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.     

Adeno-associated virus-mediated antiangiogenic gene therapy with thrombospondin-1 type 1 repeats and endostatin.

PURPOSE: Recombinant adeno-associated virus (rAAV)-mediated antiangiogenic gene therapy offers a powerful strategy for cancer treatment, maintaining sustained levels of antiangiogenic factors with coincident enhanced therapeutic efficacy. We aimed to develop rAAV-mediated antiangiogenic gene therapy delivering endostatin and 3TSR, the antiangiogenic domain of thrombospondin-1. EXPERIMENTAL DESIGN: rAAV vectors were constructed to express endostatin (rAAV-endostatin) or 3TSR (rAAV-3TSR). The antiangiogenic efficacy of the vectors was characterized using a vascular endothelial growth factor (VEGF)-induced mouse ear angiogenesis model. To evaluate the antitumor effects of the vectors, immunodeficient mice were pretreated with rAAV-3TSR or rAAV-endostatin and received orthotopic implantation of cancer cells into the pancreas. To mimic clinical situations, mice bearing pancreatic tumors were treated with intratumoral injection of rAAV-3TSR or rAAV-endostatin. RESULTS: rAAV-mediated i.m. gene delivery resulted in expression of the transgene in skeletal muscle with inhibition of VEGF-induced angiogenesis at a distant site (the ear). Local delivery of the vectors into the mouse ear also inhibited VEGF-induced ear angiogenesis. Pretreatment of mice with i.m. or intrasplenic injection of rAAV-endostatin or rAAV-3TSR significantly inhibited tumor growth. A single intratumoral injection of each vector also significantly decreased the volume of large established pancreatic tumors. Tumor microvessel density was significantly decreased in each treatment group and was well correlated with tumor volume reduction. Greater antiangiogenic and antitumor effects were achieved when rAAV-3TSR and rAAV-endostatin were combined. CONCLUSIONS: rAAV-mediated 3TSR and endostatin gene therapy showed both localized and systemic therapeutic effects against angiogenesis and tumor growth and may provide promise for patients with pancreatic cancer.     

Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies.     

Rapamycin-induced endothelial cell death and tumor vessel thrombosis potentiate cytotoxic therapy against pancreatic cancer.

PURPOSE: Despite current chemotherapies, pancreatic cancer remains an uncontrollable, rapidly progressive disease. Here, we tested an approach combining a recently described antiangiogenic drug, rapamycin, with standard gemcitabine cytotoxic therapy on human pancreatic tumor growth. EXPERIMENTAL DESIGN: Tumor growth was assessed in rapamycin and gemcitabine-treated nude mice orthotopically injected with metastatic L3.6pl human pancreatic cancer cells. H&E staining was performed on tumors, along with Ki67 staining for cell proliferation and immunohistochemical terminal deoxynucleotidyl transferase-mediated nick end labeling and CD31 analysis. Rapamycin-treated tumor vessels were also directly examined in dorsal skin-fold chambers for blood flow after thrombosis induction. Cell death in human umbilical vein endothelial cells was assessed by flow cytometry after annexin-V staining. RESULTS: Rapamycin therapy alone inhibited tumor growth and metastasis more than gemcitabine, with remarkable long-term tumor control when the drugs were combined. Mechanistically, H&E analysis revealed tumor vessel endothelium damage and thrombosis with rapamycin treatment. Indeed, dorsal skin-fold chamber analysis of rapamycin-treated tumors showed an increased susceptibility of tumor-specific vessels to thrombosis. Furthermore, terminal deoxynucleotidyl transferase-mediated nick end labeling/CD31 double staining of orthotopic tumors demonstrated apoptotic endothelial cells with rapamycin treatment, which also occurred with human umbilical vein endothelial cells in vitro. In contrast, gemcitabine was not antiangiogenic and, despite its known cytotoxicity, did not reduce proliferation in orthotopic tumors; nevertheless, rapamycin did reduce tumor proliferation. CONCLUSIONS: Our data suggest a novel mechanism whereby rapamycin targets pancreatic tumor endothelium for destruction and thrombosis. We propose that rapamycin-based vascular targeting not only reduces tumor vascularization, it decreases the number of proliferating tumor cells to be destroyed by gemcitabine, thus introducing a new, clinically feasible strategy against pancreatic cancer.     

Monitoring response to anticancer therapy by targeting microbubbles to tumor vasculature.

