Fig. 6
Activation of α-defensin expression during the development of the small intestinal and colonic adenomas. A Presence of Defa6-tdTom⁺ cells (red signal) in small intestinal adenomas of Apc+/Min mice. These cells are mislocalized from the crypts but mostly retain lysozyme positivity (green signal). Scale bar: 100 µm." B The appearance of Defa6-tdTom cells is associated with tumorigenesis in the colon. Representative fluorescence microscopy images of cells stained with an antibody against PCNA (green signal) and tdTomato (red signal). Note that the red fluorescent signal was observed either in mice treated with the mutagen azozymethane (AOM) in combination with colitis-inducing sodium dextran sulfate (DSS) or in the colon tumor developed in Apc+/Min mice. In contrast, "red" cells are present neither in the healthy colonic epithelium (wt) nor in the epithelium 8 days after the DSS treatment, i.e., without mutagen. The red glandular structures are formed 6 and 9 weeks after AOM/DSS treatment and in tumors of the Apc+/Min mice (white arrowheads). Scale bar: 150 µm. C Single-cell RNA-seq analysis of epithelial cells from dissected colon tumors in Apc+/Min mice. Cell clusters are numbered according to the cell abundance; UMAP visualizations show all epithelial tumor cells (top) and Defa6-tdTom cells (bottom). The top right diagram is an overlay of the UMAP clusters with arrows representing the lineage relationships between the cell types (RNA velocity). For better visibility, the diagram has been expanded spatially and cluster 9 (immune cells) is not shown. A complete list of cluster marker genes can be found in Additional file 6: Supplementary Table S6. D Left, the expression of marker genes in different cell clusters is shown in the violin plots, with the level of gene expression indicated on the y-axis. Genes with increased expression in Defa6-tdTom tumor cells are shown in green. Right, analysis of the Wnt signaling pathway components in individual cell clusters. Each violin plot represents the mean expression level for the Wnt signaling target genes, the genes encoding the Wnt signaling nuclear mediators Tcf/Lef or genes activated upon the Hippo pathway inhibition. Expression in all epithelial tumor cells is shown in gray, Defa6-tdTom tumor cells are shown in red. E Heatmap showing scaled expression of indicated genes in sorted Defa6-tdTom tumor cells; cluster numbers were obtained from the combined analysis of total tumor cells and Defa6-iCre-labeled cells. Expression levels are color-coded, with yellow indicating high expression. F Representative microscopy images of cells stained with an antibody against Tacst2 and tdTomato. Note the Tacstd2-positive cells in the red glandular structure (white arrows). Scale bar: 150 µm