李鲁远（美国乔治城大学医学院，华盛顿2000 7） ［摘要］异常活跃的血管发育是恶性肿瘤生长的一个重要条件。肿瘤血管有 3个要素使它成为开发抗癌药物的一个较好的靶标。第一，同基本处于静止状态的正常血管相比，肿瘤血管内皮细胞处于高度生长状态，因此成为突出目标。第二，肿瘤血管细胞是从正常组织进人肿瘤的正常细胞，基因组稳定，不象基因组极不稳定的癌细胞那样容易产生多种抗药性，因此可能较易控制。第三，尽管各种肿瘤细胞差异极大，其血管细胞因为是正常细胞，差异较小，因此同一药物如果针对肿瘤血管则可能对不同肿瘤均有疗效。本文对目前国际上对肿瘤血管发育机理的了解，常用的研究手段，及抗血管生长药物开发等方面的 概况作一简介。 【关键调】肿瘤血管；肿瘤；治疗 ［中囹分类号］ R730．5［文献标识吗］ A Targeting Tumor Vasculature for Anti-Cancer Therapy Lu-Yuan H (tombardi Cancer Center, ffeorgetown University Medical Center, 3970 Reservoir Road, washington DC, 20007, USA). Introduction Tumor vascularisation is a vital process for the progression of all solid tumors from a small, localized focus to an enlarging tumor with the capability to metastasize. This process, illustrated in Figure 1, involves the tumor inducing capillary sprouts from surrounding vasculature by altering the balance of angiogenic promoters and inhibitors. This leads to the formation of a large anastamosing vascular network, whieh is structurally and physiologieally different from native vessels. This allows for tumor intrasavation, embolisation and metastatic spread. Tumors that either fail, or are prevented from undergoing that process remain at the stage of a small 2 ~ 3 rum3 focus. There is great interest in developing new therapeutic approaches that target tumor neo-vasculature, given that the overall standardized mortality from the majority of solid tumors has altered only marginally over the last two decades with traditional treatment regimens. Anti-angiogenesis as a therapeutic concept was largely developed in the early 1970s after a body of experimental work had confirmed the biological importance of tumor angiogenesis. Therapeutic approaches aimed at preventing the process of neo-vascularisation were termed anti- angiogenic, and excluded therapies that would target existing tumor vasculature by infarction or vasoconstriction. Vascular attack is a term used to describe therapeutic strategies thst target differences between normal vasculature and tabor vasculature. Such differences include the greatly increased rate of endothelial cell proliferation in tumor vasculature as compared to quiescent normal vasculature and approaches directed at differential expression of specific endothelial markers on tumor and normal vessels. Importance of vascularity in tllmor development It has long been recognized that tumors possess an abundant vasculature and that this vasculature plays an important role in the biology of a growing cancer. In 1904, Ribbert postulated that Tumors have a superior vascular supply and in 1908 Bowen theorized that eomplete obliteration of the blood supply to a tumor means sphacelation, separation and cure. AlgUire drew attention to this phenomenon as being an active process instigated by a tumor, noting that an outstanding characteristic of the rapidly growing tumor is its capacity to elicit the production of a new eapillary endothelium from the host. The process of tumor vascularisation was visualized in experimental animals by transplanting tumors into transparent chambers in the cheek or ear, or directly into the aqueous humor of the eye. Warren visualized the growth of tumor vessels into transplanted metastatic melanoma and noted that these vessels were morphologically distinct from host vessels, being tortuous, thin walled, with sluggish flow. Using such models, the phenomenon of tumor dorfnancy was described. Tannock demonstrated that the mitotic index of tumor cells decreased with distance from the supplying blood vessel, by measuring cell division by tritiated thymidine uptake in a murine tumor with a partieularly orderly vascular tree. By altering ambient oxygen tensions he demonstrated that a mathematical model of oxygen diffusion could predict the change in mitotic index. The overall rate of tumor growth approximated to the rate of endothelial cell proliferation, and not to the much higher rate of tumor cell proliferation. He concluded that the nutrient supply to the tumor, as governed by the proliferation of the endotheliuml limited tumor size by balancing tumor cell proliferation with concomitant tumor necrosis. Folkman noted that tumors transplanted into isolated perfused organ systems such as the rabbit thyroid or dog intestinal segment, were uniformly size limited. Though not immediately aPParent, the size limitation occurred because the perfusate in these systems prevented neo- vascularisation due to the lack of platelets. By comparing the