Fig. 1: ESR1 mutant models exhibited oestrogen-independent growth and metastatic propensity.

a MTT growth assay of Tam and Ful-treated MCF-7 and b T47D cells. Percent survival is normalised to no treatment controls. Graphs represent mean + standard deviation (N = 3 replicates) one-way ANOVA was used for statistical analysis. c MCF-7 cells were cultured in charcoal-stripped serum supplemented media, underwent whole cell lysis, and analysed by immunoblot for ER-regulated proteins (left). Values below PR, CCND1 and c-Jun represent normalised densitometry of each protein compared to GAPDH. PR was not detected in WT cells; thus, relative expression was normalised to Y537S cells. Cells were cultured in charcoal-stripped supplemented media and underwent cellular fractionation (right). Nuclear fraction is shown. d T47D cells were cultured in charcoal-stripped serum supplemented media and analysed by immunoblot for ER-regulated proteins (left). Values below PR, CCND1 and c-Jun represent normalised densitometry of each protein compared to GAPDH. PR was not detected in WT cells. Cells were cultured in charcoal-stripped supplemented media and underwent cellular fractionation (right). e Venn diagram of upregulated MSigDB Hallmark gene sets in MCF-7 ESR1 Y537S and LTED cells compared to MCF-7 ESR1 WT, and T47D ESR1 Y537S compared to T47D ESR1 WT. The numbers shown are of Hallmarks with a false discovery rate < 0.25. f In vivo individual tumour growth curves of MCF-7 ESR1 WT, ESR1 Y537S and LTED tumours. g In vivo primary tumour growth of MCF-7 WT, ESR1 Y537S and LTED tumours shown by time to tumour halving after E2 withdrawal. The Mantel-Cox test was used for statistical analysis (WT N = 5, Y537S N = 8, LTED N = 9). h Metastatic frequency in MCF-7 ESR1 WT-, ESR1 Y537S- and LTED-transplanted mice (WT N = 5, Y537S N = 8, LTED N = 9). Metastatic frequency is represented by the total number of mice per group that exhibited a macrometastasis. Fisher’s Exact test was used for statistical analysis. P < 0.05 was considered statistically significant in all tests. (*p < 0.05, **p < 0.01, ***p < 0.001).