PURPOSE: New strategies to detect tumor angiogenesis and monitor response of tumor vasculature to therapy are needed. Contrast ultrasound imaging using microbubbles targeted to tumor endothelium offers a noninvasive method for monitoring and quantifying vascular effects of antitumor therapy. We investigated the use of targeted microbubbles to follow vascular response of therapy in a mouse model of pancreatic adenocarcinoma. EXPERIMENTAL DESIGN: Microbubbles conjugated to monoclonal antibodies were used to image and quantify vascular effects of two different antitumor therapies in s.c. and orthotopic pancreatic tumors in mice. Tumor-bearing mice were treated with anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and/or gemcitabine, and the localization of microbubbles to endoglin (CD105), VEGF receptor 2 (VEGFR2), or VEGF-activated blood vessels (the VEGF-VEGFR complex) was monitored by contrast ultrasound. RESULTS: Targeted microbubbles showed significant enhancement of tumor vasculature when compared with untargeted or control IgG-targeted microbubbles. Video intensity from targeted microbubbles correlated with the level of expression of the target (CD105, VEGFR2, or the VEGF-VEGFR complex) and with microvessel density in tumors under antiangiogenic or cytotoxic therapy. CONCLUSIONS: We conclude that targeted microbubbles represent a novel and attractive tool for noninvasive, vascular-targeted molecular imaging of tumor angiogenesis and for monitoring vascular effects specific to antitumor therapy in vivo.     

Inhibition of SRC tyrosine kinase as treatment for human pancreatic cancer growing orthotopically in nude mice.

PURPOSE: The Src family comprises a family of nonreceptor intracellular tyrosine kinases that mediate a variety of cellular pathways. Src kinases are overexpressed in a variety of human tumors, including cancer of the colon, breast, and pancreas, and they are an integral part of tumor cell signaling pathways associated with migration, proliferation, adhesion, and angiogenesis. EXPERIMENTAL DESIGN: We investigated whether the blockade of Src kinase by daily oral administration of the novel Src tyrosine kinase inhibitor AZM475271 [kindly provided by AstraZeneca (Macclesfield, United Kingdom)], alone or in combination with intraperitoneal gemcitabine, can inhibit growth and metastasis of orthotopically implanted human pancreatic carcinoma cells in nude mice. RESULTS: Treatment with AZM475271 alone reduced the primary pancreatic tumor volume by approximately 40%, whereas AZM475271 plus gemcitabine reduced tumor volume by 90%. Furthermore, treatment with AZM475271 and gemcitabine significantly reduced metastasis: none of eight animals who received the combination treatment had lymph node or liver metastases, compared with five of five and three of five animals, respectively, in the control group (P = 0.001). Src inhibition by AZM475271 (alone or with gemcitabine) was associated with significantly reduced tumor cell proliferation, decreased tumor microvessel density, and increased apoptosis in vivo. Moreover, these effects were all significantly increased when gemcitabine was combined with AZM475271 compared with gemcitabine alone. CONCLUSIONS: Src inhibition by AZM475271, either alone or in combination with gemcitabine, demonstrated significant antitumor and antimetastatic activity in an orthotopic nude mouse model for human pancreatic cancer. The combination of AZM475271 with gemcitabine sensitized tumor cells to the cytotoxic effect of gemcitabine.     

Matuzumab short-term therapy in experimental pancreatic cancer: prolonged antitumor activity in combination with gemcitabine

