Expression of vascular endothelial growth factor in renal cell carcinoma and the relation to angiogenesis and p53 protein expression

BACKGROUND AND OBJECTIVES: Vascular endothelial growth factor (VEGF) seems to play an important role in tumor angiogenesis. The tumor-suppressor gene p53 has been thought to regulate VEGF expression. We investigated the effect of VEGF expression on renal cell carcinoma (RCC) and the correlation between the expression of VEGF and tumor angiogenesis and p53 protein expression. METHODS: Sixty-two RCCs were examined by immunohistochemical studies with anti-VEGF, anti-p53, and anti-CD34 antibodies. RESULTS: Forty tumors (80.6%) were classified as VEGF positive, and 28 tumors (45.2%) were positive for p53 protein. The microvessel density was 75.3 +/- 33.5. A significant correlation was found between VEGF expression and both the nuclear grade (P < 0.05) and the TNM stage (P < 0.05). The tumors with VEGF expression had a significantly higher microvessel density than those without VEGF expression (P < 0.01). There was no statistically significant correlation between p53 protein and VEGF expression. No statistically significant differences in survival were found to be associated with microvessel density, VEGF expression or p53 protein expression. By using multivariate survival analyses, nuclear grade (P < 0.05) and TNM stage (P < 0.05) were the only independent prognostic factors. CONCLUSIONS: Our data do not show a direct regulation of VEGF expression by p53. We suggest that VEGF expression plays a role in the promotion of angiogenesis in RCC.     

Relationship between vessel density and expression of vascular endothelial growth factor and basic fibroblast growth factor in small cell lung cancer in vivo and in vitro.

In 21 human small cell lung cancer (SCLC) cell lines, we determined the expression of mRNA and secreted protein levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The VEGF expression was highly variable between cell lines, with a > 100-fold variation, under identical in vitro conditions. The bFGF expression in cell lines was generally very low. Nine of the cell lines were further analyzed during growth as solid tumor xenografts in nude mice (in vivo). A more uniform VEGF protein expression was present in vivo. Compared with the variable in vitro expression, VEGF was relatively up-regulated in the tumor lines CPH 54A and CPH 54B and down-regulated in GLC 3. One line, DMS 79, had a high VEGF expression in vivo as well as in vitro. The vessel density was determined by Chalkley point counting on CD31 immunostained cryosections of tumors of each of the nine SCLC lines. We found a strong positive correlation between vessel density and tissue VEGF protein expression (r(s) = 0.75; P = 0.02) and a comparatively strong negative correlation (r(s) = -0.80; P = 0.01) between vessel density and tissue bFGF expression. No significant correlation was present between vessel density and in vitro VEGF expression. We conclude that VEGF and bFGF expression is dependent on microenvironmental conditions, as well as cell line-specific factors, and that a strong positive correlation exists between in vivo VEGF expression and vessel density, whereas high tissue levels of bFGF are not correlated with higher vessel densities in SCLC xenografts.     

Successful Combination of Sunitinib and Girentuximab in Two Renal Cell Carcinoma Animal Models: A Rationale for Combination Treatment of Patients with Advanced RCC

