Overexpression of vascular endothelial growth factor isoforms drives oxygenation and growth but not progression to glioblastoma multiforme in a human model of gliomagenesis

Vascular endothelial growth factor (VEGF) is thought to promote tumor growth and angiogenesis. Whereas VEGF is up-regulated in only a portion of anaplastic astrocytoma (AA), it is overexpressed in most glioblastoma multiforme (GBM), and the level of expression is correlated with grade of glioma. To explore the possibility that VEGF may act as a driving force in the progression of AA to GBM, the VEGF isoforms VEGF(121) and VEGF(165) were overexpressed in genetically modified, mutant H-Ras-transformed human astrocytes that on intracranial implantation form AA-like tumors. The ability of the VEGF isoforms to stimulate growth, angiogenesis, oxygenation, and the formation of necrotic GBM-like tumors was then monitored. The parental mutant H-Ras-modified astrocytes expressed four times more endogenous VEGF than normal human astrocytes, but on intracranial implantation formed hypovascular, hypoxic, small AA-like tumors. Whereas these modest levels of VEGF overexpression were insufficient to drive oxygenation and GBM formation, an additional 8-fold increase in VEGF expression mediated by retroviral infection with constructs encoding either VEGF (121) or VEGF (165) resulted in cells which, after intracranial implantation, formed tumors that were larger, more vascular, and better oxygenated than those formed by the mutant H-ras parental cells. However, the tumors formed by the cells expressing exogenous VEGF (121) or VEGF (165) retained the phenotype of AA, lacking areas of necrosis that are the hallmark of the GBM phenotype. These results suggest that whereas the VEGF(121) and VEGF(165) isoforms can contribute to glioma vascularization, oxygenation, and growth, they do not in and of themselves drive the formation of the GBM phenotype.     

p73 Overexpression increases VEGF and reduces thrombospondin-1 production: implications for tumor angiogenesis.

Tumor neovascularization is controlled by a balance between positive and negative effectors, whose production can be regulated by oncogenes and tumor suppressor genes. The aim of this study was to investigate whether the angiogenic potential of tumors could also be controlled by p73, a gene homologous to the tumor suppressor p53, whose involvement in tumor angiogenesis is known. We have studied the production of proangiogenic (VEGF, FGF-2, PIGF and PDGF) and antiangiogenic (TSP-1) factors in two p73 overexpressing clones obtained from the human ovarian carcinoma cells A2780. TSP-1 was downregulated in both clones compared to mock transfected cells, both at mRNA and protein level. Conversely, both clones showed an increased production of VEGF mRNA and protein. For both TSP-1 and VEGF, regulation of expression was partially due to modulation of the promoter activity, and was dependent on p53 status. Production of the other angiogenic factors FGF-2, PIGF and PDGF-B was also increased in p73 overexpressing clones. The two clones were more angiogenic than parental cells, as shown in vitro by their increased chemotactic activity for endothelial cells, and in vivo by the generation of more vascularized tumors. These findings suggest a potential role of p73 in tumor angiogenesis.     

Overexpression of vascular endothelial growth factor-A165 enhances tumor angiogenesis but not metastasis during beta-cell carcinogenesis.

The pivotal role of vascular endothelial growth factor (VEGF-A) in the regulation of angiogenesis, in particular in the onset and maintenance of tumor angiogenesis, has been demonstrated repeatedly in experimental model systems and, more recently, in clinical trials. Experimental evidence has also suggested that up-regulated expression of VEGF-A may cooperate with other genetic or epigenetic changes to induce or accelerate tumor progression to invasive and metastatic cancers. Here we report the generation of transgenic mouse lines that express human VEGF-A165 under the control of the rat insulin promoter in the beta cells of pancreatic islets of Langerhans (Rip1VEGF-A). These mice do not exhibit detectable changes in islet development, vascularization, or physiology. Intercrosses of these mice with a transgenic mouse model of pancreatic beta cell carcinogenesis (Rip1Tag2) result in an earlier onset of tumor angiogenesis and with it accelerated tumor growth and mortality. The transition from benign tumors (adenoma) to malignant tumors (carcinoma) is modestly accelerated; however, tumor metastases are not observed. Our findings indicate that in beta-cell tumorigenesis, overexpression of VEGF-A165 accelerates the onset of tumor angiogenesis and with it tumor progression but is not sufficient to induce tumor metastasis.     

