Multiomic spatial atlas shows deleted in malignant brain tumors 1 (DMBT1) glycoprotein is lost in colonic dysplasia

Abstract

Colorectal cancer (CRC) is responsible for over 900,000 annual deaths worldwide. Emerging evidence supports pro-carcinogenic bacteria in the colonic microbiome are at least promotional in CRC development and may be causal. We previously showed toxigenic C. difficile from human CRC-associated bacterial biofilms accelerates tumorigenesis in Apc^Min/+ mice, both in specific pathogen-free mice and in gnotobiotic mice colonized with a defined consortium of bacteria. To further understand host-microbe interactions during colonic tumorigenesis, we combined single-cell RNA-sequencing (scRNA-seq), spatial transcriptomics, and immunofluorescence to define the molecular spatial organization of colonic dysplasia in our consortium model with or without C. difficile. Our data show a striking bipartite regulation of Deleted in Malignant Brain Tumors 1 (DMBT1) in the inflamed versus dysplastic colon. From scRNA-seq, differential gene expression analysis of normal absorptive colonocytes at 2 weeks postinoculation showed DMBT1 upregulated by C. difficile compared to colonocytes from mice without C. difficile exposure. In contrast, our spatial transcriptomic analysis showed DMBT1 dramatically downregulated in dysplastic foci compared with normal-adjacent tissue. We further integrated our datasets to generate custom colonic dysplasia scores and ligand-receptor mapping. Validation with immunofluorescence showed DMBT1 protein downregulated in dysplastic foci from three mouse models of colonic tumorigenesis and in adenomatous dysplasia from human samples. Finally, we used mouse and human organoids to implicate WNT signaling in the downregulation of DMBT1 mRNA and protein. Together, our data reveal cell type-specific regulation of DMBT1, a potential mechanistic link between bacteria and colonic tumorigenesis. Published 2025. This article is a U.S. Government work and is in the public domain in the USA. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: colorectal; colorectal adenocarcinoma; microbiome; tumorigenesis.

Figure 1

Cell type identification and analysis of scRNA‐seq from colonic epithelial cells in C. difficile‐colonised Apc ^Min/+ mice demonstrated unique cell states. (A) Schematic of experimental design: 8‐week‐old germ‐free Apc ^Min/+ mice were gavaged with C. difficile‐containing bacterial consortium or control consortium as previously published [13], and mice were euthanized at 10 weeks. (B) Epithelial crypts were dissociated from the colonic tissue for scRNA‐seq; n = 3 mice/group. Spatial transcriptomics was performed on formalin‐fixed, paraffin‐embedded whole colon; separate experiment with n = 3 mice/group. (C) UMAP plot with cell type assignments for clusters based on canonical mouse gene markers (supplementary material, Figure S2). (D–F) UMAP plots colored by inoculation (C. difficile or control), mouse, or cell cycle phase. (G–K) UMAP plots colored by scaled gene expression (a.u. = arbitrary units) of canonical gene markers for different cell states: fetal reversion (Marcksl1), autophagy (Sqstm1), senescence (Cdkn1), intestinal plasticity (Prom1), and regenerative stemness (Anxa1).

Figure 2

Differential gene expression analysis of absorptive colonocytes from scRNA‐seq indicated increased immune activity and upregulation of Dmbt1 expression in C. difficile‐inoculated mice. (A) Violin plot of Dmbt1 expression from C. difficile‐inoculated mice versus control within each merged cell type, scaled expression, arbitrary units (a.u.). (B) Volcano plot of differentially expressed genes from absorptive cells based on inoculation. Labeled genes were among the most differentially expressed. (C) We used computational gene set enrichment analysis to determine whether the predefined set of genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed statistically significant concordance in absorptive cells. Listed pathways contain genes that were differentially upregulated in C. difficile‐inoculated versus control mice; FDR = false discovery rate. (D) Gene enrichment plot of absorptive cells for the KEGG cell adhesion molecules pathway showed the degree to which upregulated genes correlated with C. difficile‐inoculation (Pos) compared to control (Neg) and the ranked metric quantification of the signal‐to‐noise ratio. NES = normalized enrichment score.

Figure 3

Spatial transcriptomic analysis of C. difficile‐inoculated mice versus controls. (A and B) Dimensionality reduction with UMAP demonstrated segregation based on gene expression for all segments. Each point represents an individual segment, and the shape of each point represents the tissue in which the segment was chosen. The color of the point signifies the mouse of origin (A) or the inoculation received (B). (C and D) Volcano plots of differential gene expression between normal and dysplastic tissue for (C) lamina propria and (D) epithelium; top genes with high absolute log₂ fold‐changes are labeled. (E and F) Volcano plots of differential gene expression compared segments from (E) lamina propria and (F) epithelium of control mice versus C. difficile‐colonized mice.

