We have developed a fast seven-day workflow to test how tumor cells respond to drugs in using zebrafish embryos. Being close to pediatric oncology, we have access to rare cancer samples, and we started with B-cell precursor leukemia, but we are adopting the protocol now for solid pediatric tumors to see whether the assay would work in a clinical setting as well. We can successfully grow patient-derived leukemia cells in zebrafish embryos, which makes them a terrific preclinical model.
However, pharmacokinetics can be tricky, since some drugs struggle to cross biological barriers, and therefore we make sure to test the penetration before we start experiments. Our approach is unique because it blocks innate immune cell differentiation, which helps leukemia's graft cells to survive better. Plus, we are using flow cytometry to measure single cell viability and proliferation in graft cells in pools of 10 host fish.
This allows us to have very precise drug response data and big statistical power. To begin, dissolve 1%agarose in 100 milliliters of E3 medium in a microwave. For the transplantation plate, pour about 20 milliliters of the medium into a 10 centimeter Petri dish, filling it halfway.
Gently swirl the plate to distribute the liquid evenly. For morpholino injection plates, place the injection mold onto the liquid agarose in two Petri dishes, ensuring no bubbles form. Tilt the lids to cover the dishes and leave them at room temperature until the agarose solidifies.
Once the agarose has solidified, store the plates upside down at four degrees Celsius in a sealed plastic bag. Next, generate the needles from 10 centimeter capillaries using a needle puller. Break off the tips of the needles to achieve an estimated diameter of 10 micrometers.
To inject the morpholinos, transfer batches of 100 fertilized zebrafish eggs with minimal liquid into the previously prepared injection plate. Align the embryos carefully in the grooves of the plate. Inject one nanoliter of a 50 micromolar mix of spi1 and csf3r morpholinos into the cell or the yolk sack just beneath the cell during the one-cell stage.
Incubate the injected eggs at 28 degrees Celsius overnight. On the third day, to initiate dechorionation, using a Pasteur pipette, remove any dead embryos that show no heartbeat or movement, appear opaque, or have irregular shapes from the dish. Using two precision forceps, pinch the chorion to hold it in place.
Pinch near the tip of the forceps while securing the embryo with a second set of forceps. Carefully pull the chorion apart to release the embryo. Incubate dechorionated embryos at 28 degrees Celsius overnight.
To begin, prepare two 10 centimeter Petri dishes filled with E3 medium supplemented with penicillin streptomycin or E3/P/S. Place the dishes at 28 degrees Celsius for 30 minutes to pre-warm. After fluorescent cell labeling with CellTrace Violet, or CTV, centrifuge the BCP-ALL cells.
Then add PBS to achieve the desired cell concentration and keep it on ice. Load four microliters of cell suspension into the transplantation needle using a microloader tip. Add tricaine to a Petri dish with E3/P/S medium and fish embryos to achieve a final concentration of four grams per liter for anesthetizing the embryos for at least two minutes prior to injection.
Next, transfer 15 to 20 dechorionated embryos onto an agarose-coated injection dish and take off excess liquid to prevent embryos from slipping. Arrange the embryos on the injection dish. Introducing the needle at a 45-degree angle from the dorsocaudal direction, inject approximately 1, 000 CTV-positive BCP-ALL cells into the pericardial cavity.
Once the 15 to 20 embryos are injected, transfer them to the pre-warmed Petri dish filled with E3/P/S medium. Incubate both the transplanted embryos and non-transplanted controls at 28 degrees Celsius for one to three hours. Under a fluorescent stereo microscope, screen the embryos to confirm successful engraftment and ensure that the yolk is intact.
Add 100 microliters of E3/P/S medium and 0.5%DMSO to each well of a 96 well plate. Using a glass Pasteur pipette, pick up the engrafted embryo with as little E3 medium as possible. Tilt the pipette to allow the embryo to sink to the bottom of the pipette tip and release it into the well using capillary forces.
Fill 24 wells of the 96 well plate with 100 microliters of either the vehicle control solution or two times concentrated solutions of venetoclax. Maintain the embryos at 35 degrees Celsius for 72 hours, including the non-transplanted embryos as controls. At the end of the third day, remove the 96 well plate containing engrafted zebrafish embryos from the incubator.
Screen the plate using stereo microscopy to identify and select healthy zebrafish embryos. Randomly pool 10 host embryos from each condition into a 1.5 milliliter microcentrifuge tube, ideally resulting in two tubes per condition. Remove as much liquid as possible from each tube.
Add 500 microliters of calcium and magnesium-free HBSS to each tube. Mechanically dissociate the embryos and graft cells by trituration using a 200 microliter micropipette tip, pipetting up and down approximately 15 times. Centrifuge the tubes at 350 g for five minutes at room temperature to pellet the tissue fragments.
Prepare FACS tubes with a 35 micrometer fine mesh filter strainer cap containing two milliliters of PBS per condition. Then resuspend the pellet in 500 microliters of enzyme mix and incubate for 15 minutes at room temperature. During incubation, pipette the mixture up and down every five minutes using the same pipette tip for each tube to avoid tissue loss.
Transfer the dissociated cells into the 35 micrometer fine mesh filter strainer cap of the FACS tubes. Centrifuge the tube at 350 g for five minutes. Conduct flow cytometry analysis to evaluate the total number of graft cells.
Start the data analysis with the CTV and CD19 stained 3dpi culture cells. Create a dot plot with CD19 and CTV to ensure the signal is overlapping and to confirm the presence of the double-stained cancer cells. Next, create a dot plot with FSC-A and SSC-A to distinguish intact cells from debris.
Then use the intact cell population to create a new plot with SSC-A and SSC-H to identify single cells. Create a dot plot with 7AAD and Annexin V using the single cells population to distinguish cells into four populations. Open the file of an engrafted zebrafish.
Separate human graft cells from host cells using a dot plot with CTV and CD19. Identify and isolate CTV, CD19 double positive graft cells. Copy and apply the same gating strategy described for 3dpi culture cells to the graft cell population.
Merge all Annexin V and 7AAD negative cell populations into a histogram with CTV values on the x-axis. Calculate the geometric mean for all five samples to determine proliferation rates. Flow cytometry data showed that fresh BCP-ALL single cells engrafted in zebrafish embryos had 95.2%viability after three days, 1.5 times higher than the 61.9%viability of dish-cultured cells.
The CTV fluorescence intensity in both in vivo and in vitro conditions decreased similarly over three days, indicating comparable cell division rates. Intact graft cells per embryo after three days were quantified in each pool, indicating consistent engraftment.
