Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

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Our zebrafish xenograft drug screening workflow allows for automated imaging and quantification of human cancer cells and their response to drug treatment. It can be used to rapidly test the response of a patience's cancer cells to known drugs or to identify new anticancer compounds. Using zebrafish xenograph models and drug screening allows for Inviva replicant numbers that were previously only obtainable with in vitro systems.

It is also much more cost effective than mouse models at screening through large libraries of compounds. This workflow has several steps from interjection of cancer cells in zebrafish to imaging of drug response that are learned best by visual demonstration. To begin, centrifuge a minimum of two times 10 to the sixth cells in five millimeters of PBS at 200 times g for five minutes, and aspirate the supernatant.

Resuspend the cell palette in five millimeters of dii staining solution. Incubate the cells at 37 degrees Celsius protected from light for 20 minutes. Then vortex gently and incubate the cells on ice for 15 minutes protected from light.

Then centrifuge the cells at 200 times g for five minutes and aspirate the supernatant. Wash the cells by adding five milliliters of PBS, centrifuging at 200 times g for five minutes, and aspirating the supernatant. Repeat the wash one additional time.

Resuspend the cells in one microliter of PBS per 250 thousand live cells and transfer them to a 1.5 millimeter micro centrifuge tube. Keep the resuspended cells on ice in the dark. Prior to staining cells and injecting, make agarose plates for injecting by pouring 25 millileiers of three percent agarose in 1X TBE into a petri dish and allow it to solidify.

Store the plates at four degrees Celsius for up to two weeks. Also prior to staining cells and injecting, dechlorinate two day post fertilization zebra fish using forceps under a dissecting microscope. Pull from the opposite ends of the protective chorion of the zebra fish with forceps until the chorion tears and the zebra fish becomes unenveloped.

To prevent clumping of cells during microinjection, pre chill non filamentous borosilicate glass needles at four degrees Celsius or on ice. Immediately after staining cells, use a microloader pipette tip to load five microliters of stained cells into the chilled needle. Load the needle into the microinjector arm.

Level the needle tip using a sterile razor blade. Using a stage micrometer, measure the droplet size in mineral oil. Keep the droplet volume consistently at two nanoliters, which is around 1.5 millimeters in diameter.

One minute after anesthesia, transfer the anesthetized larvae to a prepared flat surface injection plate with three percent agarose, and inject the larvae with one pump of stained cells at the desired injection site. All injections should be completed within one to three hours after staining the cells to prevent clumping of the needle. Wash the larvae off the injection plate into a 10 centimeter petri dish containing E3 media without methylene blue and incubate at 28 degrees Celsius for a one hour recovery period.

Move the dish of the injected larvae into a 34 degree Celsius incubator. With an empty petri dish placed between the shelf and the petri dish of larvae to act as a buffer. At 48 hour post injection, screen zebra fish larvae for fluorescent tumor cell engraftment and health.

Remove any dead or malformed zebra fish, and select zebra fish with similar engraftment. Remove unengrafted zebra fish. For yolk injected fish, remove fish where borders of the yoke cannot be seen around the engrafted cell mass.

For pericardium injected fish, remove fish where injected cell mass encroaches into the yolk sack. To transfer the selected fish to a 96-well plate, first cut the tip off a 200 microliter pipette tip, just large enough for a four day post fertilization zebra fish to fit through. Aspirate 150 microliters of E3 media with one zebra fish from the plate using the P200 pipette and add it to an empty well of a flat bottom 96-well plate.

Add 150 microliters of 2X diluted drug solution to each well containing zebra fish larvae to reach a final volume of 300 microliters with 1X drug solution per well. Incubate the plate at 34 degrees Celsius. Check for dead zebra fish daily.

After two days, if desired, refresh the drug by replacing 200 microliters of liquid from each well with 1X drug solution or DMSO in E3 media. In the Zen blue software, remove all unwanted fluorescent channels in the imaging software and add the Dii channel. Check the desired fluorescent channel.

In the same window, select how the images will be taken, Z stacks, automation, loops and series, et cetera. Image the DMSO control fish before the drug treated fish. And set the appropriate exposure time in the imaging software.

To quantify the fluorescence, open imagej software. Go to file, open, and select the desired CZI file. The software brings up an import options window.

For stack viewing, select hyperstacks, check open files individually, check auto scale, and check split channels. For the color option, select colorized. Then click plugins, macros, record.

Click image, adjust, threshold. Select image type as red in the drop down menu on the right side of the threshold window. Adjust the minimum threshold until the software is only highlighting areas with fluorescence, and then click apply.

The software converts the photo to black and white with the selected area in black. Click analyze and measure. The software pulls up a results window containing the fluorescent area for that image.

Click create on the macro recorder window. This opens up a new window with the code for the macro. Highlight all of the desired images for analysis and open according to stacking viewing or color option.

Select run on the window with the macro. The results window now contains the area for each image. In this protocol, at 48 hour post injection, xenografted fish were screened for fluorescently labeled tumor cells and treated with chemotherapy or DMSO.

Overall, xenografted fish treated with Vincristine showed the largest and most consistent decrease in xenografted cell mass compared to DMSO treated fish. Dexamethasone treated fish showed about half the reduction in tumor area compared to Vincristine. But still showed a reduction in tumor area compared to DMSO.

This procedure can be used to screen any cancer cell line or patient sample for response to different drugs or specific targeted therapies giving insight into each patient's tumor.

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Journal (Cancer Research)

Journal (Cancer Research)

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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