Antitumor treatment efficacy by targeting epidermal growth factor receptor and vascular endothelial growth factor receptor-2 in an orthotopic human glioblastoma model

Using an orthotopic intracerebral model from our established HM55-BGIV-101 tumor line, we investigated the antitumor effect on the angiogenesis and growth of human glioblastoma after treatment with monoclonal antibody DC101 against the vascular endothelial growth factor receptor-2 and monoclonal antibody C225 against the epidermal growth factor receptor. Nude mice bearing intracerebral glioblastoma xenografts were treated intraperitoneally with DC101 and C225 either alone or in combination. Histopathological analysis of solid tumor volume, satellite tumor number, microvessel density, tumor cell proliferation, and apoptosis was performed. In the DC101-treated group, solid tumor volume and microvessel density were reduced by 59.7 and 64%, respectively; tumor cell proliferative activity was reduced by 53.2% and the apoptotic index (AI) was increased by 66.7%; satellite tumor number was enhanced by 84.4%. C225 alone reduced satellite tumor number by 43.3%, but had no effect on solid tumor volume, microvessel density, tumor cell proliferation, and apoptosis. C225 combined with DC101 not only reduced solid tumor volume, microvessel density, tumor cell proliferative activity, and increased AI, but also reduced satellite tumor number. Inhibition of angiogenesis achieved by DC101 can cause increased tumor cell invasiveness. In our studies this increased tumor cell invasiveness was inhibited simultaneously by C225, which provides a theoretical basis for treatment of glioblastoma by the method of combining drugs with different pharmacological activity.     

Inhibition of glioblastoma angiogenesis and invasion by combined treatments directed against vascular endothelial growth factor receptor-2, epidermal growth factor receptor, and vascular endothelial-cadherin.

PURPOSE: Inhibition of angiogenesis can influence tumor cell invasion and metastasis. We previously showed that blockade of vascular endothelial growth factor receptor-2 (VEGFR-2) with the monoclonal antibody DC101 inhibited intracerebral glioblastoma growth but caused increased tumor cell invasion along the preexistent vasculature. In the present study, we attempted to inhibit glioma cell invasion using a monoclonal antibody against the epidermal growth factor receptor (EGFR), which in the context of human glioblastomas, has been implicated in tumor cell invasion. In addition, we analyzed whether blockade of vascular endothelial (VE)-cadherin as a different antiangiogenic target could also inhibit glioblastoma angiogenesis and growth. EXPERIMENTAL DESIGNS: Nude mice who received intracerebral glioblastoma xenografts were treated using monoclonal antibodies against VEGFR-2 (DC101), EGFR (C225), and VE-cadherin (E4G10) either alone or in different combinations. RESULTS: Increased tumor cell invasion provoked by DC101 monotherapy was inhibited by 50% to 66% by combined treatment with C225 and DC101. C225 inhibited glioblastoma cell migration in vitro, but had no effect on the volume of the main tumor mass or on tumor cell proliferation or apoptosis in vivo, either alone or in combination with DC101. The anti-VE-cadherin monoclonal antibody E4G10 was a weaker inhibitor of tumor angiogenesis and growth than DC101, and also caused a weaker increase in tumor cell invasion. CONCLUSIONS: Inhibition of angiogenesis achieved by blocking either VEGFR-2 or VE-cadherin can cause increased glioma cell invasion in an orthotopic model. Increased tumor cell invasion induced by potent inhibition of angiogenesis with DC101 could be inhibited by simultaneous blockade of EGFR.     

Inhibition of glioma angiogenesis and growth in vivo by systemic treatment with a monoclonal antibody against vascular endothelial growth factor receptor-2

