Antiangiogenic treatment with thrombospondin-1 enhances primary tumor radiation response and prevents growth of dormant pulmonary micrometastases after curative radiation therapy in human melanoma xenografts

Thrombospondin-1 (TSP-1) is a potent antiangiogenic factor that has been shown to inhibit tumor growth by preventing endothelial cells from responding to a wide variety of angiogenic stimulators. We have demonstrated previously that D-12 primary tumors (human melanoma xenografts) suppress the growth of their spontaneous pulmonary micrometastases by secreting TSP-1 into the blood circulation. The same tumor model was used in the present work to study antitumor effects of combined radiation therapy and antiangiogenic treatment with TSP-1. Curative radiation treatment of D-12 primary tumors resulted in rapid growth of previously dormant micrometastases. Growth of dormant micrometastases could be prevented by treating the host mice with exogenous TSP-1 after the radiation treatment. Treatment with exogenous TSP-1 after subcurative radiation treatment reduced the growth rate of recurrent primary tumors in addition to suppressing metastatic growth. TSP-1 suppressed tumor growth at both primary and metastatic sites by inducing apoptosis in tumor-associated microvascular endothelial cells. Treatment with exogenous TSP-1 before radiation treatment enhanced the antitumor effect of the radiation treatment. The radiopotentiation by TSP-1 involved at least two distinctly different mechanisms, i.e., TSP-1 reduced the fraction of radiobiologically hypoxic parenchymal tumor cells and increased the radiation sensitivity of the tumor microvasculature by promoting radiation-induced endothelial cell apoptosis. In conclusion, the present preclinical study showed that TSP-1 has antiangiogenic, antimetastatic, and radiopotentiating properties that merit additional investigation in clinical studies.     

Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies.     

A primary tumor promotes dormancy of solitary tumor cells before inhibiting angiogenesis.

Mechanisms that regulate the transition of micrometastases from clinically undetectable and dormant to progressively growing are critically important but poorly understood in cancer biology. Here we examined the effect of a primary tumor on the growth of solitary tumor cells in the mouse liver, as well as on the development of tumor angiogenesis in a dorsal skin-fold chamber. s.c. placement of a CT-26 (BALB/c-derived mouse colon carcinoma) primary tumor markedly inhibited development of liver metastasis in BALB/c mice after subsequent intraportal injection of tumor cells. Dorsal skin-fold chamber experiments showed that this growth inhibition paralleled a strong antiangiogenic effect by the primary tumor. Furthermore, intravital microscopy of the liver after intraportal injection of green fluorescent protein-expressing tumor cells showed that primary tumors promoted dormancy of single tumor cells for up to 7 days. Immunohistological staining for Ki-67 confirmed that these solitary cells were indeed dormant. In contrast, in the absence of a primary tumor, GFP-expressing tumor cells quickly developed into micrometastases. Thus, primary CT-26 tumor implants nearly abrogated tumor metastasis by inhibition of angiogenesis and by promoting a state of single-cell dormancy. Knowledge of the mechanism underlying this dormancy state could result in the development of new therapeutic tools to fight cancer.     

Vascular endothelial growth factor, interleukin 8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in human melanoma xenografts

Angiogenesis is a significant prognostic factor in melanoma, but the angiogenic factors controlling the neovascularization are not well defined. The purpose of this study was to investigate whether the angiogenesis and metastasis of melanoma are promoted by vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), platelet-derived endothelial cell growth factor (PD-ECGF), and/or basic fibroblast growth factor (bFGF). Cells from human melanoma lines (A-07, D-12, R-18, and U-25) transplanted to BALB/c nu/nu mice were used as tumor models. Expression of angiogenic factors was studied by ELISA, Western blotting, and immunohistochemistry. Angiogenesis was assessed by using an intradermal angiogenesis assay. Lung colonization and spontaneous lung metastasis were determined after i.v. and intradermal inoculation of tumor cells, respectively. The specific roles of VEGF, IL-8, PD-ECGF, and bFGF in tumor angiogenesis, lung colonization, and spontaneous metastasis were assessed in mice treated with neutralizing antibody. The melanoma lines expressed multiple angiogenic factors, and each line showed a unique expression pattern. Multiple angiogenic factors promoted angiogenesis in the most angiogenic melanoma lines, whereas angiogenesis in the least angiogenic melanoma lines was possibly promoted solely by VEGF. Tumor growth, lung colonization, and spontaneous metastasis were controlled by the rate of angiogenesis and hence by the angiogenic factors promoting the angiogenesis. Lung colonization and spontaneous metastasis in A-07 were inhibited by treatment with neutralizing antibody against VEGF, IL-8, PD-ECGF, or bFGF. Each of these angiogenic factors may promote metastasis in melanoma, because inhibition of one of them could not be compensated for by the others. Our observations suggest that efficient antiangiogenic treatment of melanoma may require identification and blocking of common functional features of several angiogenic factors.     

