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Argonne technologies win R&D 100 Awards
Serendipity in lab turns blood into stem cells Cutting blood supply to tumors could stop them Argonne Update
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Cutting blood supply to
tumors could stop them
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Creating capillaries
Capillaries are made up of endothelial cells that form little hollow tubes. When a tumor lacks oxygen, it sends protein signals toward existing capillaries. The endothelial cells in those capillaries break off, releasing enzymes that allow them to chew a hole through body tissue. They crawl toward the tumor and make a new capillary. The exact formation process is still unclear.
"The process is four-dimensional," says Rodi. "It's not static. The endothelial cells start out in one place, move to another and have to find other endothelial cells to make capillary tubes."
 CAPILLARY STUDY — Colleen Kuemmel adjusts a light microscope to get a clearer view of endothelial cells four hours after adding growth factor. This image, shown on the screen in the background, and others provide a visual timeline of capillary growth. |
Rodi's team is using a petri-dish system to mimic natural capillary formation. Endothelial cells that have been isolated from human tissue and mixed with vascular-endothelial-cell growth factor, basic fibroblast growth factor and fetal-bovine serum are placed in a protein gel. Eventually the endothelial cells recognize the protein gel as body tissue and begin to release the enzymes that enable them to chew through the tissue.
To coax the cells to grow outside the body, the plated endothelial cells are kept in a carbon-dioxide filled incubator at 98.6 degrees Fahrenheit, body temperature. Cells are fed rich food containing glutamine, an essential amino acid. The food is supplemented with penicillin and streptomycin to keep bacterial contamination to a minimum.
Researchers monitor the process over the eight hours it takes the capillaries to form. They use a light microscope with a digital camera attachment to take snapshots every 30 minutes. At about 2 1/2 hours, cells crawl through the gel, find each other and begin building networks. At five hours, most cells are in networks and by eight to nine hours most cells have formed capillaries.
Every half hour, researchers isolate the cell's ribonucleic acid (RNA).
Working with the Functional Genomics Facility at the University of Chicago, Argonne's Angiogenesis Group uses microarray analysis to identify the isolated RNA and, since RNA codes for proteins, determine what proteins the cells are making at each time interval.
In microarray analysis, scientists apply a sample of the RNA to be identified to a microscope slide with thousands of samples of known RNA. Biologists identify the unknown RNA by observing which known RNA it interacts with.
Since proteins are responsible for the cellular structure and communications, blocking them could stop endothelial cells from forming capillaries.
Argonne's researchers have identified a list of 280 proteins produced in large quantities during capillary growth.
The microarray analysis revealed that some of these 280 proteins are usually seen only in brain cells and neurons. Neurons grow processes, or protrusions, by putting out little feet that attach to tissue proteins. The Angiogenesis Group plans to determine how closely capillary growth mimics that of neurons.
In addition to microarray analysis, the Angiogenesis Group is using bioinformatic analysis to learn more about the 280 proteins. This new technique combines biology and computer research. As biologists across the world learn about proteins, they share their knowledge in Internet-accessible databases. Argonne researchers are combing these sources to find out if the proteins on the list have been studied and what functions they perform.
Fighting cancer
Rodi's group, which includes colleagues Lee Makowski, Suneeta Mandava, Wen Zhang, Frank Collart and David Glesne, wants to identify which of the 280 proteins are involved only in capillary formation from endothelial cells. Identifying these proteins would be a key step toward developing drugs to knock them out and stop tumor metastisization.
To do this, Rodi plans on working with Glesne, a cell biologist. They will systematically knock out proteins during their in vitro experiments to find if any stop angiogenesis.
Eventually, Rodi says, the group plans to collaborate with crystallographers in the Biosciences Division.
This collaboration will enable them to crystallize the isolated proteins and examine them with the Western hemisphere's most powerful X-rays for research at Argonne's Advanced Photon Source. X-ray images provide 3-D views of the protein's structure and reveal the way it works.
For more information, please contact Catherine Foster (630/252-5580 or cfoster@anl.gov) at Argonne.
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