All angiogenesis is not the same: Distinct patterns of response to antiangiogenic therapy in experimental neuroblastoma and Wilms tumor

BACKGROUND/PURPOSE: Neuroblastoma and Wilms tumor exhibit different patterns of metastasis, invasion, and therapeutic response. Vascular endothelial growth factor (VEGF) is an angiogenic factor expressed in both tumors. The authors hypothesized that because the clinical behavior of these tumors differs, the response to anti-VEGF therapy would be distinct, and tumor vascular architectures would reflect this distinction. METHODS: Xenografts were induced by intrarenal injection of cultured cells in athymic mice. After 1 week, anti-VEGF antibody or vehicle were administered for 5 weeks before sacrifice. Additional animals were maintained for 3 weeks after termination of antibody injections to assess rebound growth of tumors. Fluorescein angiography was performed in selected animals. RESULTS: Neuroblastoma control and treated tumor weights were not significantly different (1.48 g v 0.77 g, P =.34). By comparison, as previously reported, antibody-treated Wilms tumors were growth inhibited. Angiograms of treated (but not control) neuroblastomas displayed novel rounded structures at vessel branches, which the authors term terminal vascular bodies (TVBs). Wilms tumor vessels displayed no such alteration. CONCLUSIONS: Neuroblastoma xenografts are less effectively suppressed by anti-VEGF antibody than Wilms tumors. Neuroblastoma vascular architecture displays a novel alteration during antibody administration, which attenuates when antibody is withdrawn. These studies suggest that angiogenesis is differently regulated in experimental neuroblastoma and Wilms tumor.     

Combination antiangiogenic therapy: increased efficacy in a murine model of Wilms tumor

BACKGROUND/PURPOSE: Antibody to vascular endothelial growth factor (anti-VEGF) suppresses tumor growth and metastasis in experimental Wilms tumor. However, tumor growth accelerates if antibody is withdrawn. As recently shown, low-dose, frequently administered topotecan, a topoisomerase-1 inhibitor, has anti-angiogenic activity. The authors hypothesized that combined topotecan/anti-VEGF therapy would suppress tumor growth and metastasis more durably than either agent alone. METHODS: Xenografts were induced by intrarenal injection of human Wilms tumor cells in athymic mice (n = 59). Mice were divided into control (n = 10), anti-VEGF (n = 16), topotecan (n = 17), and topotecan plus anti-VEGF (n = 16) groups. All control and half the treated mice were killed at week 6. Remaining ("rebound") mice were maintained without treatment until week 8. Tumor vasculature was mapped by fluorescein angiography/PECAM immunostaining. Endothelial apoptosis was assessed by TUNEL assay. RESULTS: 6 weeks: Tumor weights were reduced significantly in treated mice (P <.003 v control). Seven of ten control and 1 of 25 treated mice displayed lung metastases (P <.003). Rebound tumors were largest in topotecan-only, intermediate in antibody-treated, and smallest in combination-treated mice. Immunostaining and angiography results showed sparse vascularity in treated xenografts. Endothelial apoptosis was observed only in treated tumors. CONCLUSION: Combination low-dose topotecan and anti-VEGF antibody therapy is antiangiogenic and suppresses tumor growth and metastasis in experimental Wilms tumor more durably than either agent alone.     

Neutralizing anti-vascular endothelial growth factor antibody inhibits further growth of established prostate cancer and metastases in a pre-clinical model

