Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

The overall goal of this procedure is to assess the malignant and invasive behavior of cancer cells in a zebrafish embryo model. This method helps researchers determine if cancer cells can invade from a functional circulatory system, thereby modeling a key step in the metastatic cascade. The main advantage of this technique is that the zebrafish embryo model provides this assessment and shorter turnaround time than similar experiments often conducted in mice.

Demonstrating the procedure will be Doctor Eric Glasgow who runs the zebrafish core facility at Georgetown University. After preparing cancer cells, reagents, and zebrafish embryos according to the text protocol, attach the micro injection dispense system to a pressurized air source, and turn on the power source. Test the pressure by depressing the foot pedal.

A brief pulse of air should emit from the needle holder. Add 20 milliliters of 2X Tricaine Solution to an injection plate, and place the plate on a shaker to equilibrate it. Then repeat the equilibration with a second aliquot of 2X Tricaine.

Next, use a plastic pipette to transfer a group of embryos to a small dish, containing 2X Tricaine Solution. Using a gel loading tip, backfill the micro-injection needle with cancer cells. Place the needle in an electrode storage jar with the pointed end facing down so cells settle near the tip.

Transfer 20 to 30 anesthetized embryos to an injection plate by collecting embryos with plenty of 2X Tricaine in a plastic pipette. Allow the embryos to set on the tip of the pipette then gently expel the embryos into the trough of the injection plate, spreading the embryos along the length of the trough. Align the embryos with heads facing up and bellies facing the steep wall of the trough.

Attach the needle to the needle holder of the micro manipulator. Then, position the injection plate under the stereoscope with the 60 degree wall to the left and focus on the top embryo at 25X magnification. Position the micro manipulator so that when extended the needle will pierce the embryo.

Then extend the needle by eye until it is nearly touching the embryo. Now, while looking under the microscope, align the needle up against the embryo. To inject the cells, pierce the embryo trough through the yolk sac.

Depress the foot pedal, placing the tip just at, but not in, the precardiac sinus. The force of the injection will expel the cells into the cardiac sinus. Alternatively, injection of cells into the yolk sac allows for the study of different facets of vascular invasion as described in the manuscript.

Using the right hand, extend and retract the injection needle. With the left hand, make fine adjustments to position the next embryo. When the entire plate is injected, return the needle to the electrode storage jar.

To transfer the embryos to the recovery dish, tilt the injection plate to pull the embryos at the bottom, washing any embryos out of the trough, and using the plastic pipette to collect them. Allow the embryos to settle in the bottom of the pipette, then transfer the embryos to the recovery dish in a minimal amount of Tricaine. Incubate the recovery dish at 28 degrees Celsius for one hour, before separating the viable embryos from dead embryos and debris.

Incubate the dish at 33 degrees Celsius until ready for scoring. After anesthetizing a batch of embryos to be scored, according to the text protocol, place an anesthetized larva on a depression microscope slide in a drop of Tricaine. Orient larvae laterally for optimal imaging of the caudal region.

Place the slide on a compound fluorescence microscope with a 10X objective lens. Score the larvae by counting the number of cancer cells that have successfully invaded out of the vasculature by focusing up and down through the tail region to clearly discern intact cells. Melt 1.5%agarose Tricaine solution and bring it to 37 degrees Celsius.

After anesthetizing the embryos as before transfer an embryo in a drop of Tricaine solution to the imaging surface. Use a glass pipette to remove the excess Tricaine solution retaining the embryo on the imaging surface. Then overlay one drop of melted agarose solution over the embryo.

Working quickly, before the agarose polymerizes use a delicate tool like an eyelash brush to orient the embryo laterally for imaging, taking extra care to ensure the embryo is flattened along the imaging surface. When the agarose drop has polymerized, submerge it in Tricaine solution. Finally, subject the live zebrafish embryo to microscopic imaging.

Of the seven breast cancer cell lines tested for their vascular invasive ability in zebrafish embryos, the MDA-MB-231 cell line exhibited the greatest number of extravasated cancer cells per embryo when assessed, 96 hours after injection. It was also observed that BT-474 cells readily invaded into the caudal region of the embryos while other cell lines such as MCF-7, SK-BR-3, and T-47D cells were minimally invasive. In a modified asay that utilizes a different injection site, a competition experiment was carried out where two populations of MDA-MB-231 cells, that differ in their vascular invasive ability, were simultaneously injected into the yolk sac of developing embryos.

Upon scoring, it was determined that MDA-MB-231 cells propagated under confluent culture conditions prior to injection, exhibited a reduction in intravasation into the circulation, and therefore, had a reduced presence in the caudal region of the embryos. Once mastered, the zebrafish embryo injections can be done in four hours if performed properly. While attempting this procedure, it's important to remember to employ rigorous and consistent criteria when scoring cancer cell extravasation.

Following this procedure, results can be further validated in a murine model. After watching this video, you should have a good understanding of how to perform the injections and score extravasation, allowing one to test the vascular invasive ability of cancer cells in zebrafish embryos.