Figure 7. Effect of combined inhibition of BRAFV600E and PLK1 on CD133+ cells and tumor growth in vivo.
(A) Ex vivo flow cytometry analyses of subcutaneous DBTRG xenografts treated for five days with PLX4720 daily at 20mg/kg, BI2536 twice at 50mg/kg or a combination of both inhibitors (Combo). Dissociated tumor cells were analyzed for incorporation of EdU (S-phase) or G2/M following an EdU pulse 30 min before tumors were harvested. (N=5 individual experiments; 2-way ANOVA with Bonferroni post-hoc test, values compared to vehicle treated conditions for each cell type.)
(B) Quantification of Ki67+, CD133+ and CD133+Ki67+ double positive cells by IF performed on intracranial xenograft tumor samples of treated mice. (N ≥ 3 individual tumors from each treatment group; a minimum of three sections for each treatment group were counted.)
(C) Quantification of cleaved caspase 3 (CC3)+, CD133+ and CD133+CC3+ double-positive cells by IF performed on intracranial xenograft tumor samples of treated mice. (N ≥ 3 individual tumors from each treatment group; a minimum of three sections for each treatment group were counted) *P≤0.05; **p≤0.005 in A–C.
(D) Normalized bioluminescence (BLI) readings from intracranial DBTRG-05MG tumors at 7–9 days post-treatment initiation. Mice were either untreated (control) or treated with either daily PLX4720 at 20mg/kg, twice-weekly BI2536 at 50mg/kg, or a combination (n=12–13 animals per treatment group, 1-way ANOVA with Tukey post-hoc test).
(E) Model for a PLK1 inhibitor-sensitive polarity checkpoint in a heterogeneous GBM tumor where CD133+ cells co-exist with CD133−NG2+ (NG2+) cells. NG2+ cells are outlined in grey; polarized CD133 are black crescents.