PURPOSE: The formation of new blood vessels from the pre-existing vasculature is necessary for support of primary tumor growth and appears coincident with the development of metastasis. In previous studies, inhibition of vascular endothelial growth factor (VEGF), a potent angiogenic factor and mediator of vascular permeability, inhibited tumor neovascularization with consequent inhibition of both primary tumor growth and micrometastases when administered at the time of tumor inoculation. In the present study, we examined the effect of inhibiting VEGF on primary tumor growth and metastases in an in vivo model of established metastatic prostate cancer. MATERIALS AND METHODS: The human prostate cancer cell line DU-145 was found to secrete VEGF. DU-145.luciferase, a subclone stably transfected with an expression vector encoding the luciferase gene, injected subcutaneously, consistently formed tumors in C.B.-17 scid/scid mice. After 6 weeks, assay of whole lung lysates showed significant luciferase activity, consistent with the presence of micrometastasis. RESULTS: Twice weekly treatment of the animals with a monoclonal anti-VEGF neutralizing antibody, A4.6.1, not only suppressed primary tumor growth, but inhibited metastatic dissemination to the lung. When treatment was delayed until the primary tumors were well-established, further growth was still inhibited, as was the progression of metastatic disease. CONCLUSION: Inhibition of tumor-secreted VEGF by a neutralizing antibody is sufficient to significantly impair prostate tumor growth and its subsequent metastasis in an in vivo model of established advanced prostate cancer. These data suggest a critical role for VEGF in initiation and maintenance of tumor angiogenesis in prostate cancer. Inhibition of VEGF in patients with VEGF-secreting prostate cancers may prove an effective approach for inhibiting disease progression even after micro-metastatic dissemination has occurred.     

Pathological angiogenesis in a murine model of human Wilms' tumor.

BACKGROUND/PURPOSE: Pathological vascular architecture is a feature of neoangiogenic processes such as diseases of the retina and tumor growth. The authors hypothesized that experimental human Wilms' tumors would display a vascular architecture similar to retinal diseases that are driven by vascular endothelial growth factor (VEGF). METHODS: Human Wilms' tumors were established in the right kidneys of nude mice. After 4.5 weeks of tumor growth, fluorescein angiograms were performed before death. Representative sections of tumors and contralateral, control kidneys were evaluated by fluorescent microscopy. RESULTS: Fluorescein angiograms demonstrated a characteristic pathological architecture. Vascular tortuosity, capillary tufting, and hemorrhage were noted. These features were not present in normal kidneys. CONCLUSIONS: Vascular architecture of Wilms' tumor displays the specific features previously described in diseases of the retina, which have been shown to be driven by VEGF, suggesting that neoangiogenesis in this model is also VEGF driven.     

抗血管内皮生长因子抗体对裸鼠原位种植人胃癌的影响

目的 研究抗血管内皮生长因子（ＶＥＧＦ）对胃癌生长和转移的抑制作用。方法 采用人胃癌ＳＧＣ－７９０１组织原位种植裸鼠模型,观察抗ＶＥＧＦ抗体和丝裂霉素Ｃ（ＭＭＣ）的抗肿瘤和抗转移作用。     

Distinct response of experimental neuroblastoma to combination antiangiogenic strategies

Background: The authors have shown previously that experimental neuroblastoma is partially inhibited (48%) by antivascular endothelial growth factor (anti-VEGF) antibody. The topoisomerase-I inhibitor, topotecan, has been shown to have antiangiogenic activity when administered in a low-dose, high-frequency regimen. We hypothesized that combining topotecan with anti-VEGF would suppress neuroblastoma more effectively than either agent alone. Methods: A total of 10⁶ neuroblastoma cells were implanted intrarenally in athymic mice. Animals received vehicle, topotecan, anti-VEGF, or topotecan plus anti-VEGF (n = 9, 20, 20, 20, respectively). All control and half the treated mice were killed at 6 weeks. Remaining (rebound) mice were maintained without treatment for 3 more weeks. Patterns of vasculature and apoptosis were determined immunohistochemically. Results: Tumor weights at 6 weeks were reduced significantly in topotecan-only (0.07g) and combination-treated animals (0.08 g), compared with controls or anti-VEGF[ndash ]treated mice (1.18 g, 0.53 g; P [lt ] .0007, all). At 9 weeks, rebound tumor weights were greatest in anti-VEGF (2.82 g), intermediate in topotecan (1.82 g), and least in combination-treated animals (1.47 g); however, the only significant difference was between anti-VEGF and combination therapy (P = 0.04). All treated tumors were vascularized sparsely in comparison with controls at 6 weeks, but exhibited brisk neoangiogenesis at 9 weeks. Conclusions: Topotecan either with or without anti-VEGF antibody significantly suppresses neuroblastoma xenograft growth in comparison with controls or anti-VEGF antibody alone. Combining topotecan with anti-VEGF antibody significantly inhibited rebound tumor growth in comparison with anti-VEGF antibody alone. Combination therapy may improve durability of antiangiogenic inhibition of neuroblastoma.     

