Mesenchymal Specific Inhibition Of Vascular Endothelial Growth Factor (VEGF) Attenuates Growth In Neonatal Mice

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Introduction: Numerous antibodies have been developed against tumor specific antigens on the cancer cell surface. For this reason, radiolabeled antibodies have the potential to be sensitive and specific PET imaging agents. Intact antibodies, however, are limited by a long serum half-life (10-20days) preventing adequate contrast between the tumor and surrounding blood. Smaller engineered antibody fragments overcome this problem by exhibiting shorter serum half-lives (4-20hrs).the diabody is the smallest antibody fragment which retains the divalency of the intact antibody. It is composed of the variable heavy and light chain and is approximately 55 kDa. Our goal was to develop and characterize the Anti-CA19-9 Diabody fragment and determine its ability to provide sensitive and specific imaging of pancreas cancer. Methods: Expression of CA19-9 was evaluated using flow cytometry on human cancer cell lines (BxPC3, Capan-2, MiaPaca-2). The diabody DNA construct was created by isolation of the variable region mRNA and utilization of RT-PCR. This construct was ligated into the pEE12 vector for transfection into NSO mouse myeloma cells for screening and production of clones that secrete the anti-CA19-9 diabody. The diabody was purified using HPLC and evaluated in vitro with flow cytometry and immunofluorescence to confirm binding ability. The diabody was labeled with a positron emitting radionuclide (Iodine-124) and injected into mice harboring an antigen positive xenograft (BxPC3 or Capan-2) and a negative xenograft (MiaPaca). MicroCT and MicroPET were performed at successive time intervals after injection. Radioactivity was measured in blood and tumor to provide objective confirmation of the microPET images. Results: Immunofluorescence and flow cytometry showed specific binding of the Anti-CA19-9 Diabody comparable to that of the parental intact antibody. Pancreas xenograft imaging of BxPC3/MiaPaca-2 with the Anti-CA19-9 Diabody demonstrated an average tumor:blood ratio of 5.0 and positive:negative tumor ratio of 8.8. Additionally, imaging of pancreatic xenografts, Capan-2/MiaPaca-2, demonstrated an average tumor:blood ratio of 2.04 and positive:negative tumor ratio of 6.2. See Figure 1. Conclusions: Molecular imaging of pancreas cancer should improve our ability to evaluate extent of disease as well as the effect of systemic therapies. To this end, we have created and characterized an Anti-CA19-9 Diabody. Furthermore, we demonstrate that Anti-CA19-9 Diabody fragment PET imaging achieves adequate tumor to blood ratios to provide sensitive and specific molecular imaging of pancreas cancer in tumor xenograft models. Additionally, the diabody fragment has the potential to provide specific tumor targeting of therapeutic nanoparticles.

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been elucidated. We sought to determine the effect of mesenchymal specific VEGF inhibition in the neonatal mouse using a novel transgenic animal model that demonstrates mesenchymal specific expression of the soluble VEGF receptor, sFlt-1. Methods: We generated triple transgenic mice that express the soluble VEGF receptor, sFlt1 specifically in the mesenchyme (Dermo-1/rtTA/sflt-1; N¼ 15; Figure 1A right panel) by selectively crossing transgenic mice. Mothers of the resulting pups (transgenic and littermate controls) were fed doxycycline chow at birth for transgene activation via breastmilk. At P21 the mice were sacrificed. Serum VEGF levels were quantified with ELISA using the Bradford protocol. Paraffin sections stained for Hematoxylin and Eosin were taken of the brain, lungs, liver, spleen, stomach, small intestine, kidney, pancreas, and colon. To test the reversibility of the system, a triple transgenic and littermate control mouse were switched to normal chow at P50 and monthly weights were measured for 9 months. Results: The Dermo-1-Cre/rtTA/ sFlt-1 mice were significantly smaller compared to littermate controls (6.3361.25 vs. 12.43 62.02 g; P<0.05; Figure 1A, left panel). There was a significant reduction in serum VEGF levels following sFlt-1 expression in these mice (Figure 1B). The rate of growth was also reduced (Figure 1C) but did not appear to impact overall survival after 9 months (85.7% transgenic, 87.5% control). A significant reduction in organ size as a% body weight was seen in the kidney and stomach (Figure 1D); however, no gross histologic difference was observed (not shown). The intestine of the Dermo-1-Cre/rtTA/sflt-1 mice demonstrated a striking difference in villus number and height per high-powered field (128.8646.8 vs. 270.57666.03 um; P<0.05). The native histologic architecture was preserved in all other organs. After 6 months on a normal chow diet, the Dermo-1-Cre/rtTA/sFlt-1 mouse’s weight doubled, indicating reversibility of transgene activation. Conclusions: Mesenchymal specific inhibition of VEGF in neonatal mice attenuates whole body and organ growth, most notably in the small intestine. In our transgenic model, this process is reversible and does not affect overall survival at 9 months.

31.2. Mesenchymal Specific Inhibition Of Vascular Endothelial Growth Factor (VEGF) Attenuates Growth In Neonatal Mice. J. A. Matthews, F. G. Sala, A. L. Speer, Y. Li, T. C. Grikscheit; Childrens Hospital Los Angeles, Los Angeles, C

31.3. Alagebrium (ALT-711) Attenuates Gastric Aspiration Induced Pulmonary Inflammatory Response In Mice Fed With High AGE Diet. J. M. Ottosen,1 B. Mullan,2 B. A. Davidson,2 M. T. Dayton,1 P. R. Knight,2 W. A. Guo1; 1Dept of Surgery, State University of New York - Buffalo, Buffalo, NY; 2 Dept. Of Anesthesiology, State University of New York Buffalo, Buffalo, NY

Introduction: VEGF is an endothelial cell mitogen that is a key mediator of angiogenesis and vasculogenesis. Previous studies have shown a significant mortality in VEGF knockout mice by postnatal day 7 (38%; near knock-out efficiency). However, the effect of mesenchymal specific inhibition of VEGF in the neonatal mouse has not

Introduction: Our previous experiments have demonstrated that high dietary AGEsexacerbate the pulmonary inflammatory response in mice following gastric aspiration, as evidenced by increased neutrophilic infiltrate and enhanced myeloperoxidase (MPO) activity. The role of AGEs in numerous disease processes has led to the