Fig. 1: CM from D492HER2 enhances endothelial network and induces endothelial feedback increasing D492HER2 migration/invasion.

a Enhanced endothelial network formation in D492HER2-CM after 4 h. Top: representative phase-contrast images of HUVEC endothelial network after 4 h on top of rBM in control (unconditioned) medium and conditioned medium from D492, D492M, and D492HER2. Each condition included 4–6 wells as replicates and 1–2 images were taken per well at ×10 magnification (scale bar = 100 µm). Bottom: corresponding angiogenesis images (ImageJ, angiogenesis analyzer plugin) showing branches, junctions, master junctions, master segments, and meshes. b Endothelial network formation—quantification. Quantification of master junctions, master segments total master segment length and meshes for each image using angiogenesis analyzer plugin (average and standard deviation per condition). Statistical analysis performed in R, one-way ANOVA, ***p < 0.001, **p < 0.01, *p < 0.05, <0.1. c Endothelial feedback—schematic overview. Schematic workflow of conditioning of ECs (see “Materials and methods”). d D492HER2-induced ECs promote D492HER2 migration and invasion. Transwell-migration and invasion assays of D492HER2 treated with unconditioned medium, unconditioned HUVEC-CM, D492-conditioned HUVEC-CM, and D492HER2-conditioned HUVEC-CM. Number of migratory/invasive cells is shown for the different treatments (Studentʼs t test, ***p < 0.001, **p < 0.01, *p < 0.05, <0.1). e Migration toward ECs—schematic overview. Schematic setup of the transwell-migration assay of D492, D492M, and D492HER2 toward HUVECs seeded below the transwell filter. Migration toward HUVECs (in H14 + EGM1) was compared with migration toward 10% FBS in H14 + EGM5 (pos. ctrl) and plain H14 + EGM5 (neg. ctrl). f Increased D492HER2 migration toward ECs. Migration of D492, D492M, and D492HER2 toward HUVECs. Number of migratory cells is shown for the different treatments (Studentʼs t test, ***p < 0.001, **p < 0.01, *p < 0.05, <0.1).