Fig. 5

MSI2-driven IL-6 secretion in CAFs regulates NSCLC cell migration and invasion. A Transwell (2D) assay was performed to evaluate NSCLC cell migration and invasion. NSCLC NCI-H292 (upper) and NCI-H460 (lower) cells were cultured in media or Ctrl CAF-CM and gMSI2 CAF-CM supplemented with recombinant IL-6, control IgG Ab, or neutralizing anti-IL-6 Ab, as indicated. See also Additional file 2: Fig. S12 for representative micrographs of migrating/invading cells. Bar graph depicts relative cell migration and invasion. Data are mean ± SD (n = 3); ^####P < 0.0001 versus media; ^$$$$P < 0.0001 versus Ctrl CAF-CM; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 versus indicated group; one-way ANOVA with Tukey’s multiple comparison test. ns, not significant. Ab, antibody. B CAF MSI2 promotes NSCLC cell motility through IL-6 in a 3D invasion assay. NSCLC NCI-H292 (upper) and NCI-H460 (lower) cells were processed to spheroids in either Ctrl CAF-CM or gMSI2 CAF-CM supplemented with control IgG Ab, neutralizing anti-IL-6 Ab, or recombinant IL-6, as indicated. Spheroids were embedded in extracellular matrix for 72 h, and invasion and dissemination events were evaluated. Representative micrographs of 3D spheroids are shown; arrows indicate invasive distance projecting from the spheroids; boxes indicate dissemination events. Scale bar = 50 μm. C Plots showing the longest invasive distance measured from a spheroid. Data are mean ± SD (n > 85 spheroids per group; four biological replicates); ^####P < 0.0001 versus Ctrl CAF-CM; ^$$$$P < 0.0001 versus Ctrl CAF-CM + IL-6 Ab; ^****P < 0.0001 versus indicated group; one-way ANOVA with Tukey’s multiple comparison test. D Frequency of dissemination events are shown as a pie chart for better visualization. Data are mean ± SD (n = 3 or 4); ^*P < 0.05, ^**P < 0.01 versus Ctrl CAF-CM with IgG; ^#P < 0.05, ^##P < 0.01 versus gMSI2 CAF-CM; ^$$P < 0.01 versus Ctrl CAF-CM with IL-6 Ab; one-way ANOVA with Tukey’s multiple comparison test. Number of total events were also shown