Amending microbiota by targeting intestinal inflammation with TNF blockade attenuates development of colorectal cancer

Abstract

Intestinal inflammation and microbiota are two important components of colorectal cancer (CRC) etiology. However, it is not clear how tuning inflammation using clinically relevant anti-inflammatory treatment impacts microbiota or whether this consequently influences CRC outcome. Here, using chemically induced (DSS/Apc min/+) and spontaneous (Apc min/+ ;Il10 -/-) mouse CRC models colonized by colibactin-producing Escherichia coli, we established the role of microbiota in mediating the antitumorigenic effect of anti-tumor necrosis factor (TNF) therapy. We found that TNF blockade attenuated colitis and CRC development. Microbiota community structure and gene activities significantly changed with disease development, which was prevented by TNF blockade. Several microbiota functional pathways underwent similar changes in patients following anti-TNF therapy. Under cohousing condition, TNF blockade failed to prevent colitis, cancer development and disease-associated microbiota structural changes. Finally, microbiota transplantation showed reduced carcinogenic activity of microbiota from anti-TNF-treated mice. Together, our data demonstrate the plasticity of microbiota, which could be reverted to noncarcinogenic status by targeting inflammation.

Figures

Extended Data Fig. 1 ∣. TNF blockade attenuates colonic inflammation and DNA damage response.

a, Timeline for short-term TNF blockade in NC101-colonized DSS/Apcmin/+ mice. Experiment was done once. b, Representative pictures of H&E stained colon Swiss roll section (distal). Scale bars = 500 μm. c, Distal colon histological scores. d, Representative pictures of colon Swiss roll sections stained for γH2AX. Scale bars = 100 μm. e, Association between treatment and incidence of γH2AX+ regenerating crypts. Fisher’s exact test was used to determine association. f, Fecal NC101 relative abundance in PBS- and anti-TNF-treated mice at day 14. g, Cytokine expression in distal colon snips of NC101-colonized Apcmin/+;Il10−/− mice treated with PBS or anti-TNF antibody (related to Fig. 2h). Data are mean ± s.d. Two-tailed Mann-Whitney U test was performed for paired comparisons. The number of animals (n) used for each graph was indicated.

Extended Data Fig. 2 ∣. Fecal microbiota and NC101 changes in DSS/Apcmin/+ mice treated with PBS or anti-TNF antibody.

a, Alpha diversity (Chao1) and beta diversity (PCoA) comparison of microbiota in PBS-treated mice between indicated time points. The box horizontal lines are the 25th (lower) and 75th (upper) percentile. The middle line is the 50th percentile (the median). The lower whisker is 25th percentile - 1.5 times the interquartile range and the upper whisker is 75th percentile + 1.5 times the interquartile range. b, Alpha diversity (Chao1) and beta diversity (PCoA) comparison of microbiota in anti-TNF-treated mice between indicated time points. Sequencing data analysis was described in details in Methods. The box horizontal lines are the 25th (lower) and 75th (upper) percentile. The middle line is the 50th percentile (the median). The lower whisker is 25th percentile - 1.5 times the interquartile range and the upper whisker is 75th percentile + 1.5 times the interquartile range. c, NC101 abundance by metagenome analysis. One-way ANOVA followed by Holm-Sidak’s multiple comparisons test was performed for multi-group comparisons. d, Normalized counts of transcripts aligned to clb genes. Two-tailed Mann-Whitney U test was performed for paired comparisons. Data are mean ± s.d. The number of animals (n) used for each graph was indicated.

Extended Data Fig. 3 ∣. Microbiota community structure changes in NC101-colonized Apcmin/+;Il10−/− mice treated with PBS or anti-TNF antibody.

a, Alpha diversity (Chao1) and beta diversity (PCoA) comparison of microbiota in PBS-treated mice between indicated time points. The box horizontal lines are the 25th (lower) and 75th (upper) percentile. The middle line is the 50th percentile (the median). The lower whisker is 25th percentile - 1.5 times the interquartile range and the upper whisker is 75th percentile + 1.5 times the interquartile range. b, Alpha diversity (Chao1) and beta diversity (PCoA) comparison of microbiota in anti-TNF-treated mice between indicated time points. The box horizontal lines are the 25th (lower) and 75th (upper) percentile. The middle line is the 50th percentile (the median). The lower whisker is 25th percentile - 1.5 times the interquartile range and the upper whisker is 75th percentile + 1.5 times the interquartile range. c, Alpha diversity (Chao1) and beta diversity (PCoA) comparison between PBS- and anti-TNF-treated mice in longitudinal stool samples and endpoint colon tissues. The box horizontal lines are the 25th (lower) and 75th (upper) percentile. The middle line is the 50th percentile (the median). The lower whisker is 25th percentile - 1.5 times the interquartile range and the upper whisker is 75th percentile + 1.5 times the interquartile range. ns, not significant. Sequencing data analysis was described in details in Methods. The number of animals (n) used for each graph was indicated.

