Most cancers become fatal when cells break away from the primary tumor and travel through the bloodstream to other parts of the body, where they settle and divide once again. Pharmaceutical companies use 2-D assays to investigate drugs that can combat cancer cell migration. In these assays, researchers have found that the cells move across the surface of a matrix in a single plane using counteracting tractile and adhesion forces. However, 3-D confocal images now have shown that something different might be happening. Muhammad H. Zaman and his colleagues, working at Whitehead Institute for Biomedical Research in Cambridge, Mass., believed that investigations on artificial 2-D plates might not provide all the crucial information about cell motility. According to Zaman, understanding how cancer cells migrate through the bloodstream will enable researchers to determine which individual receptors are responsible for the cancer cells’ increased migration. He believes that they can target those receptors and find a way to control metastasis. Zaman and his team decided to look at the trends of prostate cancer cells’ movement in the 3-D environment of gels derived from mouse sarcomas. Because cancer cells use clawlike proteins called integrins to pull themselves forward through their surrounding matrix, Zaman disabled some of these integrins to see what the 3-D imaging would reveal. Researchers have discovered that 3-D imaging reveals information about a cancer cell’s motility that 2-D imaging does not. The confocal image on the left shows a prostate cancer cell in 3-D with all of its integrins intact and expressing. The right 3-D image shows the change in a cancer cell’s shape when some of its integrins are blocked, which means that the cell’s ability to migrate also is different. Images reprinted with permission of PNAS. They labeled the cells with a green tracker dye from Molecular Probes (now Invitrogen Corp.) and embedded them inside the gel to mimic a tissue environment. After the gel polymerized, they imaged the cells with a confocal microscope — from PerkinElmer Inc. of Wellesley, Mass. — with a spinning disk at 25x and 40x magnifications. Zaman explained that only a confocal instrument could provide high-resolution imaging that would allow them to look at the details of one cell at a time as well as of a population of cancer cells over a long period. Bright-field microscopy would only enable them to image the whole field at once, resulting in photobleaching after just a few hours. The researchers collected images every 15 minutes for six hours, with each image consisting of a 100-μm Z-stack at 0.5-μm intervals. They also collected 2-D data on all of the same cancer cells, so that they could provide a good comparison with their 3-D results. Zaman said that this was perhaps the most challenging part of the experiment, as it took them a long time to acquire both 2- and 3-D data. He had been working simultaneously on a mathematical model that would help explain how drugs influence metastasis. The model predicted that the density and stiffness of the extracellular matrix would significantly affect a cancer cell’s motility. The imaging revealed contrasting results to the well-established findings from 2-D assays. Instead of compensating for a decrease in the number of integrins by binding in areas of higher matrix densities (as found in 2-D systems), the cells in 3-D appeared to find areas that were less dense. They also took on a different shape as they moved through pores of varying sizes. Zaman believes that their findings help explain why 2-D assays for metastasis-inhibiting drugs do not effectively predict their effects in tissue. He hopes to see 3-D assays, accompanied by appropriate computational models, used to predict how drugs will affect metastasis. Now at the University of Texas in Austin, Zaman plans to continue investigating how the cell attaches to the matrix and, specifically, to find which receptors are responsible for cancer cell migration. He also would like to study cancer cells that come directly from the patient, instead of those that have been being transformed to cell culture. PNAS, July 18, 2006, pp. 10889-10894.