EGFR antisense treatment of human HNSCC cell lines down-regulates VEGF expression and endothelial cell migration

Overexpression of the epidermal growth factor receptor (EGFR) is thought to play a key role in the development of head and neck squamous cell carcinoma (HNSCC) primarily through its effect on promoting uncontrolled cell proliferation. Blocking EGFR ligand binding might also inhibit angiogenesis and down-regulate the production of angiogenic factors. Angiogenesis is increased in various human tumors, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. The vascular endothelial growth factor (VEGF) is thought to be the most important angiogenic factor. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human EGFR mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell migration was measured using a modified Boyden chamber. EGFR antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense oligonucleotide control. Addition of conditioned medium from EGFR antisense-treated tumor cells resulted in decreased endothelial cell migration. In conclusion, therapeutic strategies targeting EGFR signaling in head and neck cancer might have an antitumor effect mediated in part by inhibition of tumor angiogenesis.     

Abrogation of VEGF expression in human head and neck squamous cell carcinoma decreases angiogenic activity in vitro and in vivo

Angiogenesis is increased in various human cancers, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. Vascular endothelial growth factor (VEGF) has been shown to be a key regulator of angiogenesis. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro and of HNSCC xenografts in vivo. Established human HNSCC cell lines were screened for VEGF expression at both mRNA and protein levels. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human VEGF mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell (EC) proliferation was determined by cell count and EC migration was measured using a modified Boyden chamber. Mice with HNSCC xenografts were treated with PBS, VEGF antisense or sense oligonucleotides (10 mg/kg; i.p. injection), respectively and tumor volumes were measured for 5 weeks. VEGF antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense control. Although the growth rate of the tumor cell lines was not affected, addition of conditioned medium from VEGF antisense-treated tumor cells resulted in decrease of endothelial cell proliferation and migration. VEGF antisense oligonucleotide treatment of HNSCC xenografts resulted in a significant tumor growth suppression. These results suggest that downmodulation of VEGF using antisense oligonucleotides may be a potential therapy for the inhibition of angiogenesis in HNSCC.     

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.     

Expression of the neurotrophin receptor TrkA down-regulates expression and function of angiogenic stimulators in SH-SY5Y neuroblastoma cells

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors. Mechanisms regulating the expression of angiogenic factors in tumor cells are largely unknown. High expression of the neurotrophin receptor TrkA in neuroblastomas (NBs) is associated with a favorable prognosis, whereas TrkB is mainly expressed on aggressive, MYCN-amplified NBs. To investigate the biological effects of TrkA and TrkB expression on angiogenesis in NB, we examined the expression of angiogenic factors in the human NB cell line SY5Y and its TrkA and TrkB transfectants. In comparison with parental SY5Y cells, mRNA and protein levels of the examined angiogenic factors were significantly reduced in SY5Y-TrkA cells, whereas SY5Y-TrkB cells did not demonstrate a significant change. Conditioned medium of TrkB transfectants and parental SY5Y cells induced endothelial cell proliferation and migration, but this effect was completely absent in SY5Y-TrkA cells. TrkA expression also resulted in severely impaired tumorigenicity in a mouse xenograft model and was associated with reduced angiogenic factor expression and vascularization of tumors, as determined by immunohistochemistry and an in vivo Matrigel assay. TrkA expression inhibits angiogenesis and tumor growth in SY5Y NB cells by down-regulation of angiogenic factors, whereas expression of TrkB does not down-regulate the production of these angiogenic factors. The biologically different behavior of TrkA- and TrkB-expressing NBs may be explained in part by their effects on angiogenesis.     

High-level expression of angiogenic factors is associated with advanced tumor stage in human neuroblastomas.

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors by tumor cells. Neuroblastoma (NB) is a common pediatric tumor of neural crest origin, which is biologically and clinically heterogeneous. Increased tumor vascular index correlates with poor outcome of NB. To determine which angiogenic factors contribute to NB angiogenesis and thereby support tumor progression, we examined the expression of eight angiogenic factors [vascular endothelial growth factor (VEGF), VEGF-B, VEGF-C, basic fibroblast growth factor, angiopoietin (Ang)-1, Ang-2, transforming growth factor alpha, and platelet-derived growth factor (PDGF)] by semiquantitative RT-PCR in 37 NB primary tumors and in 22 NB cell lines. We also analyzed the relationship between angiogenic factor expression and clinicopathological factors as well as patient survival. All eight angiogenic factors examined were expressed at various levels in NB cell lines and tumors, suggesting their involvement in NB angiogenesis. The expression levels of most angiogenic factors were correlated with each other, suggesting their synergy in regulating the angiogenic process. Significantly higher expression levels of VEGF, VEGF-B, VEGF-C, basic fibroblast growth factor, Ang-2, transforming growth factor alpha, and PDGF-A (P < 0.0001-0.026) were found in advanced-stage tumors (stages 3 and 4) compared with low-stage tumors (stages 1, 2, and 4S). Expression of PDGF-A was significantly associated with patient survival (P = 0.04). The redundancy in angiogenic factor expression suggests that inhibition of VEGF bioactivity alone might not be a sufficient approach for antiangiogenic therapy of human NB.     