Overexpression of SCLIP promotes growth and motility in glioblastoma cells

SCLIP, a microtubule-destabilizing phosphoprotein, is known to be involved in the development of the central nervous system (CNS). It has been well established that there are notable parallels between normal development and tumorigenesis, especially in glioma. However, no studies have examined the significance of SCLIP in gliomagenesis. To address this, we investigated the expression of SCLIP and its roles in the development of gliomas. Notably, we found that SCLIP was highly expressed in various grades of glioma samples, as compared with normal brain tissues. Overexpression of SCLIP dramatically stimulated tumor cell migration and invasion as well as proliferation and downregulation of SCLIP showed opposite effects, establishing an important oncogenic role for this gene. Furthermore, we revealed that STAT3 was required to maintain SCLIP stability, suggesting that overexpression of STAT3 may be a critical step to facilitate microtubule dynamics and subsequently promotes migration and invasion of glioma cells. Taken together, our findings demonstrate that SCLIP plays an important role in glioma pathology, and may represent a novel therapeutic strategy against human glioma.     

Inhibition of glioblastoma growth in a highly invasive nude mouse model can be achieved by targeting epidermal growth factor receptor but not vascular endothelial growth factor receptor-2

PURPOSE: Major shortcomings of traditional mouse models based on xenografted human glioblastoma cell lines are that tumor cells do not invade and that genetic alterations, such as amplification of the epidermal growth factor receptor (EGFR) gene, are not maintained. Such models are thus of limited value for preclinical studies. We established a highly invasive model to evaluate the effect of antibodies against EGFR (cetuximab) and vascular endothelial growth factor receptor-2 (antibody DC101). EXPERIMENTAL DESIGN: After short-term culture, glioblastoma spheroids were implanted into the brains of nude mice. Animals were treated either i.c. with cetuximab or i.p. with DC101. Tumor burden was determined histologically using image analysis of 36 different landmark points on serial brain sections. RESULTS: Invasive xenografts were obtained from nine different glioblastomas. Three of seven cases treated with cetuximab responded with significant tumor growth inhibition, whereas four did not. All responsive tumors were derived from glioblastomas exhibiting EGFR amplification and expression of the truncated EGFRvIII variant, which were maintained in the xenografts. All nonresponsive tumors lacked EGFR amplification and EGFRvIII expression. The proportion of apoptotic cells was increased, whereas proliferation and invasion were decreased in responsive tumors. None of four xenograft cases treated with DC101 responded to treatment, and the diffusely invading tumors grew independent of angiogenesis. CONCLUSIONS: Inhibition of glioblastoma growth and invasion can be achieved using i.c. delivery of an anti-EGFR antibody, but tumor response depends on the presence of amplified and/or mutated EGFR. Antiangiogenic treatment with DC101 is not effective against diffusely invading tumors.     

Pancreatic carcinoma cells express neuropilins and vascular endothelial growth factor, but not vascular endothelial growth factor receptors