Extended Data Fig. 4 ∣. Cohousing abolishes CRC-preventive effect of TNF blockade in NC101-colonized Apcmin/+;Il10−/− mice.

a, Schematic diagram for TNF blockade under cohousing condition. b, Macroscopic colon tumor counts. c, Distal colon histological scores. d, Fecal NC101 relative abundance in cohoused PBS- and anti-TNF-treated mice. Data are mean ± s.d. Two-tailed Mann-Whitney U test was performed for paired comparisons. Experiment was done once. The number of animals (n) used for each graph was indicated.

Fig. 1 ∣. E. coli NC101 promotes CRC development via colibactin activity in mice harboring a complex microbiota.

a, Timeline for the DSS/Apcmin/+ model. b, Representative pictures of mouse colon (tumors marked by arrowheads). Scale bars, 1 cm. c, Macroscopic tumor counts with the number of mice indicated in each group. d, Tumor size. e, Endpoint fecal Enterobacteriaceae loads. SPF mice were shown as comparison. f, E. coli relative abundance in DSS/Apcmin/+ mice associated with various E. coli strains. Quantification cycle differences (ΔCq) are shown between E. coli (Ec) 16S and universal bacterial (univ.) 16S qPCR. g, Timeline for the Apcmin/+;Il10−/− model. h, Representative pictures of mouse colon. Scale bars, 1 cm. i, Macroscopic tumor counts with the number of mice indicated in each group. j, H&E-stained colon Swiss roll sections (distal). Scale bars, 500 μm. k, Distal colon histological scores. l, Endpoint fecal Enterobacteriaceae loads. Conventionalized (Ctrl) mice without E. coli inoculation were shown for comparison. m, E. coli relative abundance in Apcmin/+;Il10−/− mice. Due to difficulty of collecting stools from certain mice, which did not correlate with tumor or inflammation status, the number of samples for Enterobacteriaceae and E. coli quantification does not always match the number of mice used for tumor characterization. Data are mean±s.d. One-way ANOVA followed by Dunn’s multiple comparisons test was performed for multi-group comparisons, two-way ANOVA followed by Tukey’s multiple comparisons test was used for tumor size comparison and two-tailed Mann–Whitney U-test was used for paired comparisons. Experiments comparing effects of different E. coli strains on colitis and tumorigenesis in DSS/Apcmin/+ and Apcmin/+;Il10−/− models were done once, with mice staggered due to limited mouse availability per experimental setup. The number of animals (n) used for each graph was indicated. GF, germ-free.

Fig. 2 ∣. TNF blockade attenuates NC101-mediated CRC development.

a, Timeline for TNF blockade in the DSS/Apcmin/+ model. b, Representative pictures of mouse colon. Scale bars, 1 cm. c, Macroscopic tumor counts with number of mice in each group. d, Tumor size. e, Fecal Enterobacteriaceae loads at indicated time points. f, Fecal NC101 relative abundance. Due to difficulty of collecting stools from certain mice, which did not correlate with tumor or inflammation status, the number of samples for Enterobacteriaceae and E. coli quantification do not always match the number of mice used for tumor characterization. g, Colon tissue-associated NC101 levels at the endpoint in DSS/Apcmin/+ mice. Cq, quantification cycles from PCR. h, Timeline for TNF blockade in the Apcmin/+;Il10−/− model. i, Macroscopic tumor counts with number of mice in each group. j, Representative pictures of H&E-stained colon Swiss roll sections (distal). Scale bars, 500 μm. k, Distal colon histological scores. l, Endpoint fecal Enterobacteriaceae loads. m, Fecal NC101 relative abundance. n, Colon tissue-associated NC101 levels in Apcmin/+;Il10−/− mice. Data are mean±s.d. Two-tailed Mann–Whitney U-test was performed for paired comparisons, and two-way ANOVA followed by Sidak’s multiple comparisons test was used for tumor size comparison. Anti-TNF treatments in DSS/Apcmin/+ and Apcmin/+;Il10−/− models were performed twice, with similar results. Data show results from one representative experiment. The number of animals (n) used for each graph was indicated.