Antiangiogenesis and damaging blood flow by antisense vascular endothelial growth factor oligodeoxynucleotides to suppress lung cancers.

Angiogenesis plays a key role in the growth and metastasis of lung cancers, and vascular endothelial growth factor (VEGF) is one of the major angiogenic factors. The study aims to investigate whether phosphoro thioate-modified antisense VEGF oligodeoxynucleo tides (ASODN) formulated in cationic liposome could inhibit the growth of Lewis lung carcinoma (LLC) tumors by antiangiogenesis. The study demonstrated that ASODN downregulated the expression of VEGF in LLC cells at levels of protein and mRNA in vitro and in vivo. The conditioned media obtained from LLC cells treated with ASODN significantly inhibited the proliferation of bovine aortic endothelial cells. The ASODN therapy significantly suppressed the growth of established subcutaneous LLC tumors in mice by inhibiting angiogenesis and damaging the blood flow of tumors. In conclusion, our results suggest that ASODN targeting VEGF presents a potent therapeutic strategy to combat lung cancers.     

Inverse correlation of apoptotic and angiogenic markers in squamous cell carcinoma of the head and neck

Angiogenesis is essential for tumour growth and metastasis. The induction of tumour vascularization is mediated by the release of angiogenic peptides. Among these factors, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) are thought to be the most important. Previous experimental studies indicate that the process of apoptosis, the programme of cell death, may be related to angiogenesis in head and neck carcinogenesis. Therefore, cryostat sections of 49 head and neck squamous cell carcinomas (HNSCC) were investigated immunohistochemically for pro-apoptotic factors caspase-3 and Fas ligand (FasL) using a standard streptavidin-biotin complex procedure. Expression of bFGF, VEGF and MMP-9 served as angiogenic markers. Additionally, intratumoral microvascular density (MVD) was counted by immunostaining of endothelial cells using anti-vWF antibody. Comparing the expression of apoptotic and angiogenic factors, a statistically significant inverse correlation of caspase-3 expression and VEGF and MMP-9 expression was found. Concerning FasL, the correlation of its expression with expression of VEGF, bFGF and MMP-9 was inversely correlated. With respect to vWF-immunostaining, statistical analysis gave a clear inverse correlation between the tumour vascularity and the expression of FasL (p = 0.0008) and caspase-3 (p = 0.0068). Our results suggest that HNSCC tumour angiogenesis contributes to a reduction of apoptosis in tumour cells. This may be explained by the activation of pro-apoptotic factors caused by hypoxia.     

抗肿瘤血管生成靶向治疗进展

血管生成是肿瘤生长和转移的关键。抗血管生成是一种癌策略,其目标是抗为活跃增殖的肿瘤细胞提供氧气和营养的新生血管。通过阻断新血管的生成抑制癌症的生长和转移。目前,最确定的抑制肿瘤血管生成的方法是阻断血管内皮生长因子（vascular endothelial growth factor,VEGF）通路。但最近,一些非VEGF因子如血小板衍生因子（platelet-derived growth factor,PDGF）、成纤维细胞生长因子（fibroblast growth factor,FGF）、肝细胞生长因子（hepatocyte growth factor,HGF）及血管生成素（angiogenin,Ang）等也参与肿瘤血管生成,强调有必要发展药物针对多种促血管生成途径。     

Regulation of the expression of pigment epithelium-derived factor,an anti-angiogenic factor in human oral squamous cell carcinoma cell lines

Angiogenesis plays an important role in tumor growth and metastasis and is regulated by a balance between angiogenic stimulators and inhibitors. We investigated the gene expression profile of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), a potent endogenous anti-angiogenic factor, in human oral squamous cell carcinoma (SCC) cell lines. The treatment of SCC cells with hypoxia increased the expression of PEDF as well as VEGF. Moreover, the treatment of SCC cells with VEGF enhanced the expression of PEDF mRNA and secretion of PEDF. In LMF-4, a SCC clone producing abundant VEGF and PEDF, the addition of neutralizing VEGF antibody substantially blocked PEDF expression. These data suggest that human oral squamous cell carcinoma cells produce VEGF, which in turn regulates PEDF production, and this balance may be contributing to neovascularization in tumors.     

