The overall goal of this microinjection of human breast cancer cells into embryonic zebrafish is to investigate the invasive behavior and assess the metastatic potential of human breast cancer cells. This method can help to answer key questions regarding intravasation and extravasation to critical processes in cancer metastasis. The main advantage of this technique is that it provides an elegant, rapid, and cheap in vivo model to measure the ability of cancer cells to invade and metastasize.
The implications of this technique extend towards the development of new pharmacological compounds with anti-metastatic activity. Though this measure can provide insight into cancer emission and the metastasis, it can also be applied to study some other phenomena, such as peritumoral angiogenesis and cancer cell dormancy. To begin, culture transduced human breast cancer MDA-MB-231 cells, breast epithelial MCF10A cells, and breast epithelial MCF10A-Ras cells in a T75 flask.
Once the transduced cells reach 80%confluence, remove the medium from the T75 flask, wash the cells with phosphate-buffered saline, and detach the cells with 0.5%Trypsin-EDTA solution. Then neutralize the Trypsin with medium. Next, centrifuge the cells at 1, 000 RPM for five minutes to remove the medium, wash them with PBS, and centrifuge the sample one more time.
After washing, resuspend the cells in approximately 200 microliters of PBS to obtain a suspension of no more than 1 x 10 to the eighth cells per milliliter. Store the cell suspension in four degrees Celsius for no longer than five hours. To prepare zebrafish embryos for implantation, incubate approximately 60 embryos in a petri dish filled with egg water, supplemented with 60 micrograms per milliliter of sea salts at 28 degrees Celsius.
Then, using fine tweezers, remove the coriands from the embryos at 48 hours post-fertilization. To anesthetize the embryos for the implantation procedure, transfer them to a 40 microgram per milliliter solution of tricaine in egg water. Prior to the implantation, prepare injection needles.
To do this, load a borosilicate microcapillary tube into a micro-pipette puller and pull the needle. Store the prepared needles in a needle-holder plate until needed. Aspirate 15 microliters of the prepared cell suspension into an injection needle.
Next, attach the needle to a micromanipulator, and, using fine tweezers, break its tip to five to 10 microns. Adjust the pneumatic PICO pump and inject cells onto the petri dish pre-coated with sterile 1%agarose. Then, transfer approximately 10 anesthetized embryos at two to three days post-fertilization onto a flat injecting plate coated with 1%agarose.
Arrange the position of the embryos with a hair loop tool so that they are oriented in the same direction. While injecting, manually adjust the injection plate position to place the embryos in a diagonal orientation, which is preferred for a precise needle insertion. Direct the needle tip at the injection site and gently insert it into the perivitelline space localized between the yolk sac and the paraderm of the zebrafish embryo.
Lastly, inject approximately 400 tumor cells labeled with mCherry. Make sure not to rupture the yolk sac to avoid a misplaced implantation. To inject the cells into the duct of Cuvier, approach the vein from the dorsal side of the embryo with the needle kept at a 45-degree angle.
Insert the needle at the starting point of the duct of Cuvier, localized dorsally to the point where the duct starts broadening over the yolk sac. Then, inject approximately 400 cells. Finally, transfer the injected zebrafish embryos to the egg water, and incubate them at 33 degrees Celsius until further analysis.
To visualize metastasis, with a Pasteur pipette, transfer the injected zebrafish embryo to a glass-bottom polystyrene dish and remove excess egg water. Image the whole body of the embryo with a confocal microscope set to low magnification to obtain a general pattern of tumor cell dissemination. Use a 488 nanometer laser to visualize the zebrafish embryo vasculature, and a 543 nanometer laser to visualize implanted tumor cells labeled with red fluorescent marker.
Next, scan the embryo in eight to 10 steps to acquire a high-quality image. Presented here are photographs of zebrafish embryos that were taken with a confocal microscope three days after perivitelline injection of MDA-MB-231 cells. MDA-MB-231 cells widely disseminated within the zebrafish body, manifesting highly invasive potential.
MCF10A-Ras cells, when implanted to zebrafish embryos, revealed moderate invasive capacity with only a few cells spread to distant parts of the body. Upon transplantation, MCF10A cells remained non-invasive and concentrated within the injection site, confirming that all studied cells reveal different invasive potential. The highly invasive character of MDA-MB-231 cells was reflected in their enhanced migration capacity observed within the perivitelline space.
To some extent, such migration was also observed for MCF10A-Ras cells. On the contrary, MCF10A cells did not migrate in the perivitelline space. Distinct metastatic behavior of MDA-MB-231 and MCF10A-Ras cells was also observed when both cell types were implanted into the embryos'duct of Cuvier.
As shown, MDA-MB-231 cells migrated independently of one another, while MCF10A-Ras were prone to migrate in clusters. Following this procedure, other measures like immunofluorescence can be performed in order to answer additional questions, such as how cancer cells proliferate in the zebrafish, and how they affect the surrounding host of cells. After watching this video, you will have a good understanding of how to use zebrafish model to study an invasive behavior and assize intravasation and extravasation capacity of human breast cancer cells.
