However, tumors treated with PF-04217903 alone for 3 weeks were similar in size to age-matched, vehicle-treated controls (data not shown). their smaller size, tumors treated with anti-VEGF antibody or sunitinib appeared to be more invasive, as judged by the irregularity of the tumor border and the abundance of clusters of amylase-positive acinar cells of the exocrine pancreas trapped inside tumors (Determine 1, E-G). Quantitative steps of the tortuosity of the tumor border (Invasion index, see Methods) and the Rabbit polyclonal to ACMSD number of trapped acinar cells were significantly greater (Physique 1, H Punicalagin and I). The relevance of amylase-positive Punicalagin cells within tumors, as an indicator of invasion, was assessed by comparing amylase staining to the basement membrane protein type IV collagen and to type I collagen, a known constituent of the capsule of RIP-Tag2 tumors (4). The three approaches gave complementary results (Supplemental Physique 1). Tumors with abundant amylase cells inside had strong staining for type IV collagen around the trapped exocrine cells, as in normal pancreatic acini, but the border had little or no type IV collagen or type I collagen (Supplemental Physique 1, A-C, G-I). Tumors that had few or no amylase-stained cells inside had type IV collagen around blood vessels, and the border had a layer of type IV collagen and a capsule of type I collagen (Supplemental Physique 1, D-F, J-L). Tumors of 14-week aged RIP-Tag2 mice treated with normal goat IgG for 1 or 3 weeks resembled those of mice treated with vehicle (data not shown). Tumor cell changes in RIP-Tag2 tumors after VEGF inhibition Proliferating cells marked by phosphohistone H3 immunoreactivity were abundant throughout vehicle-treated tumors (Supplemental Physique 2A). After treatment with anti-VEGF antibody for 3 days, proliferating cells were still abundant at the tumor border (area density: 14.7% vs. 14.3% for vehicle) but were half the control value at the tumor center (6.8% vs. 13.3% for vehicle, 0.05) (Supplemental Figure 2B). Abundant phosphohistone H3-positive cells in finger-like projections of tumor contrasted with rare dividing cells in the surrounding exocrine pancreas (Supplemental Physique 2C). Apoptotic cells identified by activated caspase-3 immunoreactivity were more abundant after anti-VEGF antibody for 3 days, but were less numerous than proliferating cells under all conditions (Supplemental Physique 2, D-F). Apoptotic cells were no more frequent in finger-like projections than elsewhere in tumors. Snail1, N-cadherin, and vimentin as markers of mesenchymal phenotype had stronger bands in western blots of tumors after treatment with anti-VEGF antibody or sunitinib than in corresponding mice treated with vehicle from age 14 to 15 weeks (Physique 1J). Densitometry values for Snail1, N-cadherin, and vimentin were 3, 5, and 10 occasions greater, respectively, after anti-VEGF antibody ( 0.05) and 3, 10, and 5 occasions greater after sunitinib ( 0.05). E-cadherin, as a marker of epithelial phenotype, was weaker in tumors of RIP-Tag2 mice at age 17 weeks (Physique 1K) than at age 10 weeks (data not shown), but was even less in tumors treated with anti-VEGF antibody (age 14 to 17 weeks), where tumor cell identity was verified by insulin staining (Physique 1, L and M). E-cadherin staining was inversely related to staining for vimentin (Physique 1, K-O) and c-Met (Supplemental Shape 2, G-H). E-cadherin was more powerful in automobile treated mice, and vimentin Punicalagin and c-Met had been more powerful after anti-VEGF antibody (Supplemental Shape 2, G-J). Hypoxia and c-Met in RIP-Tag2 tumors after VEGF inhibition Tumors in RIP-Tag2 mice treated with anti-VEGF antibody or sunitinib from age group 14 to 17 weeks got fewer arteries than in related vehicle-treated tumors (Shape 2, A-C), as discovered previously after inhibition of VEGF signaling (19, 34). The decreased vascularity was followed by higher hypoxia, shown by staining for pimonidazole, carbonic anhydrase IX (CA-IX), or blood sugar transporter 1 (Glut1) (Shape 2, A-C, Supplemental Shape 3, A-B, D-E). The staining patterns for the three markers was identical: staining was patchy in charge tumors and was wide-spread and most powerful in parts of vascular pruning in VEGF inhibitor-treated tumors. Measurements verified an inverse romantic relationship.