PURPOSE: The epidermal growth factor receptor ErbB-1 is commonly expressed in pancreatic cancer and ErbB-1 targeting has shown promising results. We wanted to evaluate matuzumab (EMD72000), a fully humanized ErbB-1-specific monoclonal antibody in combination with gemcitabine in experimental pancreatic cancer. EXPERIMENTAL DESIGN: Using the human pancreatic cancer cell line L3.6pl, we investigated matuzumab in vitro and in vivo. ErbB-1 phosphorylation and downstream pathway activation were evaluated by Western blot. Proliferation and migration assays and fluorescence-activated cell sorting analysis were done. For in vivo studies, we used an orthotopic nude mice model in which 40 mg/kg of matuzumab+/-100 mg/kg of gemcitabine were administered twice weekly. Different treatment durations (7, 14, 21, and 25 days) and varying time points of treatment initiation (days 8, 15, 22, and 29) were evaluated. Ki67, CD31, and phosphorylated p44/42 mitogen-activated protein kinase (MAPK) immunohistochemistry were done. RESULTS: ErbB-1 phosphorylation and downstream MAPK and AKT signaling were significantly reduced by matuzumab. Matuzumab significantly inhibited proliferation and migration in vitro, and induced tumor cell apoptosis in a dose-dependant manner. Matuzumab therapy significantly lowered tumor volume in vivo, reduced lymph node and liver metastases, and decreased microvessel density and tumor cell proliferation. These effects were significantly enhanced when gemcitabine was added. A significant and prolonged antitumor activity was even evident with short-term therapy (7 days) and with a late onset of therapy (day 22 after tumor cell injection). CONCLUSIONS: Matuzumab is an effective agent with long-lasting antiproliferative, proapoptotic, antiangiogenic, and antimetastatic activity in human pancreatic cancer models. These effects might be potentiated by gemcitabine.     

Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma

We determined whether down-regulation of the epidermal growth factor-receptor (EGF-R) signaling pathway by oral administration of a novel EGF-R tyrosine kinase inhibitor (PKI166) alone or in combination with gemcitabine (administered i.p.) can inhibit growth and metastasis of human pancreatic carcinoma cells implanted into the pancreas of nude mice. Therapy beginning 7 days after orthotopic injection of L3.6pl human pancreatic cancer cells reduced the volume of pancreatic tumors by 59% in mice treated with gemcitabine only, by 45% in those treated with PKI166 only, and by 85% in those given both drugs. The combination therapy also significantly inhibited lymph node and liver metastasis, which led to a significant increase in overall survival. EGF-R activation was significantly blocked by therapy with PKI166 and was associated with significant reduction in tumor cell production of VEGF and IL-8, which in turn correlated with a significant decrease in microvessel density and an increase in apoptotic endothelial cells. Collectively, our results demonstrate that oral administration of an EGF-R tyrosine kinase inhibitor decreased growth and metastasis of human pancreatic cancer growing orthotopically in nude mice and increased survival. The therapeutic effects were mediated in part by inhibition of tumor-induced angiogenesis attributable to a decrease in production of proangiogenic molecules by tumor cells and increased apoptosis of tumor-associated endothelial cells.     

Continuous administration of the three thrombospondin-1 type 1 repeats recombinant protein improves the potency of therapy in an orthotopic human pancreatic cancer model

Thrombospondin-1 is one of most important natural angiogenic inhibitors. The three thrombospondin-1 type 1 repeats (3TSR), an anti-angiogenic domain of thrombospondin-1, is a promising novel agent for anti-angiogenic treatment. In the present study, we showed 3TSR was biologically stable at least for 7 days in mini-osmotic pumps in vivo, and continuous administration of 3TSR decreased the dosage and improved the potency of therapy in an orthotopic pancreatic cancer model. By using different dosage and delivery routes, we proved that the anti-tumor efficacy of 3TSR was correlated with its anti-angiogenic efficacy. 3TSR treatment also decreased tumor vessel patency and blood flow. The results indicate the advantage of continuous administration of angiogenic inhibitors and provide rationale for using such delivery methods for cancer treatment.     

Direct effects of recombinant human endostatin on tumor cell IL-8 production are associated with increased endothelial cell apoptosis in an orthotopic model of human pancreatic cancer

PURPOSE: Recombinant human endostatin (rhES) is an antiangiogenic agent derived from collagen XVIII which inhibits tumor growth in subcutaneous models of various human malignancies. However, its effectiveness in an orthotopic xenograft model of an abdominal neoplasm has not been demonstrated. DESIGN: An orthotopic model of pancreatic cancer was established in 6-week-old male athymic mice from either of 2 human cell lines (L3.6pl or BxPC3). Established tumors were treated with 40 mg/kg rhES or vehicle controls for up to 3 weeks. Tumors were analyzed by immunohistochemistry for TUNEL/CD31, IL-8, VEGF, and bFGF. We also measured direct effects of rhES on tumor cell angiogenic factor production by ELISA in vitro. RESULTS: Overall tumor burden was not reduced with rhES treatment in mice inoculated with either cell line. Peritoneal carcinomatosis in the L3.6pl mice was greater in those treated with rhES than in those treated with normal saline or citrate buffer (p < 0.05). Treatment with rhES lowered IL-8 levels 32-47% in vivo (p < 0.001) and 40-65% in vitro (p < 0.05) in the fast-growing L3.6pl tumors but not in the slow-growing BxPC3 tumors (p < 0.05). rhES also increased the levels of endothelial cell apoptosis 16- to 24-fold in vivo in the L3.6pl mice, but not in the BxPC3 mice (p < 0.05). CONCLUSIONS: rhES downregulated IL-8 levels and induced endothelial cell apoptosis in the more aggressive cell line in a xenograft model of pancreatic cancer. Nonetheless, these effects were not sufficient to produce significant inhibition of tumor growth.     