Orthotopic human melanoma xenograft model systems for studies of tumour angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern.

Adequate tumour models are a prerequisite in experimental cancer research. The purpose of the present work was to establish and assess the validity of four new orthotopic human melanoma xenograft model systems (A-07, D-12, R-18, U-25). Permanent cell lines were established in monolayer culture from subcutaneous metastases of four different melanoma patients by using an in vivo-in vitro procedure, and cells from these lines were inoculated intradermally in Balb/c nu/nu mice to form tumours. Individual xenografted tumours of the same line differed substantially in growth and pathophysiological parameters, probably as a consequence of differences between inoculation sites in host factors which influence tumour angiogenesis. Nevertheless, xenografted tumours of different lines showed distinctly different biological characteristics. Several biological characteristics of the donor patients'' tumours were retained in the xenografted tumours, including angiogenic potential; growth, histopathological and pathophysiological parameters; and sensitivity to radiation, heat and dacarbazine treatment. Moreover, the organ-specific metastatic pattern of the xenografted tumours reflected the pattern of distant metastases in the donor patients. The organs of preference for distant metastases were lungs (A-07, D-12), lymph nodes (R-18) and brain (U-25). R-18 lymph node metastases and U-25 brain metastases developed in the absence of lung involvement. The four orthotopic human melanoma xenograft model systems show great promise for future studies of tumour angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern.     

Hypothesis: primary antiangiogenic method proposed to treat early stage breast cancer

Background  

Women with Down syndrome very rarely develop breast cancer even though they now live to an age when it normally occurs. This may be related to the fact that Down syndrome persons have an additional copy of chromosome 21 where the gene that codes for the antiangiogenic protein Endostatin is located. Can this information lead to a primary antiangiogenic therapy for early stage breast cancer that indefinitely prolongs remission? A key question that arises is when is the initial angiogenic switch thrown in micrometastases? We have conjectured that avascular micrometastases are dormant and relatively stable if undisturbed but that for some patients angiogenesis is precipitated by surgery. We also proposed that angiogenesis of micrometastases very rarely occurs before surgical removal of the primary tumor. If that is so, it seems possible that we could suggest a primary antiangiogenic therapy but the problem then arises that starting a therapy before surgery would interfere with wound healing.     

Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models

BACKGROUND: There is a paucity of information about events that follow immediately after tumor cells are triggered to initiate the process of angiogenesis (the formation of new blood vessels). Such information is relevant to the issue of when micrometastases vascularize and has implications for the accessibility of micrometastases to various treatments. In this study, we attempted to monitor events at the initiation of angiogenesis at the earliest possible stage of tumor growth in vivo. METHODS: Two different rodent mammary tumor cell lines, R3230Ac from the Fischer 344 rat and 4T1 from the BALB/c mouse, were stably transfected with a gene that encodes an enhanced version of green fluorescence protein (GFP). GFP-labeled R3230Ac or 4T1 cells (about 20-50 cells) were implanted into dorsal skinfold window chambers of Fischer 344 rats or BALB/c mice, respectively. Tumor angiogenesis was then monitored serially and noninvasively for up to 4 weeks. RESULTS: Clear evidence of modification of the host vasculature was observed when tumor mass reached approximately 60-80 cells, and functional new blood vessels were seen when tumor mass reached roughly 100-300 cells. Individual tumor cells exhibited a chemotaxis-like growth pattern toward the pre-existing host vasculature. When ex-flk1 (a soluble, truncated vascular endothelial cell growth factor receptor protein known to be antiangiogenic) was injected with the tumor cells, the initial angiogenic and tumor growth activities were inhibited considerably, indicating that angiogenesis inhibitors may halt tumor growth even before the onset of angiogenesis. CONCLUSION: Angiogenesis induced by tumor cells after implantation in the host begins at a very early stage, i.e., when the tumor mass contains roughly 100-300 cells. Identification of chemotactic signals that initiate tumor cell migration toward the existing vasculature may provide valuable targets for preventing tumor progression and/or metastases.     

