Fig. 6: Combined fulvestrant, CDK4/6i and AKTi is effective in tumor xenografts resistant to combined CDK4/6i and fulvestrant.

A Tumor growth curves of orthotopic MPF-R tumors treated with a CDK4/6 inhibitor (CDK4/6i, palbociclib, 50 mg/Kg bodyweight) combined with fulvestrant (Fulv, 100 mg/Kg bodyweight; N = 9) or in combination with both AKT inhibitor (AKTi, capivasertib, 100 mg/Kg body weight) and fulvestrant (Fulv, 100 mg/Kg bodyweight; N = 10), or vehicle (castor oil and 25% w/v HPB cyclodextrin; N = 10). CDK4/6i and AKTi were administered by oral gavage once daily for 5 days a week, whereas fulvestrant was administered subcutaneously once a week. Treatment was initiated when tumors reached 100 mm³ and continued for up to 7 weeks. Mice from the control group were sacrificed and tumors excised on week 6 due to their large size, while mice from double and triple combination groups were sacrificed and tumors excised on week 7. Data are shown as mean tumor volume ± SEM. Asterisks indicate significant differences in the two-tailed t-test at the endpoint (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and ****p < 0.0001). B Western blot analysis of key signal transduction proteins in 3 tumors of each treatment group excised when mice were sacrificed. GAPDH was used as a loading control. A representative of two independent experiments is shown. C Tumor volumes over time of the parental PDX KCC_P_3837 untreated (blue) and treated with combined CDK4/6i palbociclib (25 mg/kg in 2.5% DMSO, 25% β-cyclodextrin, 5 days per week by oral gavage) and fulvestrant (100 mg/kg in castor oil, once weekly via subcutaneous injection) (orange), and of the derivative PDX KCC_P_3837-FPR resistant to combined palbociclib and fulvestrant (red) at the third passage of continuous exposure to combined palbociclib and fulvestrant. D Kaplan-Meier survival plot of progression of PDX KCC_P_3837-FPR (resistant to combined CDK4/6i and fulvestrant) under treatment with combined CDK4/6i and fulvestrant with or without AKTi capivasertib (100 mg/kg in 2.5% DMSO, 25% β-cyclodextrin, 5 days per week by oral gavage) (N = 5 and N = 4, respectively). Progression was defined as tumors growing to at least 5 mm in the shortest dimension. A two-sided p-value (p < 0.05) was calculated using log-rank testing. E, F Evaluation of metastasis area > 2500 µm² relative to lung area at the endpoint (6 weeks) using an experimental metastasis model. MPF-R tumors were treated with fulvestrant (100 mg/Kg body weight) combined with CDK4/6i (palbociclib, 25 mg/Kg bodyweight; N = 10), fulvestrant combined with AKTi (capivasertib, 100 mg/Kg bodyweight; N = 10), or triple combination (N = 9). TPF-R tumors were treated with the same dosage of fulvestrant and CDK4/6i and 50 mg/Kg bodyweight of AKTi (N = 7 in fulvestrant and CDK4/6i group, N = 10 in fulvestrant and AKTi and N = 10 in triple combination group). CDK4/6i and AKTi were administered by oral gavage once daily for 5 days a week, while fulvestrant was administered subcutaneously once a week. Treatment was initiated 3 days before injection of cells in the tail vein and continued for up to 6 weeks. Data are shown as mean ± SEM. Significant differences were evaluated by the two-tailed Mann-Whitney test. G Representative micrographs of MPF-R and TPF-R tumors in mice lungs of each treatment group showing cytokeratin expression by immunohistochemistry (scale bars, 200 µm).