Zebrafish: Cell metastasis is the major cause of death in cancer patients. Our understanding of how metastatic cells invade tissues and gain access the blood system has been severally limited due to the inability to image these dynamic processes in high resolution in vivo. Therefore, much of our knowledge of metastasis has been obtained from static fixed specimens of advance-stage tumors. Consequently, many fundamental questions remain has to how cells metastasize in time and space. To this end, our lab has developed a new animal model that combines the optical clarity and power of zebrafish genetics with high resolution confocal microscopy and GFP technology. We have devised a method for growing highly metastatic human cancer cells in optically translucent green vascular zebrafish (Tg (fli1:egfp)) (see figures and movies below). This unique animal system provides a visual window into the metastastic process in a live vertebrate animal, with unprecedented clarity (see related information). Using this model system and the power of zebrafish transgenic technology, we are exploring the mechanisms of how metastatic cancer cells invade through living tissues, home to and remodel blood vessels, and intravasate through the vessel wall into the vascular system. We are currently investigating how the activation of the metastatic genes src and RhoC mediate this process in conjunction with the angiogenic and vascular permeability factor VEGF using intravital confocal and 2-photon microscopy.

Figure 1: Schematic showing the experimental design for growing human cancer cells in zebrafish for high resolution confocal and 2-photon imaging. Control MDA-435 adenocarcinoma cells stably expressing CFP (blue) and highly metastatic MDA-435 cells expressing the metastic gene RhoC and the marker protein DsRed were injected into the peritoneal cavity of immunosuppressed optically transparent Tg(fli1:egfp) transgenic vascular zebrafish. Mosiac tumors rapidly develop in the abdominal wall and can be directly visualized daily in live animals using confocal or 2-photon microscopy. This model of early cancer progression provides an optical window for the visualization of microtumor formation, tissue invasion, tumor-induced angiogenesis, intravasation, and extravasation of metastatic cells with unprecedented clarity. This animal model is also cost effective and readily amenable to pharmacological testing of therapeutic agents.

Figure 2. Low magnification, dual color confocal image of a microtumor (human MDA-435 adenocarcinoma cells) developing in Tg(fli1:egfp) transgenic vascular zebrafish. The tumor cells (red) are growing in the body wall between the intersegmental vessels (green). 10X magnification.

Figure 3. High magnification, three color confocal image of a mosaic tumor (MDA-435 cells with (red) or without (blue) the metastatic gene RhoC) developing in Tg(fli1:egfp). The tumor cells are growing in the body wall in close association with the intersegmental vessels (green). 40X magnification.

Figure 4. High magnification 3D reconstruction of intravasating MDA-435 cell. MDA-435 cells (red, labeled with DsRed ) were engineered to overexpress metastatic gene RhoC and human Vascular Endothelial Growth Factor (VEGF). Cells were injected into the peritoneal cavity of Tg(fli1:EGFP) zebrafish and imaged 2-5 days post injection. Fish blood vessel is shown in green (labeled with GFP). A representative image of intravasating MDARhoCVEGF cell is shown. 120x magnification.

Figure 5. High magnification 3D reconstruction of human tumor cells invading within the zebrafish tissue. MDA-435 cells that were mock transfected (blue) or engineered to overexpress metastatic gene RhoC (red, labeled with DsRed) were injected into the peritoneal cavity of Tg(fli1:EGFP) zebrafish and imaged 48h post injection. Fish blood vessel is shown in green (labeled with GFP). Note that RhoC overexpressing cells have amoeboid morphology while control cells have elongated mesenchymal morphology. 100X magnification.