Fig. 6

AD0157 suppresses lymphangiogenesis in vitro by interfering with LEC tubulogenesis, migration, invasion, and sprouting. a Tube-like structures formed by LECs in a collagen matrix, in the absence (control) or presence of different AD0157 concentrations. Scale bars represent 200 μm. b Computerized quantification of the tube area density and the number of branchings (common point between 2 or more tubes). c Pictures showing the area recovered by LECs after 48 h of wounded LEC monolayers, in the absence or presence of different AD0157 doses. Dashed lines in pictures indicate the initial (time 0) wound edges. Scale bars represent 100 μm. d Percentage of the initial cell-free area recovered by endothelial cells. e Representative images of the invaded LECs across a Transwell chamber coated with 0.2% gelatin, after 48 h of treatment. f Percentage of invaded cells. g LEC sprouting from spheroids embedded in a collagen-methyl cellulose gel under different experimental conditions. Scale bars represent 100 μm. h Computerized quantifications of the convex envelope area (minimal convex polygon area containing the spheroid core and all sprouting cells), the migrated cell area (area containing migrating cells), the LEC density (number of pixels belonging to cells that intersect the circle “i”, N_i, normalized by the corresponding perimeter) at different distances from the spheroid core and values of the LEC density at the specific distance of 0.13 mm from the spheroid centre, in control and AD0157-treated spheroids. Arrows indicate the maximal length (Lmax). Values are expressed as mean ± s.e.m., and the Wilcoxon-Mann-Whitney significance tests were used to compare the differences between control and AD0157 treatment. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5 independent tests)