The BEHAV3D-TP small-scale phenotyping correlates DMG behavioral profiles with microenvironmental composition at a single-cell resolution.
a, Overview of the small-scale TME phenotyping module. During IVM imaging, information about spatial distribution of SR101+ (purple), CD20r+ (purple) and CD31+ (red) cells is collected and processed by the BEHAV3D-TP small-scale phenotyping module. Single DMG cell spatial TME features like distance to its neighbors, number of cells in a radius or minimal distance to a cell type were measured. b, Heatmap depicting DMG cell small-scale niches’ features across distinct DMG behavioral clusters. Arbitrary units in respect to maximal and minimal values for each feature. Bubble size according to significance levels, from smallest (P < 0.05 (**)) to medium (P < 0.01 (**)) to biggest (P < 0.001 (***)): dist_10_cell, Distance to nearest 10 cells, P < 0.01 (**); n_SR101, number of SR101 cells, P < 0.05 (*); min_SR101, minimal distance to a SR101 cell, P < 0.05 (*); n_CD20r, number of CD20r cells, P < 0.05 (*); min_Cd20r, minimal distance to a CD20r cell, P < 0.01 (**); min_BV_dist, minimal distance to a Blood vessel, P < 0.001 (***). P-values represent the significance of differences across clusters tested through ANOVA for each feature. c, e, Boxplot depicting the Minimal distance to a CD20r+ TAMM (µm) (c) and the Minimal distance to a blood vessel (µm) (e) across DMG behavioral clusters. c, Invading versus Erratic, P < 0.01 (**); Invading versus Slow, P < 0.05 (*). e, Static versus Erratic, P < 0.01 (**); Static versus Retreating, P < 0.001 (***); Static versus Invading, P < 0.001 (***); Slow versus Retreating, P < 0.1 (·); Slow invading versus Retreating, P < 0.1 (·); Slow versus Invading, P < 0.1 (·); Slow invading versus Invading, P < 0.1 (·). d, Fixed multispectral image showing the movement of DMG cells (grey) relative to CD20r+ TAMM (blue) and the Tumor core. The trajectories show a path from the initial (blue) to the final position (red). Scale bar, 10 µm. f, Time lapse multispectral images (left to right) showing the DMG cells (blue) displacement along CD31 blood vessel (purple) away from the tumor edge. Scale bars, 30 µm.