Figure 3

DNA-PK inhibitors sensitize 3D tumor cell spheroids to etoposide treatment. (A) Clonogenic survival of FaDu cell spheroids treated with etoposide ± 1 µM M3814 for 24 h (mean ± SD). (B) Survival fraction of HCT-116, A549, FaDu, and SiHa spheroids treated with 25 µM etoposide + 1 µM M3814 for 24 h (mean ± SD) (p ≤ 0.0001 for all cell lines). Experiments were performed at least 3 times except for FaDu cells. (C) Representative images of EdU, EU, Top2α, and Top2β (red) staining in HCT-116, A549, FaDu, SiHa, U87 and BT474 tumor cell spheroids. Cell nuclei were stained using Hoechst 33342 (grey). (D) Evaluation of EdU and EU signal intensity versus cell depth in untreated HCT-116 spheroids. Dashed lines represent individual experiment repeats while solid lines represent group averages. (E) Analysis of p53BP1 signal intensity (mean ± SD) at different depths into tumor cell spheroids (0–50 µm, 50–100 µm and 100–150 µm). HCT-116 spheroids were treated with 25 µM of etoposide in the presence of 1 µM M3814, 1 µM AZD7648 or vehicle for 6 h before cryosectioning and staining. Addition of M3814 and AZD7648 both significantly increased p53BP1 intensity in 50–100 µm and 100–150 µm layers of spheroids (p ≤ 0.01 for AZD7648, p ≤ 0.001 for M3814).