BACKGROUND: Neuropilins (NRPs) are characterized as coreceptors of vascular endothelial growth factor (VEGF). In the current study, the authors assessed the expression of NRPs, VEGF, and vascular endothelial growth factor receptors (VEGFRs), as well as VEGF-induced cell proliferation, in pancreatic carcinoma cell lines and tissue specimens. METHODS: Human pancreatic carcinoma cell lines (Panc-1 and MIA PaCa-2), normal human pancreatic ductal epithelial cells (HPDE), and human umbilical vein endothelial cells (HUVECs) were cultured. Human pancreatic adenocarcinoma tissue specimens were also studied. Expression levels of NRPs, VEGFRs, and VEGF were determined by real-time polymerase chain reaction analysis and immunostaining. Cell proliferation was examined using a [3H]thymidine incorporation assay. RESULTS: Both NRP-1 and NRP-2 were expressed in Panc-1 cells, HPDE cells, and HUVECs but were expressed minimally in MIA PaCa-2 cells. Panc-1 expressed 30 times more NRP-1 mRNA than NRP-2 mRNA. NRP-1 levels in Panc-1 cells were 5.3 times higher than in HPDE cells but were similar to NRP-1 levels in HUVECs. NRP-2 levels in Panc-1 cells were similar to NRP-2 levels in HPDE cells but lower than NRP-2 levels in HUVECs. Expression of all three VEGFRs was observed only in HUVECs. However, VEGF mRNA was detected in all cell types except for HUVECs. NRP-1 immunoreactivity levels were much higher than NRP-2 immunoreactivity levels in Panc-1 and human pancreatic adenocarcinoma tissue specimens, whereas VEGFRs were not detected in either of these two settings. In response to VEGF165, [3H]thymidine incorporation in Panc-1 cells increased significantly (by 61%; P < 0.01). A monoclonal antibody against human NRP-1 significantly blocked VEGF-induced cell proliferation in Panc-1 cells. CONCLUSIONS: The pancreatic carcinoma cell line Panc-1 and adenocarcinoma tissue specimens expressed high levels of NRP-1 and VEGF, but not VEGFRs, and exogenous VEGF significantly increased NRP-1-mediated, but not VEGFR-mediated, Panc-1 cell proliferation. These data suggested that NRP-1 may be involved in the pathogenesis of pancreatic carcinoma.     

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.     

Transforming growth factor-[beta]1 -509T reduces risk of colorectal cancer, but not adenoma in Koreans

The proliferation of colorectal epithelial cells is regulated by various stimuli including cytokines and growth factors, thus the variants of those genes can modify the colorectal cancer risk. TGF-[beta]1 can act as both a tumor suppressor and a stimulator of tumor progression. TGF-[beta]1 C-509T polymorphism in the promoter sequence has been associated with increased levels of plasma TGF-[beta]1 in individuals with T allele. To evaluate the potential influences of this polymorphism on colorectal adenoma and cancer risk, a case-control study was conducted in Korea. A total of 646 subjects were prospectively enrolled in Seoul National University Hospital. Risk of colorectal neoplasms was evaluated separately for 244 patients with colorectal adenoma, 152 patients with colorectal cancer relative to 250 healthy controls. Genotypes were determined by the PCR-RFLP method. ORs and 95% CIs were calculated by a multivariate logistic regression analysis. The TGF-[beta]1 -509T allele containing genotypes posed a reduced risk of colorectal cancer (adjusted OR = 0.59, 95% CI = 0.28-0.92). But there was no association between this polymorphism and colorectal adenoma. Our results suggest that the TGF-[beta]1 -509T allele may have a protective role in the development of colorectal cancer, possibly consistent with its role as an inhibitor of epithelial malignant transformation.     

Prognostic but not predictive role of platelet‐derived growth factor receptors in patients with recurrent glioblastoma

Platelet‐derived growth factor receptor (PDGFR) signaling has been implicated in the pathogenesis of glioblastomas and represents a target for the tyrosine kinase inhibitor imatinib. To examine the prognostic or predictive role of PDGFRs in recurrent glioblastomas, expression was examined in tumor samples of 101 patients of CSTI571BDE40, a randomized trial comparing hydroxyurea monotherapy and a combination of hydroxyurea and imatinib. Furthermore, PDGFRα phosphorylation was investigated using in situ proximity ligation assay. PDGFRα protein was expressed in 33% of tumors and was associated with male sex, young age, presence of R132H mutated isocitrate dehydrogenase 1 protein and short median survival (142 vs. 187 days, p = 0.028). Tumor PDGFRα phosphorylation was also associated with short survival (p = 0.030). The subset of patients with PDGFRα positive glioblastoma did not have longer survival on treatment with hydroxyurea and imatinib compared with hydroxyurea monotherapy. In conclusion, both PDGFRα protein expression and phosphorylation status had a prognostic role in recurrent glioblastomas but did not define a group that showed benefit from the combination therapy consisting of hydroxyurea and imatinib.     

