Structural deficits of vessels in mammary tumors in NG2 null mice. Py8119 fat pad tumors (2-3 mm diameter) in wild type (WT) and NG2 null (NG2 KO) recipients were used to evaluate several types of structural deficits in tumor vessels due to ablation of NG2. A-C. Pericyte coverage of endothelial cells was evaluated in wild type (A) and NG2 null (B) tissue sections immunostained for desmin (red) and CD31 (green). Since desmin and CD31 are on distinct cell types, they cannot overlap in a single optical section. However, because of the intimate interaction between pericytes and endothelial cells, the two labels appear to overlap when viewed in three-dimensional space. Analysis of confocal z-stacks therefore allows quantification of the extent to which desmin appears to overlap with CD31 labeling. Arrowheads in A indicate areas of overlap between desmin and CD31. Arrows in B show vessel segments without pericyte coverage. The extent to which CD31 pixels are overlapped by desmin pixels provides a measure of pericyte ensheathment of endothelial cells (C). Data were collected from eight tumors per genotype, evaluating four sections per tumor. D. Vascular densities are not significantly different in Py8119 tumors in wild type and NG2 null mice, as quantified by counting CD31-positive vessels in a 10,000 μm2 area. Data were collected from eight tumors per genotype, evaluating four sections per tumor. E-G. Pericyte maturation was evaluated via double immunostaining for desmin (green, all pericytes) and αSMA (red, mature pericytes). Mature pericytes express both desmin and αSMA (arrows), while immature pericytes express only desmin (arrowheads). Pericyte maturation is calculated as the % of desmin-positive pericytes that are αSMA-positive (G). Data were collected from four tumors per genotype, evaluating three sections per tumor. H. Endothelial ensheathment by mature pericytes was quantified by double immunostaining for CD31 and αSMA. Because the number of mature pericytes is reduced in tumor vessels in NG2 null hosts, only vessels with αSMA-positive pericytes are included in this analysis. Endothelial investment by mature pericytes is quantified as % overlap of CD31 pixels by αSMA pixels. For each genotype, five selected vessels each were examined in three different sections from each of three tumors. I-K. Basal lamina assembly was evaluated by immunostaining for CD31 (green) and collagen IV (red) in wild type (I) and NG2 null (J) tumor sections. Arrowheads in I indicate areas of collagen IV/CD31 overlap. Arrows in J show vessel segments with poor basal lamina deposition. Confocal Z-stacks were used to determine the percentage of CD31-positive pixels covered by collagen IV pixels (K). Data were collected from six tumors per genotype, evaluating four sections per tumor. L-M. Endothelial cell sprouting was evaluated by double immunostaining for CD31 (green) and VEGFR-3 (red) in wild type (L) and NG2 null (M) tissue sections. Arrowheads in M indicate VEGFR-3 high/CD31-low structures that characterize sprouting endothelial cells. Confocal z-stacks were used to determine the number of sprouting tip cells per 100 mm2 of CD31-positive vessel area (N). Data were collected from four tumors per genotype, evaluating three sections per tumor. Scale bars = 20 μm (A, B), 90 μm (E, F), 30 μm (I, J), and 12 μm (L, M). * P = 0.02; ** P = 0.006. NG2, nerve-glial antigen 2; αSMA, α-smooth muscle actin; VEGFR-3, vascular endothelial growth factor receptor-3.