Kerbel and Folkman have proposed a classification scheme that places agents that target tumor neovasculariza-tion into two classes. Direct inhibitors, such as endostatin, angiostatin, and vitaxin, function by preventing endothelial cell proliferation, migration, and initiation of antiapoptotic programs in response to various angiogenic proteins. Indi rect inhibitors, on the other hand, block production of protein products (bFGF, VEGF) from tumor cells or interfere with endothelial cell receptor tyrosine kinase signaling cascades. Perhaps one of the most valuable lessons learned from the preclinical evaluations of these agents is that their effectiveness at limiting tumor growth is much more pronounced if they are administered in combination with chemotherapy or radiotherapy.
One of the most active areas of antiangiogenic investigation has centered on the VEGF system. Although several of the small-molecule inhibitors of VEGFR2, such as SU5416 and SU6668, demonstrated efficacy in limiting human tumor xenograft growth in mice, these agents were found to produce unacceptable toxicity profiles in humans that has halted their development. Adverse side effects observed in patients receiving these agents have included pulmonary emboli, myocardial infarction, and cerebrovascular events. rhuMAB VEGF (avastin) is a recombinant humanized monoclonal antibody to VEGF that has undergone initial evaluation in the treatment in two different types of human tumors. In a Phase II study conducted with patients harboring stage IIIB/IV nonsmall-cell lung cancer, individuals were randomized to standard chemotherapy with carbo-platin and paclitaxel alone or in combination with either 7.5 or 15mg/kg rhuMAB. Although the response rates were slightly increased in the group receiving chemotherapy and high-dose rhuMAB, median survival was only improved by 3 months in this group. A randomized trial employing rhuMAB has also been completed in a group of 116 patients with metastatic clear-cell renal carcinoma . Interestingly, in this study design, patients were assigned to groups receiving placebo or 3 or 10mg/kg rhuMAB that was administered every 2 weeks. Clear-cell renal carcinoma is perhaps an ideal candidate for VEGF therapy in that the von Hippel-Lindau tumor suppressor gene is usually mutated in this cancer and, as a result, VEGF is overproduced because of a mechanism involving HIF1-a. Unfortunately, monotherapy with rhuMAB failed to demonstrate an increase in survival in this patient population.
Many preclinical studies are based on rapidly growing subcutaneous neoplasms. Moreover, treatment of human tumor xenograft models is usually initiated at early time points. This is in sharp contrast to the clinical setting, where patients usually present with advanced disease and are eligible to receive antiangiogenic therapies only after failing one or more conventional treatment modalities. In addition, because tumors are made up of heterogeneous populations of cells, treatment with only one angiogenic agent may result in the selective expansion of cells expressing different proangiogenic proteins.
Although there remain many unanswered questions regarding which angiogenic inhibitor is appropriate for a given tumor, there have been some recent advances in determining which signaling cascades are operative in some of the preferred sites of metastasis. The bone is the fourth most common site of tumor metastasis, and recent estimates predict that approximately 350,000 cancer patients die each year with evidence of tumor in the skeleton. Consequently, much effort has been extended toward identifying those components in the bone microenvironment that support tumor growth. Recently, members of our laboratory evaluated the efficacy of STI571, a small-molecule inhibitor of the PDGF-R tyrosine kinase, in a human tumor xenograft model of androgen-independent prostate cancer implanted into the bone . In that model, inhibition of PDGF-R activation with STI571, when administered in combination with taxotere, produced a profound reduction in both tumor mass and lymphatic metastases. It was determined that the effectiveness of the combination therapy was directly related to apoptosis of tumor-associated endothelial cells. The promising results obtained in this study have prompted the initiation of a clinical trial to evaluate the efficacy of this treatment regime in men with refractory prostate cancer bone metastasis. In addition, these findings may have important implications for other cancers that metastasize to bone such as renal, breast, and lung tumors. However, it will be necessary to determine whether these tumors are operating through a PDGF-dependent pathway. Indeed, a recent report has shown that renal cell carcinoma may preferentially exploit EGF signaling cascades in the bone microenvironment in order to promote their vascularization and ensure their growth .
Paracrine EGF signaling from tumor cells to microvas-cular endothelial cells has also been shown to be an important component for the growth of pancreatic metastases in the liver. Using either a monoclonal antibody blocking strategy (C225) or small molecule inhibitor of EGFR signaling in combination with gemcitabine, Bruns and coworkers were able to dramatically reduce both primary pancreatic tumor growth and liver metastatic burden. In both tumor models, combination therapy was associated with down-regulation of VEGF in the tumor region and apoptosis of tumor-associated endothelial cells. Independent studies have also identified VEGF as an important proangiogenic cytokine in the liver. Reports examining liver regeneration in rats following partial hepatectomy have shown that VEGF plays a critical role in the revascularization process by stimulating the proliferation of hepatic sinusoidal endothelial cells. VEGF also appears to play a role in colon cancer liver metastasis, as blockade of either VEGF or VEGFR2 in a human tumor xenograft model results in the induction of both tumor cell and endothelial cell apoptosis .
