Dll4 allelic deletion reduced tumor burden in RT2 mice due to increased nonproductive tumor angiogenesis. A, Number of tumors per animal, average tumor volume and overall tumor burden, are calculated as the sum of tumor volumes per mouse, in RT2 Dll4
+/+ (n = 8) and RT2 Dll4
+/- (n = 8) mice. B, Vascular response examined in RT2 Dll4
+/+ (n = 5) and RT2 Dll4
+/- (n = 5) insulinomas by PECAM immunostaining. RT2 Dll4
+/- mice showed reduced vessel calibers, increased sprouting, branching irregularities, and network disorganization (left). Dotted lines mark tumor border. I indicates insulinoma while P indicates normal pancreatic tissue surrounding the tumor. Vascular density was estimated as percentage of PECAM-positive area per tumor section surface and presented for two experimental groups (right). C, Mural cell coverage on newly formed vessels was examined by double staining of PECAM and α-SMA. SMA-positive cells lining PECAM-positive endothelium were profoundly reduced in RT2 Dll4
+/- insulinomas (left). The percentage of PECAM-positive structures covered by SMA-positive area was measured as an indicator of vessel maturation (right). D, Tumor vessel competence evaluated by lectin perfusion. Simultaneous staining of endothelial cells with PECAM and visualization of perfused vessels demonstrate a reduction in the fraction of perfused vessels in RT2 Dll4+/- vs. RT2 Dll4+/+ mice (left). The percentage of PECAM-positive area co-localized with lectin was measured to estimate the proportion of functional vessels within insulinomas (right). The two group values for each parameter were statistically analyzed using Mann-Whitney-Wilcoxon test. Error bars represent SEM. * P < 0.05 was considered significant.