Fig. 7: TNC–TLR4 axis promotes activation of perivascular macrophages and subsequent formation of a pro-metastatic endothelial niche in lung.
a, Immunofluorescence analysis of TNC (purple), macrophages (F4/80, green) and ECs (CD31, blue) in MDA231-LM2 metastasis from mouse lung. Arrowheads indicate colocalization of TNC and macrophages at perivasculature. Dashed line indicates margins of metastasis. Shown are representative images from four independent samples. Scale bars, 50 μm. b, Hierarchical clustering of 65 human metastases of breast cancer (GSE14020) according to expression of GSP58. TNC- or classically activated macrophage-signature (CAM-S)-positive metastases are indicated by red bars. c, Correlation analysis of indicated parameters in 65 metastasis samples from patients with breast cancer. Linear regression with Pearson’s r and two-tailed P values are shown. d, Expression of indicated markers in macrophages treated with recombinant TNC or a combination of TNC and TLR4i (TAK-242) for 6 h; n = 3 (Nos2) or n = 4 (Tnf, Il1b and Il6) independent experiments. Shown are means with s.e.m. e, Experimental outlines showing MDA231-LM2 cancer cells, transduced with shRNA control (shControl) or shRNA against TNC (shTNC), injected intravenously (i.v.) into mice (top) or MDA231-LM2 cells injected i.v. into mice followed by treatment with TLR4i (bottom). f, Lung metastasis based on bioluminescence in mice injected with shControl or shTNC (two independent hairpins) transduced breast cancer cells; n = 6 mice for each group. P values calculated by one-tailed Mann–Whitney test. g, Expression of perivascular niche factors in ECs isolated from metastatic lung as in f; n = 4 mice. Data are means with s.e.m., and P values were determined by one-way ANOVA with Holm–Sidak’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. h, Bioluminescence analysis of mouse lungs harboring metastases and treated with TLR4i. For MDA231-LM2 and control, n = 10 mice; TLR4i, n = 8 mice; 4T1 and control, n = 8 mice; TLR4i, n = 9 mice. P values were calculated by one-tailed Mann–Whitney test. i, Expression of indicated genes in ECs isolated from lungs harboring MDA231-LM2 metastases as in h; n = 4 mice (control and metastasis with vehicle treatment) and n = 3 mice (metastasis with TLR4i treatment). Data are means with s.e.m., and P values were determined by one-way ANOVA with Holm–Sidak’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001. j, Experimental outline of spontaneous lung metastasis in an orthotopic model. MDA231-LM2 cancer cells were implanted to the fourth mammary fat pad and their growth followed for 24 days, then tumors were removed surgically. Mice were treated with TLR4i from day 25 onwards. k, Metastasis in lungs of mice from j. Left, ex vivo bioluminescence; right, quantification of metastasis; n = 6 (vehicle) and n = 3 (TLR4i). P value was determined by one-tailed Mann–Whitney test. l, Expression of four niche factors in EC lungs isolated from mice harboring metastasis and treated with vehicle or TLR4i as in j,k. Data are means with s.e.m. obtained from n = 6 mice (vehicle) and n = 3 mice (TLR4i). P value was determined by one-tailed Mann–Whitney test. *P < 0.05. d,g,i,l, Expression was determined by qPCR. m,n, Rescue experiments where niche factors were ectopically expressed in metastatic breast cancer cells in the context of TNC knockdown (m) or TLR4i treatment (n) during lung colonization; n = 6 mice for each group. P values were determined by one-tailed Mann–Whitney test. f,h,k,m,n, Boxes show median with upper and lower quartiles, and whiskers indicate maximum and minimum values.
