Breast cancer stem cells have low expression of ER. Cells were sorted based on surface markers to enrich for CSC. Representative Western blot showing ER expression in sorted primary metastatic cells (A) and MCF7 cells (B). Anoikis resistant (AR) cells from primary metastatic and cell lines (MCF7 and T47D) were collected for protein and RNA. (C) Representative Western blot showing expression of ER in total versus AR cells, (D) mRNA expression level of ER (ESR1) and responsive genes in AR cells compared to total population. (E) Representative Western blot of ERK and phosphorylated (actived) ERK following culture for 48 hours in monolayer ± 1 nM 17β-estradiol ± 1 μM tamoxifen or gefitinib. (F) Mammosphere formation was assessed following culture with 1 nM 17β-estradiol ± gefitinib. Fold change is normalised to control, untreated cells represented as line. Means plotted ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared to E2 treated. # P < 0.05 compared to control cells.