Fig. 7: EMT cells secrete IL6 through the SIX1/ZEB1 signaling to activate the stemness of non-EMT cells.

A Western blot analysis of stemness related protein levels in 66cl4 transfected with shSix1#2 grown for 48 h in indicated CM. B Quantitation of Western blot in (A), n = 3. C Western blot analysis of stemness related protein levels in MCF7 transfected with OE Ctrl grown for 48 h in indicated CM with 1 μg/ml IL6 antibody or an equal amount of IgG. D, E Quantitation of Western blot in (C). F Representative images of mammospheres of 66cl4 transfected with shSix1#2 grown for 48 h in indicated CM, scale bar is 100 μm. G Quantitation of mammosphere-formation efficiencies in (F), n = 3. H Quantitation of relative mammosphere size in (F), n = 4. I ELDA assays showed the mammosphere-formation abilities in 66cl4 transfected with shSix1#2 grown for 48 h in indicated CM. J Representative flow cytometry plots of CD24^lowCD44^high subset in MCF7 transfected with OE Ctrl grown for 48 h in indicated CM with 1 μg/ml IL6 antibody or an equal amount of IgG. K Quantitation of CD24^lowCD44^high subset in (J), n = 3. Correlation plots showing the mRNA relative expression levels of SIX1 and ZEB1 (L) and IL6 (M) in human breast cancer samples, quantified by RT-qPCR, n = 48. N Correlation plot showing the mRNA relative expression levels of ZEB1 and IL6 in human breast cancer samples, quantified by RT-qPCR, n = 48. Correlation plots showing the mRNA relative expression levels of SIX1 and OCT4 (O), SOX2 (P), CD44 (Q), and CD24 (R) in human breast cancer samples, quantified by RT-qPCR, n = 48. Representative IHC images of breast cancer tissues from two organoids, showing the expression of SIX1 (S), ZEB1 (T), IL6 (U), and IL6R (V), scale bar is 100 μm. W Viability curves of organoids treated with neutralizing antibodies of different concentrations of IL6. The blue curve represents organoids with lower SIX1 expression, while the red curve represents organoids with higher SIX1 expression, n = 3. X Representative images of organoids treated with different concentrations of IL6-neutralizing antibodies at days 0, 2, 4, and 6, scale bar is 100 μm. Data were presented as means ± SD; ANOVA were used for statistical analysis. Correlation analysis was performed using Pearson’s correlation coefficient. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. SCR scramble, OE Ctrl overexpressing control, sh#1 #2 two independent shRNA constructs targeting genes, CM conditioned medium.