Expression of vascular endothelial growth factor is necessary but not sufficient for production and growth of brain metastasis

We investigated the molecular mechanisms of angiogenesis in experimental brain metastasis. Cells from six different human cancer cell lines (proven to produce visceral metastasis) were injected into the internal carotid artery of nude mice. Colon carcinoma (KM12SM) and lung adenocarcinoma (PC14PE6 and PC14Br) cells produced large, fast-growing parenchymal brain metastases, whereas lung squamous cell carcinoma (H226), renal cell carcinoma (SN12PM6), and melanoma (TXM13) cells produced only a few slow-growing brain metastases. Rapidly progressing brain metastases contained many enlarged blood vessels. The expression of VEGF mRNA and protein by the tumor cells directly correlated with angiogenesis and growth of brain metastasis. Causal evidence for the essential role of VEGF in this process was provided by transfecting PC14PE6 and KM12SM cells with antisense-VEGF165 gene, which significantly decreased the incidence of brain metastasis. In contrast, transfection of H226 human lung squamous carcinoma cells with sense-VEGF121 or sense-VEGF165 neither enhanced nor inhibited formation of brain metastases. Collectively, the results indicate that VEGF expression is necessary but not sufficient for the production of brain metastasis and that the inhibition of VEGF represents an important therapeutic target.     

Angiogenesis in cholangiocellular carcinoma: expression of vascular endothelial growth factor, angiopoietin-1/2, thrombospondin-1 and clinicopathological significance

Angiogenesis in cholangiocellular carcinoma (CCC) has rarely been investigated. The aim of this study was to determine the angiogenesis status of CCC and assess its relationship with angiogenic factors and clinicopathological characteristics. We examined 33 surgically resected CCC specimens. Tumor angiogenesis was assessed by microvessel density (MVD) using the anti-CD34 antibody, and the expression of VEGF, Ang-1, Ang-2, and TSP-1 was determined by immunohistochemistry. The mean (+/- SD) MVD was 87.2+/-52.6/mm2 (range, 0-229/mm2). A total of 75.6% cases were positive for VEGF expression, 36% for Ang-1, 57.6% for Ang-2 and 45.5% for TSP-1. VEGF and Ang-2 expression was associated with a significantly higher level of MVD (p=0.004 and 0.015, respectively). TSP-1 expression was associated with a significantly lower level of MVD (p=0.005) and a higher level of intrahepatic metastasis (46.7% vs. 5.6%, p=0.012). There was no significant correlation between VEGF, Ang-1, Ang-2, and TSP-1 expression and tumor size, capsule formation, infiltration of capsule, portal vein invasion, intrahepatic metastasis or CCC differentiation. There was no significant correlation between MVD levels, VEGF, Ang-1, Ang-2, and TSP-1 expression and postoperative survival. A considerable degree of angiogenesis, comparable to that of other solid tumors, was observed in CCC. VEGF and Ang-2 might play a proangiogenic role, and TSP-1 may play an inhibitory role in CCC. Although TSP-1 may increase intrahepatic CCC metastases, neither MVD levels nor the expression of VEGF, Ang-1, or Ang-2 was associated with clinicopathological factors and prognosis.     

Antisense-mediated reduction in thrombospondin-1 expression reduces cell motility in malignant glioma cells.

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein implicated in cancer cell adhesion, migration, invasion, inhibition of angiogenesis and activation of latent transforming growth factor-beta. The involvement of TSP-1 in the motility of malignant glioma cells was investigated by transfection of TSP-1 complementary deoxyribonucleic acid (cDNA) sense and antisense expression vectors into the glioblastoma cell line T98G-G7 that secretes high amounts of TSP-1. TSP-1 production in the 3 antisense cDNA-transfected clones was significantly reduced to 51%, 43% and 47% compared to the host T98G-G7 cells. Motility of the 3 clones was evaluated by invasion assay and compared to the motility of host T98G-G7 cells and 2 sense-transfected clones. Migration of cells was significantly reduced in the 3 antisense-transfected clones with reduced TSP-1 production to 56%, 61% and 43% compared to the host T98G-G7 cells. The host T98G-G7 and another TSP-1-secreting A172 and YMG5 glioblastoma cells were also treated with a synthetic peptide, WSHWSPWSSCSVTCG, which includes 3 consecutive sequences of the adhesion sites in the TSP-1 molecule and with a control peptide. The synthetic peptide significantly inhibited the migration of T98G-G7 and A172 cells in a dose-related manner. Maximum inhibition of migration was achieved by 100 microg/ml of the peptide and the reduction of cell motility compared to untreated cells was 34.6 % and 53.9 %, respectively. On the other hand, the inhibition of migration by the peptide was minimal in YMG5 cells, which secretes a smaller amount of TSP-1 than T98G-G7 and A172 cells. These results suggest that TSP-1 secreted by malignant glioma cells is involved in the motility of glioma cells.     

