Tissue Factor in the Fight Against Tumors

By Jason Socrates Bardi

"The time has come in America when the same kind of concentrated effort that split the atom and took man to the moon should be turned toward conquering this dread disease. Let us make a total national commitment to achieve this goal."

——Richard M. Nixon, discussing cancer in his 1971 State of the Union Address.

When U.S. President Richard Nixon declared war on cancer in the early 1970s, he was seeking to energize the public and the scientific community to tackle what was then one of the leading causes of death in the United States.

Nobody could have known in December 1971, when amidst great fanfare President Nixon signed the National Cancer Act, what it would take to win this war. Certainly nobody knew how long it would take. In fits of irrational exuberance that are perhaps common at the start of a war, some even predicted a quick victory—a cure for cancer in five years.

Much has been discovered and reported on cancer in the last 30-odd years —about its causes, prevention, detection, and treatment—but the battle lines are still drawn. And in the last three decades we have learned above all that cancer, like war, is hell.

Cancer is still one of the leading causes of death in the United States. It is the second leading cause of adult mortality and the leading cause of child mortality for children under the age of 15. According to statistics compiled by the National Institutes of Health, the overall cost of cancer was over $180 billion in the year 2000 alone, a figure that is dwarfed perhaps only by the human toll. One new cancer is diagnosed every 30 seconds in the United States, and every 90 seconds another American dies of cancer.

A Basic Approach to Killing Tumors

Many of our greatest successes in the struggle against cancer have come from basic research aimed at understanding the fundamental molecular and cell biology that produces the condition.

We have learned that cancer is not a single type, but rather over a hundred different errors in cells of various tissues caused by various sorts of mutations. Some mutations turn on or increase the activity of certain key genes, increasing the expression of metalloproteinases for instance; others downregulate them, shutting off production of receptor proteins. Common to tumor cells is their resistance to normal programmed cell death. Thus they continue to live and proliferate. After certain mutations occur, a cancer cell grows out of control, dividing over and over and forming a solid tumor—or, with leukemias, an every increasing number of circulating tumor cells in the blood and throughout the body. Tumors often damage the tissues where they are located and most metastasize and migrate locally and through the bloodstream—and these are the tumors that claim so many lives every year.

Whether the wish to arrive at a single cure for cancer will ever be fulfilled is doubtful. However, the basic science that has led to a current understanding of the common abnormalities of many different types of cancer in the last several decades has yielded a number of new and promising approaches to detection and treatment.

One novel approach, pioneered by scientists at The Scripps Research Institute (TSRI) and elsewhere, is to block the flow of blood to a tumor. For a tumor to grow, it requires access to growth factors, oxygen, and nutrients supplied through the bloodstream. Block the blood, the thinking goes, and you can asphyxiate and/or starve a tumor—like drying out a lake by diverting all its tributaries. There are a number of ways to do this— for instance, by inhibiting angiogenesis, the proliferation of blood vessels supplying a tumor or blocking the interactions of the required growth factors with tumor vessels.

TSRI Professor Thomas S. Edgington and members of his laboratory in the Department of Immunology have been working for several years on another strategy within this paradigm.

Basically, they are seeking to initiate thrombotic occlusion of the blood vessels in tumors, effectively blocking the local flow of blood. This produces a "gangrene" effect in the tumors. Starved of oxygen, the tumor cells undergo immediate asphyxiation and tumor cell death on a massive scale.

"You can actually watch the tumors die right in front of you," says Edgington, who has been refining the technique for a number of years.

Clearing the Cancer Through Blood Clots

Edgington's technique basically involves delivering molecules of tissue factor (TF) to tumor vascular endothelium cells, which line the blood vessels that carry the blood to the tumors. TF has the ability to initiate the formation of blood clots within the vessels—a process known as thrombosis. If released in the blood vessels of tumors, the clots interrupt the tumor's blood supply and lead to an "avalanche" of tumor cell death, as Edgington puts it.

The key is to target this "tumor vasculature" selectively. Since aberrant thrombosis causes both massive strokes and heart attacks, unleashing blood clots in a general way would be a highly dangerous approach to treating tumors—sort of like weeding a garden with napalm.

"The [tissue factor receptor] has to land on the precisely correct part of the tumor blood vessel cell surface," says Edgington. He compares this to trying to land an airplane on a narrow strip in a rainforest. The target is a tiny fraction of the total.

Edgington first encountered TF almost 20 years ago, when he was pioneering research into blood coagulation, thrombosis, and the connections between the immune system and the vasculature. In the process, he first cloned the gene for TF in 1987, and subsequently worked out the structure and how TF works.

