Fig. 10: Pro-tumorigenic role of EREG secreted from InfFib.
From: Hypoxia-induced Wnt5a-secreting fibroblasts promote colon cancer progression
a CACO-2 cells were cultured in CM prepared from WEHI-YH2 cells under 20% or 1% O2 conditions. Proliferative abilities are presented as mean ± SD. (n = 4; Unpaired t-test, Two-tailed). b, c Expression levels of EGF ligand family across fibroblast subtypes in mouse (b) and human (c) single-cell RNA-seq data related to Fig. 4d or 3g, respectively, are shown in dot plots. Dot size indicates the proportion of expressing cells, colored by mean expression values in each cell type. d UMAP plot of cells from mouse colon cancer models related to Fig. 2b for Ereg. e Expression levels of Ereg between control and Wnt5a cKO-derived AOM/DSS-induced tumors are shown in a bar plot. mRNA expression values were obtained from bulk RNA-seq data used in Fig. 5d. (n = 4; Unpaired t-test, Two-tailed). f WEHI-YH2 cells were exposed to 1% O2 or CoCl2 for 48 h, or overexpressed with HIF2α. The mRNA levels of Ereg are represented in bar plots. (n = 3; Unpaired t-test, Two-tailed). g CACO-2 cells were cultured in CM prepared from WEHI-YH2 cells expressing EREG. Proliferative abilities are presented as mean ± SD. (n = 4; Unpaired t-test, Two-tailed). h Organoids established from AOM/DSS-derived tumors were treated with rEREG or rEGF and cultured for 6 days. Representative phase-contrast images are shown. Scale bars, 500 μm. i Organoids established from AOM/DSS-derived tumors treated with or without rEREG were exposed to 20% or 1% O2 for 48 h. The left panels are phase-contrast images. Mean area of organoids in each condition was measured for three experiments, and the results are represented in a bar plot. Scale bars, 500 μm. (n = 3; One-way ANOVA, Holm-Sidak’s multiple comparisons test). j Differentially expressed genes between control and Wnt5a cKO-derived AOM/DSS-induced tumors were subjected to pathway activity inference using decoupleR. mRNA expression values were obtained from bulk RNA-seq data used in Fig. 5d. k Organoids established from AOM/DSS-derived tumors were treated with 200 ng/mL rEREG for 2 days and EdU. Organoids were stained with indicated antibodies and DAPI, and representative images are shown. Scale bars, 20 μm. l Scatter plots showing the correlation between signature scores of MAPK (x-axis) and WNT5A gene expression levels (y-axis) were obtained from TCGA-COAD patient dataset. A regression line was fitted using linear regression, and the shaded area represents the 95% confidence interval. The Spearman R- and P-values are shown on the top. m Violin plots showing the mRNA expression levels of indicated genes among CMS subtypes in TCGA-COAD patient dataset. The width of each violin indicates the kernel density estimation of the data distribution. The central white dot represents the median, the thick bar in the center corresponds to the interquartile range (IQR), and the thin line extending from the bar denotes the data range within 1.5 times the IQR. (CMS1, n = 65; CMS2, n = 133; CMS3, n = 51; CMS4, n = 95; One-way ANOVA, Holm-Sidak’s multiple comparisons test). n Graphical abstract of this study. Created in BioRender87. ***, P < 0.001; **, P < 0.01; *, P < 0.05; Source data are provided as a Source Data file.
