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. 2018 Feb 2;359(6375):592-597.
doi: 10.1126/science.aah3648. Epub 2018 Feb 1.

VSports - Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria

Christine M Dejea  1   2 , Payam Fathi  1   2   3 , John M Craig  4 , Annemarie Boleij  1   5 , Rahwa Taddese  5 , Abby L Geis  1   2 , Xinqun Wu  1   3 , Christina E DeStefano Shields  1   2 , Elizabeth M Hechenbleikner  6 , David L Huso  7 , Robert A Anders  8 , Francis M Giardiello  2   3 , Elizabeth C Wick  6 , Hao Wang  1   2 , Shaoguang Wu  1   3 , Drew M Pardoll  1   2 , Franck Housseau  1   2 , Cynthia L Sears  9   2   3
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Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria

Christine M Dejea et al. Science. .

Abstract

Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown VSports手机版. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of Escherichia coli and Bacteroides fragilis Genes for colibactin (clbB) and Bacteroides fragilis toxin (bft), encoding secreted oncotoxins, were highly enriched in FAP patients' colonic mucosa compared to healthy individuals. Tumor-prone mice cocolonized with E. coli (expressing colibactin), and enterotoxigenic B. fragilis showed increased interleukin-17 in the colon and DNA damage in colonic epithelium with faster tumor onset and greater mortality, compared to mice with either bacterial strain alone. These data suggest an unexpected link between early neoplasia of the colon and tumorigenic bacteria. .

