Fig. 4: Combined fulvestrant, CDK4/6i and AKTi is effective in breast cancer cell lines resistant to combined CDK4/6i and fulvestrant.

The effect of fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM) and AKT inhibitor (AKTi, capivasertib, 250–500 nM in the MCF-7 cell model; 100 nM in the T47D cell model), as single agents or in the double and triple combination, was assessed in all cell lines by crystal violet growth assay (A, B, E, and F) and CellTiter-Blue viability assay (C, D, G, and H) performed over 6 days. Outgrowth of resistant colonies was investigated in MPF-R (I) and TPF-R (J) cells by weekly evaluation of the percentage of 48 wells at 50% or greater confluence (positive wells) over 12 weeks. Experiments were conducted in three biological replicates and data are shown as mean ± SEM. Asterisks indicate significant differences in one-way ANOVA tests at day 6 (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and ****p < 0.0001).