groWth of transplanted autologous tumors in the avascular aqueous humor with the vascular iris he could show distinct avascular and vascular phases of tumor growth, Which was confirmed by intravenous fluroscein injections. Commencement of vascularisation in the iris coincided with tumor growth beyond 2 ~ 3 nun3 and an increase in the rate of tumor doubling size by approximately-20 fold. Tumors implanted in the avascular aqueous humor failed to grow beyond 2 ~ 3 mms. He concluded that vascularisation of a tumor was an essential step in the progression of a tumor beyond a small primary focus and inferred that Preventing this process, which he termed anti-angiogenesis was a viable therapeutic approach. Angiogenesis assays The are a number of bioassays that are used for measuring experimental and clinical angiogenesis. As noted above transparent chambers techniques for visualizing tumor vascularization were developed in the early twentieth century. The long-term cultivation of human and bovine capillary endothelial cells allowed for assays of groWth, proliferation, chemotaxis and tubule formation assays. Development of chorio~allantoic membrane (CAM) assay and the intra-comeal assay allowed for measurement of the total angiogenic response to purified angiogenic proteins or inhibitors. In the CAM assay, a chick embryo can be maintained in a petri dish and angiogenie agents or tissue samples can be placed directly onto the avascular allantoic membrane where new blood vessel formation can be quantitated. In the intra-comeal assay a small pocket is created surgically in the eornea of an experimental animal and the sample to be assayed is inserted in this pocket. New vessel growth can readily be observed. Other angiogenesis models have been reviewed comprehensively elseWhere. Angiogenic switch in tumor development Adult endothelium is essentially quiescent. In response to both physiological stimuli (as seen in the proliferative endometrium and ovary) and pathological circumstances (injury, tumor growth and diabetic retinopathy) this quiescent endothelium can alter to a rapidly proliferating and organizing population of cells. Physiological angiogenesis can also be rapidly curtailed indicating that the process is held in check physiologically and can be activated in response to appropriate stimuli I in a fashion analogous to the clotting cascade. A considerable number of positive and negative regulatory molecules have been implicated in the control of this process.’’ By contrast the vasculature of a tumor is not quiescent but is rapidly proliferating. Endothelial cell turnover in tumor vasculature has been shown to be up to fifty fold greater than that seen in quiescent adult vasculature. A tumor induces this proliferative vascular response from host vessels by altering the balance of positive and negative innuences in its local vicinity and may utilize a number of different strategies to effect this change. The acquisition of a pro-angiogenic phenotype has been termed the angiogenic switch and may he a rate-limiting step in tumor progression. Evidence exists that tumors undergo a switch from a non-angiogenic Phenotype to an angiogenic phenotype as they progress from pre-malignant stages to frankly invasive cancers. In human tumors the development of vascularity within a tumor can be associated with progression of a tumor from a non-invasive pre-malignant stage through defined stages to an invasive carcinoma. In cervical carcinoma staining of micro-vessels with an antibody to Von Willebrand factor shows a clear increase in the number of micro-vessels apposed to the basement membrane between cervical intra-epithelial neoplasia (CIN) l&II as compared with CIN Ill lesions. Furthermore, micro-vessel density, as a surrogate for angiogenic phenotype, is a powerful independent prognostic indicator both of distant metastasis and survival. After the initial reports of micro-vessel density as a prognostic indicator in melanoma and breast carcinomal a substantial body of evidence now exists that micro-vessel density in a primary tumor can predict metastasis and survival in a variety of cancers j including nonsmall cell lung carcinoma, prostate carcinoma, bladder carcinoma, head and neck carcinoma, rectal carcinoma and CNS tumors. Angiogeulc factors At least thirteen angiogenic polypeptides that have positive effects on standard assays have now been identified, not all of which have been imPlicated in the of tumor