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Figure 6 | Breast Cancer Research

Figure 6

From: Early vascular deficits are correlated with delayed mammary tumorigenesis in the MMTV-PyMT transgenic mouse following genetic ablation of the NG2 proteoglycan

Figure 6

Vessel diameter and macrophage phenotype in wild type and NG2 null mice. A-C. Whole mounts of #4 mammary glands from 14-week old wild type (WT) and NG2 null (KO) MMTV-PyMT females were immunostained for CD31 (red) and αSMA (blue) to investigate vascularization of incipient neoplasms. Although not shown in B and C, αSMA staining was used as illustrated in A to restrict analysis of CD31-positive vessels to areas within developing MINs. Thus, we only analyzed tumor-associated vessel segments and not vessels associated with normal areas of the mammary gland. Tumor-associated vessels in wild type mice (B) appear thicker and are more robustly stained for CD31 than vessels in NG2 null mice (C). To quantify vessel size, ten vessel diameters were measured in each of five fields from each of 12 MINs for each genotype to yield the data plotted in the left-hand side of panel D (spontaneous tumors). Sections of CD31-immunolabeled Py8119 fat pad tumors (at day 12) were used to obtain the data shown on the right-hand side of panel D (Py8119 tumors). A total of 20 vessel diameters were determined in each of six sections from each of four 12-day tumors in each genotype. Scale bars = 100 μm in A and 25 μm in B. * P = 0.02; ** P = 0.007. Double immunostaining for CD31 (red) and NG2 (green) was performed on sections of 17-week spontaneous tumors from wild type (E) and NG2 null (F) MMTV-PyMT females and on sections of 12-day Py8119 tumors from wild type (G) and NG2 null (H) female mice. In both types of tumors, NG2 labeling is seen in association with CD31-positive endothelial cells in wild type hosts, but not in NG2 null hosts. Bar in E = 25 μm. TAM abundance (I-K) and TEM abundance (L-N) were studied by flow cytometric analysis of tumor macrophages in wild type and NG2 null recipients transplanted with wild type or NG2 null β-actin/EGFP bone marrow. F4/80, CD11b, CD45, and Gr1 were used for analysis of TAMs, while F4/80, Tie2, CD206, and CD11c were used for analysis of TEMs. Analysis was restricted to EGFP-positive cells. FACS plots I-J show only the F4/80 and CD11b pairing, while plots L-M show only the F4/80 and Tie2 pairing. In addition, only NG2 null recipient data are shown. Numbers in each quadrant of the FACS plots indicate the percentage of the total number of EGFP-positive cells analyzed. Data for wild type recipients appear very similar. Abundance of TAMs and TEMs are indicated in panels K and N, respectively, as percentages of the total number of EGFP-positive cells. Open bars = wild type bone marrow donors. Black bars = NG2 null bone marrow donors. These data are based on the use of all four markers, not just the two shown in the FACS plot examples. All values in panels K and N are derived from FACS analysis of five separate tumors, all from different mice. Three of the eight P values approach, but do not reach, a statistical significance level of 0.05. EGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; MMTV-PyMT, mammary tumor virus-driven polyoma middle T; NG2, nerve-glial antigen 2; αSMA, α smooth muscle actin.

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