Anti-angiogenic treatment with tyrosine kinase inhibitors (TKI) has lead to an impressive increase in progression-free survival for patients with metastatic RCC (mRCC), but mRCC remains largely incurable. We combined sunitinib, targeting the endothelial cells with Girentuximab (monoclonal antibody cG250, recognizing carbonic anhydrase IX (CAIX) targeting the tumor cells to study the effect of sunitinib on the biodistribution of Girentuximab because combination of modalities targeting tumor vasculature and tumor cells might result in improved effect. Nude mice with human RCC xenografts (NU12, SK-RC-52) were treated orally with 0.8 mg/day sunitinib, or vehicle for 7 to 14 days. Three days before start or cessation of treatment mice were injected i.v. with 0.4 MBq/5 μg ¹¹¹In-Girentuximab followed by biodistribution studies. Immunohistochemical analyses were performed to study the tumor vasculature and CAIX expression and to confirm Girentuximab uptake.NU12 appeared to represent a sunitinib sensitive tumor: sunitinib treatment resulted in extensive necrosis and decreased microvessel density (MVD). Accumulation of Girentuximab was significantly decreased when sunitinib treatment preceded the antibody injection but remained unchanged when sunitinib followed Girentuximab injection. Cessation of therapy led to a rapid neovascularization, reminiscent of a tumor flare. SK-RC-52 appeared to represent a sunitinib-resistant tumor: (central) tumor necrosis was minimal and MVD was not affected. Sunitinib treatment resulted in increased Girentuximab uptake, regardless of the sequence of treatment. These data indicate that sunitinib can be combined with Girentuximab. Since these two modalities have different modes of action, this combination might lead to enhanced therapeutic efficacy.Abbreviations: mRCC, metastatic renal cell carcinoma; CAIX, carbonic anhydrase IX; cG250/girentuximab, chimeric monoclonal antibody G250; MVD, microvessel density; ccRCC, clear cell renal cell carcinoma; VEGF, vascular endothelial growth factor; RIT, radioimmunotherapy; TKI, tyrosine kinase inhibitor     

Expression of matrix metalloproteinase-7 on cancer cells and tissue endothelial cells in renal cell carcinoma: prognostic implications and clinical significance for invasion and metastasis.

PURPOSE: The expression of matrix metalloproteinase-7 (MMP-7) correlates with the malignant potential of various tumors and patient survival. We investigated the clinical and prognostic significance of MMP-7 expression in cancer cells and endothelial cells in human renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: We reviewed tissue samples of 156 patients with RCC who had undergone radical operation. MMP-7 expression was examined by immunohistochemistry. Sections containing MMP-7-positive vessels were also stained for CD34. The density of MMP-7-positive vessels was determined by a computer-aided image analysis system. Multivariate analysis was done to assess relevant variables for invasion, metastasis, and cause-specific survival. RESULTS: The proportion of MMP-7-expressing tumor cells were significantly higher (P < 0.001) than that of normal cells. MMP-7-positive vessels were considered blood vessels based on staining for CD34, and their density was increased in tumor areas. The proportion of MMP-7-expressing cancer cells and density of MMP-7-positive vessels correlated with grade, pathologic tumor stage, and metastasis. Multivariate analysis showed that MMP-7 expression on cancer cells correlated with pathologic tumor stage only, whereas MMP-7-positive vessel density correlated with metastasis only. The elevated status of MMP-7 in cancer tissues was an independent predictor for cause-specific survival (odds ratio, 8.61; P = 0.040) by multivariate analysis. CONCLUSIONS: Our results showed that MMP-7 influences tumor progression by regulating invasion and angiogenesis. Multivariate analysis showed that MMP-7 status of cancer tissues was strong predictor of poor prognosis. Our results suggest that MMP-7 targeting treatment may be a potential target against RCC.     

Tumor microenvironment modifications induced by soluble VEGF receptor expression in a rat liver metastasis model

Vascular endothelial growth factor is a potent pro-angiogenic growth factor which is also known to alter tumor microenvironment by inhibiting dendritic cell differentiation and promoting accumulation of myeloid-derived suppressor cells. In the present study, we analyzed the modifications induced by intratumoral expression of sFLT-1, a soluble VEGF receptor, in a rat metastatic colon carcinoma model. We generated colon cancer cell lines stably expressing sFLT-1 or a mock construct. Human umbilical vein endothelial cells cultured with conditioned medium from sFLT-1-expressing tumor cells exhibit a significantly decreased survival, demonstrating the functionality of the secreted sFLT-1. Invivo, sFLT-1 expression induced a 30% decrease in microvessel density in 15-day old experimental liver metastasis from colon carcinoma. Tumor growth was inhibited by 63% and 52% in left and right liver lobes respectively within 25 days. In these tumors, sFLT-1 expression was associated with a decreased myeloid cell infiltration and a modification in the expression of several cytokines/chemokines. Altogether, these results suggest that VEGF trapping by sFLT-1 intratumoral expression results in reduced vascularization, tumor growth inhibition and modification of immune tumor microenvironment.     