Metastatic dissemination of B16 melanoma: pattern and sequence of metastasis

The progressive metastatic spread from subcutaneous transplants of two subpopulations of the mouse B16 melanoma, slow-growing clone G3.5 and fast-growing clone G3.12, was examined during tumor growth in C57BL/6 mice and after surgical excision of tumors of various sizes. In addition to enumeration of visible and lethal or potentially lethal ("clinically relevant") metastases, the occurrence of visibly undetectable proliferating (occult) or nonproliferating (dormant) micrometastases was assessed by implanting lymph nodes and organs subcutaneously into normal mice and monitoring for resulting tumor growth. Occult or dormant metastases were disseminated initially to the lungs from G3.5 tumors of 3-4 mm in mean geometric diameter (MGD) and G3.12 tumors of 6-7 mm in MGD. The ipsilateral axillary lymph node (IALN), the regional draining lymph node for these tumors, received metastases after the lungs, initially from 10 to 12-mm tumors. Subsequently, occult or dormant and visible metastases first appeared in systemic organs and lymph nodes (kidneys, adrenal glands, ovaries, and contralateral axillary lymph node) at tumor sizes of about 26 mm in MGD. Systemic metastases occurred only in mice with large and numerous lung metastases and did not depend on the continuing presence of the subcutaneous tumor or on the presence of IALN metastases, which indicated that established lung metastases were a generalizing site from which systemic metastatic spread initiated. After tumor excision, death generally resulted from extensive lung metastasis. Occasional lethal or clinically relevant metastases were also observed in the IALN, kidneys, adrenal glands, ovaries, brain, eyes, and urinary bladder; liver involvement was evident exclusively as occult or dormant micrometastases. Terminal metastatic patterns of these B16 melanoma transplants were as widespread and indiscriminate as those of malignant melanoma in humans.     

Systemic inhibition of tumor growth and angiogenesis by thrombospondin-2 using cell-based antiangiogenic gene therapy

Recent studies indicate that continuous administration improves the antitumoral efficacy of angiogenesis inhibitors, as compared with intermittent dosing, suggesting a potential role of gene therapy in antiangiogenic tumor therapy. We established a tissue-engineered implant system for the continuous in vivo production of thrombospondin-2 (TSP-2), a potent endogenous inhibitor of tumor growth and angiogenesis. Fibroblasts were retrovirally transduced to overexpress TSP-2 and were seeded onto biodegradable polymer scaffolds. After transplantation into the peritoneal cavity of nude mice, bioimplants maintained high levels of TSP-2 secretion over extended time periods, resulting in increased levels of circulating TSP-2. Bioimplant-generated TSP-2 potently inhibited tumor growth and angiogenesis of human squamous cell carcinomas, malignant melanomas, and Lewis lung carcinomas that were implanted at a distant site. These results provide the first proof-of-principle for the feasibility and therapeutic efficiency of systemic, cell-based antiangiogenic gene therapy using biodegradable polymer grafts for the treatment of cancer.     

Inhibition of infiltration and angiogenesis by thrombospondin-1 in papillary thyroid carcinoma.

PURPOSE: Angiogenesis is essential for tumor growth and is controlled by the balance between angiogenic and antiangiogenic factors. We studied the expression of angiogenic factors and antiangiogenic factors in papillary thyroid carcinoma. EXPERIMENTAL DESIGN: We investigated immunohistochemically the expression patterns and levels of antiangiogenic factor and its receptor, thrombospondin-1 (TSP-1) and CD36, and four angiogenic factors, vascular endothelial growth factor (VEGF), VEGF-C, angiopoietin-2 (Ang-2), and Tie-2, in the primary tumors of 75 papillary thyroid carcinoma patients. We also examined the microvessel count (MVC), using CD31 staining. RESULTS: VEGF expression strongly correlated with other angiogenic factors. The cytoplasm of cancer cells stained positive for all factors. Tie-2 and TSP-1 receptor also appeared in endothelia of microvessels. TSP-1 inversely correlated with the degree of invasion of the primary tumor to other adjacent organs and with MVC. A higher MVC correlated with poorer survival. To clarify the balance between angiogenic and antiangiogenic factors in the same tumor, we calculated the ratio of each angiogenic factor against TSP-1 as the antiangiogenic factor. The ratios VEGF/TSP-1, VEGF-C/TSP-1, and Ang-2/TSP-1 significantly correlated with a higher MVC. Furthermore, the ratios VEGF/TSP-1 and Ang-2/TSP-1 significantly correlated with the degree of infiltration. CONCLUSIONS: To the best of our knowledge, this is the first report demonstrating that the balance between angiogenic and antiangiogenic factors correlates with distinct invasion to other organs and neovascularization of papillary thyroid carcinoma.     

