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In many ways, today’s major advances in cancer research revolve around identification and specificity. Various techniques—from labeling molecules to imaging them—help scientists identify specific kinds of cancer cells and molecules related to the progression of cancer. In addition, advancing molecular techniques make it possible to identify these cancerous components more specifically than ever.

When asked about the most important obstacles in cancer research today, Stefan Seeger, professor at the University of Zurich, Switzerland and founder of Molecular Machines & Industries, says, “I think the variability of cancer and the modification of it over time is a really big problem.” He adds, “It’s not enough to find a diagnostic marker or therapy that can be applied in general.” Instead, scientists will probably need to find ways to specifically treat individuals, but that is complicated and expensive.

Seeing Is Believing
Whatever form of cancer is being studied or treated, investigators usually want to see the diseased tissue. That image often comes from light microscopy. Many manufacturers—including Carl Zeiss, Leica, Nikon, and Olympus—have been designing and producing light microscopes for both research and clinical use for many years.

Stephen Ross, senior scientist and manager of products and technology at Nikon Instruments, says that cancer research faces two technological obstacles. One is providing longterm viewing of live cells. “This requires very stable systems,” Ross says, “so that scientists can do these experiments without worrying about focus drift or environmental problems in keeping cells alive for long periods.” Second, Ross says that high-content screening and other techniques provide so much data that bioinformatics becomes a problem.

In late January 2005, Ross attended the Photonics West show in San Jose, California. From that experience he says, “Hyperspectral imaging was a hot topic for elucidating cancerous from healthy tissue.” This technique essentially builds up an image from light across a wide range of frequencies. Ross and his colleagues recently released Nikon’s first hyperspectral, confocal microscope, called the Nikon C1si.

To help investigators keep specimens in focus for long periods, Nikon also developed an autofocus technique that uses infrared light and a feedback system. Ross says, “As long as the cells’ environment stays healthy, this technique can keep a structure in focus for days and days.”

Getting the Right Sample
Studying cancer also depends on getting access to the right tissue or even single cells. Then, scientists can better study the process of cancer and ways to treat it. In some cases, scientists can simply order the tissue. In other cases, they must isolate it themselves.

Asterand, for example, offers human tissue that can be used in cancer research. A sample comes with clinical data, including a final pathology report in many cases. This company also makes tissue microarrays—essentially grids with tissue samples at specific x-y locations—that can be made from its tissue bank or other samples.

Sometimes, though, a scientist wants to obtain a microscopic sample from a specific tissue. That can be done—and under sterile conditions—with mmi CellCut, a laser microdissection instrument from Molecular Machines & Industries. With this system, a scientist can put a tissue sample under a microscope, view it on a monitor, and select an area to remove. An ultraviolet laser cuts out the sample. Also living cells can be isolated from culture after growing in a chamber. Seeger says, “You can isolate tiny compartments of even single cells from a sample with very high precision.”

This technology can also be combined with molecular methods to extract DNA or RNA from a sample. “With amplification,” Seeger says, “you can analyze the genome or expression profile in a single cell.”

Attacking Angiogenesis
In other cases, cancer researchers want to study larger systems, such as the growth of cancer. Growth or repair of any tissue can only be sustained by building new blood vessels, which is a process called angiogenesis. Cancer cells can produce molecules that activate blood vessel formation, including vascular endothelial growth factor (VEGF), transforming growth factor-κ (TGF-κ), and others. If investigators can learn to block angiogenesis in cancerous tissue, without harming the process in healthy tissue, it could be an effective treatment. Growth factors and related products can be obtained from many companies, including Chemicon, ReliaTech, R&D Systems, and Sigma-Aldrich.

Frank Mortari, director of flow cytometry and the immunohistochemistry department at R&D Systems, says, “The struggle had largely been trying to balance between the benefits and detriments of controlling vascularization of tumors.” Many cancer therapies—such as chemotherapy—rely on blood vessels leading to and infiltrating tumors, but cancer researchers also want to reduce a tumor’s ability to develop blood vessels that support the growth of the disease.

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Richard Krzyzek, head of molecular biology at R&D Systems, says, “VEGF is one of the most potent angiogenesis factors, and we supply a large number of antibodies and recombinant proteins to VEGF family ligands and receptors.” He adds, “We also make ELISA [enzyme linked immunosorbent assay] kits for measuring soluble receptors and ligands.” R&D Systems also sells an ELISA kit that measures the tyrosine phosphorylation state of VEGF R2 and phosphospecific antibodies that recognize phosphorylated tyrosine sites on VEGF R2. In addition, R&D Systems recently introduced the Proteome Profiler Human Phospho-RTK Array, which can profile the tyrosine phosphorylation status of 42 different receptor tyrosine kinases, including many other receptors that are involved in angiogenesis.

