Role of nitric oxide in angiogenesis and microcirculation in tumors

Nitric oxide (NO) is a free radical molecule with high reactivity, a short half life and a variety of physiological activities. The role of NO in tumor microcirculation, based on the data collected to date, can be summarized as follows: 1) NO may partially mediate tumor angiogenesis; 2) endogenous NO derived from tumor vascular endothelium and/or tumor cells increases and/or maintains tumor blood flow via dilatation of arteriolar vessels, decreases leukocyte-endothelial interaction, and increases vascular permeability; 3) exogenous NO can increase tumor blood flow via vessel dilatation, and reduce vessel tone; and 4) NO production rates and vascular response to NO are heterogeneous and tumor-dependent. Modulation of NO level in tumor vessels can alter tumor hemodynamics and thus augment oxygen, drug, gene vector and effector cell delivery to solid tumors.     

Expression of inducible nitric oxide synthase is significantly correlated with expression of vascular endothelial growth factor and dendritic cell infiltration in patients with advanced gastric carcinoma

OBJECTIVE: Nitric oxide (NO) is a product of L-arginine to L-citrulline conversion by nitric oxide synthase (NOS). The inducible form of NOS (iNOS) is one of three classes of NOS and the strongest producer of NO. It has been reported that NO correlates with angiogenesis and immune responses in some types of cancer, however, the correlations between iNOS expression, angiogenesis, and immune responses are still unclear in gastric carcinoma. METHODS: iNOS expression was determined in 135 gastric cancer patients by immunohistochemical procedures and compared with the expression of vascular endothelial growth factor (VEGF), microvessel (MV) density, and dendritic cell (DC) infiltration to evaluate the effect of iNOS on angiogenesis and immune responses in gastric carcinoma. RESULTS: iNOS expression was detected in 106 (78.5%) of 135 cases. There was a close correlation between iNOS expression and VEGF expression, a correlation with MV density and an inverse correlation with DC infiltration. There was no correlation between iNOS and p53 expression. The prognoses of patients whose tumors expressed iNOS were significantly worse than those of patients whose tumors did not express iNOS. Multivariate analysis indicated iNOS expression was an independent prognostic factor. CONCLUSION: iNOS might be associated with tumor progression by stimulating angiogenesis and suppressing immune responses in gastric carcinoma.     

Nitric Oxide and Angiogenesis

The steps required for new vessel growth are biologically complex and require coordinate regulation of contributing components, including modifications of cell–cell interactions, proliferation and migration of endothelial cells and matrix degradation. The observation that in vivo angiogenesis is accompanied by vasodilation, that many angiogenesis effectors possess vasodilating properties and that tumor vasculature is in a persistent state of vasodilation, support the existence of a molecular/biochemical link between vasodilation and angiogenesis. Several pieces of evidence converge in the indication of a role for nitric oxide (NO), the factor responsible for vasodilation, in physiological and pathological angiogenesis. Data originated in different labs indicate that NO can act both as an 'actor' of angiogenesis and as a 'director of angiogenesis', both functions being equally expressed during physiological and pathological processes. NO significantly contributes to the prosurvival/proangiogenic program of capillary endothelium by triggering and transducing cell growth and differentiation via endothelial-constitutive NO synthase (ec-NOS) activation, cyclic GMP (cGMP) elevation, mitogen activated kinase (MAPK) activation and fibroblast growth factor-2 (FGF-2) expression. Re-establishment of a balanced NO production in the central nervous system results in a reduction of cell damage during inflammatory and vascular diseases. Elevation of NOS activity in correlation with angiogenesis and tumor progression has been extensively reported in experimental and human tumors. In the brain, tumor expansion and edema formation are sensitive to NOS inhibition. On this basis, the nitric oxide pathway appears to be a promising target for consideration in pro- and anti-angiogenic therapeutic strategies. The use of NOS inhibitors seems appropriate to reduce edema, block angiogenesis and facilitate antitumor drug delivery.     

Nitric oxide and its gatekeeper thrombospondin-1 in tumor angiogenesis.

