Fig. 3

BCSC-derived IL8 drives tamoxifen resistance. A Protein array analysis of factors present in CM derived from mammosphere-enriched BCSCs or parental MCF-7 cells. B MCF-7 and T47D cells were cultured in the presence of the indicated recombinant proteins (100 ng/ml) and treated with a graded concentration of tamoxifen for 5 days. Cell viability was determined by MTT assay. C Immunoblot analysis of IL8 protein levels in matched mammosphere-enriched BCSCs and parental MCF-7 or T47D cells. D ELISA quantification of secreted IL8 levels in CM from paired mammosphere-enriched BCSCs and parental MCF-7 or T47D cells. E Immunofluorescence staining of ALDH1A1 and IL8 in breast cancer tissues. Scale bar: 100 μm. F MCF-7 or T47D cells were cultured with CM derived from respective parental cells or mammosphere-enriched BCSCs in the presence or absence of 0.1 mM repertaxin and treated with a graded concentration of tamoxifen for 5 days. Cell viability was determined by MTT assay. G, H Colony formation assay G or Apoptosis (Annexin V-FITC + /PI-) H of MCF-7 or T47D cells cultured with the CM derived from respective parental cells or mammosphere-enriched BCSCs in the presence or absence of 0.1 mM repertaxin and treated with 5 μM tamoxifen. I Immunoblot showing the activity of the AKT (p-AKT) pathways in MCF-7 cells cultured with the CM derived from parental cells or mammosphere-enriched BCSCs in the presence or absence of 0.1 mM repertaxin. J, K Immunoblot showing the phosphorylation of ERα J or the ERα transcriptional activity of estrogen response element (ERE4) K in MCF-7 cells cultured with the CM derived from parental cells or mammosphere-enriched BCSCs in the presence or absence of 0.1 mM repertaxin. L The MCF-7 and BCSCs group are same as Fig. 1K. A rescue group with Repertaxin treatment was included, and in vivo bioluminescent imaging was performed of the tumor burden generated by MCF-7-luc cells. M Kaplan–Meier plots of overall survival (OS) or relapse-free survival (RFS) in breast cancer patients stratified according to their IL8 levels. The data was analyzed by Kaplan–Meier plotter software. All experiments were repeated at least three times. Results are shown as mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (Unpaired two-tailed Student’s t-test in D, one-way ANOVA followed by Tukey’s multiple comparison in H and 3 K-3L, others Two-way ANOVA test)