Figure 2 from Human Colon Cancer–Derived <i>Clostridioides difficile</i> Strains Drive Colonic Tumorigenesis in Mice

Abstract

<p><i>C. difficile</i> and the 3728T slurry induce rapid tumorigenesis in GF <i>Apc<sup>Min/+</sup></i> mice despite representing only a fraction of the luminal and mucosal microbiota. <b>A</b> and <b>B,</b> Twenty-five GF <i>Apc<sup>Min/+</sup></i> mice were gavaged with the 3728T slurry; five mice were sacrificed per time point. <b>A,</b> Colonic microadenoma and adenoma counts. <b>B,</b> Hematoxylin and eosin (H&E) staining of a representative microadenoma is shown from a mouse at 2 weeks p.i. (top), whereas a large adenoma coated by a dense BF is shown from a mouse at 4 weeks p.i. (bottom). Confocal imaging of Carnoy's-fixed mouse distal colons stained with the EUB338 universal probe (magenta), Cd198 <i>C. difficile</i> probe (yellow), or DAPI counterstain (blue). White arrows highlight individual <i>C. difficile</i> rods. <b>C</b> and <b>D</b>, Microadenomas (<b>C</b>) and FISH staining (<b>D</b>) of 2 weeks p.i. distal colons from GF <i>Apc<sup>Min/+</sup></i> mice gavaged with the 3728T isolates ± <i>C. difficile</i> CIm_3728T. Crosses represent mouse deaths. <i>n</i> = 6 mice per group, from two independent experiments. FISH images represent three separate mice per inoculum. White arrowheads denote areas with occasional mucus-invasive BFs directly interacting with epithelium in mice gavaged with the 3728T isolates without <i>C. difficile</i> (3728T isos – Cd). White arrows (bottom) represent areas with crypt-invasive BFs in the mice gavaged with the 3728T isolates + <i>C. difficile</i>. Statistical significance in <b>A</b> and <b>C</b> was calculated via Kruskal–Wallis followed by Mann–Whitney tests. Mann–Whitney <i>P</i> values are shown. <b>E,</b> Relative abundance (RA) of <i>C. difficile</i> derived from 16S rRNA amplicon sequencing of the original human slurry (3728T) or mouse stool obtained at sacrifice of mice in the time course experiment following gavage with the 3728T slurry. dpi, days post-inoculation. <b>F,</b> FISH of the original patient tumor 3728T. CRC, colorectal cancer. <b>G–M,</b> scRNA-seq and gene set enrichment analysis of 3728T slurry (<i>n</i> = 3)– versus 3979T slurry (<i>n</i> = 3)–gavaged GF <i>Apc<sup>Min/+</sup></i> mice at 2 weeks p.i. Heat maps depict genes enriched in canonical Wnt (<b>G</b>) or ROS (<b>H</b>) signaling pathways, with Wilcoxon rank-sum test <i>P</i> values above each gene. <b>I,</b> Inflammatory signaling pathways enriched in specific cell types from 3728T slurry– versus 3979T slurry–gavaged mice at 2 weeks p.i. KEGG, Kyoto Encyclopedia of Genes and Genomes. <b>J,</b> Inhibition and activation of the most significant upstream regulators in either progenitor or differentiated colonocytes. Expression log ratio is an effect size statistic. <b>K</b> and <b>L,</b> Network analysis of transcription factors from IPA upstream regulators enriched in the progenitor cell gene list revealed activation of Wnt/Myc signaling and proliferation, respectively. Lines connect transcription factors with shared target genes in the gene list. Darker lines indicate a greater number of shared target genes. <b>M,</b> Similar network analysis for differentiated colonocytes showed activation of the innate immune response.</p>

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