VEGF-mediated angiogenesis of human pheochromocytomas is associated to malignancy and inhibited by anti-VEGF antibodies in experimental tumors

BACKGROUND: Pheochromocytomas are well-vascularized tumors of the adrenal medulla. In human pheochromocytomas, angiogenesis has been associated with tumor progression. The mechanisms, however, are unknown. METHODS: Surgical specimens of benign, invasive, and metastatic human pheochromocytomas (n = 10/5/5) were immunostained for vascular endothelial growth factor (VEGF) and CD34, to determine VEGF expression and microvessel density (vascular surface density, [VSD]). In PC12-pheochromocytoma cells, VEGF messenger RNA and protein were analyzed by Northern blotting and enzyme immunoassay; biologic activity by human umbilical vein endothelial cell-proliferation assay. Inhibition of angiogenesis of PC12 xenografts by 2 neutralizing anti-VEGF antibodies (C20-pAB, M461-mAB) was evaluated by VEGF expression, VSD, and mitotic activity. RESULTS: VEGF expression and VSD were significantly higher in metastatic pheochromocytomas (VEGF 37.1 +/- 10.9% vs 20.7 +/- 9%, VSD 26.2 +/- 8 vs 13.5 +/- 3.3 1/mm). VEGF messenger RNA and protein were confirmed in PC12 cells and stimulated by nerve growth factor. Conditioned PC12 medium increased human umbilical vein endothelial cell proliferation more than 2-fold. Xentrotransplanted PC12 cells had marked VEGF expression and angiogenesis, which was inhibited by anti-VEGF antibodies (VEGF-expression by 29 and 38%, VSD by 43 and 46%, P <.05). CONCLUSION: Higher VEGF expression and microvessel density in malignant pheochromocytomas suggest VEGF-mediated angiogenesis to be related to tumor progression. Angiogenesis of PC12 xenografts is mediated by VEGF. Neutralizing anti-VEGF antibodies inhibit angiogenesis in experimental pheochromocytomas and may have potential for treating nonresectable pheochromocytomas.     

Significance of angiogenesis and clinical application of anti-angiogenesis

Angiogenesis is essential for tumor growth and metastasis and depends upon the production of angiogenic factors by host and/or tumor cells. Increased vascularity may allow not only an increase in tumor growth but also a greater chance for hematogenous metastasis. We have already reported that vessel density and vascular endothelial growth factor (VEGF) expression are higher in metastatic tumors than in nonmetastatic tumors and that VEGF and its receptor, the KDR ligand/receptor system, also correlate with metastasis. Therefore the anti-VEGF antibody and VEGF receptor antagonist are potential targets for antiangiogenesis therapy in colon cancer. Clinical trials of such agents are continuing to phase II/III in the USA and Europe. In this paper, we introduce data on antiangiogenesis agents in the treatment of metastatic colorectal cancer and point out that the strategy for antiangiogenesis is not tumor shrinkage but tumor dormancy.     

Longterm recombinant adeno-associated, virus-mediated, liver-generated expression of an angiogenesis inhibitor improves survival in mice with disseminated neuroblastoma

BACKGROUND: A gene therapy-mediated approach to the delivery of antiangiogenic agents using adeno-associated virus (AAV) vectors has a number of advantages including the potential for sustained expression. The purpose of this study was to attempt to restrict the growth of disseminated neuroblastoma through delivery of a truncated, soluble form of the vascular endothelial growth factor receptor-2 (VEGFR-2 fetal liver kinase [Flk]-1), a decoy receptor for VEGF. STUDY DESIGN: Mice underwent portal vein injection of vector, either AAV-CAGG-tsFlk-1 or control vector. Subsequent truncated soluble fetal liver kinase-1 (tsFlk-1) expression was confirmed and quantified by ELISA. After 8 weeks, mice were given human neuroblastoma cells through the tail vein to establish disseminated disease and were sacrificed after 40 days. The weight of liver metastasis was measured. Intrahepatic tumor microvessel density and levels of apoptosis were determined. Survival was assessed in a second cohort of mice. RESULTS: After intraportal injection of AAV-CAGG-tsFlk-1, high-level, stable transgene expression was generated. Sera from these mice inhibited endothelial cell activation in vitro and Matrigel plug (BD Biosciences) neovascularization in vivo, suggesting that a systemic state of angiogenesis inhibition had been established. After neuroblastoma injection, mice expressing tsFlk-1 had a smaller tumor burden in the liver when sacrificed, as compared with control mice. The decrease in tumor weight in the liver correlated with lower intratumoral microvessel density and higher levels of tumor cell apoptosis in the tsFlk-1-treated tumors, supporting the hypothesis that decreased angiogenesis had occurred. In a second cohort of mice, survival of tsFlk-1-expressing mice after tumor cell challenge was longer than in control mice. CONCLUSIONS: Longterm in vivo expression of a functional VEGF inhibitor was established using an AAV-mediated gene therapy approach, and it demonstrated antiangiogenic and anticancer efficacy in a murine model of disseminated neuroblastoma.     

