Fighting Cancer with Angiogenesis Inhibitors

From disdain to hype, to mixed results in clinical trials, a sobering reality is setting in for researchers pursuing antiangiogenesis as a treatment for cancer: It is not as straightforward as once hoped. The idea is that choking off a tumor's blood supply will slow or eliminate its growth. But several clinical trials following this line of inquiry have failed or been discontinued. This past February, for example, Sugen, a division of Peapack, NJ-based Pharmacia, announced it was aborting its Phase III colorectal cancer clinical trial of SU5416, as no clinical benefit was found. SU5416 is a small-molecule angiogenesis inhibitor that blocks the action of vascular endothelial growth factor receptor (VEGF-R) in endothelial cells, which is critical for new blood vessel formation.1 Earlier this year, Joanne Yu and colleagues at the Sunnybrook and Women's College Health Sciences Centre in Toronto provided a possible, partial explanation for the disappointing clinical results of some antiangiogenic therapies.2 They demonstrated in mice that tumors with inactive p53 tumor suppressor genes, which account for 50% of human cancers, are relatively resistant to hypoxia. Cutting off the blood supply to such tumors, therefore, is of little clinical benefit. Yet some clinical trials have produced promising results. In February, Judah Folkman's group at Children's Hospital in Boston reported successful treatment of two cases of angioblastoma, a rare and destructive pediatric tumor, using interferon k2b, which inhibits the angiogenic protein basic fibroblast growth factor (bFGF).3 And last October, the National Cancer Institute (NCI) stopped recruiting patients into its Phase II trial of South San Francisco, Calif.-based Genentech's Avastin in metastatic renal cell carcinoma because it had already "reached its prespecified efficacy endpoint at the second interim analysis." Deeper Thinking The antiangiogenesis approach to controlling cancer can succeed only if scientists deepen their understanding of angiogenesis and discover new compounds to modulate it. "Angiogenesis is a complicated process, and it's not governed by just one growth factor. If you just target one of those proteins, the tumor figures out a way to regrow," says William Figg, chief, molecular and clinical pharmacology section at NCI. Figg specializes in screening for new and innovative angiogenesis inhibitors that will function effectively in patients and not just in the lab. "I'm always a believer that better drugs will give better benefit," he says. "There's still plenty of opportunity to develop drugs with better pharmacology." Pharmaceutical companies typically depend on high-throughput screening to rapidly and cost-effectively screen their vast chemical libraries using robots and 96- or 384-well microtiter plates. But, due to the complexity of angiogenesis, high-throughput screening can be difficult. Instead, the most effective methods for testing angiogenesis inhibitor candidates are both labor intensive and difficult to quantify. "In vitro tests in general have been limited by the availability of suitable sources for endothelial cells, while in vivo assays [are] difficult to quantitate [and] limited in feasibility, and the test sites are not typical of the in vivo reality," reports Robert Auerbach, Harold R. Wolfe Professor Emeritus of zoology, the University of Wisconsin, Madison. He speaks from direct experience, having developed three of the most widely used angiogenesis assays. Despite the difficulties, however, NCI's Figg says, "We can screen hundreds a year in our lab. But we have to have a rationale for screening a compound. We look at a compound's structure and see if side chains match known antiangiogenic compounds." Angiogenesis Assays Among the simplest antiangiogenic screening assays are endothelial cell assays, which look at proliferation, migration, and tube formation as indicators of a test compound's antiangiogenic potential.4 Cell proliferation assays use cell counting, staining, and thymidine incorporation to measure endothelial cell growth or death. The endothelial cell migration assay, which is amenable to a moderately high-throughput approach, measures the ability of cells to cross a synthetic barrier. Bedford, Mass.-based BD Biosciences' BD BioCoat™ Angiogenesis System for Endothelial Cell Invasion,5 for example, uses fluorometric detection of migration to measure endothelial cells invading a Matrigel™ barrier in the presence of angiogenesis inhibitor candidates. Finally, the tube-formation assay scores the ability of endothelial cells to produce tube-like structures. Several problems bedevil endothelial cell approaches, says Auerbach. First, because it is difficult to obtain adequate numbers of endothelial cells from primary isolates to conduct a significant number of experiments, most research depends on cells grown in the lab. Such cells can acquire altered growth properties and activation states after prolonged culture. Also, endothelial cells can produce widely varying responses to angiogenesis modulators. Another confounding factor is the sensitivity of cultured endothelial cells to slight changes in culture conditions, resulting in cell loss. Of the various cell culture models, Auerbach favors the tube-formation assays, calling them "the most faithful cell culture methods