Figure 3. r84 controls tumor growth in vivo. 2.5 million human NSCLC cells were injected subcutaneously into the right flank of NOD/ SCID mice. Therapy began one day post tumor cell injection (TCI), and continued for 4–8 weeks. Tumor volumes were measured twice/week and final tumor weights were recorded at sacrifice. r84 and bevacizumab (bev) similarly control tumor growth and final weight compared to control IgG treatment in H460, H1299 models (A, B). C, In A549 NSCLC tumor bearing animals, r84’s control of tumor growth was significantly different from bev (p,0.05). D, Titration of antibody dosing r84 Inhibits VEGFR2 in A549 tumor xenografts showed no change in tumor growth and final tumor weight with increasing doses of bev, however there was increasing control of tumor growth with increasing doses of r84, with doses of r84 controlling growth better than bev (D). A–C, n = 8 mice per group; D, n = 6 mice per group. *p,0.05, **p,0.01, ***p,0.001, statistical differences compared to Ctrl treatment, unless otherwise indicated. doi:10.1371/journal.pone.0012031.g003 therapy and given recent data suggesting a role for VEGFR2 in controlling blood pressure [13,26], we investigated the effects of r84 therapy on hypertension and proteinuria in a spontaneous, immunocompetent model of pancreatic cancer. Mice (p48- Cre:LSL-KrasG12D:p16ink4a/arf+/lox) expressing a pancreas-specific Cre recombinanse activating a constitutively active Kras allele (KrasG12D) and inactivating a single copy of Ink4a/Arf that spontaneously develop pancreatic ductal adenocarcinoma (PDAC) [17] were separated into three groups receiving either saline, mouse chimeric r84 (mcr84 [21]), or sunitinib. Therapy began when mice when mice were eight weeks old, with weekly IP injections of saline 25 mg/kg/week or mcr84, or daily oral gavage of 50 mg/kg/week sunitinib. Therapy continued for a total of eight weeks, at which time all animals were sacrificed. Tumor burden, as assessed by final pancreas weight at sacrifice, was reduced in mcr84-treated animals as compared to control- and sunitinib-treated animals, although this trend failed to reach significance (Figure S2C). However, treatment with mcr84 or sunitinib resulted in decreased tumor MVD (Figure S2D, p,0.01 or p,0.05, respectively). The effects of r84 in PDAC and its possible therapeutic benefit in animal models of PDAC are active areas of research in our lab. Tail cuff blood pressure measure- ments were gathered during weeks two and seven of therapy. At week two, animals in the mcr84 and sunitinib groups displayed elevated systolic blood pressure as compared to control-treated animals (Figure 5C, left panel, p,0.001). However, at week seven neither mcr84- nor sunitinib-treated animals displayed elevated systolic blood pressures as compared to control-treated animals although blood pressures were significantly higher in sunitinib- treated animals than in those receiving mcr84 (Figure 5C, right panel, p,0.05). Thus in this model, inhibiting VEGFR2 with mcr84 or multiple receptor tyrosine kinases including both VEGFR1 and VEGFR2 with sunitinib increased systolic blood pressure after acute but not chronic therapy. During week six of therapy, metabolic cages were used to collect urine samples from all mice, which were subsequently analyzed for urine protein (Upro) and creatine levels by UT Southwestern Medical Center’s mouse metabolic phenotyping core. The Upro:creatine ratio did not differ between the three treatment groups, suggesting that long term treatment with mcr84 and sunitinib does not induce kidney damage (Figure 5D). Similar to the initial toxicity study in NOD/ SCID mice, blood was collected from all animals at sacrifice for analysis by the mouse metabolic phenotyping core, which again yielded no significant changes in any of the 18 tested analytes between mcr84-, sunitinib-, or control-treated animals (Table S2). Therefore, in a spontaneous tumor model in immunocompetent animals, chronic treatment with mcr84 failed to produce observable, lasting toxicity. Discussion Angiogenesis is a crucial process during embryonic development and normal physiology, and during tumor development, growth, and progression [2]. Anti-angiogenic therapy therefore presents an exciting and rational approach for tumor therapy. However, the clinical efficacy of anti-angiogenic therapies have been mostly PLoS ONE | www.plosone.org 7 August 2010 | Volume 5 | Issue 8 | e12031