Figure 1: Increased CD133 and IL6 expression in HT-resistant cancers.

(a) Box plot of CD133 and ERα mRNA levels (GSE1028) in breast cancer tissues pre- and post-Letrozole neoadjuvant treatment. P refers to Wald’s test; each value corresponds to a patient sample (median, maximum and minimum values are reported). (b) CD133 expression [immunohistochemical (IHC) scores 0–3] in matched primary and metastatic tissues from patients who developed HTR metastasis (Supplementary Table 1). Representative images are shown (scale bar, 50 μM). (c) CD133 and CD44 cells from vehicle (CT) and fulvestrant (Fulv)-resistant MCF7 xenografts were quantified using flow analysis (Flow Fold Increase, n=4 per group, see Methods). (d) Bar graph of percentage of mice developing tumours (n=5 mice per group, tumour engraftment) from the injection of 100 Fulv-resistant ZR751-derived CD133^hi/CD44^lo or CD133^lo/CD44^lo cancer cells (P<0.001, Monte Carlo χ²-test). (e) IL6 levels were measured by ELISA from the serum of HT-resistant tumour-bearing mice (Fulv-resistant MCF7 tumours). (f) IL6 RT–PCR analysis from FACS-sorted populations derived from TamR-MCF7 tumours. Beta2 microglobulin (β2μ) mRNA is reported as quantitative control. (g) Representative growth kinetics (by bioluminescence (BLI)) of TamR-MCF7 tumours. MCF7 (GFP/Luciferase+) xenografts were established and mice were treated (n=5 per group) with tamoxifen citrate pellets (Tam, 5 mg per pellet) with/without tocilizumab (Toci) and in combination, TamToci. The mean BLI value±s.e.m. is reported for each time point. (h) MCF7 tumours from g were analysed for the expression of CD133 and CD44 using flow analysis (Flow Fold Inc is fold-increased as compared with placebo). (i) Flow Fold Increase (log-scale) of CD133^hi cells derived from primary tumours and matched metastasis from TamR versus placebo mice. (j) Representative IHC images of CD133 protein expression in metastatic tissues (kidney, lymph node, lung) from TamR mice. Scale bar, 100 μm (inserts, 20 μm). (k) ELISA of circulating IL6 from TamR and placebo mice (malignant ascites, M-Ascites). Data are reported as mean±s.d. of three independent experiments (n=3, c,e,h,i,k). P values (*P<0.05, **P<0.0001) refer to Wilcoxon exact test (b), t-test (c,e), multiple comparisons corrected post hoc t-test after GLM analysis of variance (ANOVA; h,i,k), repeated measures GLM (g).