Using an orthotopic intracerebral model, we investigated whether systemic treatment with DC101, a monoclonal antibody against vascular endothelial growth factor receptor (VEGFR)-2, could inhibit angiogenesis and the growth of human glioblastoma cells in severe combined immunodeficient mice. Intraperitoneal treatment with DC101, control IgG, or PBS was initiated either on day 0 or, in another series, on day 6 after tumor cell implantation, and animals were killed approximately 2 weeks after tumor cell injection. Tumor volumes in animals treated with DC101 were reduced by 59 and 81% compared with IgG and PBS controls, respectively (P < 0.001), when treatment was initiated immediately, and similar results were obtained when treatment started on day 6. Microvessel density in tumors of DC101-treated animals was reduced by at least 40% compared with animals treated with control IgG or PBS (P < 0.01). We observed a reduction in tumor cell proliferation and an increase in apoptosis in DC101-treated animals (P < 0.001). However, in mice treated with DC101, we also noticed a striking increase in the number and total area of small satellite tumors clustered around, but distinct from, the primary. These satellites usually contained central vessel cores, and tumor cells often had migrated over long distances along the host vasculature to eventually reach the surface and spread leptomeningeally. We conclude that systemic antagonization of VEGFR-2 can inhibit glioblastoma neovascularization and growth but can lead to increased cooption of preexistent cerebral blood vessels. Therefore, a combination of different treatment modalities which also include anti-invasive therapy may be needed for an effective therapy against glioblastoma, and the use of an antibody against VEGFR-2 may be one effective component.     

Combined anti-fetal liver kinase 1 monoclonal antibody and continuous low-dose doxorubicin inhibits angiogenesis and growth of human soft tissue sarcoma xenografts by induction of endothelial cell apoptosis

Vascular endothelial growth factor (VEGF) and VEGF receptor 2 [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] have been shown to play a major role in tumor angiogenesis. In this study, we investigated whether anti-Flk-1 monoclonal antibody DC101 could therapeutically inhibit growth and angiogenesis of human soft tissue sarcoma, and we explored its capacity to enhance the tumoricidal effects of doxorubicin. Treatment of well-established leiomyosarcoma SKLMS-1 and rhabdomyosarcoma RD xenografts in severe combined immunodeficient mice with DC101 resulted in significant antitumor activity. In a parallel study, we compared tumor inhibition with continuous low-dose "antiangiogenic" schedule versus once-every-2-weeks high-dose standard schedule of doxorubicin. We found that continuous low-dose treatment inhibited the tumor growth of RD xenografts about 46.5% of that with standard-schedule treatment, but that continuous low-dose treatment did not inhibit the tumor growth of SKLMS-1 xenografts. Notably, combined DC101 and continuous low-dose doxorubicin resulted in more effective growth inhibition of SKLMS-1 and RD xenografts than has been observed with any agent alone in a long-term s.c. tumor xenograft model. The combination therapy was associated with no additional toxicity to the host animal compared with low-dose doxorubicin alone. Histological examination of xenografts showed significantly reduced microvessel counts in the tumors given combined therapy compared with the tumors given either agent alone. These results are consistent with an enhanced inhibition of angiogenesis in vivo by combined DC101 and doxorubicin using Matrigel plug assay. Additionally, DC101 plus doxorubicin directly exerted enhanced inhibitory effects on endothelial cell migration, proliferation, and tube-like formation in vitro. Furthermore, the combination induced an enhanced apoptosis of endothelial cells that was associated with an increase of capase-3 activity. Thus, the inhibition of angiogenesis and induction of endothelial cell apoptosis are likely important mechanisms for the antitumor activity of combined DC101 and doxorubicin. Collectively, our data suggested that anti-VEGF receptor 2 in combination with continuous low-dose doxorubicin may provide a new therapeutic approach for human soft tissue sarcoma in the clinic.     

Inhibition of angiogenesis by the antiepidermal growth factor receptor antibody ImClone C225 in androgen-independent prostate cancer growing orthotopically in nude mice.

In human androgen-independent prostate cancer (PCa), epidermal growth factor receptor (EGFR) regulates angiogenesis, tumor growth, and progression. In this study, we evaluated whether the blockade of EGFR by the anti-EGFR antibody ImClone C225 (IMC-C225) inhibited tumor growth and metastasis by inhibiting angiogenesis, and whether paclitaxel enhanced the results of therapy in androgen-independent PCa. PC-3M-LN4 PCa cells were implanted orthotopically in athymic nude mice and treated with i.p. IMC-C225 (1 mg twice a week) and/or paclitaxel (200 microg once a week). In vitro treatment of PC-3M-LN4 with IMC-C225 inhibited EGFR autophosphorylation without any significant antiproliferative effect. In contrast, in vivo therapy with IMC-C225 alone (P < 0.05) or in combination with paclitaxel (P < 0.005) significantly inhibited PCa growth and metastasis. Serum levels of interleukin (IL) 8 were lower after therapy, and IL-8 mRNA expression was down-regulated within the tumors after therapy. The down-regulation of IL-8 correlated with reduced microvessel density. IMC-C225 reduced tumor cell proliferation, enhanced p27(kip1) expression, and induced tumor and endothelial cell apoptosis. These studies indicate that IMC-C225 has significant antitumor effect in this murine model, mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. The simultaneous administration of paclitaxel enhanced this effect.     

Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors.

Tumor angiogenesis is mediated by tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] play an important role in vascular permeability and tumor angiogenesis. Previously, we reported on the development of anti-Flk-1 and antikinase insert domain-containing receptor monoclonal antibodies (mAbs) that potently inhibit VEGF binding and receptor signaling. Here, we report the effect of anti-Flk-1 mAb (DC101) on angiogenesis and tumor growth. Angiogenesis in vivo was examined using a growth factor supplemented (basic fibroblast growth factor + VEGF) Matrigel plug and an alginate-encapsulated tumor cell (Lewis lung) assay in C57BL/6 mice. Systemic administration of DC101 every 3 days markedly reduced neovascularization of Matrigel plugs and tumor-containing alginate beads in a dose-dependent fashion. Histological analysis of Matrigel plugs showed reduced numbers of endothelial cells and vessel structures. Several mouse tumors and human tumor xenografts in athymic mice were used to examine the effect of anti-Flk-1 mAb treatment on tumor angiogenesis and growth. Anti-Flk-1 mAb treatment significantly suppressed the growth of primary murine Lewis lung, 4T1 mammary, and B16 melanoma tumors and growth of Lewis lung metastases. DC101 also completely inhibited the growth of established epidermoid, glioblastoma, pancreatic, and renal human tumor xenografts. Histological examination of anti-Flk-1 mAb-treated tumors showed evidence of decreased microvessel density, tumor cell apoptosis, decreased tumor cell proliferation, and extensive tumor necrosis. These findings support the conclusion that anti-Flk-1 mAb treatment inhibits tumor growth by suppression of tumor-induced neovascularization and demonstrate the potential for therapeutic application of anti-VEGF receptor antibody in the treatment of angiogenesis-dependent tumors.     

Anti-vascular endothelial growth factor receptor 2 antibody reduces tumorigenicity and metastasis in orthotopic prostate cancer xenografts via induction of endothelial cell apoptosis and reduction of endothelial cell matrix metalloproteinase type 9 production.

PURPOSE: Vascular endothelial growth factor (VEGF), which is produced by tumor cells, is a potent endothelial cell mitogen. The aim of the present study was to evaluate the response of orthotopic prostate cancer xenografts and prostate cancer bone metastasis to anti-VEGF receptor (flk-1) antibody (DC101) treatment. EXPERIMENTAL DESIGN: Orthotopic prostate cancer models (PC-3M-MM2 and LNCaP-LN3 prostate carcinoma cells) and a prostate cancer bone metastasis model (PC-3M-MM2) were used for these experiments. Early and established tumors were treated with saline, paclitaxel, DC101, or a DC101-plus-paclitaxel combination for 5 weeks (PC-3M-MM2) and 12 weeks (LNCaP-LN3). At the end of therapy, tumors were removed and weighed. Apoptosis, tumor cell proliferation, and angiogenesis- and metastasis-related gene expression were evaluated using immunohistochemistry, in situ hybridization, and terminal deoxynucleotidyl transferase-ediated nick end labeling (TUNEL). RESULTS: After treatment of early tumors (PC-3M-MM2), median prostate tumor weights (+/-SE) were 1230 +/- 210 mg in untreated controls, 482 +/- 121 mg in mice treated with paclitaxel (P = 0.009), 148 +/- 27 mg in mice treated with DC101 (P < 0.001), and 48 +/- 10 mg in mice treated with the combination of DC101 and paclitaxel (P < 0.001). Lymph node metastasis occurred in 7 of the 9 control mice, 5 of the 9 paclitaxel-treated animals, 5 of the 12 DC101-treated animals, and 2 of the 11 animals in the combination therapy group. Treatment with DC101 alone or in combination with paclitaxel reduced tumor-induced neovascularity measured by microvessel density and tumor cell proliferation (by proliferating cell nuclear antigen) and enhanced apoptosis (measured by TUNEL) in tumor cells and endothelial cells compared with controls. In the tibial prostate cancer metastasis model, significant inhibition of tumor growth was observed. In the LNCaP-LN3 orthotopic prostate cancer model, tumors occurred in 7 of the 10 control mice, 4 of the 10 paclitaxel-treated animals, 5 of the 10 DC101-treated animals, and 2 of the 11 animals in the combination therapy group (P < 0.05). The efficacy of DC101 was much greater in the treatment of early tumors, which suggests that tumor burden may be a critical factor in determining the response to DC101. In vitro and in vivo analysis of endothelial cell function showed reduced matrix metalloproteinase type 9 production in endothelial cells treated with DC101. CONCLUSIONS: This study confirms the principle of tumor growth inhibition by targeting angiogenesis within tumors and supports the use of anti-VEGF receptor agents.     