Celecoxib reduces pulmonary inflammation but not lung tumorigenesis in mice

Cyclooxygenase (COX) enzyme expression is elevated in human and rodent lung tumors, and non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin reduce lung tumor formation in mice. These observations, along with the well-characterized protection that NSAID treatment engenders for colon cancer, have prompted clinical trials testing whether celecoxib, a COX-2-specific inhibitor, can prevent lung cancer development in populations at high risk. Protection by celecoxib in murine models of pulmonary inflammation and lung tumorigenesis has not yet been evaluated, however, and we now report such studies. Chronic administration of butylated hydroxytoluene (BHT) to mice stimulates pulmonary inflammation characterized by vascular leakage and macrophage infiltration into the air spaces, increased PGE2 production, and translocation of 5-lipoxygenase (5-LO) from the cytosol to the particulate fraction. Dietary celecoxib limited macrophage infiltration, abrogated PGE2 production and reduced particulate 5-LO content. Celecoxib and aspirin were ineffective at preventing lung tumorigenesis in a two-stage carcinogenesis protocol in which 3-methylcholanthrene administration is followed by chronic BHT. Celecoxib also did not reduce the multiplicity of lung tumors after induction by urethane; lung tumors in celecoxib-treated mice were larger than those in mice that did not receive celecoxib. Tumors induced in celecoxib-fed mice contained 60% less PGE2 than tumors in mice fed control diets, so reducing lung PGE2 levels was insufficient to prevent lung tumor formation. As the production of eicosanoids in addition to PGE2 is also inhibited by celecoxib, and as celecoxib has COX-independent interactions, its effects on tumor formation may vary in different organ systems.     

Anti-VEGF antibody treatment of glioblastoma prolongs survival but results in increased vascular cooption

Vascular endothelial growth factor (VEGF) is an important mediator of the intense angiogenesis which is characteristic of glioblastoma. While genetic manipulation of VEGF/VEGF receptor expression has previously been shown to inhibit glioblastoma growth, to date, no study has examined the efficacy of pharmacologic blockade of VEGF activity as a means to inhibit intracranial growth of human glioblastoma. Using intraperitoneal administration of a neutralizing anti-VEGF antibody, we demonstrate that inhibition of VEGF significantly prolongs survival in athymic rats inoculated in the basal ganglia with G55 human glioblastoma cells. Systemic anti-VEGF inhibition causes decreased tumor vascularity as well as a marked increase in tumor cell apoptosis in intracranial tumors. Although intracranial glioblastoma tumors grow more slowly as a consequence of anti-VEGF treatment, the histologic pattern of growth suggests that these tumors adapt to inhibition of angiogenesis by increased infiltration and cooption of the host vasculature.     

Overexpression and immunosuppressive functions of transforming growth factor 1, vascular endothelial growth factor and interleukin-10 in epithelial ovarian cancer

