Fig. 6. Alisertib and metformin synergistically suppress breast tumor growth in vivo.

A The growth curves of xenograft tumors derived from 4T1 cells. Mice were subjected to daily treatments with a vehicle, alisertib (ALS), metformin (Metf), or combination (Metf + ALS). The one-way repeated-measure ANOVA followed by the least significant difference test were used to evaluate the differences between groups. *p < 0.05; ***p < 0.001. Mean + SD, n = 8: Vehicle, ALS, and Metf; n = 10: Metf + ALS. B Eight tumors removed from mice in each group are shown. Scale bar, 1 cm. C Statistical analysis of the weights of the dissected tumors. The one-way ANOVA test, followed by the least significant difference test, was used to evaluate the differences between groups. ns not significant; **p < 0.01. Mean ± SEM, n = 8. D The growth curves of xenograft tumor formed by MDA-MB-231 cells. Mice were subjected to daily treatments with vehicle, alisertib (ALS), or in combination (Metf + ALS). Statistical analysis was performed by one-way ANOVA. ****p < 0.0001. Mean + SD, n = 6. E Six tumors removed from mice in each group are shown. Scale bar, 1 cm. F Statistical analysis of the weights of dissected tumors. One-way ANOVA followed by multiple comparison test. ns not significant; **p < 0.01. Mean ± SEM, n = 6. G Fluorescence IHC analysis for the cell proliferation marker Ki-67 in xenograft tumors derived from 4T1 cells treated as in Fig. 6A. DNA (Blue), CD44 (Green), Ki-67 (Red). Scale bars represent 20 μm. H Quantification of Ki-67 positive cells in xenograft tumors samples derived from 4T1 cells treated as in Fig. 6A. The results are given as the mean ± SD (n = 5: Vehicle and ALS; n = 4: Metf; n = 3). *p < 0.05; ****p < 0.0001. By one-way ANOVA test, followed by the least significant difference test. I Fluorescence IHC analysis for the apoptotic events using anti-Cleaved Caspase 3 antibody. DNA (Blue), CD44 (Green), Cleaved Caspase 3 (Red). Scale bars represent 20 μm. J Quantification of Cleaved Caspase 3 positive cells in xenograft tumors samples derived from 4T1 cells treated as in Fig. 6A. The results are given as the mean ± SD (n = 5: Vehicle and ALS; n = 4: Metf; n = 3: ALS + Metf). *p < 0.05; **p < 0.01; ****p < 0.0001. By one-way ANOVA test, followed by the least significant difference test. K Fluorescence IHC analysis for binucleated cells in xenograft tumors samples derived from 4T1 cell. Images show examples from 4T1 derived tumors treated with ALS + Metf. DNA (Blue), CD44 (Green). White dashed line delineates cell border. L Quantification of binucleated cells in xenograft tumors samples derived from 4T1 and MDA-MB-231. For 4T1 derived tumors, the results are given as the mean ± SD (n = 5: Vehicle; n = 4: Metf and ALS; n = 3: ALS + Metf). For MDA-MB-231 the results are given as the mean ± SD (n = 5: Vehicle and ALS; n = 3: ALS + Metf). **p < 0.01; ***p < 0.001; ****p < 0.0001. By one-way ANOVA test, followed by the least significant difference test. M Typical mitotic and nonmitotic cells revealed by fluorescence IHC analysis. Images show examples from 4T1 derived tumors treated with vehicle. DNA (Blue), CD44 (Green), Ki-67 (Red). Scale bars represent 20 μm. N Quantification of mitotic cells from 4T1 and MDA-MB-231 derived tumor samples. For 4T1 derived tumors, the results are given as the mean ± SD (n = 5: Vehicle; n = 4: Metf and ALS; n = 3: ALS + Metf). For MDA-MB-231 the results are given as the mean ± SD (n = 5: Vehicle and ALS; n = 3: ALS + Metf). *p < 0.05; **p < 0.01; ***p < 0.001. By one-way ANOVA test, followed by the least significant difference test. O Relapse-free survival (RFS) for Aurora-A/COX5A transcription levels in breast cancer patients. Patient data from KM plotter is divided into four groups according to Aurora-A/COX5A expression level. HR hazard ratio. *p < 0.05; ****p < 0.0001. Log-rank (Mantel–Cox) test.