EC-derived TNF is necessary for TC-induced EC death. (A) HUVECs with siRNA-mediated TNF knockdown were cocultured with CFSE-labeled MDA-MB231 TCs, and cells with impaired membrane integrity were determined after 6 h by EthDIII uptake. Shown are representative images and the quantification of three independent experiments. Three technical replicates per experiment with three to four images per replicate were analyzed. Scale bar indicates 50 μm. ****, P < 0.0001 by semiparametric generalized linear model based on log-normal distributions. (B) Murine MLECs with siRNA-mediated TNF knockdown were cocultured with the indicated CFSE-labeled murine TCs. Cells with impaired membrane integrity were determined after 6 h by EthDIII uptake. Shown are representative images and the quantification of four independent experiments. Three technical replicates per experiment with three to four images per replicate were analyzed. Scale bar indicates 50 μm. *, P < 0.05 by unpaired two-tailed Mann–Whitney U test. (C) Contact with TCs might induce an autocrine soluble TNF loop in ECs, leading to EC death. (D) HUVECs with indicated siRNA-mediated knockdown were cocultured with CFSE-labeled MDA-MB231 TCs, and cells with impaired membrane integrity were determined after 6 h by EthDIII uptake. Shown are representative images of three independent experiments. Three technical replicates per experiment with three to four images per replicate were analyzed. Scale bar indicates 50 μm. ***, P < 0.001 by one-way ANOVA on ranks with Dunn’s post hoc test. (E) Experimental outline as performed in F. LacZ-expressing LLC TCs were i.v. injected into the indicated mouse strains, and TC extravasation was assessed after 6 h. (F) Extravasated TCs 6 h after TC injection were quantified by β-Gal staining. Representative microscopic images are shown. Scale bar indicates 50 μm. n = 3 mice/group. ***, P < 0.001 by unpaired Student’s t test. Data represent mean ± SEM. Ctrl, control; p.i., postinjection.