Extended Data Fig. 4: Niche-derived Wnt-ligands induce extravasation and latency in experimental metastasis models.
From: Lung endothelium exploits susceptible tumor cell states to instruct metastatic latency
a-c, Representative FACS gates for quantifications of extravasated and latent tumor cells. a, Gating strategy for discriminating extravascular from intravascular tumor cells are shown for a mouse injected with 4T1 control cells (mouse 1, dark blue line) and a mouse injected with 4T1 G-O-F cells (mouse 2, orange line). Gates were set for the control group so that all EC fall into the intravascular gate (upper left panel). The quality of the gating was assessed for each individual sample (upper right panel) and adjusted if grave differences were observed. Samples were excluded if cells were insufficiently stained. EC gates were pasted to tumor cells (lower panels) and the percentage of stained (intravascular) and unstained (extravascular) cells was measured. Lower panels show analysed control TC histograms (left panel), G-O-F TC histograms (mid panel), and the overlay (right panel). b, Gating strategy to quantify latent TC in G-O-F experiments. For each treatment group, one additional mouse was included in the experiment that was euthanized at day 1.5 and served as latent control. Latent gates were set in the respective day 1.5 control samples in a way that all TC fall into the latent gate. Gates were pasted to day 3.5 samples and percentage of latent TC was measured. Left panel shows histogram of day 1.5 TC (red line), mid panel show histogram of day 3.5 TC (blue line) and right panel shows overlay of day 1.5 and day 3.5 histograms for control treated (upper panels) and G-O-F tumor cells (lower panels). c, Representative gating for L-O-F experiment. The gating occurred in principle as outlined above, with the difference that only one day 1.5 control was included, as the same TC were injected for both groups. Left panel displays overlay of day 1.5 control histogram (blue line) and day 3.5 TC extracted from control treated mouse (grey line). Mid panel shows overlay of day 1.5 control histogram (blue line) and day 3.5 TC extracted from LGK974 treated mouse (red line). Right panel shows overlay of day 3.5 TC extracted from control (red line) and LGK974 treated mouse (grey line). d, Percentage of TC with dye retention 4 days postinjection of 4T1-GFP control and Wnt-agonist CHIR99021 long-term treated cells. n = 5-6 mice. e, Percentage of extravasated TC 1.5 days postinjection of 4T1-GFP control and Wnt-agonist SKL2001 long-term treated cells and f, Percentage of TC with dye retention at day 3.5. n = 4-5 mice. g, Relative tumor cell abundance in lungs of Ctrl treated and LGK974 treated mice 2.5 days postinjection of 4T1-GFP tumor cells. Relative tumor cell abundance was calculated as percentage of lung EC, which served as nonproliferative intra-sample control. n = 6 mice. h, Representative images and quantifications of single disseminated tumor cells (sDTC) and tumor cell clusters in mice 3.5 days postinjection for Ctrl (left panels), Wnt L-O-F (mid panels) and Wnt G-O-F (SKL2001) (right panels). For all images stacks of 12 µm were acquired and z-projections using maximum intensity are displayed. All images were acquired using 40X magnification. Panels display overlays of DAPI (cell nuclei, blue) and GFP (TC, green). Upper panels show sDTC and lower panels display cell cluster. Quantification shows cell cluster to sDTC ratios, which were calculated for each lung by summing cell clusters and sDTC across all analysed sections. Quantification to the right displays ratios for each analysed slide (sum of two sections) per group. n = 3 mice. i, Schematic of experiment. j, Percentage of extravasated TC 1.5 days postinjection of D2A1-tom control and Wnt-agonist CHIR99021 long-term treated cells. n = 6 mice. k, Percentage of TC with dye retention at day 3.5 and l, metastatic outgrowth calculated as fold change of tumor cell abundance compared to the mean tumor cell abundance for each treatment group at day 1.5. n = 5-6 mice. m, Percentage of extravasated TC 1.5 days postinjection of D2A1-tom in Ctrl and LGK974 treated mice. n = 6 mice. n, Tumor cell abundance in lungs of Ctrl treated and LGK974 treated mice 3.5 days postinjection of D2A1-tom cells, and o, metastatic outgrowth of D2A1-tom cells in Ctrl or LGK974 treated mice. n = 5-6 mice. p, Relative expression of key EMT-associated genes Zeb1, Snai1, Twist1, Klf4, Vim, Cdh2, Cdh1 and canonical Wnt-downstream target Axin2 in cultured 4T1 cells or D2A1 cells. Target gene expression was normalized to Actb and relative expression to 4T1 cells was calculated using the 2-ΔΔCt-method. For genes without detectable expression in 4T1 cells 1/ΔCt is displayed. d-p Data are presented as mean values +/− s.d., p value by two-tailed t-test are shown. n.s., not significant. * p value < 0.05, ** p value < 0.01, *** p value < 0.001, **** p value < 0.0001. n = 3-4 independent experiments.
