Zebrafish Patient-Derived Xenograft (zPDX): A Cancer Model

1. Injecting Mixed Cell Suspension into the Zebrafish

1. Add 10x tricaine solution into E3 water to anesthetize zebrafish larvae and transfer the larvae (from step 2.3) to the injection plate filled by modified E3 (E3 with 1 g/L glucose and 5 mmol/L L-glutamine).
2. Fill 25 µL of mixed cell suspension into micro capillaries needle and insert the needle into the micro-injection manipulator.
3. Set injection pressure and time. Inject 50-80 cells (~8 nL) into the yolk sac of 48 hpf zebrafish.

2. Culture of the Xenografted Zebrafish (zPDX Model)

1. Transfer the post-xenografted zebrafish larvae into 40 mL of mix solution (E3 solution with 1 g/L glucose and 5 mmol/L L-glutamine) at 32 °C.

3. Drug Administration on the Xenografted Zebrafish and the Assessment of Tumor Cells/Fibroblasts Viabilities

1. Determining the optimal concentration of gemcitabine/navitoclax.
   1. Place 10 wildtype zebrafish embryos at 48 hpf into each well of a 12-well plate.
   2. Add different concentrations of gemcitabine or navitoclax in each well and incubate at 32 °C for two days.
   3. After 2 days, calculate the maximal tolerance dosage (MTD) of gemcitabine and navitoclax at which the zebrafish larvae do not shown significant malformation and abnormal behavior, and the working concentrations are set below the MTD.
2. Treatment of zPDX models with gemcitabine/navitoclax.
   1. Place 10 xenografted larvae into each well of a 12 well plate.
   2. Divide the larvae into four groups, treat the control group in E3 containing 0.1% DMSO, and treat the other groups with 5 μg/mL gemcitabine and/or 50 μM navitoclax, and incubate at 32 °C for two days.
3. Assessment of the cell viabilities and cellular composition in zPDX models.
   1. Anesthetize the xenografted larvae post-treatment and place them in 3% methylcellulose.
   2. mage the larvae from the lateral view using a fluorescence microscope or confocal microscope.
   3. Quantify the intensity of red and green fluorescence signals using ImageJ and GraphPad software.