Fig. 6

Enhanced expression of genes related to EMT in spheroid cultures compared to monolayer cultures. a Representative immunostaining images of patient-derived meningioma spheroids and matched tumour tissue (n = 4) stained for anti-E-cadherin, and anti-vimentin (Leica IM8). Scale bars: 200 µm. b Plot of immunoscores of immunostaining of E-cadherin (epithelial indicated as circle) and Vimentin (mesenchymal indicated as triangle) in spheroids (S) and matched tumour tissue (T) (n = 4). Colour represents immunoscore of 0–4 (0 = negative, 1 = weak, 2 = moderate, 3 = strong, 4 = very strong) c Relative gene expression of a panel of EMT markers: CDH1 (E-cadherin), ZO-1 (TJP1), Snai1, Snai2, Zeb1, Notch1, Hes1, and Hey1 in 2D monolayer cultures (red) compared to matched spheroids (3D) (blue) (n = 5). d Representative western blot and e quantification showing the expression E-cadherin, Notch1 NICD, Slug, N-cadherin, Hey1 in spheroids (3D) compared to patient matched monolayers (2D). Expression is shown as the relative increase compared to monolayers. f Representative phase-contrast microscopy images of WHO grade 2 spheroids with and without ECM (Matrigel) at 24 h and 48 h time points. Scale bars indicate 100 µm. g Bar graph showing the fold change of the max. diameter for spheroids embedded in ECM compared to spheroid controls that were not embedded. Max.diameter was measured using ImageJ. One-way ANOVA with Tukey’s multiple comparisons test was used for statistical evaluation. ***p < 0.001, ****p < 0.0001. h Fluorescence microscopy image showing F-actin (phalloidin, green) and nuclei (DAPI, blue) in spheroids without ECM and i with ECM showing invadopodia -like projections migrating into the ECM (Matrigel) at 48 h. Scale bars indicate 100 µm