Fig. 5

ALK inhibition drives cell-cell adhesion enhancement in CMS1 cells cultured in 3D. A Confocal analysis of fixed LoVo cultured in 3D. DAPI (blue) was used to stain nuclei, while Phalloidin was used to stain actin cytoskeleton (red). The upper panel shows maximum intensity projection and single sections (insets) from control and treated spheroids. In the lower panel a volume view with 3D rendering is shown. B Confocal analysis of clarified LoVo spheroids, stained with anti-ALK (green) and anti-E-Cadherin (grey) antibodies. Phalloidin (red) and DAPI (blue) were used to stain actin and nuclei respectively. Both 3D merges and single sections at different magnifications for each channel are shown. C Determination of vimentin (VIM) expression by qPCR in LoVo and SW48 cells cultured in 3D settings and treated with CZB for 8–10 days. Data are reported in relation to untreated control. Unpaired T- test and One-Way ANOVA were applied for statistical analysis. D IHC of LoVo spheroids collected after 15 days of growth, in full medium or under CZB treatment (250 nM or 500 nM). All specimens were formalin-fixed and paraffin-embedded. In the left column Hematoxylin and Eosin staining is provided (scale bar: 100 μm). In the central and right columns, we display respectively E-cadherin and Vimentin IHC (200X magnification)