Nitric oxide (NO) plays a central role in angiogenesis as a mediator of signaling by vascular endothelial growth factor and other angiogenic factors. Low concentrations of NO produced in response to angiogenic factors stimulate angiogenesis, whereas higher concentrations typical of inflammatory responses inhibit angiogenesis. The proangiogenic activity of NO is mediated by activation of soluble guanylyl cyclase, leading to cyclic guanosine 3',5'-monophosphate accumulation and activation of its target kinases and ion channels. The four angiogenesis inhibitors currently approved for clinical use target components of the signaling cascade upstream of NO. New research has identified components downstream of NO as the primary target of the endogenous angiogenesis inhibitor thrombospondin-1 and has shown that circulating levels of thrombospondin-1 are sufficient to limit angiogenic responses by antagonizing NO signaling. This provides new insights into the significance of the widespread loss of thrombospondin-1 expression during malignant progression. Although clinical trials suggest that blocking NO signaling can inhibit tumor angiogenesis, this approach also inactivates inhibitory signaling from thrombospondin-1. We discuss the implications of the balance between these pathways for applying thrombospondin-1 mimetics and redox modifiers as cancer therapeutics.     

Causes and effects of heterogeneous perfusion in tumors

A characteristic of solid tumors is their heterogeneous distribution of blood flow, with significant hypoxia and acidity in low-flow regions. We review effects of heterogeneous tumor perfusion are reviewed and propose a conceptual model for its cause. Hypoxic-acidic regions are resistant to chemo- and radiotherapy and may stimulate progression to a more metastatic phenotype. In normal tissues, hypoxia and acidity induce angiogenesis, which is expected to improve perfusion. However, aggressive tumors can have high local microvessel density simultaneously with significant regions of hypoxia and acidosis. A possible explanation for this apparent contradiction is that the mechanisms regulating growth and adaptation of vascular networks are impaired. According to a recent theory for structural adaptation of vascular networks, four interrelated adaptive responses can work as a self-regulating system to produce a mature and efficient blood distribution system in normal tissues. It is proposed that heterogeneous perfusion in tumors may result from perturbation of this system. Angiogenesis may increase perfusion heterogeneity in tumors by increasing the disparity between parallel low- and high-resistance flow pathways. This conceptual model provides a basis for future rational therapies. For example, it indicates that selective destruction of tumor vasculature may increase perfusion efficiency and improve therapeutic efficacy.     

Thrombospondin 1 promotes tumor macrophage recruitment and enhances tumor cell cytotoxicity of differentiated U937 cells

Inhibition of tumor growth by thrombospondin (TSP) 1 is generally attributed to its antiangiogenic activity, but effects on tumor immunity should also be considered. We show that overexpression of TSP1 in melanoma cells increases macrophage recruitment into xenograft tumors grown in nude or beige/nude mice. In vitro, TSP1 acutely induces expression of plasminogen activator inhibitor-1 (PAI-1) by monocytic cells, suggesting that TSP1-induced macrophage recruitment is at least partially mediated by PAI-1. Tumor-associated macrophages (TAM) can either promote or limit tumor progression. The percentage of M1-polarized macrophages expressing inducible nitric oxide synthase is increased in TSP1-expressing tumors. Furthermore, soluble TSP1 stimulates killing of breast carcinoma and melanoma cells by IFN-gamma-differentiated U937 cells in vitro via release of reactive oxygen species. TSP1 causes a significant increase in phorbol ester-mediated superoxide generation from differentiated monocytes by interaction with alpha(6)beta(1) integrin through its NH(2)-terminal region. The NH(2)-terminal domain of TSP2 also stimulates monocyte superoxide production. Extracellular calcium is required for the TSP1-induced macrophage respiratory burst. Thus, TSP1 may play an important role in antitumor immunity by enhancing recruitment and activation of M1 TAMs, which provides an additional selective pressure for loss of TSP1 and TSP2 expression during tumor progression.     

Ionizing radiation inhibits tumor neovascularization by inducing ineffective angiogenesis

The vascular effects of ionizing radiation were examined in K1735 murine melanoma tumors. Single-fraction and fractionated radiation virtually arrested growth of these tumors for about a week, after which they resumed more rapid growth. Tumor microvessel density (MVD) and blood perfusion was unchanged seven days after radiation but decreased at later time points after irradiation, when they had grown 10-fold or more. Together with the finding of severe tumor hypoxia and VEGF induction in the latter tumors, the evidence pointed to vascular insufficiency and inhibited neovascularization in tumors that had grown substantially after radiation. Endothelial cell (EC) death detected by TUNEL staining only transiently increased the day following radiation, whereas EC proliferation detected by Ki-67 staining was increased in irradiated tumors that had grown substantially. The fact that increased EC proliferative activity produced fewer vessels suggests that angiogenesis is defective or ineffective after radiation. These results complement recent genetic evidence that EC damage from radiation plays a major role in tissue damage and antitumor efficacy to highlight the importance of EC and vasculature in radiation response. Our studies further show that radiation impact on tumor vasculature extends beyond near-term induction of EC death to more prolonged effects on their ability to support angiogenesis.     