Effects of combination anti-vascular endothelial growth factor receptor and anti-epidermal growth factor receptor therapies on the growth of gastric cancer in a nude mouse model

We hypothesised that the combination of anti-angiogenic and anti-epidermal growth factor (EFG)-receptor (R) therapies would more effectively inhibit gastric cancer growth than single-agent therapy. TMK-1 gastric cancer cells were injected into the gastric wall of nude mice to generate tumours. After 4 days, mice were randomly assigned to the following groups: control, DC101 ([vascular endothelial growth factor (VEGF)-receptor (R)-2 antibody], C225 (EGF-R antibody), or a combination of DC101 and C225. The combination therapy significantly inhibited gastric tumour growth compared with the control group, whereas the decrease in tumour growth in mice treated with DC101 or C225 alone did not reach statistical significance. All mice administered DC101 demonstrated decreased tumour vascularity and increased endothelial cell apoptosis. C225 alone did not affect angiogenesis, but inhibited tumour cell proliferation. The combination therapy led to a further decrease in tumour cell proliferation. The combination of anti-VEGF-R and anti-EGF-R therapies was effective in inhibiting gastric cancer growth. These findings support the hypothesis that inhibiting multiple biological pathways that mediate tumour growth may be an effective therapeutic strategy.     

Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) regulate colon cancer growth and metastasis. Previous studies utilizing antibodies against the VEGF receptor (DC101) or EGF receptor (C225) have demonstrated independently that these agents can inhibit tumour growth and induce apoptosis in colon cancer in in vivo and in vitro systems. We hypothesized that simultaneous blockade of the VEGF and EGF receptors would enhance the therapy of colon cancer in a mouse model of peritoneal carcinomatosis. Nude mice were given intraperitoneal injection of KM12L4 human colon cancer cells to generate peritoneal metastases. Mice were then randomized into one of four treatment groups: control, anti-VEGFR (DC101), anti-EGFR (C225), or DC101 and C225. Relative to the control group, treatment with DC101 or with DC101+C225 decreased tumour vascularity, growth, proliferation, formation of ascites and increased apoptosis of both tumour cells and endothelial cells. Although C225 therapy did not change any of the above parameters, C225 combined with DC101 led to a significant decrease in tumour vascularity and increases in tumour cell and endothelial cell apoptosis (vs the DC101 group). These findings suggest that DC101 inhibits angiogenesis, endothelial cell survival, and VEGF-mediated ascites formation in a murine model of colon cancer carcinomatosis. The addition of C225 to DC101 appears to lead to a further decrease in angiogenesis and ascites formation. Combination anti-VEGF and anti-EGFR therapy may represent a novel therapeutic strategy for the management of colon peritoneal carcinomatosis.     

Treatment of human metastatic transitional cell carcinoma of the bladder in a murine model with the anti-vascular endothelial growth factor receptor monoclonal antibody DC101 and paclitaxel.

