Figure 9

Increased VEGF expression in mammary tumors in NG2 null mice. A, B. Because hypoxia induces VEGF expression, we used double immunostaining for VEGF (green) and CD31 (red) to localize VEGF expression relative to tumor blood vessels in wild type (WT, A) and NG2 null (NG2 KO, B) hosts. Some VEGF is associated with CD31-positive tumor blood vessels and some VEGF is dispersed in the tumor tissue. Evaluating overlap of VEGF and CD31 pixels allowed quantification of these two different pools of VEGF. Total VEGF is increased in tumors from NG2 null hosts (C). This VEGF increase in NG2 null tumors is due to non-vascular VEGF dispersed in the tumor tissue (E), rather than to VEGF closely associated with vessels (D). Data were collected from four tumors per genotype, evaluating four sections per tumor. F-H. Triple immunostaining for VEGF, CD31, and pimonidazole hypoxia probe in NG2 null tumors shows that non-vascular VEGF (arrows) is localized to hypoxic areas lacking blood vessels. Scale bars = 60 μm (A, B) and 20 μm (F-H). NG2, nerve-glial antigen 2; VEGF, vascular endothelial growth factor.