Inhibition of neuroblastoma-induced angiogenesis by fenretinide.

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor- (VEGF-) and fibroblast growth factor-2- (FGF-2)-induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor-induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy-induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF- and FGF-2-specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT-PCR analysis of VEGF and FGF-2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48-72 hr of treatment with HPR, followed by a progressive accumulation of cells in G(1) at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF-2 receptor-2 was reduced after implantation of HPR-loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.     

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.     

Inhibition of vascular endothelial growth factor in the treatment of solid tumors

Vascular endothelial growth factor (VEGF) is the term used for a family of tumor-derived angiogenic factors that mediate endothelial proliferation and vascular permeability. Preclinical models have demonstrated the essential nature of VEGF in the angiogenesis of solid tumor growth and metastasis, whereas pathologic investigations have revealed strong correlations between VEGF production, microvessel density, and overall aggressiveness of many human solid tumors. Recent advances in the understanding of the molecular mechanisms of VEGF action have led to successful models for intervention in VEGF-mediated pathways in therapy for solid tumors. These include antibodies to block the binding of VEGF to its cellular receptors, small-molecule chemical inhibitors of the tyrosine kinase functions of the VEGF receptors, and antisense nucleic acids to interfere with cellular production of VEGF. Clinical investigations are ongoing to test the value of VEGF-based intervention alone or in combination with other anticancer agents.     

Antiangiogenic activity of prostate-specific antigen.

BACKGROUND: Measurement of serum levels of prostate-specific antigen (PSA) is widely used as a screening tool for prostate cancer. However, PSA is not prostate specific, having been detected in breast, lung, and uterine cancers. In one study, patients whose breast tumors had higher levels of PSA had a better prognosis than patients whose tumors had lower PSA levels. To test the hypothesis that PSA may have antiangiogenic properties, we evaluated the effects of PSA on endothelial cell proliferation, migration, and invasion, which are key steps in angiogenesis, the process by which tumors develop a blood supply. METHODS: To assess the antiproliferative effects of PSA, we treated bovine endothelial cells and human endothelial cell lines (HUVEC and HMVEC-d) with purified human PSA (0.1-10 microM) and then stimulated them with 10 ng/mL fibroblast growth factor-2 (FGF-2). Effects on FGF-2- or vascular endothelial growth factor (VEGF)-stimulated endothelial cell migration, invasion, and tube formation were measured by use of one cell line only (HUVEC). PSA was administered to mice at 9 microM for 11 consecutive days after intravenous inoculation of B16BL6 melanoma cells to assess its ability to inhibit the formation of lung colonies (i.e., metastatic tumors). RESULTS: PSA inhibited endothelial cell proliferation, migration, and invasion at IC(50) (i. e., the concentration at which inhibition was 50%) values ranging from 0.3-5 microM. In addition, PSA inhibited endothelial cell responses to both angiogenic stimulators tested, FGF-2 and VEGF. In a mouse model of metastatic disease, daily PSA treatment resulted in a 40% reduction in the mean number of lung tumor nodules compared with phosphate-buffered saline treatment (two-sided P =.003). CONCLUSION: To our knowledge, this is the first report that PSA may function in tumors as an endogenous antiangiogenic protein. This function may explain, in part, the naturally slow progression of prostate cancer. Our findings call into question various strategies to inhibit the expression of PSA in the treatment of prostate cancer.     

Antiangiogenic activity of aplidine, a new agent of marine origin

The antineoplastic compound aplidine, a new marine-derived depsipeptide, has shown preclinical activity in vitro on haematological and solid tumour cell lines. It is currently in early phase clinical trials. The exact mechanism of action of this anticancer agent still needs to be clarified. We have previously reported that aplidine blocks the secretion of the angiogenic factor vascular endothelial growth factor (VEGF) by the human leukaemia cells MOLT-4, suggesting a possible effect on tumour angiogenesis. This study was designed to investigate the antiangiogenic effect of aplidine. In vivo, in the chick embryo allantoic membrane (CAM) assay, aplidine inhibited spontaneous angiogenesis, angiogenesis elicited by exogenous VEGF and FGF-2, and induced by VEGF overexpressing 1A9 ovarian carcinoma cells. In vitro, at concentrations achievable in the plasma of patients, aplidine inhibited endothelial cell functions related to angiogenesis. It affected VEGF- and FGF-2-induced endothelial cell proliferation, inhibited cell migration and invasiveness assessed in the Boyden chamber and blocked the production of matrix metalloproteinases (MMP-2 and MMP-9) by endothelial cells. Finally, aplidine prevented the formation of capillary-like structures by endothelial cells on Matrigel. These findings indicate that aplidine has antiangiogenic activity in vivo and inhibits endothelial cell functional responses to angiogenic stimuli in vitro. This effect might contribute to the antineoplastic activity of aplidine.     