angiogenesis. The two positive regulators that are implicated most commonly in the neo-vascularisation of tumors are vascular endothelial gtowth factor (VEGF) and basic fibroblast growth factor (FGF-2 ). In addition to their active soluble forms, both VEGF and FGF-2 appear ’’to exist as inactive molecules sequestered in extra-cellular matrix, presumably facilitating their physiological regulation. Tumors can locally up-regulate these factors both by increased production and by release of the sequestered inactive molecules. i) Vascular endothelial growth factor (VEGF) VEGF is an angiogenic factor that has been strongly implicated in tumor neo-vascularisation. This protein was first identified as a vascular permeability factor that played a role in ascites formation. Its more general angiogenic properties were identified later when it was fully characterized. VEGF is a secreted protein that is mitogenic for endothelial cells and yet has no appreciable mitogenic activity on other cell types. It has a wide range of angiogenic effects on endothelial cells,- including ehemotaxis and capillary tubule formationl’’n VI’’ho. It is positively angiogenic in the CAM and comeal pocket assay. The biology of VEGF is interesting in that it exists as four different isoforms of various lengths in their primary sequences (VEGFlzl, VEGF16s, VEGFlso, VEGFzo6 ). These isoforms are all products of the same gene but are produced by differential messenger RNA splicing. The significance of these isoforms is that the larger peptides bind with higher sanity to heparan like proteoglycans and thus are effectively sequestered by the extra-cellular matrix. They can however be cleaved into soluble shorter molecules by proteases such as plasmin. This may be an important physiological means of regUlation, and it may be particularly important in the setting of a tumor where proteolytic activity is up-regUlated. Two high affinity VEGF receptors with tyrosine kinase activity have now been identified (fib-l, KDR) and the expression of these is restricted to vascular endothelium. n) Fibroblast growth factors The fihroblast growth factors (FDFs) are a family of structurally homologous cytokines now totaling 12 in number. They are functionally diverse, with roles in embryonic development, neo-vascularisation and wound healing. The prototype molecule, basic fibroblast growth factor (FGF2) was initially identified from bovine brain and pituitary extracts and later purified from tumor cell lines as a capillary endothelial growth factor. FGF-2 1 and the other prototype molecule acidic fibroblast growth factor (FGF-1 ), are potently mitogenic for endothelial cells and for a wide variety of other cell types of mesodermal and neuro- ectodermal lineage. They are chemotactic for endothelial cells and fibroblasts and induce differentiation a variety of cells. The importance of this family of growth factors in tumor biology is demonstrated partly by the fact that later additions to the FGF family (FGF-3, FGF4 & FGF-5 ) are oncogene products that can transform cells 1’’n VI’’ho. Also transfecting cDNA coding for FGF- 1 and FGF4 into poorly tumorigenic non metastatic cell lines will convert them into angiogenic metastasizing clones. Elevated levels of basic FGF have been demonstrated in serum and urine in a significant proportion of patients with a variety of cancersl ranging from 29% in breast cancer patients to 60% in sarcoma patients. Both basic and acidic FGF lack a secretion peptide ’’ but nevertheless are transported outside of the cells. These factors are ubiquitously present in the extra-cellular matrix, bound to heparin-like proteoglycans. The transportation mechanism of fibroblast groWth factors is not yet clear. There are a number of mechanisms by which tumors release FGF s from tumor cells or the inactive extra-cellular reservoir to induce anglogenesis. Firstly FGF-3, FGF4& FGF-5 are all pl.oducts of oncogenes and possess secretion peptides and are secreted by tumor cells as mature glycosylated soluble angiogenic factors. Sdly in mouse fibrosarcomas t the tumor develops the ability to release soluble and active FGF-2, that normally is bound to proteoglycans, in a signal peptide independent fashion. This change in phenotype coincides both with progression to an angiogenic, invasive tumor from non-invasive fibromatosis. Thirdly heparanases can degrade heparin like proteoglycans, thus releasing active FGF from the extracellular matrix. Anti-angiogenic