One of the most elusive organs for targeted treatment of metastasis is the brain. Cerebral blood vessels possess highly resistant tight junctions that are further reinforced by the end feet processes of astrocytes. In addition, this highly specialized vascular network is also enriched with a number of transport proteins, such as the multidrug-resistant protein P-glycoprotein, which restrict the passage of several chemotherapeutic agents into the tissue parenchyma. In fact, it has been shown that P-glycoprotein can also mediate the efflux of small-molecule inhibitors such as Glivec. Adding to the complexity of the cerebral vasculature, Fidler and colleagues demonstrated that neovascularization of metastases in the brain occurs by a mechanism that is distinct from traditional sprouting angiogenesis. In brain metastasis, blood vessel expansion occurs by the insertion of dividing endothelial cells into the preexisting blood vessel, a process that is referred to as angioectasia. In addition, because of the detrimental consequences that ensue upon cessation of cerebral blood flow, it is likely that there is considerable redundancy in angiogenic proteins in this anatomic compartment.
Although most of the examinations into the neovascularization of tumors have focused on angiogenesis, new evidence indicates that hematopoietic stem cells (HSCs) and endothelial precursor cells (EPCs) may also play an important role in this process. Asahara et al.  were the first to demonstrate the EPCs could be isolated from human peripheral blood and incorporate themselves into active areas of angiogenesis in animal models of ischemia. EPCs may be differentiated from mature circulating endothelial cells that have been shed from the vascular wall by their significantly enhanced capacity to proliferate, and also by their unique expression of cell-surface markers that include VEGFR2, AC133, CXCR4, and CD146. Recent studies are beginning to provide insight into the mechanisms that facilitate the mobilization of HSCs and EPCs from the bone marrow and to sites of neovascularization. The recruitment process appears to be initiated by angiogenic products that are released from tumor cells and lead to the activation and secretion of matrix metalloproteinase-9 (MMP-9) by hematopoietic and stromal elements in the bone marrow. MMP-9 activation, in turn, leads to liberation of soluble KIT ligand that promotes cell proliferation and also directs the transfer of these cells into the peripheral circulation.
Direct evidence that HSCs and EPCs contribute to tumor neovascularization has come from examinations conducted in mice that are deficient in Id proteins. Mutant mice with the Id1+/"Id3_/_ phenotype possess defective angiogenic responses and, thus, are unable to support tumor growth. However, when these mice are transplanted with wild-type bone marrow or HSCs, tumor growth in the subcutaneous compartment can be restored. This process appears to require cooperation of both VEGFR1 and VEGFR2 in that treatment of Id1+/"Id3_/" mice with neutralizing antibodies against both of these receptors results in vascular disruption and tumor cell death. The contribution of HSCs and EPCs to the tumor vasculature appears to be influenced by the tumor type, but most reports suggest that these cells represent a small fraction (6 to 10%) of tumor-associated blood vessels, and that they arrive at tumor vessels during the initial phase of malignant growth. Despite the relatively low number of progenitor cells that lend support to tumor vessels, evidence suggest that their ability to traffic to tumor sites may be exploited for therapeutic targeting. Genetically modified endothelial progenitors that were stably transfected with thymidine kinase or the soluble truncated form of VEGFR2 have been shown to home to subcutaneous tumors and significantly impair tumor growth. Although these initial reports are promising, more studies are needed to determine which types of malignant lesions are critically dependent upon progenitor cells.
Over the past decade, the understanding of the cellular and molecular mechanisms that tumors utilize to promote their blood delivery and facilitate their growth has advanced. However, therapies that are directed toward the vascular compartment of malignant lesions are still in the early developmental phase. Continued investigations into the specific factors that regulate angiogenesis in different organs, coupled with detailed characterization of the products released by tumors in different tissues, should improve targeted therapy of angiogenesis. Similarly, an enhanced understanding of the biology of HSCs and EPCs could provide clinicians with a superior method of delivering therapy to tumors. The identification of tissue-specific receptors, on both normal and tumor-associated endothelial cells, will also enhance the development of vascular-based therapies. Employing orthotopic tumor models that more closely approximate the clinical reality could well facilitate translation of laboratory findings to the clinic.
Angioectasia: Blood vessel dilation that is due to endothelial cell division occurring within the wall of the blood vessel. This process is distinct from angiogenesis and has been reported in brain metastasis.
Angiogenesis: Refers to the generation of new vascular networks from preexisting blood vessels. During this process, endothelial cells elaborate proteolytic enzymes, exhibit migratory behavior, and undergo cell division. Ultimately, developing capillary sprouts will coalesce to form new vascular structures.
Endothelium: Specialized simple squamous epithelium that lines the intimal surface of all blood vessels.
The author thanks Dr. I. J. Fidler for helpful discussions and editorial assistance.
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Robert R. Langley received his Ph.D. from the Department of Molecular and Cellular Physiology, Louisiana Health Sciences Center, Shreveport, Louisiana, in 1999. He received postdoctoral training in the Department of Cancer Biology at the University of Texas M.D. Anderson Cancer Center in Houston, Texas, from 2000 to 2003. He is currently serving as an Instructor in the Department of Cancer Biology and examining the contribution of endothelial cells to the metastatic process.
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