Deletion of MyD88 in Intestinal Mesenchymal Cells Reduces Tumorigenesis in the Apc^min/+ Model of Sporadic Intestinal Cancer
(A and B) Total number of tumors per mouse (A) and number of tumors per intestinal part (B) in 4-month-old Apc^min/+Myd88^IMCko mice (n = 15) and their littermate controls (n = 18). The insert shows the number of colonic tumors.
(C and D) Size of small intestinal tumors presented as mean tumor size (C) and distribution of tumors per size (D) in the two genotypes.
(E) Representative BrdU and Tunel staining in small intestinal tumors of Apc^min/+Myd88^IMCko mice and their littermate controls. DAPI was used to stain the nuclei in the Tunel stainings.
(F and G) Quantification of the number of BrdU⁺ cells per tumor (F) and Tunel⁺ cells (G) per field in equal-sized tumors (n = 6 mice per genotype).
(H) Number of dysplastic lesions per mouse in 6-week-old Apc^min/+Myd88^IMCko and their littermate controls (n = 6–7 mice per genotype).
(I) Number of tumors per mouse in Myd88^IMCko mice (n = 8) and their littermate controls (n = 7) at the end of the AOM/DSS protocol (one representative experiment of four performed).
Data represent mean ± SEM. ^∗∗p < 0.01, ^∗∗∗p < 0.001, ns = not significant. Scale bars represent 50 μm. See also and .

Tumors from Mice with Deletion of MyD88 in IMCs Show Differential Gene Expression, Reduced STAT3 Activation, and Altered Inflammatory Cell Infiltration
(A) Gene expression analysis in the small intestine of Myd88^F/F and Myd88^IMCko mice (n = 3) and in tumors from Apc^min/+Myd88^F/F and Apc^min/+Myd88^IMCko mice (n = 6). Hprt was used for normalization.
(B) Representative immunohistochemical staining for pSTAT3 in the normal small intestine and tumors of Apc^min/+Myd88^F/F and Apc^min/+Myd88^IMCko mice (n = 5 mice per genotype).
(C) Quantification of pSTAT3 staining in tumors from Apc^min/+Myd88^F/F and Apc^min/+Myd88^IMCko mice (n = 12–14 tumors from 5 mice per genotype).
(D–F) Infiltration of CD45⁺ cells (D), CD11b⁺F4/80⁺ macrophages and CD11b⁺Gr1⁺ neutrophils (E), and CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and CD11c⁺ dendritic cells (F) in tumors from 4- to 5-month-old Apc^min/+Myd88^F/F and Apc^min/+Myd88^IMCko mice (n = 4–7), quantified by FACS analysis (from two independent experiments).
(G) Representative confocal images from tumors of Apc^min/+-ColVIcre-Myd88^F/+-mTmG and Apc^min/+-ColVIcre-Myd88^F/F-mTmG mice (n = 3). Scale bars represent 50 μm.
(H) FACS analysis of tumors from Apc^min/+-ColVIcre-mTmG mice, stained with markers for epithelial (EpCAM), immune (CD45), and endothelial (CD31) cells (n = 3 mice). Unstained controls are presented as gray histograms.
Data represent mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ns = not significant. See also .

Deletion of TLR4 in IMCs Reduces Tumorigenesis in the Apc^min/+ Model of Sporadic Intestinal Cancer
(A) IL-6 quantification by ELISA in the supernatants of IMCs stimulated for 24 h with TLRs and interleukins, as described in . One representative of three independent experiments performed in triplicates is presented.
(B) Heatmap of the differential expression of the indicated genes in CAFs versus cancer cells and leukocytes from the dataset GEO: GSE39395. Log2 fold-change of genes is shown. Red denotes high expression values, and blue denotes low expression values.
(C and D) Total number (C) and size (D) of tumors per mouse in 4-month-old Apc^min/+Il1r1^IMCko mice (n = 14) and their littermate controls (n = 24).
(E and F) Total number of tumors per mouse (E) and number of tumors per intestinal part (F) in 4-month-old Apc^min/+Tlr4^IMCko mice (n = 13) and their littermate controls (n = 20). The insert shows the number of tumors in the colon.
(G and H) Mean tumor size (G) and distribution of tumors (H) per size in the two genotypes.
(I) Representative BrdU and pSTAT3 staining in small intestinal tumors of Apc^min/+Tlr4^IMCko mice and their littermate controls. Scale bar = 50 μm.
(J) Quantification of the number of BrdU⁺ cells per tumor (n = 6 mice per genotype).
(K) Mean signal intensity (MSI) of pSTAT3 (n = 12–14 tumors from 4 mice per genotype).
(L) Gene expression analysis in tumors from Apc^min/+Tlr4^F/F and Apc^min/+Tlr4^IMCko mice (n = 6). Hprt was used for normalization.
(M) Number of tumors per mouse in Tlr4^IMCko mice (n = 8) and their littermate controls (n = 8) at the end of the AOM/DSS protocol (one representative experiment of three performed).
Data represent mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ns = not significant. See also .

TLR4/MyD88 Signaling Induces a Pro-Tumorigenic Gene Signature in IMCs
(A) Gene Ontology (GO) terms enriched in differentially expressed genes between untreated control and MyD88 knockout IMCs.
(B) Venn diagram showing the number of overlapping differentially regulated genes between untreated and LPS-treated control and MyD88 knockout IMCs.
(C) GO terms enriched in MyD88-regulated genes indicated as bold in (B).
(D) Heatmap of differentially expressed genes between control and LPS-induced WT and MyD88 knockout IMCs that belong to the GO terms shown in (C). Log2 transformed normalized read counts of genes are shown. Read counts are scaled per column. Red denotes high expression values, and blue denotes low expression values.
(E and F) Venn diagram showing the number of overlapping genes (E) and heatmap of their expression between the IMC-specific MyD88-regulated gene signature and genes overexpressed in CAFs versus cancer cells from the datasets GEO: GSE39395 and GSE35602 (F). Log2 fold-change of genes is shown.
See also .