Extended Data Fig. 8: Analysis of TNC-TLR4 axis in lung metastasis.
a, Immunofluorescence analysis of TNF in F4/80+ macrophages within MDA231-LM2 metastatic nodules from mice treated with vehicle or TLR4i. Representative examples (left) and quantification of relative signals (right) from 5 mice. P value was determined by one-tailed Mann-Whitney test. Scale bar, 20 μm. b, Immunofluorescence analysis of macrophages (F4/80) in MDA231-LM2 lung metastases from mice treated with vehicle or TLR4i. Dashed lines indicate metastases margins. Relative number of F4/80+ cells per unit area (UA) of metastatic foci was quantified, and means with s.e.m. are shown (right). Statistical analysis was performed with one-tailed Mann-Whitney test; n= 5 mice. Scale bar, 50 μm. c, Experimental outline of spontaneous lung metastasis assay in an orthotopic model where tumor-bearing mice were treated with TLR4i. d, Primary tumor volume measured on day 24, Vehicle: n= 15 mice, TLR4i: n= 12 mice. Statistical analysis was performed with one-tailed Mann-Whitney test. e, Spontaneous metastases in lungs of mice on day 24 as in c. Left, representative examples of metastases (arrows) marked by expression of human vimentin in cancer cells. Right, quantification of lung metastasis area and nodule numbers based on human vimentin marker expression; Vehicle: n= 15 mice, TLR4i: n= 12 mice. P values were calculated by one-tailed Mann-Whitney test. Scale bar, 200 μm. f, Liver metastasis in an orthotopic metastasis model where breast cancer cells were implanted into mammary fat pads and mice treated with TLR4i; n= 8 mice for each group. P value was calculated by one-tailed Mann-Whitney test. g,h, Analysis of lung metastasis in TNC knockdown orthotopic metastasis model where primary tumor was removed surgically at day 22. Diagram of experimental procedures (g). Quantification of lung metastasis based on bioluminescence in mouse lungs (h), shControl: n= 8 mice, shTNC: n= 7 mice. P value was determined by one-tailed Mann-Whitney test. i, Expression of indicated niche components in ECs isolated from mice as in panels g,h; n= 5 for each group. Shown are means with s.e.m., and P values were calculated by one-tailed Mann-Whitney test. *P< 0.05, **P< 0.01 and †P= 0.075. Boxes in panels d-f and h show median with upper and lower quartiles and whiskers indicate maximum and minimum values.
