Fig. 1: Transcriptomic analysis identifies characteristic changes in reactive ECs during metastatic colonization of the lung.
a, Immunofluorescence images showing association of lung ECs (CD31) with metastatic breast cancer cells (GFP) in mouse lung at indicated time points post intravenous injection of MDA231-LM2 breast cancer cells. Scale bars, 50 μm. Dashed lines indicate margins of metastatic foci. b, Quantification of metastatic nodules from a with intranodular ECs; n = 72 nodules (week 1), n = 76 nodules (week 2) and n = 83 nodules (week 3) from four mice were analyzed for each time point. Data are presented as means ± s.e.m. c, Size of MDA231-LM2-derived metastatic nodules in lung at weeks 1–3. A minimum of 16 nodules were analyzed for each lung; n = 4 mice per group. d, MDA231-LM2 cancer cells (left) and ECs (right) in lung at indicated time points. Data show means ± s.d; n = 4 mice per time point. P values were determined by one-way ANOVA with Dunnett’s multiple comparison test. e, Experimental setup for EC isolation from mouse lung at different stages of MDA231-LM2-derived metastasis, followed by transcriptomic analysis. f, PC analysis of gene expression profiles from ECs isolated from healthy lung (control) or lung with different stages of metastasis (as in e). g, GSEA of isolated ECs using proliferation- or inflammation-related signatures. Signatures with nominal P < 0.05 and FDR < 0.25 were considered significant. h,i, Violin plots showing z-score analysis of tumor angiogenesis and tip cell signatures11,12 (h) or patient poor-outcome gene clusters13,14 (i), calculated from transcriptomic profiles of ECs isolated from metastatic lungs at indicated time points. P values were determined using averaged z-scores of each signature by unpaired two-tailed t-test; n = 3 biological replicates per group. j, Heatmap showing expression of 58 genes of secreted proteins (GSP58) upregulated in lung ECs at week 3 post cancer cell injection. Cutoff log2(fold change (FC)) > 0.75, P < 0.05, FDRq < 0.25. k, GSEA graph showing enrichment of GSP58 in samples of human lung metastases of breast cancer, ranked according to lung metastasis-free survival. NES, normalized enrichment score. FDR was determined from P values calculated by random permutation test.
