Fig. 2

Stromal biglycan inhibition suppresses lung metastasis, by normalizing tumor vasculature in breast cancer. a Growth curves of E0771 tumor growth in WT and Bgn KO mice measured every 4 days. b Comparison of E0771 tumor weight at day 24 after inoculation. c Representative IVIS images and quantitative analysis of metastatic lungs in E0771 tumors of WT versus Bgn KO mice. d Bgn mRNA expression in E0771 tumor tissues of WT and Bgn KO mic analyzed by quantitative real-time RT-PCR. e Biglycan concentration in mouse plasma from non-tumor WT mice, E0771 tumor-bearing WT mice, and E0771 tumor-bearing Bgn KO mice detected by ELISA. f Immunofluorescent images of biglycan (green) expression in CD31+ blood vessels (red) in E0771 tumors grown in WT mice. DAPI, blue. Scale bar = 20 μm. g Bgn mRNA expression in FACS isolated CD31− cells, CD45− CD31+ cells (E0771-TECs), and dermal ECs from naïve mice by quantitative real-time RT-PCR. h Representative images of CD31+ blood vessels in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm. i Quantification of blood vessel density. j Representative images of CD31 immunostaining (red), ɑ-SMA (green), and DAPI nuclear staining (blue) in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm. k Quantification of ɑ-SMA+ CD31+ tumor blood vessels. The rate of microvessel pericyte coverage was analyzed by counting the vessels that stained positive for both CD31 and α-SMA (white arrowheads) among CD31-positive vessels. l Representative images of CD31 immunostaining (red), FITC-lectin (green), and DAPI (blue) in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm. m Quantification of lectin+ blood vessels. The rate of lectin+ blood vessels was analyzed by counting the vessels that stained positive for both CD31 and lectin (white arrowheads) among CD31-positive vessels. n Representative images of CD31 immunostaining (green), Glut1 (red), and DAPI (blue) in E0771 tumors from WT and Bgn KO mice. Scale bar = 100 μm. o Quantification of Glut1+ area. *p < 0.05, ***p < 0.001. Significance in b, d, i, k, m, and o were determined by a two-tailed Student’s t test. Significance in g was determined by a one-way ANOVA. All data represent means ± SD