Objective: Transforming growth factor-1 (TGF-β1), vascular endothelial growth factor (VEGF), and interleukin-10 (IL-10) may be critical cytokines in the microenvironment of a tumor, playing roles in immune suppression. This study was conducted to elucidate the roles and immunosuppressive functions of these cytokines in epithelial ovarian cancer (EOC). Methods: The expression levels of TGF-β1, VEGF and IL-10 in malignant tissue were evaluated by immune-histochemistry and compared with corresponding borderline, benign, and tumor-free tissues. Moreover, relationships among the levels of these cytokines and correlations between expression and the prognosis of EOC were analyzed by Pearson rank correlations and multi-factor Logistic regression. The roles of TGF-β1, VEGF, and IL-10 in the immunosuppressive microenvironment of ovarian cancer were studied through dendritic cell (DC) maturation and CD4+CD25+FoxP3+Treg generation in vitro experiments. Results: TGF-β1, VEGF, and IL-10 were expressed in 100%, 74.69%, and 54.96% of EOC patients, respectively. TGF-β1 was an independent prognostic factor for EOC. IL-10 was significantly co-expressed with VEGF. In vitro, VEGF and TGF-β1 strongly interfered with DC maturation and consequently led to immature DCs, which secreted high levels of IL-10 that accumulated around the tumor site. TGF-β1 and IL-10 induced Treg generation without antigen presentation in DCs. Conclusions: TGF-β1, VEGF and IL-10 play important roles in EOC and can lead to frequent immune evasion events.     

Overexpression of c-met proto-oncogene but not epidermal growth factor receptor or c-erbB-2 in primary human colorectal carcinomas.

The epidermal growth factor receptor (EGFR) and the protein products of c-erbB-2 and c-met proto-oncogenes belong to a family of growth factor receptors with tyrosine kinase activity. In human colonic carcinomas, the expression of the EGFR and c-erbB-2 have been studied at the protein level only, while c-met expression has not been reported. We have examined the mRNA expression of these genes in human normal colorectal mucosa and primary carcinomas. The results demonstrate that the normal mucosa shows highly variable levels of EGFR and c-erbB-2 mRNAs, but expresses consistently low amounts of c-met mRNA. Colorectal carcinomas did not express significantly higher levels of the EGFR and c-erbB-2 mRNAs than the normal mucosa. In contrast, c-met was consistently and significantly overexpressed (mean sixfold) in carcinomas as compared with normal mucosa. Seventy percent of paired normal-tumor specimens showed a tumor to normal c-met mRNA ratio of greater than 4. The expression of c-met mRNA was also enhanced in the adenomas, suggesting that over-expression of this proto-oncogene may have mechanistic significance in the early stages of human colorectal carcinogenesis.     

Heterogeneity of glioblastoma with gliomatosis cerebri growth pattern on diffusion and perfusion MRI

Journal of Neuro-Oncology - Gliomatosis cerebri (GC) is a rare growth pattern of glioblastoma whose diffuse nature is reflected by unspecific, relatively uniform findings on conventional MRI. In...     

Significance of tumour-associated macrophages, vascular endothelial growth factor and thrombospondin-1 expression for tumour angiogenesis and prognosis in endometrial carcinomas.

Angiogenesis is a key process in tumour growth and metastasis, and microvessel density has been found to influence the prognosis of endometrial carcinoma patients. Less is known about regulators of angiogenesis. Studies of other tumour types have indicated that the density of tumour-associated macrophages (TAMs) and the expression of vascular endothelial growth factor (VEGF) might stimulate vessel formation, whereas thrombospondin-1 (TSP-1) may inhibit this process. We investigated the influence of TAM (CD68+), VEGF and TSP-1 expression on tumour vascular density and prognosis among endometrial carcinoma patients and compared our findings with clinico-pathological variables and tumour markers. In a prospective study, 60 endometrial carcinoma patients with long (median 11 years) and complete follow-up were included. Intratumour density of TAMs was significantly associated with FIGO stage, histological type, histological grade, DNA index, estradiol receptor concentration, intratumour Ki-67 and p53 protein expression (all p < 0.05). Moderate or strong expression of VEGF was significantly associated with serous papillary/clear cell tumour types, high microvessel density and aneuploidy (p < 0.05). There was a tendency to strong TSP-1 expression among tumours with weak VEGF expression (p=0.09). TAM density influenced survival significantly in univariate survival analysis (Kaplan-Meier method, p<0.05) in contrast to VEGF and TSP-1 expression. In Cox regression analysis, however, no independent prognostic impact remained. In conclusion, moderate or strong VEGF expression was significantly associated with high microvessel density and TAM count was increased in a subgroup of aggressive tumours. High TAM density was significantly associated with reduced survival in univariate analysis.