Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish xenografts are being widely used for cancer research. You can take human tumor cells and inject it in little larvae zebrafish, or even into adults. Here, what we are presenting is a step-by-step protocol of the larva model, where we can study several human cancer hallmarks in a living animal.

You can study proliferation, you can study angiogenesis, you can study metastasis, and also interactions within the tumor microenvironment. And a big advantage is that you can study this in a very short time frame. Just four days, for instance.

Another great advantage is that you can study this live with single cell resolution, using a confocal or a light sheet microscope. You can study how cells migrate, how cells interact with each other, or even how they talk to each other. So it's a really powerful model to study cancer biology.

Zebrafish xenografts can be used to study response to anti-cancer therapies, like chemotherapy, radiotherapy, or even targeted therapies with antibodies. And finally, also, this protocol can also be adapted to use patient-derived cells from a surgical sample, or even from biopsies, and then generate the zebrafish avatars. Setting up for injection.

Two weeks before injection, expand the cells in culture. Start with excess cells and excess fish until becoming proficient with the technique, since many cells and fish will be lost during training. Table one of the accompanying PDF has a detailed guide of the optimal percentages of in-vitro confluence for injection of several cell lines.

Three days before the injection, cross the zebrafish of the desired background. 24 hours before injection. Clean the zebrafish embryo plates.

Discard all dead and non-developed embryos and refresh E3 medium. In the cell culture room, discard the cell culture medium. Wash once with PBS 1X to remove the dead cells and add fresh medium.

Prepare the tools for the injection procedure, such as microinjection needles, agarose plates, and hairpins to align the embryos for injection. For the plate preparation, pour one layer of dissolved 2%agarose in the lid of a clean Petri dish. Let it polymerize and with the help of a ruler, make three to four straight agarose lines for the alignment of the embryos.

Injection day. Separate the hatched embryos from unhatched eggs. Adding Pronase to the embryo medium at this stage can boost hatching.

Place the embryos into the incubator at 28 degrees Celsius until injection. Cell labeling for injection. To help in the establishment of a reproducible cell labeling technique, check tables one and two of the written protocol for a compilation of a list of cell lines and several labeling solutions.

Remove the cell culture medium and wash the flask twice with PBS 1X. Label the cells with a lipophilic dye, avoiding exposure to light, and incubate the flask at 37 degrees. You can also opt to label the cells in a 1.5 ml microcentrifuge tube upon detachment.

Remove the cell labeling solution and wash the cells with PBS 1X to take out the excess dye. Add the corresponding detachment agent, and incubate the cells at 37 degrees Celsius. Facilitate the detachment process by brushing the flask with a sterile cell scraper and collect the cells with a serological pipette.

Transfer the cells to 1.5 ml microcentrifuge tubes and centrifuge. Discard supernatant and re-suspend the pellet with cell culture medium. Quantify cell viability using a Neubauer chamber with trypan blue exclusion or other method of choice.

Centrifuge and re-suspend the cells in the injection medium. The recommended concentrations of cells in medium can be found in table one of the manuscript. From this point onwards, the cells must be kept on ice.

Injection procedures. Anesthetize the embryos in tricaine 1X for 5 minutes. Then, transfer a small amount of anesthetized embryos to an agarose plate and align them with the help of a hairpin loop.

Make sure to maintain distance between the embryos. Especially between the yolk of one and the head of the next one. To prevent mortality, ensure that the aligned embryos do not dry out in the agarose plate by carefully adding one to three drops of tricaine 1X solution to the plate.

Lightly tap the microcentrifuge tube to re-suspend the cells. Backload the injection needle with a cell suspension using a Microloader tip, avoiding air bubbles, as they can compromise the integrity of the embryos. Open the air pressure valve, set up the microinjector, and carefully place the microinjection needle into the holder.

Cut the microinjection needle close to the tip. Carefully pierce the embryo's yolk, lowering the angle of the needle and cautiously pushing until the tip of the needle reaches the perivitelline space. Once the tip is in the perivitelline space, inject the cells.

Try to inject a volume of cells similar to the size of the embryo's eye, and as far as possible from the heart to prevent cardiac edema. Adjust the microinjector pressure, if needed. Transfer the injected embryos to a clean Petri dish with tricaine 1X solution and leave them to rest for five to 10 minutes.

This will give the wound time to close. Remove the tricaine 1X solution and add fresh E3 medium. Then, incubate the embryos at 34 degrees Celsius.

