Fig. 6

MSI2 deficiency in CAFs inhibits NSCLC EMT and metastasis via IL-6. A H&E staining and IHC analysis of EMT-related markers were performed on primary tumors collected from mouse xenografts. Representative micrographs and quantitative analysis of mesenchymal marker vimentin and epithelial marker E-cadherin using ImageJ software are shown. Scale bar = 100 μm. Data are mean ± SD (n = 3); ^***P < 0.001, ^****P < 0.0001 versus NCI-H292 cells with Ctrl CAFs; two-tailed Student’s t-test. B CAF MSI2 mediates EMT in NSCLC cells. Western analysis of E-cadherin and vimentin in NSCLC NCI-H292 (left) and NCI-H460 (right) cells after exposure to Ctrl CAF-CM or gMSI2 CAF-CM for 48 h (see also Additional file 2: Fig. S14 for more EMT markers). β-actin was used as a loading control. Data are mean ± SD (n = 3); ^#P < 0.05 versus media; ^*P < 0.05 versus Ctrl CAF-CM; one-way ANOVA with Tukey’s multiple comparison test. C Inhibition of EMT in NSCLCs by MSI2-depleted CAF-CM is rescued by an addition of recombinant IL-6. Western analysis of E-cadherin and vimentin in NSCLC NCI-H292 (left) and NCI-H460 (right) cells after exposure to gMSI2 CAF-CM with or without recombinant IL-6 for 48 h. β-actin was used as a loading control. Data are mean ± SD (n = 3); ^*P < 0.05, ^**P < 0.01 versus untreated; two-tailed Student’s t-test. D Neutralization of IL-6 reverses CAF-CM-induced EMT in NSCLC cells. Western analysis of E-cadherin and vimentin in NSCLC NCI-H292 (left) and NCI-H460 (right) cells after exposure to Ctrl CAF-CM in the presence of anti-IL6 neutralizing antibody (IL-6 Ab) or isotype-matched IgG control (IgG). β-actin was used as a loading control. Data are mean ± SD (n = 3); ^*P < 0.05 versus IgG control; two-tailed Student’s t-test. E IL-6 activates EMT in NSCLC cells. Western analysis of E-cadherin and vimentin in NSCLC NCI-H292 (left) and NCI-H460 (right) cells upon direct exposure to recombinant IL-6 for 48 h (see also Additional file 2: Fig. S15 for more EMT markers). β-actin was used as a loading control. Data are mean ± SD (n = 3 or 4); ^*P < 0.05, ^**P < 0.01 versus media; two-tailed Student’s t-test