TF is the primary molecule that initiates the cascade of reactions in thrombosis, which involves about 30 interacting proteins, and ultimately results in the processing of fibrinogen molecules in the bloodstream to form the sticky clot-forming fibrin.

As a cell surface receptor TF is highly efficient, binding to its target substrate with picomolar affinity. "One molecule of TF running 100 percent can produce in one minute over a billion molecules of fibrin," says Edgington.

Because of this efficiency, TF is effective in very small quantities. In fact, its concentration in tissues is estimated at only three parts per million or less. The search for the protein responsible for the function of TF was 50 years from first description to isolation and cloning.

"TF had been the missing element in the coagulation system," says Edgington. "On paper it had to exist, but nobody could isolate it."

In 1986, Edgington and associates were the first group to sequence TF and clone it after two years of dedicated effort. "We started in 1984, working full time, six to seven days a week," says Edgington.

They eventually succeeded in isolating the elusive TF molecule by reducing 500 fresh human placentas, which, taken together gave them enough protein to isolate the trace TF protein and with a new type of amino acid analyzer that he designed and built it was possible to determine the amino acid sequence of the minute amounts of TF that could be isolated.

In the years since, Edgington and Associate Professors Wolfram Ruf and Nigel Mackman have directed much of their efforts towards characterizing TF, its gene and its regulation, the protein's structure and mechanisms of action, and the complicated cascade of physiological reactions that TF directs in hemostasis, thrombosis, inflammation, certain immune reactions, and even in tumor biology.

Hitting the Target

In a paper published this month in the journal Cancer Research, Edgington and his colleagues report that they have found a way to deliver molecules such as TF to specifically target only those vessels that are supplying blood to tumors and leave the rest of the vasculature alone. To do this, they have employed a small part of a protein called vascular endothelial growth factor (VEGF), a growth factor that regulates the growth of new blood vessels.

Certain forms of VEGF have a particular stretch of amino acids, called the heparin binding domain, that when properly folded binds to a number of sugars decorating proteins on the surface of cells. And one of the sugars it binds to seems to be only on the surface of endothelial cells local to cancer tumors.

Edgington and his colleagues used a truncated 24-amino acid stretch of this heparin binding domain and showed that when injected into the blood stream it can find and anchor a viral phage particle to the blood vessels only of a tumor.

"This really shows that you can [use the truncated part of heparin binding domain to] deliver molecules or even particles selectively to the tumor vasculature and thus to a tumor," says Edgington.

Edgington attached the heparin binding domain to an additional copy of a phage gene that codes for a coat protein displayed on the surface of a phage particle—a virus that infects bacteria. Then he carefully controlled the number of this additional gene and its heparin binding element expressed on the surface of the phage so that of the 2,000-plus proteins on the surface of the phage only one to seven will have the heparin binding domain. This low copy number is important because Edgington wants to find a targeting molecule that could strongly anchor to the tumor vasculature.

In experiments described in the paper, Edgington and his colleagues injected the modified phage particles into an in vivo cancer model, a mouse with a large solid tumor. Normally the large phage particles will circulate through the bloodstream and their levels will drop as they are progressively cleared by the body. But if the phage binds tightly to some part of the body, like the cells lining tumor vasculature, then it will remain even after the rest of the phage is cleared from the bloodstream.

Edgington found that even a single molecule of heparin binding domain on the surface of the large phage particle will localize and anchor the phage. The concentration of phage in the tumor vessels increased as the levels of phage in the bloodstream dropped. By looking for those particular heparin–phage constructs that were present in the tumors when all the phage was washed out of the bloodstream, Edginton and his colleagues were able to identify the constructs that tightly bound to the tumors selectively. Then they could recover these phage particles for analysis.

"You can anchor the phage with only one copy [of heparin binding domain]," says Edgington, "but the protein must be specific for the tumor vasculature if you are to only recover it from tumor and no other tissues."

Edgington has several hopes for this technology. The ability of heparin binding domain to target tumors may be used as the basis of a diagnostic to image the tumor vasculature—a technology that could help surgeons see the exact size and locations of tumors that could then be surgically removed.

Also, the targeting potential of the heparin binding domain might be used to direct molecules like TF to the tumor vasculature, where they could block the flow of blood and kill tumor cells.

The beauty of Edgington's technique is that it targets those cells that line the vasculature, which means that any potential therapeutic that would be derived from it would have easy access to the targets. Unlike the tumor cells, which readily mutate to resist treatment, the endothelial cells are not prone to mutations and therefore represent a more stationary target common to all solid tumors independent of the type of cancer.


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Professor Thomas S. Edgington and members of his laboratory in the Department of Immunology have been working on blocking the flow of blood to a tumor. Photo by Biomedical Graphics.
























A Tumor Targeting Molecule.