Figure 3: IL6R blockade increases ER and decreases Stat3/Notch3/CD133 expression restoring HT sensitivity.

(a) Metastatic tumour cells, isolated from Fulv-resistant metastases (MCF7:FulvR, Supplementary Table 2), were cultured in the presence of vehicle (Placebo) or Fulv (10 μM), and growth was determined after 10 days using CalceinAM fluorescent probe: partial (sensitive, HTS) or complete hormone-resistant features (HTR) were generated. Data are reported as mean (fluorescence)±s.e.m. of the last time point of the growth curve (three biological replicates with three technical replicates each). (b) Fold increase in the expression of CD133, Stat3, IL6 and Notch3 mRNA using qPCR from HTR and HTS cells treated with Fulv (see a, HTS transcript as reference). (c) Representative images of Notch3 expression by IHC of lung metastases from placebo versus TamR mice (scale bar, 20 μm). (d) Proliferation (CalceinAM fluorescence) in Stat3 or Notch3 stably silenced HTR cells (shS3, shN3) derived from FulvR-Met and propagated in vitro (a) in the presence/absence of Fulv (10 μM). Data are reported as the mean (fluorescence)±s.e.m. of each time point of the growth curve (three biological replicates with three technical replicates each). (e) Number-No- of II-MS (>100 μm) from FulvR-metastatic cells (MetMS) expressing stable shS3 and shN3 cultured in the presence/absence of Fulv (10 μM) and/or tamoxifen (1 μM, 14 days). (f) qPCR of CD133, Notch3 and ER mRNA (fold increase Fulv versus untreated cells) in shS3-FulvR-metastatic cells cultured in the presence/absence of Fulv in vitro (10 μM, 7 days). (g) Number of II-MS (diameter>100 μm) from FACS-isolated (10³ cells per well) CD44^hi and CD133^hi cells from TamR-Met and FulvR-Met (Supplementary Table 2) treated with Tam (1 μM) or Fulv (10 μM) and tocilizumab (50 μg ml⁻¹), either alone or in combination (7 days). (h) Western blot analysis and IHC images for ER expression from HTR cancer cell lines (ZR751, BT474) and xenografts (MCF7) following treatment with Fulv (10 μM, 14 days) in presence/absence of tocilizumab (50 μg ml⁻¹, treatment for 14 days) and after shS3 (scale bar, 50 μm). Data are reported as mean±s.d. of three independent experiments (n=3; b,e,f,g). P values (*P<0.05, **P<0.0001) refer to t-test (a,b,f), multiple comparisons corrected t-test after GLM ANOVA (e,g) and GLM for repeated measures (d).