factors As noted earlier, normal vasculature is essentially quiescent and physiological angiogenesis is often swiftly terminated, whereas pathological angiogenesis is often prolonged and difficult to terminate. This would imply the existence of an endogenous negative regulatory system that may be defective or overridden in tumor angiogenesis. An increasing number of endogenous compounds have been identified that have inhibitory activity on endothelial cells assessed byin Vjtro andin VI’’ro assays. i) Throlnbospondin Thrombospondin is a 160 Kd adhesive glycoprotein, found in platelet alpha granules involved in platelet aggregation. A 140 Kd fragment of thrombospondin is a specific anti-angiogenic factor and also is under transcriptional control of the tumor suppressor gene p53. Thrombospondin was identified in the conditioned media of a hamster cell line that is one step away from being tumorigenic and can be transformed into a tumorigenic cell line by addition of carcinogens. The parental cell line is non-angiogenic in a comeal assay but the transformed cell line is. The conversion to the angiogenic phenotype is caused by the loss of a single allele of p53, which caused loss of production of an inhibitory glycoprotein, which was shown to be a fragment of thrornbospondin. Subsequently other fragments of thrombospondin have been shown to have inhibitory activity. n) Angiostatin and endostatin Angiostatin was identified from a lung carcinoma cell line that is particularly effective in suppressing metastatic growth until the primary tumor is removed. Sera and urine from animals with this primary tumor have anti-angiogenic activity in in VJ’’tro assays. Animals bearing this primary tumor inhibit the growth of blood vessels to an angiogenic stimulus (FGF-2 ) implanted into the cornea, but when the primary tumor wus removed, vessel growth in response to FGF-2 was preserved. A circulating angiogenic inhibitor I subsequently named angiostatin, was purified from the urine of these animals, and found to be a 38 Kd fragment of plasminogen. Purified angiostatin when given systemically can reproduce the inhibitory effect of the primary tumor after it has been surgically removed. The anti- metastatic effects of systemic angiostatin have been coallrmed in a number of cell lines with little evidence of toxicity. A prostate cancer cell line, which also demonstrates this inhibitory phenomenon I can enzymatically cleave plasminogen to form angiostatin, which is active inin aam and in vial angiogenesis assays. Another circulatory inhihitor I endostatin I was identified using the same strategy. Endostatin is a fragment of collagen XVlll, a form of collagen that exists exclusively in blood vessels. Once again this eompound can inhibit angiogenesis in standard assays and cause complete regression of a variety of formed tumors in experimental animals I measuring up to l% of body mass. Repeated intermittent treatment with endostatin, after tumors have re-grown from their regressed dormant state, will induce regression with no sign of drug resistance. iii) Vascular endothelial growth inhibitor Vascular endothelial cell groWth inhibitor (VEGI) is a recently described cytokine that has been implicated in maintenance of normal vascular quiescence and is also as a novel anti-angiogenic agent. The physiology of this protein is interesting in that it is produced almost exclusively by endothelial cells and the target cells for activity I through a presumed cell surface receptor, are also endothelial cells. The cytokine was identified by a subtractive technique whereby messenger RNAs (mRNA) that were expressed only in endothelial cells were identified and cloned. VEGI mRNA codes for a 174-amino acid protein that exhibits considerable structural homology to tumor necrosis family (TNF ) family members. Subsequent northern blotting analysis confirmed expression only in endothelial cells. However VEGI mRNA is widely expressed in many adult human tissues suggesting a physiological role in adult vasculature. The primary sequence of the VEGI protein reselnLbles a type 11 membrane protein with a brief intracellular segment and most of the protein being an extracelhllar domain, similar to most TNF family m6mbers. Since many TNF family members are cleaved from the membrane to function as soluble paracrine factors on target cells with appropriate receptors, a similar mechanism of activation is assumed for VEGI. A recombinant soluble