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Figures

Fig. 1
Fig. 1. Fluorescent in situ hybridization (FISH) and microbiology culture analysis of FAP mucosal tissues
(A) Top panels: Representative FISH images of bacterial biofilms (red) on the mucosal surface of a FAP polyp and paired normal tissues counterstained with DAPI (4′,6-diamidino-2-phenylindole) nuclear stain (blue). Middle panels: Most of the biofilm composition was identified as B. fragilis (green) and E. coli (red) by using species-specific probes. Bottom panels: PAS (periodic acid–Schiff)–stained histopathology images of polyp and paired normal mucosal tissues demonstrating the presence of the mucus layer. Images were obtained at 40× magnification; scale bars, 50 μm. Dotted lines delineate the luminal edge of the colonic epithelial cells. Images are representative of n = 4 to 23 tissue samples per patient screened (at least 10 5-μm sections screened per patient). (B) Enterobacteriaceae (yellow) and E. coli (red) FISH probes on paired normal FAP tissue (100× magnification) revealing invasion into the epithelial cell layer at the base of a crypt (arrows). Bottom panels with insets of Enterobacteriaceae (bottom left panel) in yellow, E. coli (bottom middle panel) in red, and overlay (bottom right panel) confirming identification of the invasive species. Scale bar, 20 μm. Images are representative of n = 5 to 16 tissue samples per patient screened (at least 10 5-μm sections screened per patient). (C) FAP and control prevalence of pks+ E. coli and enterotoxigenic Bacteroides fragilis (ETBF). Chi-square P-values are shown that represent the difference in probability of detection of each bacterium in FAP versus control patients. (D) PCR detection of clbB (a gene in the pks island) and bft within laser-captured biofilms containing E. coli and B. fragilis from designated FAP patients (table S1) and controls (table S2; materials and methods). Data show a representative image from two independent experiments with two or three replicates per experiment performed.
Fig. 2
Fig. 2. Cocolonization by pks+ E. coli and ETBF increases colon tumor onset and mortality in murine models of CRC
(A) Total colon tumor numbers detected in sham (n = 9), ETBF monocolonized (n = 12), pks+E. coli monocolonized (n = 11), pks+ E. coli/ETBF cocolonized (n = 13), E. coliΔpks/ETBF (n = 9), or pks+ E. coli/ETBFΔbft (n = 10) AOM mice at 15 weeks after colonization. Data indicate mean ± SEM. Overall significance was calculated with the Kruskal-Wallis test, and the overall P value is shown; Mann-Whitney U was used for two-group comparisons; **P = 0.016, ****P < 0.0001. (B) Representative colons of monocolonized (ETBF or pks+ E. coli), cocolonized (ETBF/pks+ E. coli), E. coliΔpks/ETBF, and pks+ E. coli/ETBFΔbft mice at 15 weeks after colonization of AOM-treated mice. Images are representative of n = 9 to 13 mice for each group. (C) H&E (hematoxylin and eosin) histopathology of an invasive adenocarcinoma in a cocolonized (pks+ E. coli/ETBF) AOM mouse at 15 weeks. Main image, 10× magnification; scale bar, 1 mm. Inset image, 100× magnification; scale bar, 0.2 mm. Blue arrow depicts the disruption of the muscularis propria by the invasive adenocarcinoma, and white arrows (inset) identify invading clusters of adenocarcinoma epithelial cells. (D) Kaplan-Meir survival plot of ApcΔ716Min/+ mice (n = 30) colonized with either ETBF (blue; n = 10), pks+ E. coli (orange; n = 10), or cocolonized with pks+ E. coli and ETBF (purple; n = 10). Cocolonization significantly (P < 0.0001) increased the mortality rate. Statistics were analyzed with the log-rank test. All surviving mice (n = 19) were harvested at 110 days.
Fig. 3
Fig. 3. IL-17–induced inflammation is necessary for bacterial-driven tumorigenesis
(A) Histologic hyperplasia and (B) inflammation scores of 15-week AOM sham (n = 9), ETBF monocolonized (n = 12), pks+ E. coli monocolonized (n = 11), or pks+ E. coli/ETBF cocolonized (n = 13) mice. Data represent mean ± SEM of three independent experiments. For (A) and (B), overall significance was calculated by using the Kruskal-Wallis test, and the overall P value is shown; Mann-Whitney U was used for two-group comparisons; **P = 0.01, ***P = 0.0014, ****P = 0.0006; NS, not significant. (C) Myeloid and lymphoid lamina propria immune cell infiltrates plotted as percentage of live cells in AOM mice at day 7 (top panels) and day 21 (bottom panels) after colonization. Data represent mean ± SEM of three independent experiments (total three to five mice per group). (D) Total tumor numbers detected in IL-17–deficient AOM-treated mice (IL17−/−) versus wild-type AOM mice (WT). Both mouse strains were cocolonized with pks+ E. coli and ETBF and tumors assessed at 15 weeks. Data represent mean ± SEM of two or three independent experiments (total 6 to 13 mice per group). Significance calculated by the Mann-Whitney U test represents differences between the non-normally distributed colon tumors in the independent mouse groups. (E) IL-17–producing cell subsets and total number of IL-17–producing (IL-17tot) cells per colon harvested from germ-free C57BL/6 mice monocolonized with pks+ E. coli or ETBF or cocolonized with pks+ E. coli and ETBF for up to 60 hours. Data represent mean ± SEM of two independent experiments (total 3 to 5 mice per group). Overall significance across IL-17–producing cell types was calculated by using two-way analysis of variance testing based on log-transformed data (bold P value). For each cell subset and total number of IL-17–producing cells (gray dotted line box), the overall P value is shown and was calculated by using the Kruskal-Wallis test. Two-group cell subset and total number of IL-17–producing cell comparisons were analyzed by Mann-Whitney U test and are reported in table S7.
Fig. 4
Fig. 4. ETBF enhances pks+ E. coli colonization and colonic epithelial cell DNA damage
(A) ELISA results showing anti-pks+ E. coli (NC101) IgA and anti-ETBF (86-5443-2-2) IgA present in fecal supernatants from wild-type AOM mice under the designated colonization conditions for 4 weeks. Data represent mean ± SEM of three independent experiments (total 3 to 10 mice per group). (B) Colonization of distal colon mucosae by pks+ E. coli and ETBF under mono- and cocolonization conditions at 4 weeks in AOM mice. Data represent mean of three independent experiments (total of 15 mice per group). (C) Mucus depth (μm) of HT29-MTX-E12 monolayers under the designated colonization conditions. Data represent mean ± SEM of three independent experiments. A. muc, Akkermansia muciniphila. (D) Representative images of γ-H2AX immunohistochemistry of distal colon crypts from AOM mice (five mice per condition) mono- or cocolonized with pks+ E. coli and ETBF for 4 days with quantification (right panel) of γ-H2AX–positive cells displayed as percentage positive per crypt (see materials and methods). Data represent mean ± SEM of three independent experiments. For (A), (B), and (D), significance was calculated with the Mann-Whitney U test for two-group comparisons; for (C), overall significance was calculated with the Kruskal-Wallis test and the overall P value is shown; Mann-Whitney U was used for two-group comparisons; ****P < 0.0001.
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