Vascular endothelial growth factor mediated angiogenic potential of pancreatic ductal carcinomas enhanced by hypoxia: an in vitro and in vivo study

Angiogenesis in pancreatic ductal adenocarcinomas depends on the presence of angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and is thought to be stimulated by hypoxia. We tested the angiogenic potential of 9 cell lines of pancreatic ductal carcinoma origin by screening mRNA and protein expression of VEGF and bFGF and the release of VEGF into culture medium under normoxic and hypoxic (5% or 0.2% O2) conditions. Angiogenic activity was determined using 2- and 3-D endothelial cell assays. Furthermore, VEGF expression and tumor vascularization were studied in human pancreatic carcinoma tissues from orthotopic xenografts and resection specimens. All cell lines expressed (mRNA, protein) and secreted VEGF, whereas bFGF was only found in 3 cell lines and was secreted into the medium in low concentrations. In addition to the dominant isoforms VEGF121,VEGF165 and VEGF189, 2 isoforms described recently, VEGF145 and VEGF183, were detected. Severe hypoxia (0.2% O2), but not moderate hypoxia (5% O2) raised VEGF mRNA expression and protein secretion in 7/9 and 5/9 cell lines, respectively. Conditioned media from 7/9, 6/9, 8/9 and 7/9 cell lines stimulated endothelial cell proliferation under normoxic (24 and 48 hr) or hypoxic (24 hr, 0.2% and 48 hr 5% O2) conditions, respectively. Conditioned media from 4/9 cell lines also induced capillary-like sprouting under normoxic conditions and from 6/9 under hypoxic (0.2% O2) conditions. In xenografted carcinoma tissues microvessel density was found not to be increased around areas of ischemic necrosis. In resected ductal carcinomas showing tumor necrosis VEGF expression and microvessel density were only increased in 3/12 and 2/13 cases, respectively. In conclusion, in vitro most pancreatic ductal carcinomas show a distinct VEGF related angiogenic potential, as demonstrated by 2- and 3-D endothelial cell proliferation, which may be promoted by severe hypoxia. Surprisingly, perinecrotic tumor areas, which are supposed to be hypoxic, only rarely showed the expected increase in microvessel density and VEGF expression.     

A strategy for antitumor vascular therapy by targeting the vascular endothelial growth factor: receptor complex

Vascular endothelial growth factor (VEGF) is produced by cancer cells in response to hypoxia and is the primary stimulant of vascularization in solid tumors. Endothelial cells lining the blood vessels of these tumors have a high concentration of receptor-bound VEGF on their surface, providing a target for antibody- directed cancer therapy. To obtain a cloned antibody to this target when bound to its receptor on tumor endothelium, we used phage display technology to create a single-chain Fv (sFv) antibody library from mice immunized with the 165-amino acid isoform of human VEGF-A. We selected, purified, and characterized LL4, an anti-VEGF sFv that was shown to react with receptor-bound VEGF. LL4 bound selectively to blood vessel endothelium, as shown by immunohistochemistry on tissue sections of human tumors. Furthermore, using autoradiography and grain counting of histological sections, systemically administered LL4 was shown to localize selectively to the endothelial lining of tumor blood vessels in human colorectal carcinoma xenografts in vivo. This study demonstrates the feasibility of targeting tumor vasculature using recombinant antibodies to the VEGF:receptor complex.     

