BMC Cancer 2008, 8:352 http://www.biomedcentral.com/1471-2407/8/352 TFmuiogmdueorlreos2fizpeainscredauticecdanafcter CT-322 treatment in a syngeneic Tumor size is reduced after CT-322 treatment in a syngeneic model of pancreatic cancer. Orthotopic murine Pan02 tumors were established in C57BL/6J wild- type mice. Treatment twice weekly i.p. injections of CT-322 (30 mg/kg), gemcitabine (3.5 mg/animal/injection) (GEM), or CT-322 (CT) + GEM was started 7 days after tumor cell injection and continued for 6 weeks, at which point animals were sacrificed. Mean (+/- SEM) tumor weight at the time of sacrifice is displayed. The combination treatment of CT-322 and GEM significantly inhibited tumor growth (control 0.44 ± 0.11 g, 0.153 ± 0.01 g; n = 8/group except CT+GEM n = 10; p < 0.05). endothelial cells. Treatment with CT-322 reduced tumor microvessel density significantly in the MiaPaCa-2 xenograft model (Fig 3) and the Pan02 syngeneic model (Fig 4). Furthermore, CT-322 also decreased the level of VEGF- activated blood vessels. VEGF binding to VEGFR2 medi- ates the activation of endothelial cells and induces angio- genesis [18]. An antibody that recognizes VEGF bound to Table 1: Effect of therapy on metastatic burden in mice bearing Pan02 tumors GROUP METASTASIS Control CT GEM CT+ GEM Number 14 0 0 0 Incidence (%) 6/8 (75) 0/8 (0) 0/8 (0) 0/10 (0) Metastatic events in mice bearing orthotopic Pan02 tumors were evaluated by visual inspection of the liver, lungs, and entire gastrointestinal tract at the time of sacrifice. VEGFR (Gv39M) [17,19] was used to assess whether CT- 322 had an effect in vivo on the activation state of blood vessels in MiaPaCa-2 tumors. As shown in figure 5A, there were no blood vessels that were Gv39M positive following CT-322 treatment, whereas approximately 30% of vessels in the control treated animals were positive for a standard endothelial cell marker (MECA-32) and Gv39M (Fig 5B). Increased tumor destruction following CT-322 treatment Routine hematoxylin and eosin staining demonstrated that treatment of mice bearing orthotopic MiaPaCa-2 tumors with CT-322 resulted in a significant increase in tumor necrosis (Fig 6A). Furthermore, treatment with CT- 322 induced a significant increase in the number of TUNEL-positive cells in the tumor tissue compared to control treated animals (Fig 6B). Discussion Pancreatic cancer remains a deadly disease, with a dismal five year survival [1]. Contributing to the poor outcome is the resistance or insensitivity of most pancreatic cancers to chemotherapy. Gemcitabine, the current standard of care, extends life only minimally. Therefore, novel therapeutics agents are necessary to improve the survival associated with this disease. Angiogenesis has been described as both a hallmark of cancer [2] and as an "organizing principle" around which anti-cancer therapy can be developed [20]. VEGF is a key mediator of tumor angiogenesis that functions primarily through VEGFR2. The complexity of the VEGF signaling pathway allows for multiple levels of inhibition, as seen by the large number of lead compounds entering trials that target this pathway [8,20]. One approach for VEGF inhibition is to reduce the availability of ligand (VEGF), as demonstrated by bevacizumab, a monoclonal antibody specific for human VEGF [9]. This approach has been val- idated in clinical trials combining chemotherapy and bev- acizumab in the treatment of metastatic colorectal [10] or breast cancer [21]. However, despite initially promising results in pancreatic cancer, a subsequent randomized controlled trial failed to show clinical benefit of combin- ing bevacizumab with gemcitabine [22,23]. In this series of experiments, we chose to investigate a novel anti-angiogenic agent. The Adnectin CT-322 binds VEGFR2 and prevents activation of the receptor. This approach is different than marketed tyrosine kinase inhib- itors (e.g., sutent and sorafenib) in that CT-322 selectively blocks VEGFR2 activation without affecting other recep- tors or pathways. Additionally, the Adnectin compound utilizes E. coli-based manufacturing methods, making it well suited for clinical drug development. Page 4 of 10 (page number not for citation purposes)