Biological and clinical role of angiogenesis in breast cancer

Summary Angiogenesis, the process leading to the formation of new blood vessels, plays a central role in tumor progression of solid neoplasia. The switch from the avascular to the vascular phase is generally accompanied by rapid primary tumor growth and local invasiveness. Furthermore, angiogenesis is also necessary both at the beginning and at the end of the development of distant metastasis and is implicated in the phenomenon of dormant micrometastases.The angiogenic activity of both the primary tumor and its metastases is the result of the net balance between angiogenic peptides and natural inhibitors, and it is regulated by multiple biochemical and genetic mechanisms. In normal tissues of the adult, unlike invasive cancers, the angiogenic inhibitory pathway predominates.Several experimental and clinico-pathologic studies have confirmed that angiogenesis is specifically involved in transformation and progression of human breast cancer. In particular, clinicopathologic studies have found that the degree of vascularization of primary invasive human breast cancer is heterogeneous and correlates with the prognosis of patients.A number of antiangiogenic agents have been recently discovered, and some are under early clinical evaluation. Thus, angiogenic activity of the tumors represents a potentially novel anticancer therapeutic target.This issue ofBreast Cancer Research and Treatment reports on the most relevant basic biological aspects of angiogenesis, on its clinical role in breast cancer prognosis, and on the implications of inhibition of angiogenesis for future novel anticancer therapeutic approaches.     

Thrombospondin 1 Triggers Osteosarcoma Cell Metastasis and Tumor Angiogenesis

Osteosarcomas, especially those with metastatic or unresectable disease, have limited treatment options. The antitumor effects of pharmacologic inhibitors of angiogenesis in osteosarcomas are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here we demonstrated that thrombospondin 1 (TSP-1) is a potent inhibitor of the growth and metastasis of the osteosarcoma cell line MG-63. Moreover, we demonstrate that upregulation of TSP-1 facilitated expression of vasculostatin in MG-63 cells. In angiogenesis assays, overexpression of TSP-1 inhibited MG-63 cells and induced tube formation of human umbilical vein endothelial cells (HUVECs) in a CD36-dependent fashion. Finally, in xenografted tumors, we observed that TSP-1 overexpression inhibited angiogenesis and tumor growth. These results provided strong evidence for an important role of the TSP-1/CD36/vasculostatin signaling axis in mediating the antiangiogenic activity of osteosarcoma.     

Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice

Extracellular pH (pH(e)) is lower in many tumors than in the corresponding normal tissue. The significance of acidic pH(e) in the development of metastatic disease was investigated in the present work. Human melanoma cells (A-07, D-12, and T-22) were cultured in vitro at pH(e) 6.8 or 7.4 (control) before being inoculated into the tail vein of BALB/c nu/nu mice for formation of experimental pulmonary metastases. Cell invasiveness was studied in vitro by using Matrigel invasion chambers and angiogenesis was studied in vivo by using an intradermal assay. Protein secretion was measured by ELISA and immunocapture assays. Cells cultured at acidic pH(e) showed increased secretion of proteinases and proangiogenic factors, enhanced invasive and angiogenic potential, and enhanced potential to develop experimental metastases. Acidity-induced metastasis was inhibited by treatment with the general matrix metalloproteinase (MMP) inhibitor GM6001, the general cysteine proteinase inhibitor E-64, or blocking antibody against vascular endothelial growth factor-A (VEGF-A) or interleukin-8 (IL-8). Our study indicates that acidic pH(e) promotes experimental pulmonary metastasis in A-07, D-12, and T-22 human melanoma cells by a common mechanism involving acidity-induced up-regulation of the proteolytic enzymes MMP-2, MMP-9, cathepsin B, and cathepsin L and acidity-induced up-regulation of the proangiogenic factors VEGF-A and IL-8. One consequence of this observation is that treatment strategies involving deliberate tumor acidification to improve the efficacy of chemotherapy, photodynamic therapy, and hyperthermia should be avoided. Moreover, the possibility that the pH(e) of the primary tumor may be an important prognostic parameter for melanoma patients merits clinical investigation.     