Vitamin D binding protein-macrophage activating factor (DBP-maf) inhibits angiogenesis and tumor growth in mice

We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf) generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is antiproliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09). At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.     

ABT-510, a modified type 1 repeat peptide of thrombospondin, inhibits malignant glioma growth in vivo by inhibiting angiogenesis

Anti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown to inhibit angiogenesis in non-glioma tumor models and a modified TSR peptide, ABT-510, has now entered into Phase II clinical trials of its efficacy in non-glioma tumors. As microvascular endothelial cells (MvEC) exhibit heterogeneity, we evaluated the ability of the modified TSR peptide (NAcSarGlyValDallolleThrNvalleArgProNHE, ABT-510) to inhibit malignant glioma growth in vivo and to induce apoptosis of brain microvessel endothelial cells (MvEC) propagated in vitro. We found that daily administration of ABT-510 until euthanasia (days 7 to 19), significantly inhibited the growth of human malignant astrocytoma tumors established in the brain of athymic nude mice. The microvessel density was significantly lower and the number of apoptotic MvEC was significantly higher (3-fold) in the tumors of the ABT-510-treated animals. Similar results were found using a model in which the established tumor is an intracerebral malignant glioma propagated in a syngeneic mouse model. ABT-510 treatment of primary human brain MvEC propagated as a monolayer resulted in induction of apoptosis in a dose- and time-dependent manner through a caspase-8-dependent mechanism. It also inhibited tubular morphogenesis of MvEC propagated in collagen gels in a dose- and caspase-8 dependent manner through a mechanism that requires the TSP-1 receptor (CD36) on the MvEC. These findings indicate that ABT-510 should be evaluated as a therapeutic option for patients with malignant glioma.     

Effect of the vascular endothelial growth factor receptor-2 antibody DC101 plus gemcitabine on growth,metastasis and angiogenesis of human pancreatic cancer growing orthotopically in nude mice

Vascular endothelial growth factor (VEGF) is the major pro-angiogenic factor for most tumors. VEGF expression has been shown to be associated with a poor prognosis in human pancreatic cancer. The purpose of our study was to determine the effect of blockade of VEGF receptor-2 activity with or without gemcitabine on tumor growth and metastasis in an orthotopic model of human pancreatic cancer in nude mice. Therapy with gemcitabine or DC101, a VEGF receptor-2 antibody, resulted in a significant reduction of primary pancreatic tumor growth compared to untreated controls. The combination of DC101 and gemcitabine inhibited primary pancreatic tumor growth and lymphatic metastasis to a greater degree than either agent alone. Treatment with DC101 decreased vessel counts and increased the area of hypoxic tumor tissue compared to controls. Immunofluorescent double staining for apoptotic endothelial cells demonstrated a significant increase in the number apoptotic endothelial cells 24 days after initiation of therapy with DC101 plus gemcitabine. DC101 plus gemcitabine also increased tumor cell death and decreased tumor cell proliferation in pancreatic tumors. These findings indicate that blockade of VEGF receptor activation interferes with the survival of tumor endothelial cells, resulting in a reduction of primary pancreatic tumor growth in nude mice. Furthermore, the data demonstrate that anti-VEGF receptor-2 therapy potentiates the tumoricidal effect of gemcitabine in this model. Anti-VEGF receptor-2 therapy in combination with gemcitabine may be a novel therapeutic approach for advanced pancreatic cancer.     

Optimization for the blockade of epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma.

