Fig. 5: Analysis of 3D topology and comparison of microniche shapes in a complex organoid environment.
a, Comparison of cellular positioning on two different shapes of microniche (blue, ‘cup’; orange, ‘well’) and microniche without ECM (yellow) depicted on a scatterplot of oblate-versus-prolate ratio (one dot denotes one nucleus). Color-coded feedback (red contours denote ‘spherical’ gate, cyan denotes all other nuclei) presented on 3D segmentation views (xy and xz) and a median xy plane (indicated by white double arrows) with the percentage of nuclei exhibiting ‘spherical’ ellipsoids for each microniche (representing an isotropic neighborhood). Red arrows indicate disorganized multilayers of cells. Scale bar, 25 µm. b, Comparison of different regions of interest within a complex primary PDAC organoid. Two complementary strategies were used: a single-step data gating on the scatterplot of oblate-versus-prolate ratio, enabling feedback of three different classes—disk (magenta), rod (green) and sphere (red)—on the 3D view. Red arrows denote enriched areas of ‘sphere’ nuclei distribution (in the core of the organoid), while green arrows highlight ‘rod’ enriched areas (in several buds). ‘Disk’ (magenta) nuclei distribution is not displayed in zoomed images for improved visualization. A two-step strategy is also applied, starting with image gating on segmented images (image gating: core (red), bud (light blue/cyan), borders (orange), with corresponding nuclei counts displayed). Subsequently, the same data gating step (magenta/green/red) is applied to multi-region scatterplots. Additionally, linear regression analysis is performed, and corresponding slope values are presented. Pie charts display the corresponding nuclei percentage in each data gate (disk, rod and sphere) per image gate (regions of interest: core, bud and border). Scale bars, 150 µm.