Overexpression of thrombospondin-1 reduces growth and vascular index but not perfusion in glioblastoma

Little is known about the effects of antiangiogenic therapy on perfusion of human tumors and the mechanisms by which tumors can adapt to these treatments and recur. Here, we examined the effects of serial passaging of LN-229 human glioma xenografts overexpressing thrombospondin (TSP)-1 on tumor growth, vascularity, and perfusion. Persistence of TSP-1 overexpression was confirmed after three serial s.c. passages of small xenografted tumor blocks of cells stably transfected with TSP-1 cDNA (clones C9 and E7) or vector controls (pooled clones A7-A9) in immunodeficient nu/nu mice. The tumor vascularity was estimated by noninvasive near infrared spectroscopy measuring blood volume at 800 +/- 10 nm and by histological vessel scores in CD31-immunostained cryosections. The tumor perfusion was assessed by noninvasive laser Doppler flowmetry. Overexpression of TSP-1 significantly inhibited tumor growth. In size-matched tumors (approximately 300 mm(3)), the blood volume and the histological vessel scores were lower in the TSP-1-transfected tumors than in controls, and this effect was more pronounced in tumors derived from the clone with the highest TSP-1 expression (clone E9). Despite this clear reduction in tumor vascularity, the tumor perfusion was the same in TSP-1-transfected tumors and controls. This study shows that TSP-1 overexpression slows glioma growth in vivo but does not prevent it from reaching a large size (300 mm(3)). Whereas a clear reduction in blood volume during tumor growth and a reduced vascular index at sacrifice are observed in TSP-1-transfected tumors, this did not affect perfusion when size-matched comparisons were performed. Given the increased time needed to reach equal size, it indicates that a fixed rate of perfusion must be maintained in the tumor to allow for growth. Elucidation of the mechanisms that allow this to happen has important consequences for the understanding of tumor recurrence after antiangiogenic therapy.     

Antiangiogenic agents potentiate cytotoxic cancer therapies against primary and metastatic disease.

The formation of a blood supply (angiogenesis) is critical to the growth of solid tumors. The naturally occurring steroid tetrahydrocortisol, the synthetic cyclodextrin derivative beta-cyclodextrin tetradecasulfate, and the tetracycline derivative minocycline have antiangiogenic activity. Tetrahydrocortisol and beta-cyclodextrin tetradecasulfate in a 1:1 molar ratio by continuous infusion over 14 days and minocycline administered i.p. over 14 days from day 4 to day 18 postimplantation of the Lewis lung carcinoma significantly increased the growth delay of the primary tumor after treatment with cis-diamminedichloroplatinum(II), melphalan, cyclophosphamide, Adriamycin, bleomycin, and radiation therapy administered in standard regimens. Addition of the antiangiogenic agents to treatment with the cytotoxic therapies not only reduced the number of lung metastases formed from the primary tumor but also reduced the number of large metastases. Five of 12 animals treated with the antiangiogenic modulators and cyclophosphamide were long-term survivors (> 120 days). Thus, antiangiogenic therapies can potentiate the efficacy of standard anticancer therapies.     

Prevention of Hepatic and Peritoneal Metastases by the Angiogenesis Inhibitor FR-118487 after Removal of Growing Tumor in Mice

We established a mouse "primary tumor resection model" in which a transplanted tumor was resected after an orthotopic transplantation of colorectal cancer tissue to estimate the therapeutic effect of an angiogenesis inhibitor on metastasis. The angiogenesis inhibitor FR-118487 is a member of the fumagUlin family. Here, 1 mg/kg/day of FR-118487 was subcutaneously administered to nude mice for 1 week, 2 weeks, or 4 weeks through an osmotic pump. Liver metastasis developed in 7 of 9 control mice, 2 of 6 mice that underwent the tumor resection 2 weeks after transplantation (early resection), and in all 7 of the mice that underwent the tumor resection 4 weeks after transplantation (late resection). In the short treatment trial, the FR-118487 administration immediately after the early resection completely inhibited both hepatic and peritoneal metastases, whereas its administration after the late resection had no effect on liver metastasis. In the prolonged treatment trial, inhibitory effects of prolonged treatment with FR-118487 on both hepatic and peritoneal metastases after the late resection were clearly demonstrated. The mice of the resection-alone group all died within 106 days after tumor inoculation, due to metastases of colon carcinoma. In contrast, half of the mice that underwent resection and then received antiangiogenic therapy were alive at the end of the observation period (160 days after transplantation). In conclusion, the combination of surgery and subsequent antiangiogenic therapy may be useful to prevent the distant metastasis of colorectal cancer and to improve the prognosis of patients with colorectal cancer.     

