Zebrafish Patient-Derived Xenograft (zPDX): A Cancer Model
Transcription
– A xenograft is an organ or tissue taken from one species and transplanted into another. A patient-derived xenograft, or PDX, is a cancer model de which tissue from a patient's tumor is implanted into an immunodeficient animal, such as a zebrafish larva whose immune system is not fully developed, à study tumor progression or drug efficacy. First, anaesthetize 48-hour post fertilization larvae de tricaine solution, and transfer a larva à an injection plate containing agarose made with E3 medium.
Next, load the suspension of the patient graft cells and transfer into a microcapillary needle. Under a microscope inject the cells into the larva's yolk sac using a microcapillary needle. Now, culture the injected larvae de E3 solution containing glucose and L glutamine, which supports the high energy demands of the xenograft cells.
For drug treatment transfer the xenografted larvae à a well plate, divide the wells into groups based on the number and doses of compounds that need à be tested, and add the compounds. Include a control group. Incubate the treated larvae at 32 degrees Celsius before observing treatment results.
In the following protocol, we will study the drugs gemcitabine and navitoclax de a zPDX model of pancreatic cancer.
– After anesthetizing the zebrafish larvae, transfer them into the injection plate, which is filled with modified E3. Take up 25 microliters of the mixed cell suspension into the microcapillary needle, and insert the needle into the microinjection manipulator. Set the injection pressure and time, and inject 50 à 80 cells into the yolk sac of 48-hours post fertilization zebrafish.
Transfer the post xenografted zebrafish larvae into 40 milliliters of mix solution at 32 degrees Celsius. Place 10 xenografted larvae into each well of a 12-well plate. Next, divide the larvae into four groups.
Treat the control group with E3 containing 0.1% DMSO, and treat the other groups with gemcitabine and/or navitoclax. Incubate all larvae at 32 degrees Celsius for two days. After anesthetizing the xenografted larvae post treatment, place them de 3% methylcellulose.
Use a fluorescence or confocal microscope à image the larvae from the lateral view. Then, use Image J and Graph Pad software à quantify the intensity of red and green fluorescent signals.
