Extended Data Fig. 2: Inhibition of VEGF signaling in ECs during lung metastasis.
a, Expression of VEGF target genes in lung ECs isolated from healthy control mice or mice with lung metastasis (at week 3) treated with IgG or anti-VEGFA antibody (B20); n= 3 experiments. Means with s.e.m. are shown. b, Immunofluorescence analysis of ECs (CD31, red) in metastatic nodules in lungs at week 3 from mice treated with control IgG or B20 antibodies. MDA231-LM2 cancer cells are green (GFP). Representative images (left) and quantification of vascular area per nodule (right). Scale bar, 50 μm. P value was determined by one-tailed Mann-Whitney test; n= 4 mice (IgG) and 5 mice (B20). Boxes show median with upper and lower quartiles and whiskers indicate maximum and minimum values. c, GO analysis of differentially regulated genes (P< 0.05, log2FC> ± 0.5) in lung ECs following in vivo treatment with B20. Shown are biological processes enriched with FDR< 0.05. d,e, GSEA of “E2F targets” (Hallmark in MSigDB) and “VEGF targets”17 comparing lung ECs from mice treated with IgG or B20. NES, normalized enrichment score. f, Experimental outline for the analysis of VEGFR2 function in metastasis-associated lung ECs. MDA231-LM2 cells were injected intravenously followed by treatment with anti-VEGFR2 antibody (DC101), or control IgG. ECs were isolated from metastatic lungs and transcriptomic analysis performed. Mice harboring comparable metastatic loads in lungs, measured by in vivo bioluminescence imaging, were selected for analysis. g, Expression of VEGF target genes in lung ECs isolated from healthy control mice or mice with lung metastasis treated with IgG or DC101. Data are means with s.e.m. from 3 experiments. h, Overview of REACTOME pathway genes down-regulated by DC101. Pathways with FDR< 0.05 are shown. i, Repression of gene signatures of “Cell cycle mitotic” (REACTOME) and “Tumor angiogenesis” (tumor angiogenesis UP cluster)16 in lung ECs treated as in panel f. NES, normalized enrichment score. j-m, Violin plots showing z-scores of genes from indicated signatures expressed in ECs from lungs of mice under indicated conditions. Signatures: tip cells12, lung EC inflammation associated with metastasis (Supplementary Table 4), poor prognosis angiogenesis genes14 and GSP58. P values were determined by one-way ANOVA with Dunnett’s multiple comparison test; n= 3 for each group. NS, not significant.
