BMC Cancer 2008, 8:352 http://www.biomedcentral.com/1471-2407/8/352 Background Pancreatic cancer continues to carry a poor prognosis, with a 5-year survival rate of approximately five percent [1]. As patients typically present at an advanced stage, new chemotherapeutic strategies are necessary to improve the dismal prognosis associated with this disease. Angiogen- esis is a hallmark of cancer [2] and is required for cancer growth beyond 1–2 mm3 [2,3]. Vascular endothelial growth factor A (VEGF) is the best characterized member of the VEGF family of growth factors. VEGF is a potent angiogenic factor expressed during development and in tumors [4,5]. The effects of VEGF are mediated by binding to one of its two receptors VEGF receptor 1 or 2 (VEGFR1, VEGFR2) [4,6]. Tumor angiogenesis is driven primarily by VEGF:VEGFR2 interaction [5,6]. The effect of VEGFR1 activation is less understood, but is thought to be involved in macrophage chemotaxis [5-7]. The complexity of the VEGF pathway allows for multiple targets for inhibiting tumor angiogenesis [5,8]. For exam- ple, bevacizumab (Avastin®, Genentech, Inc., South San Francisco, CA) is a monoclonal antibody to human VEGF which binds VEGF and blocks its interaction with both VEGFR1 and VEGFR2 [9]. Bevacizumab has been shown to be effective in combination with chemotherapy for the treatment of metastatic colorectal cancer and non-small cell lung cancer [10,11]. Receptor tyrosine kinase inhibi- tors have also been developed which inhibit the VEGF receptors [5,8]. These small molecules penetrate into cells and, unlike antibodies, inhibit multiple members of the VEGF receptor family. This broad spectrum of inhibition may lead to different side effect profiles from monoclonal antibodies [5]. There are a variety of proteins being developed as new bio- logic drugs beyond the traditional biologic class of mon- oclonal antibodies [12]. Adnectins are a new class of targeted biologics among the most advanced of such pro- teins. Adnectins are well-suited to pharmaceutical discov- ery and development, based on preclinical data [13,14]. These small proteins are derived from the 10th type III domain of human fibronectin, an extracellular protein that is abundant in human serum and the extracellular matrix, and naturally binds to other proteins [13,14]. By changing the amino acid sequence of three targeting loops clustered at one end of the protein, an Adnectin can be designed to bind to a specific disease target, such as a receptor, ligand or protein with nanomolar or picomolar affinity, and potency and specificity comparable to or bet- ter than antibodies. One such Adnectin has been devel- oped that binds to VEGFR2. This construct, CT-322, has been shown previously to block the activity of murine and human VEGFR2 in vitro [13]. In the present study, we were interested in whether this novel compound would block tumor angiogenesis and subsequent growth in an orthotopic model of pancreatic cancer. In the following experiments, we demonstrate that CT-322 is effective at treating pancreatic tumors in two animal models, that CT-322 blocks tumor angiogenesis and that treatment with CT-322 induces tumor destruc- tion. Methods Cell Lines and Culture The human pancreatic carcinoma cell line MiaPaCa-2 (ATCC, Manassas, VA) and the murine cell line Pan02 also known as Panc02 (NCI, Frederick, MD) were main- tained at 37°C in a mixture of 5% CO2 and 95% air in Dulbecco's minimal essential medium (DMEM; Invitro- gen, Carlsbad, CA) supplemented with 10% fetal calf serum (Gimini Bio-Products, Woodland, CA). Cell sus- pensions for tumor cell injection (TCI) were made by removing cells from tissue culture flask with 0.2% trypsin. The reaction was stopped with serum-containing DMEM. Cells underwent centrifugation at 230 × g for 5 min and the cell pellet was resuspended to the appropriate concen- tration in PBS. Cell suspensions were used only if viability exceeded 95% by trypan blue exclusion assay. Animal Models Athymic female nude mice were purchased from Charles River (Wilmington, MA). Female C57BL/6J wild-type mice were purchased from Jackson Laboratories (Bar Har- bor, MA). Animals were housed with ad libitum access to food and water in a pathogen free facility. All procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of UT Southwestern (Dallas, TX). Orthotopic tumors were established by injection of cells directly into the pancreas as previously described [15]. Briefly, animals were anesthetized using 2–4% iso- fluorane. The abdominal wall and peritoneum were opened and the spleen and the tail of the pancreas were identified and externalized through the wound. Tumor cells were injected under the capsule the pancreas using a 30-gauge needle (2.5 × 105 Mia-PaCa-2 cells, 4 × 105 Pan02 cells, in 50 μl of PBS). The incision was closed with a 5-0 prolene suture. Treatment In the xenograft model (MiaPaCa-2) therapy began 10 days after tumor cell injection (TCI). Therapy groups con- sisted of control (vehicle provided by Adnexus, Waltham, MA) and CT-322 (30 mg/kg, provided by Adnexus) in the xenograft model. In the syngeneic model (Pan02) a gem- citabine (3.5 mg/mouse) group and a combined CT-322 + gemcitabine group were added. Therapy was given twice Page 2 of 10 (page number not for citation purposes)