肾细胞癌VEGF表达和MVD检测的临床意义

目的 探讨肾细胞癌（RCC）组织中血管内皮细胞生长因子（VEGF）的表达与肿瘤间质微血管密度（MVD）检测的临床意义。方法 采用免疫组化方法对65例肾细胞癌（RCC）及10例正常肾组织进行VEGF单克隆抗体及CD34单克隆抗体染色，观察RCC的VEGF表达与MVD之间的相关性。结果 VEGF的表达与肿瘤间质微血管密度之间存在正相关性，二者均与RCC的病理分级、临床分期及远处转移显著相关（P〈0.05，P〈O．001）。结论 VEGF与MVD可客观准确反映RCC的生物学行为，上述二项指标可作为评估RCC恶性程度、转移及预后的重要指标。     

抗人膀胱癌/抗VEGF双功能基因抗体对膀胱癌生物学行为的影响

目的:探讨抗人膀胱癌/抗VEGF双功能基因抗体对人膀胱癌生长及转移的影响。方法:通过构建人膀胱癌裸鼠皮下移植瘤模型并注射双功能抗体,观察肿瘤生长及盆腔淋巴结转移情况,同时采用免疫组化法检测肿瘤微血管密度值及凋亡的肿瘤细胞指数。结果:肿瘤大小:双抗组为(19.50±4.51)mm2,对照组为(57.62±8.31)mm2,两组比较P<0.01;肿瘤微血管密度双抗组为(2351±207)个,对照组(4356±548)个,两组相比P<0.05;凋亡指数双抗组为19.25,对照组为9.31,两者比较P<0.05;双抗组无一只发生盆腔转移,而对照组为75.0%,两组比较P<0.05。以上各项组间差异均有统计学意义。结论:抗人膀胱癌/抗VEGF双功能基因抗体对人膀胱癌具有良好的靶向性,能够通过抑制肿瘤微血管形成和加速肿瘤细胞凋亡,遏制实验性人膀胱癌的生长转移,为该抗体用于临床膀胱癌的治疗提供了一定的实验基础。     

血管生成抑制剂YH-16联合氟尿嘧啶抑制结直肠癌肝转移的研究

目的探讨血管生成抑制剂YH-16和氟尿嘧啶（5-FU）联合应用对结直肠癌肝转移的抑制作用。方法用MTT方法测定血管生成抑制剂YH-16和5-FU对血管内皮细胞和结肠癌细胞的IC50；建立小鼠肝转移模型。随机分为对照组、YH-16组（又分为低、中、高剂量3组）和5-Fu组及联合治疗组（YH-16加5-FU），术后2周观察各组小鼠肝转移瘤数目、原发灶大小和毒性反应。并检测肝转移瘤血管内皮生长因子（VEGF）的表达和肿瘤微血管密度（MVD）。结果YH-16对结肠癌细胞的IC50是血管内皮细胞的3．38倍，而5-Fu对两种细胞的IC50差别不大。高剂量YH-16组、5-FU组和联合治疗组肝转移瘤数目明显低于对照组。而联合治疗组又低于高剂量YH-16组和5-FU组（均P〈0．05）。YH-16各剂量组的脾原发瘤体积与对照组比较均P〉0．05。差异无统计学意义；而5-FU组和联合治疗组则小于对照组（均P〈0．05）。YH-16的毒性明显低于5-FU（P〈0．05），且两者联合使用其毒性与单用5-FU比较，差异无统计学意义（P〉0．05）。5-FU组和联合治疗组肝转移瘤组织中VEGF的表达明显降低，中、高剂量YH-16组和5-FU组及联合治疗组肝转移瘤组织中的MVD计数明显降低（均P〈0．05）。结论血管生成抑制剂YH-16明显抑制结直肠癌肝转移，YH-16与5-FU联合应用对结直肠癌肝转移的抑制具有协同作用。     

