Fig. 6: Assessment of the cause of the in vitro/in vivo disconnect.

a CTC174 tumours were disaggregated into single cells, seeded in 6-well plates and dosed with increasing concentrations of AZ’6421 for 24 hours before being lysed. ERα levels were assessed by immunoblotting. ERɑ levels normalised to vinculin and expressed as % DMSO control are shown underneath the immunoblot. b Representative IHC images taken from sections of tumour treated with vehicle control or 100 mg/kg AZ’6421 for 4 days. Sections were stained with an antibody against ERα (Sp1, Ventana). c Schematic of potential sites of metabolism d Free plasma exposure of AZ’6421 and its metabolite 8 following 100 mg/kg dosing of AZ’6421. e Concentration-response curves for AZ’6421 and metabolite 8. ERα levels in MCF7 cells were assessed using an immunofluorescence endpoint after 24 h exposure to compound. Individual data points were normalised to vehicle control (0% inhibition) and 100 nM fulvestrant (100% inhibition) and a concentration-response curve plotted using nonlinear regression analysis (n = 8–10). Points in bold represent the mean. f Effect of metabolite 8 on ERα degradation in CTC174 cells cultured ex vivo. Graph shows ERɑ levels normalised to vinculin and expressed as % DMSO control. g Immunoblots for ERα and vinculin loading control. MCF7 cells were pre-treated with 10 µM cpd 8 or DMSO control for 1 h before being exposed to DMSO, AZ’6421 or fulvestrant, at the concentrations indicated, for a further 24 h. A representative immunoblot from two biological replicates is shown.