 701 N 10 microliter Hamilton syringe 2 to 3 times before loading the syringe with 5 microliters of the cell suspension. After anesthetizing the irradiated recipient fish in tricaine, place it on its side on a damp Kimwipe.

Stabilize the fish with one hand and insert the needle with the bevel facing up at a 45-degree angle into the flank of the fish above the peritoneal cavity about halfway between the posterior boundary of the hindbrain and interior border of the dorsal fin. Gently depress the plunger. Allow the fish to recover in fresh fish water and observe the fish daily for tumor engraftment. If engraftment has occurred, continued growth and disease development can also be observed.