Fig. 3 ∣. TNF blockade alters microbiota composition in DSS/Apcmin/+ mice.

a, Comparison of microbiota alpha diversity (Chao1) in fecal samples collected at indicated time points and colon tissue snips at the endpoint between PBS- and anti-TNF-treated mice. The box horizontal lines are the 25th (lower) and 75th (upper) percentiles. The middle line is the 50th percentile (the median). The lower whisker is the 25th percentile–1.5 times the interquartile range and the upper whisker is the 75th percentile+ 1.5 times the interquartile range. b, PCoA comparison of microbiota community structure in fecal samples collected at indicated time points and colon tissue snips at the endpoint between PBS- and anti-TNF-treated mice. NS, not significant. Sequencing data analysis was described in detail in the Methods. The number of animals (n) used for each graph was indicated.

Fig. 4 ∣. TNF blockade alters microbiota activity in DSS/Apcmin/+ mice.

a, PCA comparison of fecal microbiota metatranscriptomes normalized by metagenomes between day 14 and day 35 in PBS- and anti-TNF-treated mice. b, PCA comparison of fecal microbiota metatranscriptomes normalized by metagenomes between PBS- and anti-TNF-treated mice at days 14 and 35. c, Differentially expressed KEGG pathways in day-14 and -35 stools from PBS-treated mice. d, Differentially expressed KEGG pathways in PBS- and anti-TNF-treated mice at day 14. e, Differentially expressed KEGG pathways in PBS- and anti-TNF-treated mice at day 35. Pathways similarly affected by anti-TNF therapy in patients are highlighted (magenta). Mice were randomly selected from each group, and sequencing data analysis was described in detail in the Methods. The number of animals (n) used for each graph was indicated. PC1, first principal component; PC2, second principal component.

Fig. 5 ∣. Cohousing abrogates TNF blockade effect on microbiota, inflammation and CRC.

a, Timeline for TNF blockade in DSS/Apcmin/+ mice under cohousing condition. b, PCoA comparison of microbiota structure in longitudinal stool samples and endpoint colon tissue snips between cohoused PBS- and anti-TNF-treated mice. Sequencing data analysis was described in detail in the Methods. c, Endpoint fecal NC101 relative abundance. d, Fecal lipocalin-2 (LCN2) levels. Randomly selected mice at day 0 (before DSS treatment) were used as uninflamed reference. e, Representative pictures of mouse colon. Scale bars, 1 cm. f, Macroscopic tumor counts. g, Tumor size from cohoused PBS- and anti-TNF treated DSS/Apcmin/+ mice. Data are mean±s.d. Two-tailed Mann–Whitney U-test was performed for paired comparisons, one-way ANOVA followed by Sidak’s multiple comparisons test was performed for multi-group comparisons and two-way ANOVA followed by Sidak’s multiple comparisons test was used for tumor size comparison. The cohousing experiment was done once. The number of animals (n) used for each graph was indicated. ABX, antibiotics.

Fig. 6 ∣. TNF blockade-associated microbiota show reduced carcinogenic activity.

a, Schematic diagram of cecal microbiota transplantation using gnotobiotic DSS/Apcmin/+ mice. Pooled cecal contents from PBS- or anti-TNF-treated mice (separately caged as described in Fig. 2a) were used to inoculate germ-free Apcmin/+ mice to generate PBS microbiota recipient (PBS.MR) and anti-TNF microbiota recipient (anti-TNF.MR) mice. b, PCoA comparison of microbiota composition in donor and recipient cohorts shows separate clustering by treatment (PBS or anti-TNF). Sequencing data analysis was described in detail in the Methods. c, Fecal NC101 relative abundance at indicated time points. d, Representative pictures of mouse colon. Scale bars, 1 cm. e, Macroscopic tumor counts. f, Tumor size in PBS.MR and anti-TNF.MR mice. Data are mean±s.d. Two-tailed Mann–Whitney U-test was performed for paired comparisons, and two-way ANOVA followed by Sidak’s multiple comparisons test was used for tumor size comparison. Microbiota transplantation was performed once due to limited materials for inoculation. The number of animals (n) used for each graph was indicated.