Figure 4

Integration of spatial and single‐cell transcriptomics revealed potential pathways to C. difficile‐associated dysplasia. (A) UMAP plot of scRNA‐seq dataset representing the 12‐gene dysplasia score (a.u. = arbitrary units) generated from the spatial transcriptomics dataset. (B) Violin plot showing dysplasia score distribution for each inoculation and cell type. (C) Scatterplot and Spearman correlation between scaled Dmbt1 gene expression and the dysplasia score for all epithelial cells from scRNA‐seq data. Shading around the trendlines indicates 95% confidence intervals. C. difficile Spearman = −0.3415, p‐value = 1.958e‐113; Control Spearman = −0.3003, p‐value = 3.155e‐90. (D) Heatmap showing scRNA‐seq expression for the 12 genes used to create the dysplasia score. Gastric precancer genes are shown with relatively no expression. (E and F) Heatmaps displaying the total number of significant ligand‐receptor interactions inferred by CellPhoneDB. (G) Venn diagrams illustrating the intersection of C. difficile‐specific interaction partners from scRNA‐seq and spatial transcriptomics datasets. (H) Chord diagrams illustrating the predicted interactions with Sema7a by cell type in C. difficile‐colonized mice and control mice. No significant Sema7a interactions were predicted in control mice.

Figure 5

Dmbt1 expression is downregulated in multiple mouse models of colonic tumorigenesis. (A) Volcano plot showing Dmbt1 among the most differentially regulated genes expressed by differentiated colonocytes from mice given protumorigenic slurry (magenta) versus nontumorigenic slurry (blue). Genes with the highest absolute log₂ fold‐changes are labeled (supplementary material, Table S11). (B) Dmbt1 normalized gene expression from spatial transcriptomics demonstrated upregulation in epithelial cells from C. difficile‐inoculated mice but downregulation in dysplastic foci. Points are segments with bar and whiskers as mean and standard error of the mean. Asterisks (*) indicate a significant difference (p < 0.05 from Wilcoxon rank sum test with continuity correction) (supplementary material, Table S11). (C) Immunofluorescence microscopy shows Dmbt1 protein expression is reduced in dysplastic foci, as indicated by altered β‐catenin distribution (decreased membranous, increased cytoplasmic and nuclear localization) compared to adjacent normal tissue in three different mouse models of colonic neoplasia. Dmbt1 was reduced in 100% of dysplastic foci (n = 57) from 11 mice. (D) Organoid formation assay using Lrig1 ^CreER/+ ;Apc ^fl/fl organoids treated with 1 μm 4‐hydroxytamoxifen (4OH‐Tam) versus vehicle control at 7 days posttreatment, shown using phase contrast microscopy. (E) Cell viability based on luminescence (RLU = relative light units). (F) RT‐qPCR analysis of Dmbt1 expression in 4OH‐Tam‐treated Lrig1 ^CreER/+ ;Apc ^fl/fl organoids compared to vehicle control where relative quantification (RQ) is 2^−ΔΔCt, and Ct is the cycle threshold. Expression is quantified relative to the reference transcript Actb. These plots represent averages from 43 independent wells of organoids from two different animals. (G) Immunoblotting analysis of vehicle control versus 4OH‐Tam‐treated Lrig1 ^CreER/+ ;Apc ^fl/fl organoids.

Figure 6

DMBT1 expression was decreased in human colonic dysplasia. (A) UMAP plot of scRNA‐seq data from human colonic precancerous polyps with coloring indicating cell types, EEC = enteroendocrine. (B) Violin plot showing DMBT1 scaled expression (a.u. = arbitrary units) in different cell types. Asterisks (*) indicate significant difference from all other cell types (p < 0.05 Kruskal–Wallis with Dunn's test, supplementary material, Table S12). (C) Heatmap of DMBT1 mean expression alongside canonical WNT signaling target genes indicated an inverse relationship between WNT activity and DMBT1 expression. (D) DMBT1 immunostaining in well‐differentiated, low‐grade dysplastic crypts (arrowhead) adjacent to high‐grade dysplasia (arrow) within a human colon adenocarcinoma. (E) Quantification of DMBT1 immunofluorescence staining in a human tissue microarray with specimens spanning the progression from normal adjacent tissue to metastatic colorectal cancer. The average fluorescence (raw integrated density) for 3 or 4 tissue cores per patient sample is shown as a single point; n = 3–15 patients/group (*p < 0.05, **p < 0.01 from Kruskal–Wallis with Dunn's test). Blue lines are means, and whiskers are standard error of the mean. (F) RT‐qPCR analysis performed on normal human colonic organoids. Each point represents the mean of three technical replicates from seven independent wells pooled together. NKD1 and AXIN2 are canonical WNT signaling target genes. Relative quantification is equivalent to 2^−ΔΔCt, and Ct is the cycle threshold. Expression is quantified relative to the reference transcript ACTB, normalized to vehicle control, and scaled to min–max of 0 to 1 (*p < 0.05 from Wilcoxon Rank Sum Test). CHIR = CHIR99021.