We seek to gain molecular insight into the control of normal and malignant hematopoiesis, and then to exploit those insights to develop new therapies for the treatment of blood cancers. Tumor xenografts are an indispensable tool for evaluating the impact of a genetic alteration or treatment on tumor behavior. However, mice xenografts are expensive, time-consuming, and limited in scope.
Using zebrafish for tumor xenotransplantation overcomes all these drawbacks. In this protocol, we demonstrate an easy and rapid mounting process for xenotransplantation, which increases throughput. We further employ a cell staining to know the quality of the inoculum and then flow cytometry to assess the tumor progression by evaluating or quantifying the disease burden.
We intend to use this approach to identify novel ways of blocking the function of the Ras/MAP kinase cascade, which drives the vast majority of human cancers. And we'll do that not by trying to block the catalytic activity of the Ras/MAP kinase cascade, but rather by preventing its ability to phosphorylate those cancer-driving substrates it modifies. To begin obtain leukemia cells grown under optimal conditions for transplantation.
After counting the cells, pellet them at 300 g for five minutes at room temperature and discard the supernatant. For CM-Dil staining, resuspend the cells in the working solution to obtain one times 10 to the power of six cells in 100 microliters and incubate them at 37 degrees Celsius for 10 minutes. Wash the cells two times with 10 milliliters of 1X HBSS at room temperature.
After pelleting the cells at 300 g for five minutes, resuspend them in PBS containing 1%FBS to achieve a density of 40, 000 cells per microliter. Then turn on the microinjector and the pump. Set the injection pressure to nine to 11 pounds per square inch and injection time to 0.5 seconds for clipping the needle and setting the orifice.
Load around five microliters of the tumor cell suspension line into the microneedle carefully in a single pass, avoiding air bubble formation. Using Dumont 5 forceps, cut the end of the needle to produce an orifice that can support the ejection. Then select healthy zebrafish embryos under the microscope, eliminating any with developmental anomalies.
Using a glass pasture pipette, pick up the anesthetized embryos and arrange 10 to 15 of them in a lateral position on the 1.5%agarose plate. Remove excess water to leave a minimum amount for embryo survival. Next, confirm under the light microscope that the needle is cut well and has cells.
Inject 10 to 15 nanoliters corresponding to 400 to 600 cells into the yoke of the embryos for 0.2 to 0.3 seconds. After injecting all the embryos, collect them in fresh embryo water. One hour after the injection, monitor the bolus of cells to separate the embryos with optimal staining or good bolus from those with inferior staining or inferior bolus.
The CM-Dil staining and fluorescent protein marking enabled the monitoring of disease progression by microscopy and the survival of embryos decreased after the injection with two genetically distinct leukemia lines. To begin, obtain zebrafish embryos injected with leukemia cells pre-stained with CM-Dil. Transfer the anesthetized embryos to a 1.5 milliliter centrifuge tube, and using a P200 pipette, dissolve the yolk in 100 microliters of calcium-free Ringer's solution for five minutes.
Incubate each sample of deyolked embryos with one milliliter of the trypsin collagenase solution at 29 degrees Celsius for 30 to 35 minutes. Using a P1000 tip, pipette the embryos up and down in this solution every five minutes until the backbone structure is no longer visible. To stop the reaction, add 200 microliters of FBS to the tube.
Mix well and incubate for an additional five minutes. Pellet the cell suspension at 300 g for five minutes at four degrees celsius. Once the supernatant is discarded, wash the cell pellet two times in chilled PBS and strain the cells through a 70 micrometer cell strainer.
After washing the cells, resuspend the cell pellet in the staining medium containing an antibody reactive with the transplanted cells. Incubate the cells at four degrees Celsius for 20 minutes. Pellet the cells.
And resuspend them in 200 microliters of staining medium containing one micromolar Helix NP Blue. Transfer the cell mix to a five milliliter round bottom polycarbonate tube and perform flow cytometry. The dual labeling allowed the measurement of differences in the tumor burden between good versus inferior bolus inoculation.
