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

Overview

This video describes the method for transplantation of tumor cells from the melanoma-bearing donor zebrafish model to an immunocompromised recipient Casper zebrafish. It helps to visualize tumor engraftment and proliferation in the recipient fish.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Transplantation Assay

1. Prepare recipient 2-3 month old Casper fish by irradiating with 25 Gy of gamma irradiation one day prior to transplantation. Allow the fish to recover in fish water.
2. Euthanize a tumor-bearing fish according to institutional guidelines. Specifically, a fish is placed into a dish with 0.6 mg/mL tricaine until gill movement stops.
3. Cut off the tumor and put it in a Petri dish with approximately 5 mL filter sterilized 0.9x PBS with 5% FBS. Dice the tumor with a razor while in solution.
4. Working at room temperature, triturate with a P1000 pipette to get single cells. Make up the volume to 25 mL with filter sterilized 0.9x PBS with 5% FBS.
5. Filter through 40 μM mesh filter.
6. Spin in an Eppendorf 5810R tabletop centrifuge at 1,500 rpm (453 rcf) for 10 min.
7. Remove the supernatant and make the cell suspension to roughly desired concentration (assume 10⁷ cells for a 100 mm³ tumor).
8. Calculate the exact cell number using a hemocytometer and dilute the cell suspension so that the final injection volume is approximately 5 μL. For example, if 50,000 cells are to be injected, the cell suspension should be diluted to a final concentration of 10,000 cells/μL. Flick the tube containing the cell suspension every few min to prevent clumping of the cells.
9. Wash a 26s gauge (bevel tip) 701 N 10 μL Hamilton syringe 2-3 times with 100% ethanol and 0.9x PBS. Load the syringe with 5 μL of the cell suspension.
10. Anesthetize the irradiated recipient fish in 0.17 mg/mL tricaine. Place fish on its side on a damp Kimwipe (Figure 1A).
11. Stabilize the fish with one hand and insert the needle with the bevel facing up at a 45° angle into the flank of the fish above the peritoneal cavity about halfway between the posterior boundary of the hindbrain and anterior border of the dorsal fin. Gently depress the plunger (Figure 1B).
12. Allow the fish to recover in fresh fish water and observe the fish daily for tumor engraftment (Figure 1C). If engraftment has occurred, continued growth and disease development can also be observed.

Representative Results

Figure 1. Transplantation of melanoma cells. A) Uninjected Casper zebrafish. Scale bar = 500 μM. B) Subcutaneous transplantation site (arrowhead) on an irradiated Casper recipient immediately after injection with 50,000 melanoma cells. Scale bar = 200 μM. C) Irradiated Casper recipient showing tumor engraftment (arrowhead) two weeks after injection with 50,000 melanoma cells.

Materials

Name	Company	Catalog Number	Comments
Gateway recombination reagents	Invitrogen
miniCoopR
Casper Zebrafish
701N 10 μl Syringe	Hamilton/Fisher	14-824
40 μM filter	BD Falcon/Fisher	352340
FBS	Invitrogen	26140079