It takes two to spread cancer, according to researchers from the University of California, San Diego, in La Jolla. They demonstrated that the vascular endothelial growth factor and the protein RhoC work together to allow cancer cells to enter blood vessels, initiating metastasis.The scientists used a combination of new and existing technology to study how these two biomolecules affect this intermediate stage of metastasis. The new tool was a transparent zebra fish, which allowed them to observe the behavior of cancer cells inside the organism.Transparent zebra fish allow researchers to follow processes occurring inside. In this case, human tumor cells secreting vascular endothelial growth factor cause an angiogenic response from the zebra fish blood vessels, which is rendered in this 3-D reconstruction. Tumor cells are red, thanks to DsRed fluorescent protein, while zebra fish blood vessels are green, as a result of enhanced GFP. Images courtesy of Konstantin Stoletov, University of California, San Diego. “The optical clarity of the tissue just makes a superior imaging system,” said Richard Klemke, a professor of pathology at the university and at the associated Moores Cancer Center. Klemke and postdoctoral fellow Konstantin Stoletov led the team.They used the existing technologies of high-resolution confocal microscopy and gene engineering, which let them produce zebra fish with tissues that expressed various fluorescent molecules.Klemke said that they developed the animal model and technique for tracking the intermediate metastasis process steps because of their frustration with other approaches. Previously, when trying to unravel how tumors enter the vascular system, the researchers could perform only end-point assays. Unfortunately, such studies do not reveal how a tumor cell in one part of the body spreads to another part.With transparent zebra fish, however, the researchers could image the process as it took place. First, though, they had to figure out how to immunosuppress the animals so that labeled human cancer cells could be xenografted into them. They were able to do so with the right drug protocol, a discovery that Klemke characterized as the breakthrough that allowed their studies to proceed. He added, however, that the protocol may not be needed in the future.“Excitingly, there are transgenic and mutant animals being developed that will be immune-compromised, including animals that are T-cell deficient,” he said. That, he added, would simplify the process and eliminate the possibility that the immune-suppressing drugs could affect the results.The researchers knew before they started that RhoC is linked to increased metastasis of various human cancers. They also knew that vascular endothelial growth factor is involved in the cancer spreading process, but it wasn’t clear how.They started by transfecting stable human cancer cell lines with a green, red or cyan fluorescent protein. They injected these into the immunosuppressed zebra fish, which had been transfected so that their blood systems expressed GFP. This gave the researchers an easy visual indicator of angiogenesis -- or blood vessel formation -- and a way to track the behavior and spread of cancer cells within the fish.During the study they kept the fish in small wells and imaged them with Bio-Rad or Nikon confocal microscopes. They used an excitation wavelength of 488 nm for GFP, 561 nm for the red fluorescent protein and 442 nm for the cyan fluorescent protein. When imaging, they acquired a three-dimensional data stack of 512 × 512 or 1024 × 1024 pixels in 0.5- to 2-μm vertical steps. They did this using 10×, 40× and 6× objectives.The researchers acquired images of the anesthetized fish sealed in a chamber, with data acquisition taking one to three minutes per step. The sedated animal could be imaged for up to four hours using standard confocal microscopy repeatedly, for many days, without a significant loss of the fish’s viability.Highly and minimally metastatic human tumor cells react differently within transparent zebra fish tissue in this 3-D reconstruction. Highly metastatic cells are labeled red with DsRed fluorescent protein, minimally metastatic cells are labeled blue with cyan fluorescent protein, and zebra fish blood vessels are green with enhanced GFP. When injected into the transparent zebra fish, the cell lines behaved as they do in humans. Those lines that are aggressive in humans tended to spread easily in the fish, while those that do not metastasize easily in humans tended to exist as tight cell clusters in the fish. On the other hand, nontumor cells displayed no tendency to invade other parts of the fish, and fluorescent 10-μm beads did not move.The researchers worked with a cell line that readily formed tumors but tended not to spread, with only about 20 percent showing nearby blood vessel remodeling four or so days after injection. They engineered some of the cells to secrete vascular endothelial growth factor at a rate of roughly 100 times as much as nonengineered cells do. When injected, the cells overexpressing the growth factor engaged in robust vessel remodeling. Quantitative analysis of the images showed a dramatic increase in the number of branch points, total vessel length and vessel diameter.Based upon the fact that the remodeled vasculature was highly permeable to red fluorescent dextran, whereas normal vessels were not, the researchers concluded that vascular endothelial growth factor induced remodeling and made the vessels permeable. However, they did not find evidence that the cells overexpressing the growth factor developed what are called invadopodia -- extrusions that allow tumor cells to penetrate the vessels and enter the bloodstream. The work was published in the Oct. 30 issue of PNAS. This 3-D reconstruction shows a single human tumor cell interacting with an angiogenic zebra fish blood vessel. Tumor cells are shown in red, and zebra fish blood vessels are shown in green. However, they discovered that amplifying RhoC did just that. Overexpressing the protein by five- to tenfold allowed cells to make specialized invadopodia and protrusions more easily. These cells then could penetrate the vascular openings present in the remodeled blood vessels.Hence, the investigators found that the combination of vascular endothelial growth factor and RhoC allowed the tumor cells to enter the blood vessels rapidly, or intravasate (one of the first steps in the metastatic cascade). Another step involves the cells exiting the blood vessels and growing in a new location. While studying the transparent animals, Klemke said he saw cells crawl out of the blood vessel and into the tissue. Preliminary studies of such extravasation using the animal model and microscopy imaging are encouraging, he reported.He added that the transparent zebra fish can be used in other important studies. In particular, to visualize what happens to a microtumor and vasculature when a cancer therapeutic is used, which makes it possible to gain detailed information about how drugs or combinations affect tumor development, data that otherwise would be difficult to obtain.“You can actually determine how they’re impacting the metastatic cascade and what step of this dynamic process they’re blocking,” Klemke said.