Figure 5. Evaluation of VEGF-A, VEGF-C, and M1- and M2-specific biomarkers in tumor-associated macrophages in Matrigel plugs in WT and Ndrg1 KO mice.
(a) Tumor angiogenesis and macrophage infiltration in Matrigel plugs containing B16/BL6 cells. Tumor angiogenesis (MVD) and infiltrating macrophages (F4/80+) were determined by IHC analysis of Matrigel plugs using CD31 (left) and F4/80 (right) antibodies (top). Matrigel plugs were quantitatively analyzed at day 7 by scoring five areas in each section for microvascular density (MVD) and F4/80-positive cells (bottom) (n = 5 per genotype). (b) Vegf-A and Vegf-C expression in macrophages isolated from Matrigel plugs was determined by qRT-PCR. The data were normalized by the WT expression levels of each factor. (c) Expression of M1-type (IL-1β, TNF-α and iNOS)- and M2-type (IL-10 and arginase)-specific biomarkers in macrophages isolated from Matrigel plugs was determined by qRT-PCR. The data were normalized by the WT expression levels of each factor. ND; not detectable. (d) Reduction of tumor angiogenesis in BM-eradicated male WT mice following the transfer of BMDMs from Ndrg1 KO mice. Matrigel plugs containing B16/BL6 cells (1 × 105 cells per plug) were subcutaneously inoculated with BMDMs (1 × 106 cells) derived from WT or Ndrg1 KO mice at day 0. Tumor angiogenesis (MVD) and infiltrating macrophages (F4/80+) were evaluated by IHC analysis of Matrigel plugs using CD31 (red) and F4/80 (green) antibodies (top). Matrigel plugs at day 7 were quantitatively analyzed by scoring five areas in each section for microvascular density (MVD) (bottom) (n = 4 per genotype). Each bar is an average ± SD, *P < 0.05; **P < 0.01 versus WT mice (two-tailed Student’s t-test). Original magnification × 200 for all panels.