Paneth cell–derived growth factors support tumorigenesis in the small intestine

Representative images of small intestine and colon adenomas of 20-wk-old Apc^Min mice. (A) Immunofluorescence staining for Lysozyme (red) and the lineage tracer, membrane-targeted GFP (green), in normal small intestine epithelium of Defa4-Cre, mTmG reporter mice. Scale bar, 100 μm. (B) Immunofluorescence staining for membrane-targeted GFP (green) in intestinal adenomas of Defa4-Cre, mTmG reporter mice carrying the Apc^Min allele. Scale bar, 100 μm.

(A) Diagram depicting the genetic strategy used to generate intestinal PC depletion (PC^del: Defa4-Cre, Rosa26^iDTA/WT) or control animals (WT: Rosa26^iDTA/WT). (B) Representative H&E staining images of small intestinal mucosa in wild-type (WT) and PC^del mice. Insets to highlight the crypt base where PCs reside (cells with large granules) are also shown. Scale bar, 100 μm. (C) Immunofluorescence staining for Lysozyme (red) and Lectin. (green). Representative images of small intestinal mucosa of WT and PCdel are shown. Scale bar, 100 μm.

(A, B, D, E) Intestinal tumor burden in 20-wk-old Apc^Min mice carrying the PC^del genotype or littermate WT control: small intestine (SI) adenoma count (A), SI adenoma size (B), colon adenoma count (D) and colon adenoma size (E) were analyzed. Data represent the aggregate of three independent experiments; Apc^Min, PC^del (n = 20), and Apc^Min, WT (n = 14) animals. Mean and SEM are graphed, each dot represents an individual animal. ***P < 0.001 (unpaired t test). (C, F) Representative H&E images of adenoma burden in the entire SI (C) and colon (F). Yellow arrows mark intestinal adenomas. Scale bar, 5 mm. (G, H) Expression in SI adenomas of PC-specific genes, Defa (G) and Lyz1 (H) was determined by RT-qPCR analysis (n = 6 per each genotype). Mean and SEM are graphed, each dot represents an individual animal. ***P < 0.001 (unpaired t test).

Relative expression of Wnt genes in small intestinal adenomas. (A, B) WT and PC^del mice at 16–20 wk were euthanized and stool (WT n = 9, PC^del n = 9), ileal content (WT n = 10, PC^del n = 8), and ileal mucosa (WT n = 10, PC^del n = 8) samples were collected for 16S microbiota analysis. (A, B) PCoA β-diversity plots (A) and phyla-level composition (B) are shown. No statistically significant differences were noted between WT and PC^del groups. (C) Heatmap presentation of top differentially expressed genes from RNA-seq of small intestine adenoma tissues from Apc^Min, PC^del, and Apc^Min, WT mice. (D) Wnt3 gene expression in small intestine adenomas by RT-qPCR analysis (n = 6 per each genotype). *P < 0.05 (unpaired t test).

(A, B, D, E) PC-specific Wnt3 knockout mice Apc^Min PC-Wnt3^−/− (n = 9) and littermate Apc^Min PC-Wnt3^+/− (n = 9) and Apc^Min (n = 5) control mice were euthanized at 20 wk of age to determine tumor burden including small intestine (SI) adenoma count (A), SI adenoma size (B), colon adenoma count (D), and colon adenoma size (E). Results represent the aggregate of two independent experiments. *P < 0.05, **P < 0.01 (one-way ANOVA analysis). (C) Representative H&E images of SI adenoma burden. Yellow arrows mark intestinal adenomas. Scale bar, 5 mm. (F) PC morphology was evaluated by immunofluorescence staining for lysozyme (red) and lectin (green) in normal SI of Apc^Min PC-Wnt3^−/− mice and control to Apc^Min PC-Wnt3^+/− Scale bar, 100 μm.