We determined the optimal administration schedule of a novel epidermal growth factor receptor (EGFR) protein tyrosine kinase inhibitor (PKI), PKI 166 (4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine), alone or in combination with gemcitabine (administered i.p.) for therapy of L3.6pl human pancreatic carcinoma growing in the pancreas of nude mice. Seven days after orthotopic implantation of L3.6pl cells, the mice received daily oral doses of PKI 166. PKI 166 therapy significantly inhibited phosphorylation of the EGFR without affecting EGFR expression. EGFR phosphorylation was restored 72 h after cessation of therapy. Seven days after orthotopic injection of L3.6pl cells, groups of mice received daily or thrice weekly oral doses of PKI 166 alone or in combination with gemcitabine. Treatment with PKI 166 (daily), PKI 166 (3 times/week), or gemcitabine alone produced a 72%, 69%, or 70% reduction in the volume of pancreatic tumors in mice, respectively. Daily oral PKI 166 or thrice weekly oral PKI 166 in combination with injected gemcitabine produced 97% and 95% decreases in volume of pancreatic cancers and significant inhibition of lymph node and liver metastasis. Daily oral PKI 166 produced a 20% decrease in body weight, whereas treatment 3 times/week did not. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased tumor cell and endothelial cell apoptosis correlated with therapeutic success. Collectively, our results demonstrate that three weekly oral administrations of an EGFR tyrosine kinase inhibitor in combination with gemcitabine are sufficient to significantly inhibit primary and metastatic human pancreatic carcinoma.     

Synergistic antitumor activity of ZD6474, an inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling, with gemcitabine and ionizing radiation against pancreatic cancer.

PURPOSE: Standard treatments have modest effect against pancreatic cancer, and current research focuses on agents targeting molecular pathways involved in tumor growth and angiogenesis. This study investigated the interactions between ZD6474, an inhibitor of tyrosine kinase activities of vascular endothelial growth factor receptor-2 and epidermal growth factor receptor (EGFR), gemcitabine, and ionizing radiation in human pancreatic cancer cells and analyzed the molecular mechanisms underlying this combination. EXPERIMENTAL DESIGN: ZD6474, ionizing radiation, and gemcitabine, alone or in combination, were given in vitro to MIA PaCa-2, PANC-1, and Capan-1 cells and in vivo to MIA PaCa-2 tumor xenografts. The effects of treatments were studied by the evaluation of cytotoxicity, apoptosis, cell cycle, EGFR and Akt phosphorylation, modulation of gene expression of enzymes related to gemcitabine activity (deoxycytidine kinase and ribonucleotide reductase), as well as vascular endothelial growth factor immunohistochemistry and microvessel count. RESULTS: In vitro, ZD6474 dose dependently inhibited cell growth, induced apoptosis, and synergistically enhanced the cytotoxic activity of gemcitabine and ionizing radiation. Moreover, ZD6474 inhibited phosphorylation of EGFR and Akt and triggered cell apoptosis. PCR analysis showed that ZD6474 increased the ratio between gene expression of deoxycytidine kinase and ribonucleotide reductase. In vivo, ZD6474 showed significant antitumor activity alone and in combination with radiotherapy and gemcitabine, and the combination of all three modalities enhanced MIA PaCA-2 tumor growth inhibition compared with gemcitabine alone. CONCLUSIONS: ZD6474 decreases EGFR and Akt phosphorylation, enhances apoptosis, favorably modulates gene expression in cancer cells, and acts synergistically with gemcitabine and radiotherapy to inhibit tumor growth. These findings support the investigation of this combination in the clinical setting.     

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.     

Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic cancer cell lines.

PURPOSE: Vascular endothelial growth factor A (VEGF-A) is a potent angiogenic agent that binds to two high affinity VEGF receptors (VEGFRs), a process facilitated by the low affinity neuropilin receptors. Although VEGF-A is overexpressed in pancreatic ductal adenocarcinoma, it is not known whether the in vivo growth of multiple pancreatic cancer cells can be efficiently blocked by VEGF-A sequestration. EXPERIMENTAL DESIGN: Four human pancreatic cancer cell lines were grown s.c. in athymic nude mice. One cell line also was used to generate an orthotopic model of metastatic pancreatic cancer. The consequences of VEGF-A sequestration on tumor growth and metastasis were examined by injecting the mice with a soluble VEGFR chimer (VEGF-Trap) that binds VEGF-A with high affinity. RESULTS: VEGF-Trap, initiated 2 days after tumor cell inoculation, suppressed the s.c. growth of four pancreatic cancer cell lines and markedly decreased tumor microvessel density. Analysis of RNA from tumors generated with T3M4 cells revealed that VEGF-Trap decreased the expression of VEGFR-1 and neuropilin-1 and -2. VEGF-Trap, initiated 3 weeks after tumor implantation, also attenuated intrapancreatic tumor growth and metastasis in an orthotopic model using PANC-1 cells. CONCLUSIONS: VEGF-Trap is a potent suppressor of pancreatic tumor growth and metastasis and also may act to attenuate neuropilin-1 and -2 and VEGFR-1 expression. Therefore, VEGF-Trap may represent an exceedingly useful therapeutic modality for pancreatic ductal adenocarcinoma.     

