TWIST1 over-expression increases invasion of SNB19 and T98G cells in organotypic brain slice. (A) Representative low magnification (4×) images of SNB19 Ctrl and TWIST1 (TW) expressing cells cultured on the brain slices (a). Magnified sub-regions indicated by boxes in the top row are shown below (b). Cells invading brain tissue are shown with arrows. Scale bars: 500 and 200 μm. (c) Quantified cell migration is shown on the bar diagram (p = 0.0002). Brain slices were imaged using laser scanning confocal microscopy at equivalent optical planes and analyzed using Metamorph software. (B) (a): Low-magnification photomicrograph of merged fluorescent and DIC channel images. (Left) T98G control (Ctrl) cells generate defined cell aggregates in the brain slice surface. (Right) T98G cells with TWIST1 (TW) over-expression are dispersed as single cells and small non-cohesive aggregates over the brain slice. (b): Representative laser scanning confocal images of T98G Ctrl and T98G TW slices. Images obtained through the entire thickness of the brain slice and 50 μm sections (as indicated by white lines) were digitally reconstructed in the orthogonal plane for analysis of invasive cells. (c) Representative orthogonal views for T98G (Ctrl) and T98G TW are presented as a maximum-intensity projection image. Arrows show cell aggregates on the surface of the slice and arrowheads show invading cells. (d) The results of the analysis of invasive cell density demonstrated a significant increase in the invasion of T98G TW (7564 +/- 1771 cells/mm3) versus T98G control cells (1695 +/- 847 cells/mm3), p = 0.007.