Fig. 3. Secreted factors produced by reactive ECs promote lung metastasis.
a, Analysis of endothelial niche candidate genes in different stromal cells isolated from metastatic lungs. Heatmap summarizes expression of genes with log2FC > 3.5. Z-scores were calculated from qPCR analysis of different stromal cells isolated from three or four mice per group. Genes highly expressed in ECs are marked in red. BMDC, bone marrow-derived cell; fibro., fibroblasts; epi., epithelial cells. b, Expression of four endothelial niche candidate genes in lung ECs isolated from indicated mouse strains with metastasis of different breast cancer cell lines. Heatmap was generated from qPCR analyses and P values calculated by unpaired one-tailed t-test. *P < 0.05, **P < 0.01, ***P < 0.001; n = 3–4 mice per group. c, Correlation analysis of expression of the four candidate genes and CDH5 in metastatic lesions of patients with breast cancer (GSE14020). Linear regression with Pearson correlation (r) and two-tailed P value is shown; n = 65. d, Immunohistochemical analysis of CD31, SCGB3A1 and LAMA1 expression in lung metastasis samples from patients with breast cancer; shown are two representative examples of metastases from analysis of 11 patients. Arrows indicate positively stained endothelial cells. Scale bars, 100 μm (patient 1) and 60 μm (patient 2). e, Proportion of lung metastasis samples expressing SCGB3A1 or LAMA1. f–h, Metastatic colonization of lungs (f) by SUM159 breast cancer cells (g) and MDA231 cells (h) overexpressing endothelial niche factors or a control vector. g,h, Bioluminescence images (left) and normalized photon flux (right) 42 days post intravenous injection by SUM159 (g) and MDA231 (h) breast cancer cells. S3A1, SCGB3A1. Boxes depict median with upper and lower quartiles. Data points show values of biological replicates and whiskers indicate minimum and maximum. g, P values were calculated by one-tailed Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001,✝P = 0.069. Control, n = 20; INHBB, n = 6; LAMA1, n = 8; SCGB3A1, n = 5; OPG, n = 5. h, Control, n = 19; INHBB, n = 10; LAMA1, n = 4; SCGB3A1, n = 10; OPG; n = 9; and four genes, n = 5. i, Histological examples of metastases marked by expression of human vimentin in lungs of mice from h. Shown are representatives from at least four independent samples. Scale bar, 200 μm. j, Kaplan–Meier analyses on compiled datasets of ER– breast cancers (KM plotter) examining association of the four endothelial niche factors with relapse-free survival (n = 347 patients) and overall survival (n = 79 patients). P values were determined by log-rank test. HR, hazard ratio.