form of this protein was found to have highly potent anti- angiogenic activity in endothelial proliferation assays, while having no effect on the proliferation of non-endothelial cells. Furthermore tubule formation on collagen gels and vessel formation on the CAM in response to either FGF-2 or VEGF were all inhibited by VEGI. To mimic the presumed physiological mechanism of activity where the extracellular domain of the protein acts as a soluble factor, a secreted form of VEGI was over- expressed in murine colon cancer cells. The transfected cancer cells had greatly decreased tumorigenicity when implanted in C57/BL syngenic mice and inhibition of tumor angiogenesis was evident from much decreased intra- tumoral microvessel density. Similar anti-tumor effects were found when this secreted form of VEGI was expressed in non tumorigenic Chinese hamster ovary cells that were then co-inoculated into athymic mice with aggressive breast cancer cell lines. The resulting inhibition of growth of the breast cancer cells indicates a paracrine anti-tumor effect by VEGI presumably by inhibition of tumor vascularisation. Taken together, these findings suggest that the protein functions physiologically as an endogenous mediator of vascular quiescence via a juxtacrine or paracrine mechanism, and also implicate VEGI as an attractive candidate molecule for anti-angiogenic therapy. Tumor endothelial cells as a therapeutic target Another anti-vascular approach is to direct toxic agents at the tumor endothelial cell. This approach has a number of theoretical advances over conventional cytotoxic therapy whose principal mechanism of action is directed principally at the tumor cell. First, endothelial cells are not transformed and as such are unlikely to acquire mutations resulting in drug resistance. Second, as native host cells, therapy directed at endothelial cells would be applicable to solid tumors generally, irrespective of tumor cell origin. Third endothelial cells are uniquely exposed to blood borne agents, circumventing a problem of drug delivery to the center of a tumor, which is a major hurdle in conventional treatment. A variety of exogenous anti angiogenic agents that are under clinical evaluation appear to have direct anti- proliferative effects on endothelial cells. Although some synthe\ic compounds exert anti-endothelial effects through endogenous growth factors (e. g. pentosan polysulfate binds to heparin binding groWth factors) such as FGF, other have direct ant-endothelial activity. The fumagillin derivative TNP470 is a potent synthetic anti-angiogenic compound that prevent’’endothelial cell entry into the cell cycle. Carboxyamido-triazole inhibits signal transduction of endothelial cell receptor tyrosine kinases. Other anti- angiogenic compounds, such as thalidoide, have nuclear mechanisms of action. There are potential endogenous target molecules for such an approach. As tumor vessels are actively proliferating, a number Of molecules are over-expressed on actively dividing endothelial cells whilst absent or weakly expressed L. quiescent vessels. The integrin av P3 is specifically expressed on proliferating vessels in healing wounds and granulation tissue and also in tumor vasculature while not on quiescent vasculature. A monoclonal antibody to integrin av P3 prevent angiogenesis in the CAM assay, and can cause melanoma tumor regression in animals as a result of endothelial cell apoptosis. Other candidate molecules that may be specific to tumor vasculature include the VEGF receptors, the EN7/44 antigen and endosialin. Condusions The process of neo-vascularisation is a pre-requisite for tumor development beyond a small primary focus. The biology of the humoral mechanisms underlying this process is increasingly understood and is the basis for a number of novel approaches to treatment of solid tumors. As a result of this research, many compounds that have a partial or complete anti angiogenic mechanisms of action are now under assessment in clinical trials. Future clinical research will focus on the potential for synergistic action with conventional ehemotherapy and also new regimens for the administration of anti angiogenic drugs. Given the role of angiogenic inhibitors in the maintenance of tumor dormancy, it is possible that these agents may be effective in the maintenance of long term remissions, an approach not currently used for solid tumors.
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