Fig. 2

Effect of siramesine on patient-derived spheroid cultures. The glioblastoma stem cell-like containing spheroid (GSS) cultures T78, T86 and T87 were exposed to siramesine (0–15 μM) for 24 h. a–b The dye propidium iodide (PI) (red fluorescence) enters dead and dying cells and was used to identify cell death in spheroids. PI uptake in all three GSS cultures was already seen by 5 μM siramesine and was pronounced at 10–15 μM siramesine. Hoechst 33324 staining (blue fluorescence) was used to stain all cells to be able to calculate a percentage of PI uptake per spheroid. c Formation of secondary spheroids from siramesine-exposed primary spheroids was reduced for all three GSS cultures already by 5 μM siramesine. d After siramesine exposure of T78 primary spheroids, the spheroids were fixed and paraffin embedded for histology. H&E staining and immunohistochemical staining with CD133, Nestin, Bmi-1, Sox 2, Ki-67, LAMP-2 and caspase 3 of 3 μm histological sections were performed. Only small fragments and single cells were found at concentrations of 10 and 15 μM, suggesting induction of pronounced cell death by siramesine. However, CD133 was expressed in both control spheroids and in siramesine treated spheroids. Nestin was expressed in control spheroids and spheroids exposed to 5 μM siramesine whereas some spheroid residues at 10 and 15 μM had lost the nestin expression. Bmi-1 was expressed in control spheroids and in the siramesine exposed spheroids and the same pattern was seen for Sox2, Ki-67, Caspase 3 and Lamp-2. These staining thus suggested a potential for recurrence. Scalebar 100 μm (a and d). Data are displayed as mean values ± SEM, and *P < 0.05, **P < 0.01, ***P < 0.001 were assessed by one-way ANOVA