This temperature will allow the survival of both the zebrafish embryos and the human tumor cells. Metastatic assay. If you want to study the metastatic potential of your cell lines, at around one hour post-injection, screen the injected embryos on a fluorescence stereomicroscope, and sort them into two groups according to the absence or presence of cancer cells in circulation.

One day post-injection. On a fluorescence stereomicroscope, carefully analyze each embryo and select those with properly injected tumors. Sort the selected xenografts according to the tumor size.

Use the size of the eye for comparison. Discard any embryos with abnormal morphology, cardiac or yolk edema, xenografts with very few cancer cells, or those with cancer cells only inside the yolk. Distribute the xenografts according to the desired experimental layout and start the drug assay.

Replace the drugs and E3 medium daily. Incubate the xenografts, maintaining the temperature of 34 degrees Celsius until the end of the assay. Four days post-injection.

On the final day of the assay, anesthetize the xenografts with tricaine 1X solution, and carefully align them on the agar plate. Discard any dead or swollen xenografts. These xenografts are not considered for engraftment quantification.

On a fluorescence stereomicroscope, carefully analyze each xenograft and assess the absence or presence of tumors in the perivitelline space. According to the experimental setup, select the xenografts of interest and euthanize them with tricaine 25X. Fix them in 4%formaldehyde for at least 4 hours at room temperature to proceed with immunostaining.

Otherwise, store overnight at four degrees Celsius and on the next day, replace formaldehyde with 100%methanol. Xenografts fixed in 100%methanol can be stored at 20 degrees indefinitely. Whole mount immunostaining for confocal imaging.

The whole mount immunofluorescence technique takes three days, divided as follows. The first day is for permeabilization of the xenografts and primary antibody incubation. The second day, for washing and secondary antibody incubation.

And the third day, for washing, fixation of xenografts and storage in the mounting medium. More details of this procedure can be found in the accompanying PDF. Mounting of xenografts.

Seal the edges of a cover slip with petroleum jelly or silicone grease, so that the mounting medium does not leak. Using a Pasteur pipette, transfer the xenografts to the cover slip. Carefully align them with a hairpin.

Add the mounting medium to another cover slip. Carefully join both cover slips and place them on top of a microscope slide to allow for easier manipulation. Confocal imaging.

An apochromatic 25X immersion objective lens with water correction is optimal for imaging tumors with a single cell resolution. Acquire the samples using the Z-Stack function with an interval of five microns between each slice. Open the raw data in Fiji software.

Select three representative slices of the tumor from the top, middle and bottom, per z-stack, per xenograft. Open the Cell Counter plugin from Fiji software. In the Cell Counter tool, click Initialize, select a counter type, and click on the image to start the counting mode manually.

For every click, the counter asks how many cells are counted. To obtain the total number of cells in the tumor, use the following formula. To assess total numbers or percentage of markers, such as immune cells, mitotic figures, activated caspase, among others, quantify manually the cells that are positive to the specific labeling, marker or transgene of interest along all the slices of the z-stack with the Cell Counter plugin.

Be aware that some cells will be located between two slices. Go back and forth in the z-stack to make sure that no cell is being counted twice. Representative results.

HCT 116 colorectal cancer cell line xenografts were generated as described in the protocol. Xenografts were randomly distributed in non-treated controls and FOLFIRI chemotherapy. Immunofluorescence was performed to detect apoptotic cells, using anti-cleaved caspase-3 antibody and DAPI for nucleic counterstaining.

Quantification of the mitotic index, apoptosis, and tumor size, revealed that FOLFIRI induces a significant decrease of mitosis and a significant induction of apoptosis, accompanied by a 54%reduction of tumor size. These features are useful in high-throughput phenotypic drug screens, as well as to test the cell intrinsic and physiological effects of several cancer treatments in a short time frame. Another great advantage of the zebrafish xenograft model is that it is possible to study the interactions of different tumor cells and analyze how each type of cell can influence the behavior of the other.

Different human cancer cells, different clones from the same tumor, or from different tumors, can be co-injected. In this example, two colorectal cancer cell lines derived from the same patient were labeled with different lipophilic dyes and mixed in a ratio of one-to-one for injection. We hope that this protocol can help researchers become real experts in generating zebrafish xenografts.

It's not easy. You need to practice, practice. Don't give up.

I'm sure you'll get there. Good luck.