The overall goal of this procedure is to enable long-term assessment of tumor cell behavior, such as invasion and dissemination, and test potential therapeutics in an immune-competent animal host. This method can help answer key questions in the field of pediatric brain cancer, including the longevity of a drug response and mechanisms driving invasion and dissemination. The main advantage of this technique is it minimizes toxicity to the host, which enables lasting engraftment of tumor cells and therefore long-term studies of brain cancer biology.
The implications of this technique extend toward therapy of brain cancer because tumor burden can be directly assessed in vivo after drug treatment. Generally, individuals new to this method will struggle if the tumor cell suspension is too concentrated or too dilute, making it difficult to inject consistent amounts into the fourth ventricle. To begin, prepare a 50-milliliter solution of 1.2%agarose dissolved in egg water.
Boil the solution until the agarose dissolves, and then supplement it by adding 05 milligrams per liter methylene blue. Pour 25 milliliters of the final solution into a 10-centimeter petri dish and allow it to solidify. Place the remaining 25 milliliters of agarose solution in a 42 degrees Celsius water bath.
Then set a two inch diameter beaker in the center of the solidified surface, and pour the remaining 25 milliliters of 1.2%agarose onto the previous layer. Maintain the injection plate at 28 degrees Celsius for at least 30 minutes prior to injection or at four degrees Celsius for long-term storage. Make the needles by using a needle puller to pull 10-centimeter capillaries with an outer dimension of 1.2 millimeters and an inner dimension of 0.9 millimeters.
Carefully place the needle on a microscope slide wrapped in plastic paraffin film. Then use a razor blade to cut the end of the needle at a 45-degree angle to create a needle with an opening at the tip. After euthanizing a brain tumor-bearing fish and dissecting the tumor according to the text protocol, manually use a P1000 to disrupt the tumor mass until a uniform, cloudy solution develops.
Use a pipette or a 40-micron cell strainer to remove large particulates. Then centrifuge the suspension at 290 times g at room temperature for five minutes, and remove the supernatant. Resuspend the tumor cell pellet in 100 microliters of sterile PBS, and transfer it to a 1.5-milliliter microcentrifuge tube.
Use 250 microliters of PBS to dilute five microliters of the cell suspension. Then use a hemocytometer to count the number of cells. Centrifuge the suspension at 290 times g for three minutes.
After removing the supernatant, use sterile PBS to resuspend the pellet to obtain the desired cell concentration. Store the tumor suspension on a 28 degrees Celsius heat block during the transplantation procedure. Using a transfer pipette, transfer 10 to 20 anesthetized embryos to the periphery of the injection plate.
The embryos should fall laterally, with the ventricles clearly visible and accessible. Use an angled probe to adjust the embryos as needed and position them away from the outer edge of the injection plate. Then use a gel-loading tip to load one to two microliters of the tumor cell suspension into the injection needle, and insert the needle into the manipulator.
Next, manually lower the manipulator, holding the needle at a 45-degree angle. Adjust the knobs of the micromanipulator in the x, y, and z directions until the needle is just above and approximately five millimeters to the right of the embryo head. Using a stereomicroscope, slowly adjust the micromanipulator in the x-direction until the needle pierces the fourth ventricle of the embryo.
Do not allow the needle to pierce the heart or the yolk. For consistent injections, the embryos must be anesthetized, the tumor must be properly dissociated into a cellular suspension, and the needle must pierce the fourth ventricle but not extend beyond into the heart. Push the microinjector foot pedal to inject the tumor cell suspension.
When finished injecting, use fresh egg water to gently rinse the injected embryos off the injection plate and into a petri dish. Now, inspect the injected embryos under a fluorescent stereomicroscope in a dark room. Confirm that the injection pressure, angle, needle size, and cell suspension viscosity result in tumor cells filling 25 to 50%of the ventricle space.
Place the embryos back into the 28 degrees Celsius incubator overnight. The next day, assess embryo survival by examining morphological and physiological features, such as normal heart and brain development, as previously described. Use a transfer pipette to add 4%methylcellulose onto the middle of a petri dish and add anesthetized embryos to the methylcellulose drop.
Use an angled probe to orient the embryos, and under the fluorescence microscope, screen for a consistent engraftment size. For maintenance of tumors, once the embryos reach eight days post-fertilization, place the transplanted embryos in tanks to grow. Image and carry out additional studies according to the text protocol.
In this experiment, the central nervous system primitive neural ectodermal tumor cells were transplanted into the mitfa zebrafish mutant that lacks melanophores. As early as 24 hours post-transplantation, tumor cells can be seen invading into surrounding brain tissue. Tumor grafts continue to grow in the ventricle and surrounding brain tissue over the next week, and fluorescence can also be observed in the region of the kidneys.
Tumor cells continue to proliferate and invade, so by 28 days post-fertilization, a tumor mass can be seen throughout the zebrafish brain. A representative group of zebrafish transplanted with mCherry-labeled brain tumor cells is shown in this figure. Tumor cell engraftment is typically achieved in 80 to 90%of zebrafish, and tumor transplants persist into adulthood.
In this experiment, an NRAS-driven, mCherry-labeled zebrafish CNS-PNET from the optic tectum and a GFP-labeled CNS-PNET from the cerebellum were harvested by whole-tumor dissociation, processed into a single-cell suspension, then mixed in a one-to-one ratio before transplantation. This representative embryo that was imagined four days after transplantation demonstrates that the optic tectum tumor cells, in red, migrate more extensively into the host brain than the cerebellar tumor. Once mastered, approximately 300 embryos can be injected in three to four hours.
Following this procedure, additional methods, such as live animal imaging and drug administration, can be performed to answer additional questions related to tumor cell biology, drug response, and chemoresistance. After its development, this technique paved the way for researchers in the zebrafish cancer modeling field to explore novel inhibitors for treating pediatric brain cancers.