Stimulating Cell Death
In development and maintenance of an organism, the death of cells is just as important as the creation of new cells. (For more on aging see the accompanying Keeping Up with Aging.) For example, programmed cell death—also known as apoptosis—plays a crucial role in the development of the brain. Some investigators also suspect that cancer might be killed if the process of apoptosis can be directed at diseased tissue. One way to do that is with tumor necrosis factor (TNF)-Related Apoptosis Inducing Ligand, or TRAIL. This transmembrane protein forms trimers that can interact with two receptors, TRAIL-R1 and TRAIL-R2, and that connection triggers apoptosis. Most important, TRAIL-stimulated apoptosis kills a broad range of tumor cells but seems to bypass most healthy cells. As a result of that, a variety of companies—including Alexis Biochemicals, R&D Systems, and STI Signal Transduction Products—offer TRAIL and related products.

Scientists at R&D Systems developed a wide range of products related to TRAIL. For instance, Mortari points out that TRAIL interacts with at least four different receptors—two that actually get activated and two decoys—and R&D Systems offers soluble versions of all four receptors and TRAIL, plus antibodies to these proteins.

Other molecules—including caspases—also participate in apoptosis. Michelle Moore, application and technical service consultant at Roche Applied Science, says, “There are more than 20 caspases involved in apoptosis at some point.” These proteases can turn on or off steps in apoptosis. Cytokeratin 18 is one of the first molecules cleaved in the cascade that drives apoptosis. To help investigators track this early apoptotic event, Roche provides its M30 CytoDeath antibody. Moore states, “M30 detects the cleavage products of cytokeratin 18 in cells and tissue of tumor or epithelial origin where the cytokeratin molecule is freely expressed.” In addition, this antibody can be used with flow cytometry to visualize apoptotic specific substrate cleavage. Moore adds, “The M30 antibody can also be used with paraffin embedded or frozen tissue sections. The immunoreactivity of M30 CytoDeath antibody stain is confined to the cytoplasm of the apoptotic cell. Therefore, it can be used in combination with immunohistochemical counter staining and other nuclear staining techniques.”

Moore also points out that Roche makes other products that can help cancer researchers. For example, she says, “Our Cell Death Detection ELISA can differentiate between apoptosis, necrosis, and healthy cells in one assay by analyzing the relative distribution of DNA fragmentation.”

Nuclear Warfare
Short of cell death, inflammation might also tell researchers something about cancer, because of nuclear factor-κB (NF-κB) proteins. Jeff Till, scientific director of phosphorylation at Upstate, says, “There is an apparent link between cancer and inflammation, and researchers have identified the molecular and cellular hallmarks of inflammation at the site of tumors.” That made scientists wonder if the family of NF-κB proteins—all of which are transcription factors—might be related to cancer. In addition, many studies show over expression of NF-κB proteins in leukemias, lymphomas, nonsmall cell lung carcinoma, and other cancers. Recent research shows an increase in pro-apoptotic factors in the absence of NF-κB activity. Many current antitumor therapies seek to block NF-κB activity—either directly or through upstream activators, such as the I-κ-B kinase—to inhibit tumor growth or sensitize tumor cells to conventional therapies. Companies like Active Motif, Chemicon, and Upstate offer these products.

According to Till, when NF-κB proteins are activated, they translocate from the cytoplasm to the nucleus. So Upstate provides reagents, such as antibodies, to help scientists monitor the localization of NF-κB proteins. “The antibodies allow you to visualize NF-κB proteins translocated to the nucleus using immunocytochemistry,” Till says. “Then, you can perform cell based assays with inhibitors to see if the compound prevents NF-κB proteins from getting to the nucleus.”

Controlling Kinases and Cytokines
In addition to proteins, environmental cues can trigger cancer. The flow of such external signals generally gets to the machinery of a cell through kinases. The signal reaches the outside of a cell, binds to a receptor, and then a cascade of phosphorylation events—all driven by kinases—carry the signal to subcellular compartments. Kinases are offered by many companies, including Alexis Biochemicals, Cell Signaling Technology, GloboZymes, and Stratagene.