Novel angiogenesis inhibitors: addressing the issue of redundancy in the angiogenic signaling pathway

Angiogenesis, the formation of new blood vessels from established vasculature, is a fundamental process in the growth and metastasis of solid tumours. It is a complex, tightly regulated process that requires the coordinated action of antiangiogenic and proangiogenic factors, the balance of which becomes disturbed during tumour development. Vascular endothelial growth factor (VEGF) and its receptor are the key mediators of angiogenesis and targets for multiple pharmacologic agents. Many patients treated with VEGF inhibitors survive for a longer period; however, eventual resistance is associated with progressive disease and death. Multiple approaches to overcome resistance have been investigated with varying success, including the use of agents that target multiple angiogenic factors or co-administration of angiogenesis inhibitors with standard chemotherapy or radiotherapy. It would appear that the future of angiogenic inhibitors lies in the intelligent combination of multiple targeted agents with other angiogenic inhibitors, as well as more conventional therapies to maximise therapeutic effect.     

Antitumor effects of ZD6474 on head and neck squamous cell carcinoma

Angiogenesis is required for tumor growth and metastasis and, therefore, represents a target for cancer treatment. While many factors have been implicated in promoting angiogenesis, vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis. ZD6474 is a potent VEGF receptor-2 (VEGFR-2) tyrosine kinase inhibitor which also has activity against the epidermal growth factor receptor (EGFR) tyrosine kinase. The purpose of this study was to investigate the sensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines to ZD6474, and to evaluate its antitumor efficacy on HNSCC xenografts. This is the first demonstration of antitumor effects of ZD6474 on HNSCC. In vitro ZD6474 displayed antiproliferative effects on HNSCC cells and inhibition of VEGFR-2 and EGFR pathways. In vivo ZD6474 displayed antitumor activity, induced apoptosis and antiangiogenic activity on nude mice bearing an established xenograft of YCU-H891 cells. These results suggest that ZD6474 has the potential to inhibit two key pathways in tumor growth via inhibition of VEGF-dependent tumor angiogenesis and via inhibition of EGFR-dependent tumor cell proliferation.     

Expression of vascular endothelial growth factor in patients with testicular germ cell tumors as an indicator of metastatic disease

BACKGROUND: Angiogenesis is essential for tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP)/platelet-derived endothelial cell growth factor (PD-ECGF) are involved in increased angiogenic activity and disease progression in solid tumors. However, there is no information regarding the association of these angiogenic factors with clinicopathologic findings in testicular germ cell tumors (GCTs). METHODS: The authors examined the expression of VEGF and TP as well as microvessel density in GCTs and their association with clinicopathologic findings. Expression of VEGF and TP and microvessel density were examined immunohistochemically in 80 GCTs, including 33 seminomas (25 tumors with organ-confined disease and 8 with metastasis) and 47 nonseminomatous testicular GCTs (NSGCTs) (20 tumors with organ-confined disease and 27 with metastasis). Expression of VEGF also was examined in four GCTs and one nonneoplastic testis by immunoblotting. RESULTS: VEGF protein was expressed more highly in GCTs compared with nonneoplastic testes. VEGF expression in GCTs was correlated significantly with microvessel count (P < 0.001). Both VEGF expression and microvessel count were correlated with metastasis in seminoma (P = 0.008 and P < 0.001, respectively), but only VEGF expression was identified as statistically significant by multiple regression analysis (P = 0.006). Conversely, four variables (VEGF expression, microvessel count, the presence of venous invasion, and the presence of embryonal carcinoma elements in the primary tumor) were correlated with metastasis in NSGCT (P < 0.001, P < 0.001, P = 0.004, and P = 0.029, respectively). However, multiple regression analysis revealed that only VEGF expression and microvessel count were significant factors for metastasis (P < 0.007 and P < 0.001, respectively). In contrast, high levels of TP were observed in infiltrating cells, but not in the majority of cancer cells. CONCLUSIONS: The findings of the current study suggest that VEGF expression is involved in tumor development, angiogenesis, and metastasis in GCT.