for angiogenesis."6 The aortic ring assay is another widely used in vitro assay. Rat aortas are sliced crosswise and placed in Matrigel. After three to five days, microvessel sprouting can be roughly quantified by manual counting or by using image analysis. The aortic ring assay is low-cost, easy to use, and offers many assays from a single animal, but it can be difficult to quantitate. A relatively new variant uses embryonic chick aortic arches.7 The human saphenous vein, which runs from the groin to the ankle and is routinely harvested for coronary bypass surgery, is an excellent ex vivo model for angiogenesis. The vein is sectioned in 2-mm thick cross-sections and embedded into 12-well plates coated with Matrigel. The rings are cultured for 14 days before growth analysis. The limitations to this model, according to NCI's Figg, are the amount of tissue available and donor-to-donor variation. The mouse corneal angiogenesis assay offers a vascularization model in a normally avascular tissue.8 Pellets are inserted into surgically created micropockets in the cornea. This model has the distinct advantage of measuring only new vessel growth, which can be easily visualized. But because the cornea is normally avascular, it may not be an ideal testbed, as it lacks many of the blood-borne substances that modulate endothelial cell growth. Finally, in the chick embryo chorioallantonic membrane (CAM) assay, a window is cut into the egg shell, exposing the vessels below.9 Alternatively, the embryo can be transferred to a petri dish, where the CAM grows as a flat membrane, thus allowing several test sites. This assay's caveats include critical timing for observing effects, confounding inflammatory and vasodilation effects, and an inability to assess drugs requiring metabolic activation. Introspection Researchers are not confining their search for new angiogenesis inhibitors to the world at large; they are also looking to the body within. "There may be a bag of inhibitors in our body, a whole bunch of them, maybe 25 or 30, and they collectively do this job," says Raghu Kalluri, associate professor of medicine at Harvard Medical School. "The question is, can they do this job against an enemy that's too strong?" What the body may need is a massive influx of inhibitors to counteract the pathological levels of angiogens that tumors release. And different inhibitors may be needed at varying concentrations to thwart the tumor's adaptation to the counterassault. Kalluri recently identified one new inhibitor, Tumstatin, a 28-kDa fragment of type IV collagen, which binds to the kVb3 integrin molecule and halts its ability to accelerate blood vessel formation.10 Harvard transferred the Tumstatin technology to ILEX Oncology, headquartered in San Antonio, Texas, for drug development and clinical trials. According to Surender Kharbanda, ILEX's senior director of research, the company has identified a single 27-amino-acid peptide that retains all of Tumstatin's antiangiogenic activity. ILEX is running this fragment through a battery of xenograft models, in which human tumor cells are implanted into mice. So far, the peptide appears to actively suppress tumor growth, Kharbanda says. It is clear from early failures of many antiangiogenic substances in clinical trials that more precise preclinical compound screening will be required to make this approach a real force in cancer care. Increasingly, clinicians are combining antiangiogenics with standard radiation and chemotherapy protocols to achieve results. To that end, the NCI is sponsoring a conference in San Diego in November to review and standardize angiogenesis assays. With standardization, perhaps the next round of angiogenesis inhibitors will finally deliver the benefits promised by earlier research. Scott Veggeberg (sveggeberg@attbi.com) is a freelance writer in Winchester, Mass. References 1. H. Black, "The goal: Control blood vessel development," The Scientist, 16[6]:31, March 18, 2002. 2. J.L. Yu et al., "Effect of p53 status on tumor response to antiangiogenic therapy," Science, 295:1526-8, Feb. 22, 2002. 3. J.J. Marler et al, "Successful antiangiogenic therapy of giant cell angioblastoma with interferon alfa 2b: Report of two cases," Pediatrics, 109:E37, February 2002. 4. E. A. Kruger et al., "Approaches to preclinical screening of antiangiogenic agents," Seminars in Oncology, 28:570-6, December 2001. 5. W. Gloffke, "Counting endothelial cell invasion," The Scientist, 15[22]:26, Nov. 12, 2001. 6. R. Auerbach et al., "Angiogenesis assays: Problems and pitfalls," Cancer and Metastasis Reviews, 19:167-72, 2000. 7. V. Muthukkaruppan et al., "The chick embryo aortic arch assay: A new, rapid, quantifiable in vitro method for testing the efficacy of angiogenic and anti-angiogenic factors in a three-dimensional, serum-free organ culture system," Proceedings of the American Association for Cancer Research Annual Meeting, 2000. 8. V. R. Muthukkaruppan, R. Auerbach, "Angiogenesis in the mouse cornea," Science, 205:1416-8, 1979. 9. R. Auerbach et al., "A simple procedure for the long term cultivation of chicken embryos," Developmental Biology, 41:391-4, 1974. 10. Y. Maeshima et al., "Tumstatin, an endothelial cell- specific inhibitor of protein synthesis," Science, 295:140-3, Jan. 4, 2002.

Fighting Cancer with Angiogenesis Inhibitors

Interested in reading more?

Become a Member of