Interleukin-4 cytotoxin therapy synergizes with gemcitabine in a mouse model of pancreatic ductal adenocarcinoma

Targeting cell surface receptors with cytotoxins or immunotoxins provides a unique opportunity for tumor therapy. Here, we show the efficacy of the combination therapy of gemcitabine with an interleukin-4 (IL-4) cytotoxin composed of IL-4 and truncated Pseudomonas exotoxin in animal models of pancreatic ductal adenocarcinoma (PDA). We have observed that 42 of 70 (60%) tumor samples from patients with PDA express moderate- to high-density surface IL-4 receptor (IL-4R), whereas normal pancreatic samples express no or low-density IL-4R. IL-4 cytotoxin was specifically and highly cytotoxic [50% protein synthesis inhibition (IC50) ranging from >0.1 to 13 ng/mL] to six of eight pancreatic cancer cell lines, whereas no cytotoxicity (IC50>1,000 ng/mL) was observed in normal human pancreatic duct epithelium cells, fibroblasts, and human umbilical vein endothelial cells (HUVEC). We also showed that IL-4 cytotoxin in combination with gemcitabine exhibited synergistic antitumor activity in vitro. To confirm synergistic antitumor activity in vivo and monitor precise real-time disease progression, we used a novel metastatic and orthotopic mouse model using green fluorescent protein-transfected cancer cells and whole-body imaging system. The combination of both agents caused complete eradication of tumors in 40% of nude mice with small established PDA tumors. In addition, combined treatment significantly prolonged the survival of nude mice bearing day 14 advanced distant metastatic PDA tumors. Similar results were observed in mice xenografted with PDA obtained from a patient undergoing surgical resection. These results indicate that IL-4 cytotoxin combined with gemcitabine may provide effective therapy for the treatment of patients with PDA.     

Administration of optimal biological dose and schedule of interferon alpha combined with gemcitabine induces apoptosis in tumor-associated endothelial cells and reduces growth of human pancreatic carcinoma implanted orthotopically in nude mice.

PURPOSE: We determined whether chronic administration of IFN-alpha at optimal biological dose inhibits angiogenesis of human pancreatic carcinoma growing in the pancreas of nude mice. EXPERIMENTAL DESIGN: Cells of the human pancreatic cancer cell line L3.6pl were implanted into the pancreas of nude mice. Seven days later, groups of mice received s.c. injection with IFN-alpha alone (50,000 units biweekly or 10,000 units daily), i.p. injection with gemcitabine alone (125 mg/kg biweekly), or injection with both daily IFN-alpha and biweekly gemcitabine for 35 days. In a survival study, the mice were treated until they became moribund. RESULTS: Biweekly treatments with 50,000 units of IFN-alpha alone were ineffective. In contrast, daily injections of IFN-alpha (10,000 units/day) alone, biweekly injections of gemcitabine alone, or the combination of IFN-alpha and gemcitabine reduced tumor volume by 53%, 70%, and 87%, respectively. Immunohistochemical analysis revealed that treatment with IFN-alpha alone or with IFN-alpha plus gemcitabine inhibited expression of the proangiogenic molecules basic fibroblast growth factor and matrix metalloproteinase 9 more than did treatment with gemcitabine alone. These treatments also decreased the staining of proliferating cell nuclear antigen within the tumor and induced apoptosis in tumor-associated mouse endothelial cells (staining with CD31/terminal deoxynucleotidyl transferase-mediated nick end labeling), leading to a decrease in microvessel density. CONCLUSIONS: These data show that administration of IFN-alpha at optimal biological dose and schedule in combination with gemcitabine induced apoptosis in tumor-associated endothelial cells and decreased growth of human pancreatic cancer cells in the pancreas, leading to a significant increase in survival.