VEGF is upregulated in a neuroblastoma and hepatocyte coculture model

BACKGROUND: We hypothesize that angiogenic factors are altered by the interaction between neuroblastoma cells and host tissues. MATERIALS AND METHODS: Human Chang hepatocytes and human neuroblastoma cells are cultured separately and in a noncontact, coculture system. Immunostaining for VEGF is performed on the cells. ELISA is used to detect vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and interleukin-8 in the conditioned media. Human umbilical vein endothelial cells (HUVEC) are cultured with standard medium (control) and hepatocyte, neuroblastoma, and coculture conditioned media. After 48 and 72 h, cells are counted to determine proliferation. Finally, VEGF-blocking antibody is added to the HUVEC cultures with the conditioned media. RESULTS: VEGF is markedly elevated in the coculture medium compared to the media from hepatocytes or neuroblastoma grown alone [412.2 +/- 52 vs 235 +/- 35 or 74.5 +/- 28.5 (pg/10(6) cells), P < 0.05]. Other growth factors are almost undetectable in any of the media. Immunostaining for VEGF in the cocultured hepatocytes is decreased by almost 50%, but VEGF immunostaining is increased fourfold in the cocultured neuroblastoma cells. A significant increase in cell proliferation is seen at both 48 and 72 h when HUVEC are cultured with the coculture media. Cell proliferation is blocked with the addition of anti-VEGF antibody. CONCLUSION: The interaction of neuroblastoma with hepatocytes results in an increased production of VEGF. It stimulates endothelial cell proliferation and may enhance the tumor's metastatic potential in an autocrine fashion.     

Vascular invasion and potential for tumor angiogenesis and metastasis in gastric carcinoma

BACKGROUND: Hematogenous metastasis occurs when cancer cells released from the primary site enter blood vessels and are transported to distant organs, where they attach and proliferate. Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors by tumor cells. METHODS: We analyzed data on 1184 Japanese adult men and women with gastric cancer with respect to the relation between vascular invasion and the potential for tumor angiogenesis and metastasis. All these patients were treated from 1976 to 1995 in the Department of Surgery II, Kyushu University. In 300 patients, the expression of vascular endothelial growth factor (VEGF) and p53 protein in tumor tissues was examined by using an immunohistochemical staining method or Northern blotting or both. Intratumoral microvessels were stained with anti-CD31 monoclonal antibody. RESULTS: Vascular invasion was evident in 254 patients (21.5%), and in these patients lymphatic invasion was more frequent and the rate of lymph node metastasis was higher in relation to the extent of vascular invasion. The positive findings were directly related to the depth of invasion and the presence of lymph node and liver metastasis. Tumor invasive and metastatic rates increased in relation to the extent of vascular invasion. Expressions of VEGF and p53 protein were higher and microvessel density was more prominent in tumor tissues in relation to the extent of vascular invasion. A close relation between VEGF and p53 protein expressions was also noted in tumors that showed vascular invasion. The expression of VEGF is one of the independent risk factors for vascular invasion. The postoperative outcome was poorer in patients with vascular invasion in relation to the extent of vascular invasion. CONCLUSIONS: Our findings show that gastric cancers with characteristics of vascular invasion have greater intratumoral angiogenesis and that VEGF and p53 overexpression is associated with intratumoral angiogenesis and metastases to distant organs.     

