Extended Data Fig. 1: Characterization of cell shape and invasion depth in ex vivo brain cultures.
(a) Percentage of 1205Lu cells adhered to vessels after 24h of ex vivo culture (n=7 slices, 4 experiments). Graph shows mean ± standard deviation. (b) Brain slice immunostaining of laminin (magenta), lifeact-GFP (green) and nuclei (hoechst, blue) of a 1205Lu cell spreading over the outer surface of a vessel. 3D view,11µm-thick image stack. Scale bar 10µm. (c) Left: single image plane of the cell in (b), including isolectin-IB4 labelling of endothelial cells (grey). Scale bar 10µm. Right: laminin (magenta), lifeact-GFP (green) and isolectin-IB4 (grey) fluorescence profile along the dashed yellow line. (d) Example of cell invasion depth quantification. Left: immunostaining of laminin (magenta) and lifeact-GFP (green). Yellow dashed lines outline a cell in the bulk (cell 1) and a cell spreading along a vessel (cell 2). Scale bar 50µm. Right: region 2 (centred on the cell of interest) is used to average laminin intensity across a 60µm stack and define the reference point for invasion depth (0µm). Because of the opacity of brain tissue, averaged laminin intensity shows a peak at the top of the slice and decays as imaging deeper into the tissue. The origin of invasion depths is defined as \({z}_{{peak}\_{region}2}-6\mu m\). Region1 (that follows the cell 2D shape), is used to average both cell and laminin fluorescence. The intensity of the GFP signal is used to determine the cell depth within the slice, and local laminin intensity is used to determine if a cell is in the bulk or on a vessel (if observing a local peak of laminin intensity). (e) Violin plots of 1205Lu cell shape circularities when adhered to vessels or to the bulk brain tissue 4 and 24h after seeding. (f) Violin plots of 1205Lu invasion depths within the slice when adhered to vessels or to the bulk brain tissue 4 and 24h after seeding. For e-f, n=562 for Bulk, n=621 for Vessel at 4h, n=337 for Bulk, n=706 for Vessel at 24h, from 6 slices, 3 experiments. Graphs show median ± interquartile range. Dots represent the median values for each slice analysed. Statistical analysis: Kruskal–Wallis test followed by a post hoc Dunn’s test.