Vascular endothelial cell growth factor (VEGF) regulates angiogenesis and metastasis of bladder cancer (transitional cell carcinoma, TCC) through binding to VEGF receptor-2 (VEGFR-2). In this study, we evaluated whether the anti-VEGFR monoclonal antibody (Mab) DC101 in combination with paclitaxel inhibited tumorigenesis, angiogenesis, and metastasis of human TCC growing within the bladder of athymic nude mice. In vivo therapy with Mab DC101 and paclitaxel induced significant regression of bladder tumors compared with either agent alone. Median bladder weights were reduced from 601 mg in untreated controls, 422 mg in mice treated with paclitaxel alone (P < 0.005), 361 mg in mice treated with DC101 alone (P < 0.005), and 113 mg in mice that received combination therapy (P < 0.0005). Only one of nine mice developed spontaneous lymph node metastasis after combined treatment, compared with seven of seven untreated controls (P < 0.0005), six of eight after DC101 (P < 0.01), and five of eight mice after paclitaxel (P < 0.05). Combined treatment with both paclitaxel and DC101 inhibited tumor-induced neovascularity compared with all other groups (P < 0.005), without altering the expression of VEGF or flk1. Mab DC101 and paclitaxel combined enhanced apoptosis in the tumor and endothelial cells compared with other treatment (P < 0.005). These studies indicate that Mab DC101, which blocks VEGFR-2 function, has significant efficacy against human TCC, especially when combined with the chemotherapeutic agent paclitaxel. The antitumor effect was mediated by inhibition of angiogenesis and induction of both tumor cell and endothelial cell apoptosis.     

Paclitaxel enhances the effects of the anti-epidermal growth factor receptor monoclonal antibody ImClone C225 in mice with metastatic human bladder transitional cell carcinoma.

Previously we reported that when cells from the human transitional cell carcinoma cell line 253J B-V growing orthotopically within the bladder of athymic nude mice were treated with the anti-epidermal growth factor receptor monoclonal antibody C225, angiogenesis was inhibited, resulting in regression of the primary tumor and inhibition of metastasis. In this study, we evaluated whether paclitaxel enhanced this therapeutic effect of C225. In vitro, the proliferation of 253J B-V cells was inhibited more by the combination of C225 and paclitaxel than with either agent alone. In vivo therapy with C225 and paclitaxel resulted in significantly greater regression of tumors compared with either agent alone. Median bladder tumor weight was 85 mg (range, 69-133 mg) compared with 168 mg (range, 72-288 mg) after C225 alone (P < 0.05), and 273 mg (range, 83-563 mg) after paclitaxel alone (P < 0.005). The incidence of spontaneous lymph node metastasis was also reduced by the combination of C225 with paclitaxel, although this result did not significantly differ from results after the use of C225 alone. Treatment with paclitaxel and C225 down-regulated the expression of basic fibroblast growth factor, vascular endothelial cell growth factor, interleukin-8, and matrix metalloproteinase type 9 and inhibited tumor-induced neovascularity compared with untreated controls (P < 0.005). Moreover, the combination of C225 and paclitaxel enhanced apoptosis in tumor and endothelial cells compared with either agent alone (P < 0.005). These studies indicate that therapy with paclitaxel increases the ability of C225 to inhibit tumorigenicity and metastasis. This effect is mediated by inhibition of angiogenesis and induction of apoptosis.     

Effects of an antibody to vascular endothelial growth factor receptor-2 on survival, tumor vascularity, and apoptosis in a murine model of colon carcinomatosis

Vascular endothelial growth factor (VEGF) is the predominant regulator of colon cancer angiogenesis and is associated with a poor prognosis and the development of metastases. We hypothesized that DC101, an antibody against the VEGF receptor-2 (flk-1), may be efficacious in the therapy of colon cancer peritoneal carcinomatosis in a murine model. BALB/c mice underwent intraperitoneal injection of CT-26 colon cancer cells to generate peritoneal metastases. Mice received control solvent or DC101 for up to 60 days. In parallel studies, mice were sacrificed at sequential time points to determine the effect of DC101 on tumor angiogenesis, tumor cell proliferation and apoptosis, and endothelial cell apoptosis. Mice treated with DC101 demonstrated a 30% increase in mean survival. In addition, DC101 also led to a significant decrease in tumor vascularity, growth and tumor cell proliferation. In sequential studies, anti-VEGF-R therapy led to a progressive increase in endothelial cell apoptosis followed by an increase in tumor cell apoptosis. These findings suggest that anti-flk-1 therapy may prolong survival in patients with colon cancer carcinomatosis. The temporal studies demonstrating that anti-flk-1 therapy lead to an increase in endothelial cell apoptosis that in turn lead to an increase in tumor cell apoptosis confirms the role of VEGF as an endothelial cell survival factor.     