Interstitial fluid pressure in cervical cancer: guide to targeted therapy

Interstitial fluid pressure (IFP) is elevated in most malignant tumors, mainly as a result of the abnormal tumor vasculature that develops from unregulated angiogenesis. Theoretical models predict that IFP should correlate with capillary flow resistance in tumors, and therefore also with perfusion and oxygenation. However, a prospective clinical study in patients with cervical cancer at Princess Margaret Hospital failed to demonstrate a relationship between IFP and oxygenation. Despite this, high IFP was strongly associated with inferior survival after radiotherapy independent of clinical prognostic factors and tumor oxygen status. This suggests that IFP and direct needle oxygen measurements may provide information about different aspects of tumor oxygenation, such as chronic versus intermittent hypoxia. Alternatively, IFP may reflect an aspect of tumor biology that is largely unrelated to perfusion and oxygenation. One possibility is that tumors with high pretreatment angiogenesis levels, as indicated by high IFP, may be more radioresistant because the vascular endothelium is more likely to survive during and after treatment. The mechanistic link between elevated IFP and the abnormal tumor vasculature and the strong prognostic effect of IFP in our cervix study together suggest that drugs targeted at angiogenesis, when combined with radiotherapy, may lead to improved tumor control and patient survival.     

Vascularization of melanoma by mobilization and remodeling of preexisting latent vessels to patency

Tumors must manipulate the host vasculature to provide a blood supply adequate for their proliferation. Although tumors may arise as avascular masses, there is increasing evidence that some tumors begin to proliferate by first co-opting preexisting host blood vessels. By fluorescent vascular imaging, we provide evidence that the vasculature in orthotopically implanted melanoma arises from a preexisting red cell-deficient vascular network that remodels to patency to accommodate the requirements of the expanding tumor mass. Topical application of vascular endothelial growth factor to vascular beds generated immediate and robust vascular transitions that were morphologically similar to tumor-induced transitions. N(phi)-nitro-L-arginine, a nitric oxide inhibitor, significantly inhibited the growth of a syngeneic K1735M2 melanoma by reducing blood supply to the tumor by a mechanism independent of endothelial cell proliferation. These findings suggest that tumor-induced remodeling of red cell-deficient vessels to patency contributes to tumor vascularization and growth.     

Use of fluorine-19 nuclear magnetic resonance spectroscopy and hydralazine for measuring dynamic changes in blood perfusion volume in tumors in mice.

BACKGROUND: One method of evaluating the mechanism of action of agents which alter tumor oxygenation is to determine their effects on tumor blood flow. PURPOSE: This study tests applicability of a new approach using an emulsion of the inert fluorocarbon perfluorooctylbromide (PFOB) at nontoxic doses as a tracer in fluorine-19 (19F) nuclear magnetic resonance (NMR) spectroscopy to evaluate dynamic changes in vascular perfusion volume in transplanted tumors. METHODS: The PFOB emulsion (100% wt/vol) was injected into the tail vein in tumor-bearing C3H/He or nu/nu mice immobilized in a magnet interfaced to a spectrometer, either as a single bolus injection of 8 mL/kg body weight or in multiple injections to a total dose of 24 mL/kg. A 7-mm external surface coil was placed over the tumor. Signal from the PFOB in the tumor volume seen by the coil rapidly reached equilibrium and was maintained for at least 2 hours, and multiple doses of PFOB emulsion resulted in a linear increase in 19F signal strength. Since the 19F signal strength was directly proportional to the perfusion volume of the tumor vasculature, reduction of signal intensity should correspond directly to any reduction in volume caused by a change in the tumor blood flow. To investigate this hypothesis, the vasoactive agent hydralazine (5 mg/kg) was injected intravenously after administering the PFOB emulsion to induce changes in tumor blood supply. KHT and RIF-1 murine sarcomas, the HT29 human colon carcinoma, and the HX118 human melanoma tumors were studied. In a comparative analysis of changes in blood flow induced by hydralazine, we studied Xe-133 clearance in KHT murine sarcoma and SCCVII/Ha (SCCVII) murine squamous cell carcinoma. RESULTS: Hydralazine significantly reduced the 19F signal intensity in the murine tumors RIF-1 and KHT and in the HT29 human tumor, with little reduction in the SCCVII/Ha murine and HX118 human tumors. Hydralazine induced a statistically significant 64% decrease in mean clearance rate in the KHT tumor, while SCCVII/Ha tumors showed no significant change, indicating that hydralazine restricted blood flow to a greater extent in the tumor type that showed reduced 19F signal from the PFOB emulsion. CONCLUSION: These data demonstrate the potential of PFOB emulsion as a tracer in NMR spectroscopy for studying tumor vasculature.     

