Fig. 5: Inhibition of endothelial NOTCH signaling prevents increased tube formation and feedback induction. | Laboratory Investigation

Fig. 5: Inhibition of endothelial NOTCH signaling prevents increased tube formation and feedback induction.

From: ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion

Fig. 5

a ECM1 increases endothelial NOTCH1 and NOTCH3. Increased NOTCH1 and NOTCH3 expression in HUVECs treated with D492HER2-CM and rECM1 but not D492HER2-kdECM1-CM on gene expression level (left) and protein expression level (IF staining, scale bar = 50 µm). Studentʼs t test, ***p < 0.001, **p < 0.01, *p < 0.05, <0.1. b NOTCH inhibition blocks ECM1-induced increase of endothelial network formation. Top: HUVEC endothelial network in H14 (ctrl), 60 ng/ml rECM1, 60 ng/ml rECM1 + DMSO and 60 ng/ml rECM1 + DAPT (20 µM) after 4 h. 4–6 wells as replicates per condition and 1–2 images taken per well at ×10 magnification (scale bar = 100 µm). Bottom: corresponding angiogenesis images (ImageJ angiogenesis analyzer). c Endothelial network formation—quantification. Quantification of master junctions, master segments, total master segment length, and meshes for each image using angiogenesis analyzer plugin. One-way ANOVA, ***p < 0.001, **p < 0.01, *p < 0.05, <0.1. d Endothelial feedback reduction—schematic overview. Schematic workflow of treating ECs using 60 ng/ml rECM1 and blocking NOTCH signaling using DAPT (20 µM). e NOTCH inhibition blocks rECM1-induced endothelial feedback on migration and invasion. Transwell-migration and invasion assays of D492HER2 treated with unconditioned medium, unconditioned HUVEC-CM, DMSO-treated HUVEC-CM, DAPT-treated HUVEC-CM, 60 ng rECM1-treated HUVEC-CM, 60 ng rECM1 + DMSO-treated HUVEC-CM, and 60 ng rECM1 + DAPT-treated HUVEC-CM (NOTCH inhibition). Number of migratory/invasive cells for different treatments (Studentʼs t test, ***p < 0.001, **p < 0.01, *p < 0.05, <0.1).

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