Pathophysiological effects of vascular endothelial growth factor receptor-2-blocking antibody plus fractionated radiotherapy on murine mammary tumors

Although clinical trials of antiangiogenic strategies have been disappointing when administered as single agents, such approaches can play an important role in cancer treatment when combined with conventional therapies. Previous studies have shown that DC101, an antiangiogenic monoclonal antibody against vascular endothelial growth factor receptor-2, can produce significant growth inhibition in spontaneous and transplanted tumors but can also induce substantial hypoxia. Because DC101 appears to potentiate radiotherapy in some tumors, the present studies were undertaken to characterize pathophysiological changes following combined therapy and to determine whether radioresponse is enhanced despite the induction of hypoxia. MCa-4 and MCa-35 mammary carcinomas were treated with: (a) DC101; (b) 5 x 6 Gy radiation fractions; or (c) the combination. Image analysis of frozen tumor sections was used to quantitate: (a) hypoxia; (b) spacing of total and perfused blood vessels; and (c) endothelial and tumor cell apoptosis. For MCa-4, combination treatment schedules produced significant and prolonged delays in tumor growth, whereas single-modality treatments had minor effects. For MCa-35, radiation or the combination led to equivalent growth inhibition. In all tumors, hypoxia increased markedly after either radiation or DC101 alone. Although combination therapy produced no immediate pathophysiological changes, hypoxia ultimately increased after cessation of therapy. Preferential increases in endothelial apoptosis following combination treatment suggest that in addition to blocking tumor angiogenesis, DC101 enhances radiotherapy by specifically sensitizing endothelial cells, leading to degeneration of newly formed blood vessels.     

Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms.

Both epidermal growth factor receptor (EGF-R) signaling mechanisms and angiogenesis have been evaluated as independent targets for therapy of human pancreatic carcinoma, but a link between the two processes has been identified only recently. This study evaluated whether EGF-R blockade therapy with anti-EGF-R antibody C225 inhibits pancreatic carcinoma growth and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whether gemcitabine potentiates this effect. In vitro treatment of human pancreatic carcinoma L3.6pl cells with C225 inhibited EGF-R autophosphorylation, producing a maximum of 20% cytostasis. Treatment with C225 plus gemcitabine resulted in additive cytotoxic effects that increased with increasing gemcitabine concentrations. Dose-dependent decreases in expression of the angiogenic factors vascular endothelial growth factor and interleukin 8 (but not basic fibroblast growth factor) were observed in the C225-treated cells (mRNA and protein levels). In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with gemcitabine resulted in growth inhibition, tumor regression, and abrogation of metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm3, respectively. Gemcitabine treatment alone reduced median tumor volume from 538 to 152 mm3. Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and 20% of C225-treated animals. No macroscopically visible liver metastases were observed in the combination treatment group. As early as 11 days after C225 treatment, the median percentage of proliferating cell nuclear antigen-positive cells was substantially reduced compared with gemcitabine treatment alone (26% versus 73%, respectively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after 11 days treatment, production of vascular endothelial growth factor and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 days after C225 or combination treatments (but not gemcitabine alone) in direct correlation with the difference in percentage of apoptotic endothelial cells, as visualized by double immunofluorescence microscopy. These experiments indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6pl pancreatic carcinoma growing in nude mice which is mediated in part by inhibition of tumor-induced angiogenesis, leading to tumor cell apoptosis and regression. Furthermore, this effect is potentiated in combination with gemcitabine.     

Antiangiogenic treatment with the three thrombospondin-1 type 1 repeats recombinant protein in an orthotopic human pancreatic cancer model.

