Fig. 3. Lung endothelial cells are a major source of latency-inducing Wnt ligands.
a, Schematic of the experimental G-O-F and L-O-F (loss-of-function) strategy. G-O-F was achieved by treatment with either CHIR99021, a selective GSK-3 inhibitor that leads to the accumulation of β-catenin via inhibition of its proteasomal degradation, or SKL2001, which directly interacts with β-catenin and thereby stabilizes the protein. L-O-F was achieved by treatment with LGK974, a selective inhibitor of Porcupine, an enzyme that is crucially involved in the secretion of Wnt ligands. Gray bar indicates time span (in days) of daily treatment with LGK974. b, Percentage of extravasated TCs for control, G-O-F and L-O-F 1.5 days postinjection. Data presented as mean ± s.d.; P values by one-way ANOVA with Tukey post test; n = 3–6 mice. c, Quantification of absolute TC number per lung 3.5 days postinjection for control, G-O-F and L-O-F. Data presented as mean ± s.d., P values by one-way ANOVA with Tukey post test; n = 5–6 mice. d, Quantification of relative TC number normalized to EC abundance in lungs 7 days postinjection for control, G-O-F and L-O-F. Data presented as mean ± s.d., P values by one-way ANOVA with Tukey post test; n = 5–6 mice. e, Quantification of absolute TC number per lung 14 days postinjection for control and L-O-F. Data presented as mean ± s.d., P value by two-tailed t-test; n = 6 mice. f, Schematic of experiment. Gene recombination was induced by tamoxifen administration: 2 × 105 E0771-GFP cells were injected into the tail vein of EC-specific knockout (iECKO) and control animals. Gray bar indicates time span (in days) of daily tamoxifen treatment (left). Total number of TCs per mg lung tissue of control and iECKO mice 2 weeks postinjection of E0771-GFP (right). Data presented as mean ± s.d., P value by two-tailed t-test; n = 9 mice. g, Left, schematic of the experiment. 1 × 106 4T1-GFP cells were implanted into the mammary fat pad of NOD-SCID mice. Once tumors had reached a size of 100 mm³, mice were treated with LGK974 for 5 days until tumor resection. Following resection, mice were treated for an additional 2 days and left to develop metastases. Gray bar indicates time span (in days) of daily treatment with LGK974. Weights of resected primary tumors (middle) and total number of TCs per mg lung tissue of control and LGK974-treated mice 2 weeks postresection (right). Data presented as mean ± s.d., P value by two-tailed t-test; n = 12–14 mice.