Extended Data Figure 10: Effects of loss of APP on TCs and on metastasis formation.
From: Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis
a, Analysis of knockdown efficiency in B16 and LLC1 TCs using different siRNAs against mRNA encoding APP (siAPP). Shown is the relative mRNA expression normalized to GAPDH levels and to the level detected in scramble siRNA-treated samples (siCTRL). b–d, Knockdown of APP in B16 or LLC1 TCs (B16siAPP and LLC1siAPP) and evaluation of cell proliferation (b), viability (c) and migration (d). e, f, Evaluation of APP-deficient TC-induced EC death in vitro in C57BL/6 WT primary MLECs (e) and the ability of APP-deficient TCs to migrate over an endothelial layer (f). g, Quantification of lung metastases 12 d after i.v. injection of LLC1 TCs with silenced APP expression (LLC1siAPP) into WT animals. Shown are representative data of three independent experiments with mean values ± s.e.m. (b, c, e) or ± s.d. (d, f, g) from biological sextuplicates (n = 6) (b, c, e, f), triplicates (n = 3) (d) or from n = 5 animals per condition (g). *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. Unpaired, two-tailed Student’s t-test (b, c, f) or one-way ANOVA and Bonferroni’s post hoc test (e, g). h, Model: TCs induce RIPK1/RIPK3/MLKL-dependent necroptosis in ECs via APP-DR6. TCs then may directly pass through the emerging gap after EC death. Alternatively, or in parallel, damage-associated molecular pattern molecules (DAMPs) released from necroptotic ECs could act on TCs and/or non-necroptotic endothelial as well as other cells to promote TC extravasation and metastasis.