PURPOSE: This study investigates the antiangiogenesis and antitumor efficacy of a recombinant protein composed of the three type 1 repeats (3TSR) of thrombospondin-1 in an orthotopic human pancreatic cancer model and provides useful preclinical data for pancreatic cancer treatment. EXPERIMENTAL DESIGN: Human pancreatic cancer cells (AsPC-1) were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR (3 mg per kg per day) or PBS for 3 weeks. Subsequently, the effects of 3TSR on tumor growth, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. The in vitro effects of 3TSR on human pancreatic cancer cells were also studied. RESULTS: 3TSR treatment significantly reduced angiogenesis and tumor growth of orthotopic pancreatic cancer. 3TSR-treated mice had a 69% reduction in tumor volume (316.6 +/- 79.3 versus 1,012.2 +/- 364.5 mm(3); P = 0.0001), and a significant increase in tumor necrotic area. After 3TSR treatment, both the vessel number and average microvessel size were significantly decreased, and microvessel density was decreased from 8.0% to 3.7% (P < 0.0001). The apoptotic rate of tumoral endothelial cells in 3TSR-treated tumors increased to 14.7% comparing to 4.2% in control tumors (P < 0.0001). 3TSR showed no direct effects on pancreatic cancer cell proliferation or apoptosis either in vivo or in vitro. CONCLUSION: 3TSR, a domain of a natural occurring angiogenesis inhibitor, showed potent therapeutic effect in pancreatic cancer by inhibiting tumor angiogenesis and may prove to be a promising agent for clinical pancreatic cancer treatment.     

Combination celecoxib and temozolomide in C6 rat glioma orthotopic model

The purpose of this study was to determine whether a combination treatment of temozolomide with celecoxib is effective in the rat orthotopic glioma model. After stereotactic injection of C6/LacZ rat glioma cells into the Sprague Dawley rat brain, the rats were randomly assigned to four treatment groups [group 1, control treatment; group 2, celecoxib (25 mg/kg p.o. everyday) alone; group 3, temozolomide (7.5 mg/kg i.p. for 5 days at 2nd week) alone; group 4, a combination of celecoxib and temozolomide]. Rats were sacrificed 18 days after treatment, and the body weight, tumor volume, tumor cell proliferation, microvessel densities, and apoptosis were evaluated. There was a significant reduction of tumor volume in combination group compared to control or single-agent therapy. The median tumor volume was estimated to be 111.5 mm(3) (control), 65.0 mm(3) (celecoxib), 71.8 mm(3) (temozolomide) and 18.7 mm(3) (combination). In the combination group, there was increased tumor cell apoptosis as well as decreased microvessel density and tumor cell proliferation relative to the control and single-agent therapy (P<0.05). Collectively, the data suggest that the combination celecoxib and temozolomide may provide a novel and effective approach to the treatment of glioblastoma.     

抗新生血管内皮细胞单克隆抗体BVE-1治疗实体瘤的实验研究

目的研究以肿瘤新生血管为靶,用抗血管内皮细胞单克隆抗体治疗实体肿瘤的可行性。方法裸小鼠移植人平滑肌肉瘤细胞,人肝癌细胞及人胰腺癌细胞,以抗血管内皮细胞的单克隆抗体ＢＶＥ１或１３１Ｉ标记抗体ＢＶＥ１腹腔给药,并与正常鼠ＩｇＧ及１３１ＩＩｇＧ治疗对比,观察肿瘤体积变化,死后及活杀鼠进行病理学检查,计算瘤内微血管密度。结果肝癌、胰腺癌、平滑肌肉瘤组经抗体ＢＶＥ１治疗后,抑瘤率分别为７０．５％、４８．７％和４９．８％,并抑制了平滑肌肉瘤转移和降低了死亡率。１３１Ｉ标记抗体ＢＶＥ１治疗组一次给药后抑瘤率达８６．６％～８２．２％,抑瘤生长率较单纯抗体组明显提高。病理检查证实,抗体ＢＶＥ１治疗组肿瘤区毛细血管管壁变性,管腔阻塞,周围肿瘤细胞大片坏死。瘤内微血管密度明显低于未治疗组及鼠ＩｇＧ组。结论抗增生血管内皮细胞抗体ＢＶＥ１具有杀伤及抑制肿瘤区血管内皮细胞生长作用,从而导致肿瘤区毛细血管阻塞,阻断血供,抑制了肿瘤生长和转移,降低了死亡率。以之为载体偶联核素可增强此作用。     

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.