多层螺旋CT灌注成像在肾肿瘤中的应用及与分子病理学的相关性

[目的]探讨多层螺旋CT（MSCT）灌注成像在肾肿瘤诊断中的价值。[方法]59例肾肿瘤行MSCT灌注扫描．应用perfusion2软件包计算血流量（BF）、血容量（BV）、表面通透性fPS）及平均通过时间（MTT）．同时检测肾肿瘤及肿瘤旁正常组织中血管内皮生长因子（VEGF）的表达及微血管密度（MVD）,[结果]59例肾肿瘤术后病理证实为。肾癌48例,肾盂癌6例,肾血管平滑肌脂肪瘤5例。在不同级别的肾细胞癌中．其灌注参数BF、BV和PS均明显低于正常肾皮质（P〈0．01）,且高级别的肾癌灌注参数BF、BV和PS均明显高于低级别的肾癌（P〈0．01）。肾血管平滑肌脂肪瘤和肾盂癌的灌注值均明显低于肾癌,有显著性差异（P〈0．01）。肾癌的BF、BV、PS值与肿瘤的VEGF表达呈显著正相关（P〈0．01）,MTT、值与VEGF表达呈显著负相关（P〈0．01）。[结论]MSCT灌注成像能定量评价肿瘤血管生成,血管灌注及血管通透性改变,有助于肾细胞癌的术前分级．并在肾肿瘤的鉴别诊断方面具有一定的临床应用价值。     

Renal cell carcinoma does not express argininosuccinate synthetase and is highly sensitive to arginine deprivation via arginine deiminase

Recently, pegylated arginine deiminase (ADI; EC 3.5.3.6) has been used to treat the patients with hepatocellular carcinoma or melanoma, in which the level of argininosuccinate synthetase (ASS) activity is low or undetectable. The efficacy of its antitumor activity largely depends on the level of intracellular ASS, which enables tumor cells to recycle citrulline to arginine. Thus, we examined the expression levels of ASS in various cancer cells and found that it is low in renal cell carcinoma (RCC) cells, rendering the cells highly sensitive to arginine deprivation by ADI treatment. Immunohistochemical analysis revealed that in biopsy specimens from RCC patients (n = 98), the expression of ASS is highly demonstrated in the epithelium of normal proximal tubule but not seen in tumor cells. Furthermore, RCC cells treated with ADI showed remarkable growth retardation in a dose dependent manner. ADI also exerted in vivo antiproliferative effect on the allografted renal cell carcinoma (RENCA) tumor cells and prolonged the survival of tumor-bearing mice. Histological examination of the tumors revealed that tumor angiogenesis and vascular endothelial growth factor (VEGF) expression were significantly diminished by ADI administration. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of RCC in ways of inhibitions of arginine availability and neovascularization.     

Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production

Ganglioside GD3 is overexpressed in many types of tumors and may be associated with tumor progression and the development of metastatic potential. In our previous study (G. Zeng et al., Biochemistry, 38: 8762-8769, 1999), we established a subclone of the rat dorsal root ganglion-derived F-11 cells in which the expression of ganglioside GD3 was inhibited by stable transfection of the antisense vector against CMP-NeuAc: GM3 alpha2-8 sialyltransferase (GD3-synthase) gene. This cell line exhibits markedly reduced rate of tumor growth in vivo. Here, we further characterized the antisense-transfected cell line, and the results showed that these cells formed small, minimally vascularized tumors exhibiting extensive necrosis. In vivo Matrigel assay revealed reduced vascularization and low hemoglobin content in the antisense xenografts. Significantly fewer new vessels were found on the antisense xenografts and the skin around them than those on/around the xenografts formed by the sense-transfected and untransfected F-11 cells. The hemoglobin content of the antisense xenografts was much lower than that of the xenografts formed by the control cells. The reduced angiogenesis in the antisense xenografts was correlated with a decrease in vascular endothelial growth factor (VEGF) production. The expression of VEGF was suppressed in the antisense xenografts and the conditioned culture media of the antisense-transfected F-11 cells as determined by Western blotting analysis. This was further confirmed by immunohistochemistry of the tumors using antibodies against VEGF and platelet/endothelial cell adhesion molecule (PECAM-1). Therefore, our results demonstrate that reduced tumor growth in nude mice by suppression of GD3-synthase expression in F-11 cells results from minimal angiogenesis of the tumors through down-regulation of the VEGF expression, which indicates an important role for GD3 in tumor angiogenesis.