Researchers at the Massachusetts General Hospital (MGH) have discovered that a cellular growth factor called TGF-beta-1 has a previously unsuspected role in regulating the growth of blood vessels associated with metastatic gallbladder cancer. They also report finding that, in animal studies of the development of blood vessels (angiogenesis) associated with cancer, model systems in which tumor cells are grown in an unnatural location - for example, gallbladder cancer cells grown under the skin - may produce misleading results.
The study's results, appearing in the October issue of Nature Medicine, may someday lead to strategies to prevent the growth of metastases from gallbladder cancer, a serious problem in treatment of the disease. They also add further complexity to current knowledge about factors that regulate angiogenesis and their role in the growth and spread of cancer.
"We discovered that suppression of the growth of a secondary tumor [metastases] by antiangiogenic factors released by the primary tumor depended on the primary tumor's growing in the right environment," says Rakesh Jain, PhD, director of the Steele Laboratory for Tumor Biology at the MGH and the study's senior author. "That may have implications for the identification of anti-angiogenesis agents that may be useful for cancer treatment."
The research team focused on gallbladder cancer, which is diagnosed in more than 6,000 Americans each year, because of a frustrating property of the disease. As long as the primary tumor remains in the gallbladder, metastases - pockets of cancer that develop in other parts of the body - are few and very small. But soon after the primary tumor is surgically removed, metastases appear and grow quickly. As a result, only about 5 percent of patients having the surgery, which currently is the only treatment for the disease, survive for five years or more.
This apparent suppression of the growth of metastases by the presence of a primary tumor is seen in other types of cancer and suggested to researchers that primary tumors emitted some kind of antimetastatic signals. Work in animal models led by Judah Folkman, MD, of Children's Hospital and other researchers pointed to factors that suppress angiogenesis, the growth of blood vessels required to supply tumors, as being these antimetastatic agents. But exactly what factors were associated with spread of gallbladder cancer were unknown.
Takeshi Gohongi, MD, the study's first author, is a surgeon who treated patients with gallbladder cancer in his native Japan and is now a research fellow at the MGH Steele Laboratory. "This is a disease with a very poor prognosis, and basic research in the area is not well developed. I came to this country to work with Dr. Jain, who has been studying the physiology and vasculature [blood vessels] of tumors using specialized animal models."
In the first step of their study, the research team implanted human gallbladder carcinoma cells in either the gallbladder wall or a pocket under the skin (subcutaneous) of immunodeficient mice. The cells implanted in the gallbladder grew more rapidly, and no metastases were observed in either group of animals.
To measure suppression of angiogenesis at a secondary tumor site, the researchers implanted either gallbladder tumor cells or gels containing a known angiogenesis promoter (bFGF) into cranial windows - transparent compartments placed into the brain - in both groups of mice, as well as in a control group with no implanted tumors. In mice with tumors in their gallbladder, growth of blood vessels in the cranial windows was significantly less than seen in mice with subcutaneous tumors or with no tumors.
To identify which factors might be involved in the observed suppression of angiogenesis, the researchers analyzed blood levels of five factors known to either stimulate or inhibit angiogenesis. The only one showing a significant difference among the groups was TGF-beta-1, which was three times higher in the mice with tumors in their gallbladders than in the subcutaneous or control groups, suggesting that it was the factor suppressing angiogenesis in the secondary tumors.
"We were very surprised to find that TGF-beta-1 was the only growth factor produced in higher concentrations by the orthotopic [in the right place] tumors," says Jain. "This factor is what we call a pluripotent cytokine - it can have many different effects depending on the concentrations at which it is present, its location and other circumstances. Some studies have shown that it promotes angiogenesis. Finding that it has an opposite effect in this situation is provocative."
Jain adds that TGF-beta-1 suppressed both growth of the tumors in the cranial windows and the development of blood vessels in windows with the bFGF gels, confirming that it acted by suppressing angiogenesis and not just killing the cancer cells. To confirm that the observed activity was the result of TGF-beta-1 and not an unmeasured factor, the researchers added an antibody to TGF-beta-1, which successfully blocked angiogenesis suppression.
Finally the researchers studied samples of human gallbladder tumors from patients treated at the MGH and analyzed them for the presence of TGF-beta-1. In all samples studied, even though they were different grades of cancer, TGF-beta-1 was present. It also was found in metastatic cells found in the patients' lymph nodes but not in normal gallbladder tissue. "This finding is quite fascinating, because it shows this is not just something that occurs in animals. TGF-beta-1 is also present in human tumors," say Jain.
"Finding out that TGF-beta-1 is important in inhibiting angiogenesis in the spread of gallbladder cancer gives us an opportunity to develop postsurgical treatments," says Gohongi. "If we can learn how to modulate TGF-beta-1 levels after removal of the primary tumor, we may be able to prevent the rapid growth of metastases that proves fatal for so many of these patients."
Additional co-authors of the study are Dai Fukamura, MD, Yves Boucher, PhD, and Chae-Ok Yun, PhD, of the Steele Laboratory; Gerald Soff, MD, of Northwestern University; Carolyn Compton, MD, of the MGH Pathology Department; and Takeshi Todoroki, MD, of the University of Tsukuba in Japan. The research was supported by a grant from the National Cancer Institute.
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