反应停对人肝细胞癌生长侵袭的作用

目的 观察反应停对人肝细胞癌（HCC）血管生成和肿瘤生长侵袭的干预作用。方法 建立高转移潜能人肝癌裸鼠肝原位移植模型，随机分成4组。各组模型自建立次日起分别经腹腔注射0．5％羧甲基纤维素钠（CMC）、反应停（200mg／kg体重）、紫杉醇（13mg／kg体重）、反应停（200mg／kg体重）＋紫杉醇（13mg／d）。给药第30天处死裸小鼠，观察肿瘤生长、转移情况，分别用免疫组织化学和半定量逆转录．聚合酶链反应（RT—PCR）的方法检测癌组织CD34和血管内皮生长因子（VEGF）mRNA的表达并记录微血管密度（MVD）。结果 反应停组肿瘤重量、体积均与空白对照组差异无统计学意义（P〉0．05）。各药物处理组肺转移灶计数、MVD和VEGF均低于对照组，以联合用药组最为显著。结论 反应停对HCC生长无明显抑制作用，但能抑制肿瘤血管生成并降低肺转移。     

Anti-VEGF antibody in experimental hepatoblastoma: suppression of tumor growth and altered angiogenesis

Background: Hepatoblastoma is the most common primary hepatic malignancy of childhood, frequently presenting as advanced disease. Vascular endothelial growth factor (VEGF) is an endothelial mitogen and survival factor critical to growth and angiogenesis in many human cancers. Inhibition of VEGF effectively suppresses tumorigenesis in multiple experimental models. The authors hypothesized that anti-VEGF antibody would alter vascular architecture and impede tumor growth in experimental hepatoblastoma. Methods: The Institutional Animal Care and Use Committee of Columbia University approved all protocols. Xenografts were established in athymic mice by intrarenal injection of cultured human hepatoblastoma cells. Anti-VEGF antibody (100 [mu ]g/dose) or vehicle was administered intraperitoneally 2 times per week for 5 weeks. At week 6, 10 control/treated mice were killed and remaining animals maintained without treatment until week 8. Tumor weights were compared by Kruskal-Wallis analysis, and vascular alterations ascertained by fluorescein angiography and specific immunostaining. Results: Anti-VEGF antibody significantly inhibited tumor growth at 6 weeks (1.85 g +/[minus ] 0.60 control, 0.05 +/[minus ] 0.03 antibody, P [lt ] .0003). In comparison with controls, treated xenografts showed decreased vascularity and dilated surviving vessels with prominent vascular smooth muscle elements. Conclusions: Specific anti-VEGF therapy inhibits neoangiogenesis and significantly suppresses tumor growth in experimental hepatoblastoma. Surviving vasculature displays dilation and increased vascular smooth muscle. Anti-VEGF agents may represent new therapeutic alternatives for children with advanced disease. J Pediatr Surg 38:308-314.     

SO05THE NATURE OF NEARBY LYMPHATICS DICTATES WHETHER A VASCULAR ENDOTHELIAL GROWTH FACTOR‐D (VEGF‐D) INDUCES TUMOR LYMPHATICS AND METASTASIS

Purpose Tumors secreting VEGF‐D or VEGF‐C proteins exhibit increased lymphatic vessel density and lymph node spread (1), however tumor depth and anatomical location also influence metastasis in some tumors. It is unclear whether tumor lymphatics mirror one of the normal lymphatic subtypes or if they form an independent phenotype, and how this may vary in different anatomical locations. We aimed to study lymphatics induced in a VEGF‐D+ tumor model (1), xenografted in different locations, to better understand the vessels that facilitate tumor spread. Methods We analysed lymphatics formed in mouse skin wounds and tissue from VEGF‐D+ or VEGF‐D‐ tumors in the ear or flank, using immunohistochemical lymphatic marker LYVE‐1 and confocal imaging of fluorescent‐labelled antibodies. Results VEGF‐D+ tumors that were adherent to skin induced abnormal lymphatic structures that promoted lymph node metastasis (89%) and exhibited morphologically abnormal lymphatics; whereas size‐matched tumors that were adherent to the underlying body wall (similar VEGF‐D levels) metastasised minimally (19%, p < 0.001) and contained no morphologically abnormal lymphatics. Analysis of ear wounds and tumors suggested that lymphatics sprout mainly from the ‘pre‐collector’ lymphatic subtype within skin. Conclusion Lymphangiogenic growth factors are important but tumor location also plays a role in metastasis. A distinct tumor lymphatic subtype may alsooffer new diagnostic or therapeutic molecular targets.