Role of nitric oxide in tumor progression: Lessons from experimental tumors

Nitric oxide (NO), a potent biological mediator, plays a key role in physiological as well as pathological processes, including inflammation and cancer. The role of NO in tumor biology remains incompletely understood. While a few reports indicate that the presence of NO in tumor cells or their microenvironment is detrimental to tumor cell survival and consequently their metastatic ability, a large body of clinical and experimental data suggest a promoting role of NO in tumor progression and metastasis. We suggest that tumor cells capable of very high levels of NO production die in vivo, and those producing or exposed to lower levels of NO, or capable of resisting NO-mediated injury undergo a clonal selection because of their survival advantage; they also utilize certain NO-mediated mechanisms for promotion of growth, invasion and metastasis. The possible mechanism(s) are: (a) a stimulatory effect on tumor cell invasiveness, (b) a promotion of tumor angiogenesis and blood flow in the tumor neovasculature, and (c) a suppression of host anti-tumor defense. In this review, we discuss these mechanisms on the basis of data derived from experimental models, in particular, a mouse mammary tumor model in which the expression of eNOS by tumor cells is positively correlated with invasive and metastatic abilities. Tumor-derived NO was shown to promote tumor cell invasiveness and angiogenesis. The invasion-stimulating effects of NO were due to an upregulation of matrix metalloproteases and a downregulation of their natural inhibitors. Treatment of tumor-bearing mice with NO-blocking agents reduced the growth and vascularity of primary tumors and their spontaneous metastases. We propose that selected NO-blocking drugs may be useful in treating certain human cancers either as single agents or as a part of combination therapies.     

Overexpression of Notch ligand Dll1 in B16 melanoma cells leads to reduced tumor growth due to attenuated vascularization

Notch signaling plays an important role in vascular development and tumor angiogenesis. It has been shown that disruption of Dll4-triggered Notch signal activation effectively inhibits tumor growth, but this treatment also results in the formation of vascular neoplasms. In this study, we investigate the effects of over-expressing Notch ligand Dll1 in B16 melanoma cells on tumor cell proliferation and tumor growth in vitro and in vivo. Our results showed that over-expression of Dll1 could activate Notch signaling in tumor cells, and promote tumor cell proliferation in vitro. In contrast, growth of Dll1-over-expressing tumors in vivo was reduced, due to abnormal tumor vessel formation. Impaired tumor vasculature enhanced hypoxia and necrosis in tumor tissues, leading to retarded tumor growth. These results suggest that activation of Notch signaling may serve as an anti-angiogenesis strategy in the treatment of malignant tumors.     

Evidence for the involvement of ET<Subscript>B</Subscript> receptors in ET-1-induced changes in blood flow to the rat breast tumor

