Bv8 expression in RIP-Tag mice. Total RNA was extracted from BMMNCs (A) and pancreata (B) of WT, tumor-free (4 weeks old), and tumor-bearing (10 and 13 weeks old) RIP-Tag mice. Bv8 expression was measured by qRT-PCR as described in Materials and Methods. Data shown are means ± SEM (n = 4). * indicates significant difference by t test (P ≤ 0.05) when comparing Bv8 expression with the corresponding WT group.

Bv8 plays a role in tumor angiogenesis in early but not in late stages. (A) RIP-Tag mice were treated with control mAb (n = 12), anti-Bv8 (n = 9), or anti-VEGF (n = 7) mAbs for 5 weeks, starting at 5 weeks of age. Angiogenic islets were counted in each treatment group by using a dissection microscope. Bars represent the mean ± SEM of angiogenic islets. (B) Representative images from each treatment group, as indicated. Arrows point to the hyperemic lesions. (C and D) RIP-Tag mice received control mAb (n = 8), anti-Bv8 (n = 6), or anti-VEGF (n = 10) for 3 weeks, starting at 10 weeks of age. Tumor burden (C) and number of tumors (D) were measured in each treatment group. (E) Representative images are shown from each treatment group. Data are representative of two independent experiments, with similar results. * indicates significant difference when compared with control mAb-treated group (P ≤ 0.05). (Magnification: B and E, ×0.8.)

Anti-Bv8 treatment reduces the frequency of CD11b+Gr1+ cells in PB and pancreas at the end of prevention trials. RIP-Tag mice were treated with control mAb, anti-Bv8, or anti-VEGF, in prevention and intervention trials, as described in Materials and Methods. Measurements were performed at the end of such therapeutic intervals. (A–C) Mononuclear cells were isolated from PB (A), BM (B), and spleen (C). Cells were stained with fluorochrome-conjugated anti-CD11b and anti-Gr1 antibodies as described in Materials and Methods. The frequency of CD11b+Gr1+ cells in each tissue was measured as described. (D) Single-cell suspensions were obtained from pancreata and then stained with the same anti-CD11b and anti-Gr1 antibodies as described above. Bar graphs represent the means ± SEM of the frequency of CD11b+Gr1+ cells in each tissue. * indicates significant difference when compared with control mAb-treated group (P ≤ 0.05). (E) Representative images from prevention and intervention trials illustrating immunostaining for CD11b (green) and Gr1 (red) in the spleen, angiogenic islets (prevention) and tumors (intervention) from each treatment cohort. Arrows point to CD11b+Gr1+ cells in and around angiogenic islets and CD11b+ cells in and around tumors. Dotted lines show boundaries between neoplastic lesions and surrounding exocrine tissue. (Magnification: ×20.)

Anti-Bv8 treatment reduces VSA and neutrophil infiltration in hyperplastic and angiogenic lesions in prevention but not in intervention trials. RIP-Tag mice were treated with control, anti-Bv8, or anti-VEGF mAbs in prevention or intervention trials, as described. Evaluations were performed at the end of such periods. Sections (6 μm) were cut from frozen tumors and stained with anti-CD31 (shown in red) and anti-Gr1 (shown in green) antibodies as described. (A) VSA was assessed on the basis of the percentage of CD31+ areas relative to the entire lesion in each treatment group. Bars represent the mean VSA ± SEM. Data represent VSA from at least three mice per treatment. (B) Representative images of hyperplastic, angiogenic, and tumor lesions from prevention trials. RIP-Tag mice were treated with control, anti-Bv8, or anti-VEGF mAbs for 5 weeks. Tumor sections were stained with anti-CD31 (shown in red) to label blood vessels and anti-Gr1 (shown in green) to label neutrophils. Nuclear counterstaining with DAPI is shown in blue. Arrows point to Gr1+ cells in hyperplastic and angiogenic lesions. Dotted lines show the boundaries between hyperplastic islets and the surrounding exocrine tissue; angiogenic islet and tumor photomicrographs are focused within the core of the lesion. Anti-Bv8 treatment reduces neutrophil infiltration in the exocrine pancreas. (C) RIP-Tag mice were treated in an intervention trial with control, anti-Bv8, or anti-VEGF mAb, and VSA was measured in tumor sections (n = 3) as described. (D) Images are representative of hyperplastic, angiogenic, and tumor lesions at the end of intervention trials. Vascular structures and neutrophils are shown in red and green, respectively. DAPI-counterstained nuclei are shown in blue. * indicates significant difference (P ≤ 0.05) when comparision with the control mAb-treated groups. (Magnification: B and D, ×20.)