The tumor bed effect: increased metastatic dissemination from hypoxia-induced up-regulation of metastasis-promoting gene products

Cancer patients with recurrent local disease after radiation therapy have increased probability of developing regional and distant metastases. The mechanisms behind this observation were studied in the present work by using D-12 and R-18 human melanoma xenografts growing in preirradiated beds in BALB/c-nu/nu mice as preclinical models of recurrent primary tumors in humans. D-12 tumors metastasize to the lungs, whereas R-18 tumors develop lymph node metastases. Based on earlier studies, we hypothesized that metastasis was governed primarily by the proangiogenic factor interleukin-8 (IL-8) in D-12 tumors and by the invasive growth-promoting receptor urokinase-type plasminogen activator receptor (uPAR) in R-18 tumors. Pimonidazole was used as a hypoxia marker, and hypoxia, microvascular hotspots, and the expression of IL-8 and uPAR were studied by immunohistochemistry. The metastatic frequency was significantly higher in tumors in preirradiated beds than in control tumors in unirradiated beds, and it increased with the preirradiation dose. D-12 tumors showed increased fraction of hypoxic cells, increased fraction of IL-8-positive cells, and increased density of microvascular hotspots in preirradiated beds, and R-18 tumors showed increased fraction of hypoxic cells and increased fraction of uPAR-positive cells in preirradiated beds. Strong correlations were found between these parameters and metastatic frequency. IL-8 was up-regulated in hypoxic regions of D-12 tumors, and uPAR was up-regulated in hypoxic regions of R-18 tumors. Daily treatment with anti-IL-8 antibody (D-12) or anti-uPAR antibody (R-18) suppressed metastasis significantly. Our preclinical study suggests that primary tumors recurring after inadequate radiation therapy may show increased metastatic propensity because of increased fraction of hypoxic cells and hypoxia-induced up-regulation of metastasis-promoting gene products. Two possible mechanisms were identified: hypoxia may enhance metastasis by inducing neoangiogenesis facilitating hematogenous spread and by promoting invasive growth facilitating lymphogenous spread. The aggressive behavior of postirradiation local recurrences suggests that they should be subjected to curative treatment as early as possible to prevent further metastatic dissemination. Moreover, the possibility that patients with a high probability of developing local recurrences after radiation therapy may benefit from postirradiation treatment with antiangiogenic and/or anti-invasive agents merits clinical investigation.     

Endogenous inhibitors of angiogenesis in malignant gliomas: nature's antiangiogenic therapy

Angiogenesis is necessary for tumor growth beyond a volume of approximately 2 mm(3). This observation, along with the accessibility of tumor vessels to therapeutic targeting, has resulted in a research focus on inhibitors of angiogenesis. A number of endogenous inhibitors of angiogenesis are found in the body. Some of these are synthesized by specific cells in different organs, and others are created by extracellular proteolytic cleavage of plasma-derived or extracellular matrix-localized proteins. In this review, we focus on angiostatin, endostatin, PEX, pigment epithelial-derived factor, and thrombospondin (TSP)-1 and -2, either because these molecules are expressed in malignant glioma biopsies or because animal studies in malignant glioma models have suggested that their therapeutic administration could be efficacious. We review the known mechanisms of action, potential receptors, expression in glioma biopsy samples, and studies testing their potential therapeutic efficacy in animal models of malignant glioma. Two conclusions can be made regarding the mechanisms of action of these inhibitors: (1) Several of these inhibitors appear to mediate their antiangiogenic effect through multiple protein-protein interactions that inhibit the function of proangiogenic molecules rather than through a specific receptor-mediated signaling event, and (2) TSP-1 and TSP-2 appear to mediate their antiangiogenic effect, at least in part, through a specific receptor, CD36, which initiates the antiangiogenic signal. Although not proven in gliomas, evidence suggests that expression of specific endogenous inhibitors of angiogenesis in certain organs may be part of a host antitumor response. The studies reviewed here suggest that new antiangiogenic therapies for malignant gliomas offer exciting promise as nontoxic, growth-inhibitory agents.