PURPOSE: Structure, growth, and function of the blood vessels in breast tumors are markedly different from those in normal breast tissue due to changes in the production of growth factors such as vascular endothelial growth factor (VEGF), vasoactive substances such as endothelin-1 (ET-1) and cytokines. The role of ET-1 in breast tumor angiogenesis is not adequately understood. Studies have shown that the expression of proET-1, proET-3, and ET(B) receptors is increased in breast tumor. However, it is unclear whether there are any changes in ET-1-induced vascular responses in breast tumor. Hence, in the present study we investigated systemic hemodynamics and regional circulatory effects of ET-1 in rats with breast tumors. METHODS: Female Sprague-Dawley rats weighing 180-200 g were divided into the following groups: (1) normal rats treated with saline ( n=6), (2) tumor-bearing rats treated with methylnitrosourea (MNU) ( n=6), (3) normal rats treated with saline plus the specific ET(B) receptor antagonist BQ 788 ( n=5), and (4) tumor-bearing rats treated with MNU plus BQ 788 ( n=5). Tumor development was monitored by regular palpation and measurement of tumor size. Once tumors had reached approximately 2-4 cm in diameter, the rats were anesthetized with urethane (1.5 g/kg i.p.) and their cardiovascular parameters were measured using a radioactive microsphere technique. Simultaneously, blood perfusion to the breast tissue was also measured using a laser Doppler technique. RESULTS: ET-1 produced a significant increase in mean arterial pressure in normal and tumor-bearing rats. Blood flow to the tumor tissue increased significantly in response to ET-1 as compared to breast tissue in normal rats. This response was accompanied by a concomitant decrease in vascular resistance in the tumor tissue. These results were confirmed by laser Doppler flowmetry, which showed a significant increase in blood perfusion to breast tumor compared to normal breast tissue. This increase in blood perfusion was attenuated by pretreatment with BQ 788, suggesting an ET(B) receptor-mediated vasodilator action of ET-1 in rat breast tumor. CONCLUSIONS: The results indicate that ET-1 induced an increase in blood flow to breast tumor tissue mediated through ET(B) receptors.     

SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth

In the past few years, several laboratories have developed antiangiogenic molecules that starve tumors by targeting their vasculature and we have shown that, when produced in tumors, the antiangiogenic molecule thrombospondin-1 (TSP1) reduces the vascularization and delays tumor onset. Yet over time, tumor cells producing active TSP1 do eventually form exponentially growing tumors. These tumors are composed of cells secreting unusually high amounts of the angiogenic stimulator vascular endothelial growth factor (VEGF) that are sufficient to overcome the inhibitory TSP1. Here, we use short double-stranded RNA (siRNA) to trigger RNA interference and thereby impair the synthesis of VEGF and ask if this inability to produce VEGF prevents the development of TSP1 resistance. Systemic in vivo administration of crude anti-VEGF siRNA reduced the growth of unaltered fibrosarcoma tumor cells, and when the anti-VEGF siRNA was expressed from tumor cells themselves, such inhibition was synergistic with the inhibitory effects derived from TSP1 secretion by the tumor cells. Anti-VEGF siRNA delayed the emergence of TSP1-resistant tumors and strikingly reduced their subsequent growth rate.     

Effects of the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 on the tumor vasculature: implications for clinical imaging

Dysregulated angiogenesis and high tumor vasculature permeability, two vascular endothelial growth factor (VEGF)-mediated processes and hallmarks of human tumors, are in part phosphatidylinositol 3-kinase (PI3K) dependent. NVP-BEZ235, a dual PI3K/mammalian target of rapamycin (mTOR) inhibitor, was found to potently inhibit VEGF-induced cell proliferation and survival in vitro and VEGF-induced angiogenesis in vivo as shown with s.c. VEGF-impregnated agar chambers. Moreover, the compound strongly inhibited microvessel permeability both in normal tissue and in BN472 mammary carcinoma grown orthotopically in syngeneic rats. Similarly, tumor interstitial fluid pressure, a phenomenon that is also dependent of tumor permeability, was significantly reduced by NVP-BEZ235 in a dose-dependent manner on p.o. administration. Because RAD001, a specific mTOR allosteric inhibitor, was ineffective in the preceding experiments, we concluded that the effects observed for NVP-BEZ235 are in part driven by PI3K target modulation. Hence, tumor vasculature reduction was correlated with full blockade of endothelial nitric oxide (NO) synthase, a PI3K/Akt-dependent but mTORC1-independent effector involved in tumor permeability through NO production. In the BN472 tumor model, early reduction of permeability, as detected by K(trans) quantification using the dynamic contrast-enhanced magnetic resonance imaging contrasting agent P792 (Vistarem), was found to be a predictive marker for late-stage antitumor activity by NVP-BEZ235.     

