(A) To assess pericyte investment of blood vessels, sections of B16F10 tumor-bearing wild type (wt) and NG2 null (ko) brains at 7 days post-injection were double-stained for CD31 and PDGFRβ. Confocal z-stacks of images were processed by image analysis to quantify several aspects of the relationship between endothelial cells (red) and pericytes (green). Bar = 100 μm. Data in panels B-D are derived from 4 independent tumors of each genotype, sampling 4 fields from 3 sections in each tumor (12 fields × 4 tumors). (B) Quantification of green pixels allowed a comparison of the total number of pericytes per field. (C) The extent to which pericytes were not associated with endothelial tubes (detached pericytes) was assessed by quantifying the percentage of green pixels not in physical contact with red pixels. (D) The extent of pericyte ensheathment of endothelial cells was determined by quantifying the percentage of red pixels that were covered by green pixels. These values were determined both at day 5 and day 7 of tumor growth, revealing a deficit in the NG2 null mouse at both days.
* P<0.01. (E) To evaluate pericyte maturation in tumor blood vessels, sections of 7-day tumors were stained for CD31 to visualize blood vessels (not shown), and for PDGFRβ (green) and αSMA (red) to identify nascent and maturing pericytes, respectively. (F) Data in panel F are derived from 4 independent tumors for each genotype, sampling 4 fields from 3 sections in each tumor. Confocal z-stacks of images were processed by image analysis to quantify the extent to which the PDGFRβ label is coincident with αSMA. Bar = 100 μm. * P < 0.0003

(A) Patency of 7-day tumor vessels was determined following intravenous injection of FITC-LEA lectin (green), followed by perfusion to clear unbound lectin. Sections were then stained for VEGFR-2 (red) to allow comparison of the total number of blood vessels with the number of patent vessels. Arrows denote VEGFR-2-positive vessels without LEA lectin labeling. Bar = 100 μm. (B) Data in panel B are derived from 4 independent tumors for each genotype, sampling 4 fields in 3 sections in each tumor. Image analysis of z-stacks of confocal images allowed determination of the percentage of VEGFR-2 pixels covered by LEA lectin pixels. * P<0.001.

Tissue sections were prepared from animals that had been injected with pimonidazole hypoxia probe at 3 (A) and 7 days (C) of tumor development. Sections were double-stained for pimonidazole (green) and CD31 (red). Data in panels B and D were derived from 3 independent tumors for each genotype, sampling 4 fields in 3 sections from each tumor. Hypoxia levels at 3 (B) and 7 (D) dpi were determined as the percentage of total tumor area covered by pimonidazole pixels. Dashed lines illustrate brain-tumor boundaries. * P = 0.192, ** P<0.0001. Bar = 100 μm.

Tumor sizes were measured in wild type and NG2 null mice that had been injected in the corpus callosum with 1 × 10⁴ CT2A, B16F10/NG2, or B16F10 cells. (A) Nine wild type and nine NG2 null mice were injected with CT2A cells and analyzed for tumor size at 26 days post-injection. On average, CT2A tumors in NG2 null mice are 7-fold smaller than in wild type mice. (B) Seven wild type and seven NG2 null mice were injected with B16F10/NG2 cells and analyzed for tumor size at 11 days post-injection. The average tumor size was 10-fold smaller in NG2 null mice. (C) Five mice in each group were analyzed at days 3, 5, 7, 11, and 17 after injection of B16F10 cells. Average tumor sizes are plotted versus days post-injection. The inset panel shows the first week after tumor initiation on an expanded scale, revealing that tumors in wild type and NG2 null mice begin to diverge in size between 3 and 7 days post-injection. (D) Fifteen mice in each group were analyzed at 17 days after injection of B16F10 cells. B16F10 tumors in NG2 null mice are at least 3-fold smaller than in wild type mice. Dashed lines show average tumor sizes. * P<0.001. (E) Double immunostaining for NG2 (green) and CD31 (red) shows that in both B16F10 and B16F10/NG2 tumors, NG2 is expressed by pericytes associated with CD31-positive endothelial cells. Apart from the vasculature, NG2 is also strongly expressed by B16F10/NG2 tumor cells, but is absent from B16F10 tumor cells. Bar = 100 μm.

(A) To evaluate basal lamina assembly associated with blood vessels, sections of 7-day tumors were double-stained for CD31 (red) and collagen IV (green). Bar = 100 μm. (B) Data in panel B are derived from 3 independent tumors for each genotype, sampling 4 fields from 3 sections in each tumor. Both day 5 and day 7 tumors were analyzed. Confocal z-stacks of images were processed by image analysis to quantify the extent to which collagen IV labeling overlapped with CD31 labeling. Basal lamina deposition is reduced in the NG2 null mouse at both time points. * P< 0.002. (C) Association of collagen VI with 7-day tumor blood vessels was evaluated in wild type (wt), NG2 null (NG2 ko), and collagen VI null (coll VI ko) mice by double-staining for CD31 (red) and collagen VI (green). Bar = 100 μm. (D) Data in panel D are derived from 3 tumors for each genotype, sampling 4 fields from 3 sections in each tumor. Image analysis of z-stacks of confocal images allowed determination of the percentage of CD31 pixels covered by collagen VI pixels. * P<0.03. ND, not detectable. (E) Assembly of collagen IV-positive basal lamina was compared in tumor vessels in wild type, NG2 null, and collagen VI null mice. ** P< 0.007 *** P< 0.002

(A) Leakiness of tumor vessels at 3 (top panels) and 7 (lower panels) days post-injection (dpi) was visualized by intravenous injection of FITC-dextran (green) followed by a 10-minute circulation period and tissue fixation. Sections were stained for CD31 (red) to define blood vessel boundaries. Dashed lines show the brain-tumor boundaries at 3 dpi. Bar = 100 μm. z-stacks of confocal images were processed by image analysis to quantify FITC-dextran pixels lying external to CD31-positive vessels. (B) Data in panel B are derived from 3 independent tumors for each genotype, sampling 4 fields in 3 sections from each tumor. The extravascular FITC-dextran area is expressed as the percentage of the total tumor area at 3 dpi (left panel) and 7 dpi (right panel). * P<0.001. (C) Pericyte investment of tumor vessels in wild type mice at 3 days post-injection was evaluated by double-staining for CD31 (red) and PDGFRβ (blue) in sections from FITC-dextran-perfused (green) tumors at 3 dpi. Dashed lines mark brain-tumor boundaries. Bar = 100 μm.