Fig 2: DKK3 is upregulated in the stroma of breast, colon and ovarian cancers. a Tukey boxplots showing z-score values of DKK3 mRNA expression in normal and cancerous stroma from breast, colorectal and ovarian cancers (Breast: normal, n = 6; cancer, n = 53. Colon: normal, n = 4; cancer, n = 13. Ovary: normal, n = 8; cancer, n = 31). b Representative images of DKK3 staining in breast, colorectal and ovarian cancers and normal tissues. Scale bar, 100 µm. c Tukey boxplots showing quantification of DKK3 staining (Histoscore) in breast, colorectal and ovarian cancers and normal tissue counterparts (Breast: normal/adjacent, n = 9; cancer, n = 109. Colon: normal/adjacent, n = 14; cancer, n = 107. Ovary: normal/adjacent, n = 8; cancer, n = 138). d Tukey boxplots showing DKK3 Histoscore in non-invasive breast cancers (Stage 1&2), invasive breast-cancers (Stage 3&4) and normal tissue counterparts. Left graph shows all cancers irrespective of their subtype (normal, n = 9; Stage 1&2, n = 74; Stage 3&4, n = 74). Middle graph shows ER-negative breast cancers (normal, n = 9; Stage 1&2, n = 40; Stage 3&4, n = 40). Right graphs shows ER-positive breast cancers (normal, n = 9; Stage 1&2, n = 21; Stage 3&4, n = 21). e Disease-free survival of breast cancer patients stratified on stromal DKK3 gene expression (GSE9014, ER-negative patients). f Images show DKK3 (green), vimentin (VIM; red) and DAPI (blue) staining of two representative human breast cancer tissues. Scale bar, 50 µm. g Tukey boxplot shows Dkk3 mRNA expression levels (relative to Gapdh) in Cancer cells (Epcam+), immune cells (Cd45+), endothelial cells (Cd31+) and fibroblasts (Pdgfra+) from MMTV-PyMT tumorus (n = 4). h Graphs show correlations between the expression of DKK3 and ACTA2, FAP and COL1A2 in normal and cancerous stroma from mammary gland (GSE9014), colorectal (GSE35602) and ovarian (GSE40595) human tissues. Pearson correlation coefficient (r) is shown. Each dot represents z-score values from individual patients. i Kaplan–Meier curves of recurrence-free survival, disease-specific survival and progression-free survival of ER-negative breast cancer, colorectal cancer and ovarian cancer patients, respectively, based on DKK3 and DKK2 gene expression. Where indicated, individual p values are shown; alternatively the following symbols were used to describe statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001; n.s., non-significant

Fig 3: Identification of CD49f (Itga6) as a differentially expressed cell surface marker in OHBBN-induced bladder tumors.a Bulk RNA-seq analysis showing differentially expressed genes belonging to various lineage subtypes in the normal bladder (n = 5) and OHBNN induced bladder cancer (n = 5). The surface antigen, CD49f (Itga6, arrows), is overexpressed in bladder tumors and found within the basal mRNA cluster. b tSNE cell clusters 3, 5, 8, and 11 defining Epcam (left) and CD49f (Itga6) expression. c Strategy for discriminating GFP-positive, CD45-negative OHBBN-induced cancer cells from non-tumor host cells in FVB/NJ-transplanted mice. Tumor cells are evaluated for expression of Epcam and CD49f, and define lineage populations within defined quadrants (Q1, Q2, Q3, and Q4). d Differential gene expression for lineage subtypes using Epcam-CD49f expression in cell populations of GFP-positive;CD45-negative tumor cells sorted from quadrants Q1, Q2, Q3, and Q4. Individual data points are represented by circles, the error bars represent mean ± SD of the three independent replicates. Each experiment was performed at least in triplicate.

Fig 4: Characterisation of pulmonary pericytes during alveologenesis. a, b Three-dimensional reconstruction (a) and high magnification thin optical section (b) of confocal images with Airyscan detection showing AQP5-stained type 1 alveolar epithelial cells (green), PDGFRß-stained pulmonary pericytes (PCs) (red) and PECAM1-stained endothelial cells (ECs) (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets (a) or single channels (b). Scale bar, 30 µm (a, left), 15 µm (a, right), 10 µm (b, left) and 5 µm (b, right panels). c, d Three-dimensional reconstruction (c) and thin optical section (d) of confocal images showing SFTPC-stained, cuboidal AT2 cells (green), PDGFRß-stained PCs (red) and RAGE-stained AT1 cells (blue) in lung at 4 weeks. Panels on the right show higher magnification of corresponding insets. Scale bar, 20 µm (left), 10 µm (right). e Quantitation of EC to PC ratio as measured in peripheral lung sections at different developmental stages. Data represents mean ± s.e.m. (n = 5 for P7, n = 6 for P21 and Adult mice). f Scheme showing the time points of tamoxifen administration (P1–3) and analysis for the Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. g–k High magnification images of Pdgfrb(BAC)-CreERT2 R26-mT/mG lung sections at indicated stages showing pulmonary GFP+ PCs (green), PECAM1+ ECs, PDGFRß+ PCs, PDGFRa+ fibroblasts or aSMA+ bronchial smooth muscle cells/myofibroblasts (red, as indicated). Arrows in g indicate PC cell bodies, in g–k GFP-positive PDGFRß+ cells (g, h), GFP-negative PDGFRa+ cells (i) or aSMA+ cells (j, k) are marked. Scale bar, 20 µm (g, h), 15 µm (i, k) and 30 µm (j). l RT-qPCR analysis of Pdgfrb (pericytes), Pecam1 (endothelium), and Sftpc (epithelium) expression in freshly sorted GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG lung. Data represents mean ± s.e.m. (n = 4 mice)

Fig 5: No apparent change in thymus development in ß-cat loss-of-function (LOF) embryos.(A) Thoracic cavity of control mice and ß-cat LOF mice at E15.5. Dotted lines show the outline of the thymic primordium. Representative data from three independent experiments are shown. TH: thymus, H: heart. Bar: 1 mm. (B) Immunofluorescence staining for ß-catenin and Foxn1 (left) or K5 and K8 (right) on sagittal sections of the thymus from control mice and ß-cat LOF mice at E15.5. Shown are merged images with nuclear counterstaining (TO-PRO3) (top) and images obtained in each channel (middle, bottom). Representative data from three independent experiments are shown. Bar: 100 µm. (C) Intracellular staining of ß-catenin in CD45-EpCAM+ thymic epithelial cells (TECs) from control mice and ß-cat LOF mice at E15.5. Histograms show ß-catenin expression in control TECs (blue line) and ß-cat LOF TECs (red line). Shaded area and black line represent the fluorescence in the absence of anti-ß-catenin antibody in control TECs and ß-cat LOF TECs, respectively. Plots show net median fluorescence intensity (MFI) values for ß-catenin (means and standard error of the means [SEMs], n = 4). The numbers in parentheses indicate percentage of control value. (D) Flow cytometric analysis of enzyme-digested thymic cells from indicated mice at E15.5. Shown are profiles of EpCAM and CD45 expression in PI- viable cells (left) and UEA1 reactivity and Ly51 expression in CD45-EpCAM+ cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. (E) Plots show the number of total thymic cells (left) and the frequency and the number of total TECs (middle) and cortical thymic epithelial cell (cTECs; right) from control mice and ß-cat LOF mice at E15.5 (means and SEMs, n = 4). **p < 0.01; N.S., not significant.