BMC Cancer 2008, 8:352 http://www.biomedcentral.com/1471-2407/8/352 weekly by intraperitoneal (i.p.) injection and was contin- ued for six weeks. Animals were sacrificed at this time as control mice developed lethargy and failure to properly groom. Immunohistochemistry Tissue sections were either snap frozen in liquid nitrogen or fixed in formalin (Sigma, St Louis, MO) and paraffin embedded. Hematoxylin and eosin (H&E) staining was done by the Molecular Pathology Core at UT Southwest- ern (Dallas, TX). Paraffin embedded sections were first de- paraffinized by heating to 60°C followed by xylene immersion. Sections were re-hydrated with sequential ethanol submersion. Non-specific binding was blocked with 20% Aquablock (East Coast Biologics, North Ber- wick, ME) for 30 minutes. Primary antibody (MECA-32 [16], Gv39M [17]) was added for one hour at room tem- perature or overnight at 4°C and subsequently developed with the appropriate secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). TUNEL analysis was done per manufacturer's recommendations (Promega, Madison, WI). Immunofluorescent sections were quantified using Nikon Elements imaging software (Melville, NY). Statistics Data was collected into a spreadsheet program (Microsoft Excel) and then imported into GraphPad (GraphPad Prism version 4.00 for Windows; GraphPad Software, San Diego CA, http://www.graphpad.com). Differences between means of two groups were calculated using a Mann-Whitney test; differences between three or more groups were determined with non-parametric one-way ANOVA (Kruskal-Wallis test) with Dunn's post test for multiple comparisons. Statistical significance was estab- lished as p < 0.05. Results CT-322 inhibits the growth of orthotopic MiaPaCa-2 pancreatic tumors To test whether direct inhibition of VEGFR2 with CT-322 inhibits pancreatic tumor growth, we used an orthotopic model of pancreatic cancer. In this model, MiaPaCa-2 human pancreatic cancer cells were injected into the tail of the pancreas of nude mice and tumors were allowed to develop prior to treatment [15]. Therapy consisted of vehicle control or CT-322 given twice weekly and was ini- tiated ten days after tumor cell injection. At sacrifice, tumor weights were measured. CT-322 reduced pancreatic tumor growth by approximately 50% in this model (Fig 1). In this model CT-322 did not reduce metastatic events and the incidence of metastasis was the same (8/9, 88%) in the control and CT-322 treated animals and the mean number of metastases was similar in both groups (1.7, CT- 322; 2.1, control). aFCtiTigc-u3cra2en2c1reerduces tumor size in a xenograft model of pancre- CT-322 reduces tumor size in a xenograft model of pancreatic cancer. Human MiaPaCa-2 tumors were estab- lished in the tail of the pancreas in athymic nude mice. Ther- apy was initiated 10 days after tumor cell injection. Treatment with the anti-VEGFR2 Adnectin CT-322 (30 mg/ kg) was given by i.p. injection twice weekly for 6 weeks (until sacrifice). Mean (+/- SEM) tumor weight at the time of sacri- fice is displayed. CT-322 significantly reduced tumor size compared to control treated animals (Control 1.11 ± 0.17 g, CT-322 0.64 ± .010 g; n = 9 animals per group. *p < 0.05). CT-322 is effective in combination with gemcitabine in a syngeneic model of pancreatic cancer One advantage of CT-322 compared to human mono- clonal antibodies specific for VEGF or VEGFR2 is the fact that CT-322 binds to both murine and human VEGFR2. This allows for testing in a syngeneic model using immu- nocompetent animals. Additionally in this experiment, we tested the combination of CT-322 and gemcitabine, the standard chemotherapy for pancreatic cancer. Tumors were established in C57BL/6J mice using the murine pan- creatic carcinoma cell line Pan02. Therapy was started one week later and continued for 6 weeks. The combination of CT-322 and gemcitabine was an effective therapeutic reg- imen, reducing tumor growth by >50% (Fig 2). Both CT- 322 and gemcitabine alone decreased tumor growth, but combination therapy had a greater effect and was the only treatment group to reach statistical significance compared to control-treated animals. Metastatic disease was assessed by visual inspection of abdominal organs, lymph nodes and lungs. In this model all treatment groups showed decreased metastatic burden compared to control (Table 1). CT-322 reduces microvessel density VEGF inhibition is thought to exert anti-tumor effects in part by inhibiting the development of blood vessels in the tumor. To assess the effect of CT-322 on tumor angiogen- esis, sections of tumor were analyzed by immunohisto- chemistry with MECA-32, an antibody specific for mouse Page 3 of 10 (page number not for citation purposes)