The effects of tumor-derived platelet-derived growth factor on vascular morphology and function in vivo revealed by susceptibility MRI

Platelet-derived growth factors (PDGF) play a major role in pericyte recruitment in tumor capillaries. Pericytes are required for proper vessel development, and contribute to tumor angiogenesis by promoting stabilization and maturation of newly formed vessels. To investigate the effects of pericyte coverage on tumor vessel morphology and function in vivo, tumors derived from B16 melanoma cells transfected with either control plasmid (B16/ctr) or plasmid encoding full-length PDGF-BB (B16/PDGF), the latter previously shown to have enhanced blood vessel pericyte coverage and an increased tumor growth rate, were assessed using histopathological methods, Hoechst 33342-based perfusion analyses, and two noninvasive susceptibility magnetic resonance imaging (MRI) methods. Susceptibility-contrast MRI, incorporating the use of ultrasmall superparamagnetic iron oxide particles, revealed a significant (p < 0.05) reduction in vessel size index (R(v)) of B16/PDGF tumors, and which was validated histologically by the presence of significantly smaller (p < 0.001), more punctate blood vessels identified by fluorescence microscopy of the perfusion marker Hoechst 33342. Intrinsic-susceptibility MRI was used to measure the transverse MRI relaxation rate R(2)*, sensitive to changes in endogenous paramagnetic [deoxyhaemoglobin], and used to probe for vascular maturation and function. Hypercapnia (5% CO(2)/95% air) induced a negligible Delta R(2)* response in the B16/ctr and B16/PDGF tumors. In contrast, hyperoxia (5% CO(2)/95% O(2)) induced a significantly greater R(2)* reduction in the B16/PDGF tumors (p < 0.02). Together the susceptibility MRI-derived biomarkers reveal novel pericyte-dependent changes in the morphology and function of the perfused tumor vasculature in vivo.     

Nitric oxide synthase II gene disruption: implications for tumor growth and vascular endothelial growth factor production

The expression of a primary initiator of tumor angiogenic responses, vascular endothelial growth factor (VEGF), may be induced by nitric oxide (NO) in carcinoma cells. However, the net impact of NO on carcinogenesis remains unclear, because manipulation of NO levels has been shown to either stimulate or inhibit tumor growth. We have investigated the relationship between inducible NO synthase (NOS II), VEGF expression, and growth of B16-F1 melanoma over 14 days in wild-type (NOS II+/+) mice and in those in which the gene for NOS II has been deleted (NOS II-/-). B16-F1 tumor growth was measured as wet weight of the excised tissue. Tumor NOS II and VEGF localization were evaluated by immunohistochemistry, and VEGF mRNA levels were measured by Northern blot analysis. In NOS II+/+ mice inoculated with B16-F1 melanoma cells, macroscopic tumors were always observed at 14 days; however, 22% of NOS II-/- mice had no detectable tumor mass. Immunoreactive NOS II was detected in tumor cells of tumors grown in NOS II+/+ but not in NOS II-/- mice. Although immunoreactive VEGF was detected in the granules of tumor-associated mast cells from both NOS II+/+ and NOS II-/- mice, VEGF mRNA expression in tumors from NOS II-/- was half that in NOS II+/+ mice. Neither NOS II inhibition, exogenous NO, nor peroxynitrite influenced DNA synthesis in culture B16-F1 melanoma cells. The NO donor did not alter either VEGF mRNA levels or degranulation in cultures of the mast cell line RBL-2H3, but peroxynitrite increased both VEGF mRNA expression and degranulation. We conclude that host expression of NOS II contributes to induction of NOS II in the tumor and to melanoma growth in vivo, possibly by regulating the amount and availability of VEGF.     

Thrombospondin-1 triggers cell migration and development of advanced prostate tumors

The antitumor effects of pharmacologic inhibitors of angiogenesis are hampered in patients by the rapid development of tumor resistance, notably through increased invasiveness and accelerated metastasis. Here, we reevaluated the role of the endogenous antiangiogenic thrombospondin 1 (TSP1) in prostate carcinomas in which angiogenesis is an active process. In xenografted tumors, we observed that TSP1 altogether inhibited angiogenesis and fostered tumor development. Our results show that TSP1 is a potent stimulator of prostate tumor cell migration. This effect required CD36, which also mediates TSP1 antiangiogenic activity, and was mimicked by an antiangiogenic TSP1-derived peptide. As suspected for pharmacologic inhibitors of angiogenesis, the TSP1 capacities to increase hypoxia and to trigger cell migration are thus inherently linked. Importantly, although antiangiogenic TSP1 increases hypoxia in vivo, our data show that, in turn, hypoxia induced TSP1, thus generating a vicious circle in prostate tumors. In radical prostatectomy specimens, we found TSP1 expression significantly associated with invasive tumors and with tumors which eventually recurred. TSP1 may thus help select patients at risk of prostate-specific antigen relapse. Together, the data suggest that intratumor disruption of the hypoxic cycle through TSP1 silencing will limit tumor invasion.