Fig. 2
The MCT-1/IL-6/IL-6R pathway activates THP-1 differentiation that stimulates cancer cell invasion. (a and b) The cytokine arrays were incubated with the CM of the MDA-MB-231 cells (control vs. MCT-1) and IV2–3 subline (scramble vs. shMCT-1#3) to identify the secreted factors specific to the indicated cellular conditions. (c) Quantitative RT-PCR evaluated IL-6, CCL2 and GM-CSF mRNA levels in the MDA-MB-231 cells. (d) The IL-6 secreted from the MDA-MB-231 cells (control vs. MCT-1) and IV2–3 subline (scramble vs. shMCT-1#3) were quantified by ELISA using the anti-IL-6 mAb. The results were normalized with the plated cell numbers (2 × 105). (e) PD-L1, IL-6/IL-6R, EGFR, Stat3 and EMT molecules were analyzed upon different doses of IL-6 simulation for 24 h. Different blots with normalized internal control were used to present the data of the experiment. (f) The effect of IL-6 knockdown (shIL-6) on EMT signaling molecules were characterized. (g) The proteins in the cytosol and membrane fractions were isolated from 5 × 106 cells and then compared between control (c) and MCT-1 (m) groups. (h) The conditioned media of the MDA-MB-231 cells with different MCT-1 expression conditions were harvested after 48 h culture. The ELISA measured soluble IL-6R (sIL-6R) levels, and the results were normalized with the plated cell numbers. (i) The interaction of gp130 and IL-6R in vivo were identified by IL-6R immunoprecipitation assay. (j) The clinical relevance of MCT-1 and IL-6 was examined in TNBC (Curtis database) and in metastatic breast cancer (Bos dataset) using the Oncomine database. Pearson’s correlation coefficient indicates the statistical significance. (k) MCT-1 associated with IL-6/IL-6R was studied using the breast cancer cDNA arrays (n = 124). The Chi-square test was used to calculate the statistical significance. (l) The MDA-MB-231 cells were pretreated by IL-6, anti-IL-6R mAb or tocilizumab for 24 h followed by THP-1 coculture in the transwell for 48 h. Parental MDA-MB-231 invasiveness was examined as cocultured with the preconditioned THP-1 cells in a Boyden chamber-coating Matrigel matrix for 24 h. The results are expressed as the mean ± SD of three independent assays (n = 3). One-way ANOVA with a post hoc two-tailed t-test was used to calculate the statistical significance of pairwise comparisons. (*p < 0.05; **p < 0.01; *** p < 0.001)