Chris Bunker, director, new business development at Cell Signaling Technology, says, “Many of the kinases were initially described in energy and glucose metabolism, but many more act as agents involved in cells progressing along cancerous pathways.” The cancer connection arises, at least in part, because many kinases participate in growth regulation. In fact, many growth factors work through receptor tyrosine kinases. “These kinases can get turned on by point mutations or translocation events,” says Bunker, “and there is no way to shut off the growth factor pathway.”

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Scientists at Cell Signaling Technology develop antibodies that detect phosphorylation events. Bunker says, “These can be used to see if a kinase or pathway is active in a particular type of cancer.” In addition to phosphorylation-specific antibodies to monitor kinase activation, Cell Signaling Technology offers over 90 recombinant, human kinases. Bunker says, “These can be used for high throughput screening, to identify kinase inhibitors, and for lead optimization. Cell Signaling Technology has also developed the publicly accessible PhosphoSite Resource [http//:www.phosphosite.net], which provides a wealth of tools and information for kinase and protein phosphorylation research.”

Many cancer researchers also explore the application of cytokines, which are hormone-like proteins made by a variety of cells. For example, lymphocytes make cytokines called interleukins. According to Xin Xiao Zheng, assistant professor of medicine at Harvard Medical School, “In cancer, cytokines play two very important roles: they can activate the immune system against a tumor, and they can also induce a tumor.” Consequently, cytokines could be useful in many aspects of cancer research. For example, Terry B. Strom of Beth Israel Deaconess Medical Center and Zheng created a variety of immunoglobulin-based chimeric cytokine fusion proteins, which facilitate the study of cytokines in vivo by increasing the usually very short circulating half-lives of these proteins. Chimerigen licensed this technology.

As Zheng explains it, “We fuse a cytokine with the Fc, or constant, domain of an antibody. The cytokine domain of the fusion protein provides specificity.” In this fused molecule, a cytokine maintains its biological function for days or weeks in vivo. The cytokine mediates immune responses to attack the cell, or tumor. In addition, the Fc domain can target the cell or tumor recognized by the cytokine moiety of the fusion proteins.

Going with the Flow
With so many potential markers to follow, cancer researchers need high throughput techniques. One of the most reliable and fastest techniques for keeping track of cells and their function is flow cytometry. This technique can take measurements in many cells in a very short time and also profile a population of cells, based on one or more parameters of particular interest. Several companies including BD Biosciences, Beckman Coulter, and CompuCyte offer flow cytometers.

Kurtis R. Bray, director of research, development, and applications support at Beckman Coulter, says, “In flow cytometry, cells can be marked with antibodies or other labels, so that an investigator can perform functional and morphological analysis of cells, identify them, assess their activation state, and so on.” Bray adds that there have been recent advances in automation of flow cytometry and the reagents. For example, Tandem dyes allow half a dozen colors to be followed with a single laser.

A variety of Beckman Coulter tools can be applied to cancer research. For example, Bray says, “Our CMV [cytomegalovirus] specific MHC tetramers are a really new tool to monitor one of the chief complications of stem cell transplantation, which is used to treat many lymphoid cancers.” Cancer patients often become immunosuppressed during treatment, and can easily suffer reactivation of CMV. “With these MHC tetramers specific for CMV,” Bray says, “you can see which patients are at higher risk of a problem and which ones aren’t.” He adds,” We have a clinical trial under way that is determining how the CMV tetramers could be used clinically.”

Running Interference
A pathway from research to the clinic could also lie ahead for RNA interference (RNAi), which can be used to suppress a gene. Jie Kang, vice president of research and development at Qiagen, says, “In the old days, scientists used transgenic mice to understand how a gene works. Now, you can do it much faster and easier with RNAi.” She adds, “People can knock down every gene with this technique.”

Kang also points out that RNAi fits perfectly with cancer research. For example, Qiagen’s new HiPerFect transfection reagent works with a very low concentration of siRNA (small interfering RNA), routinely around 5 nanomolar, which is important to reduce side effects associated with RNAi. Kang says, “This reagent is quite robust with different cell lines, even difficult ones like breast cancer.” In addition, Qiagen offers siRNA for the entire human, mouse, and rat genomes.

A combination of tools—from antibodies and imaging to cytokines and siRNA—could be just what researchers and clinicians need to take a more personalized approach to fighting cancer. These tactics should eventually give investigators a better idea of how cancer works, from one person to the next. For the moment, cancer researchers aim at earlier diagnosis and more specific therapies. Those goals alone provide significant challenges, as well as powerful promise.

Mike May (mikemay1{at}verizon.net) is a communications consultant for science and technology based in Madison, Indiana, U.S.A. Gary Heebner
(gheebner{at}cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.