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Endogenous murine microbiota member Faecalibaculum rodentium and its human homologue protect from intestinal tumour growth

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Abstract

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigenesis. We identified two anti-tumourigenic strains of the microbiota—Faecalibaculum rodentium and its human homologue, Holdemanella biformis—that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymethane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endogenous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

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Fig. 1: F. rodentium is under-represented during the early phases of tumour development.
Fig. 2: F. PB1 loss coincides with mucus changes and reduces tumour growth when reintroduced.
Fig. 3: F. PB1 reduces tumour cell proliferation without a major impact on immune cells.
Fig. 4: F. PB1 releases SCFAs that have anti-proliferative activities.
Fig. 5: The metabolic products of F. PB1, particularly butyrate, have an anti-proliferative activity in vivo that is independent of the microbiota.
Fig. 6: H. biformis is the equivalent of F. PB1 in humans.

Data availability

Source Data for the figures and Extended Data figures are provided in the online version of the paper. Raw sequencing data and metadata associated to samples are available online at the NCBI under the accession number PRJNA564752.

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Acknowledgements

We thank M. Bugatti and S. Licini (supported by Fondazione Beretta, Brescia, Italy) for their technical support in the histological analyses; A. Thomas and E. Pasolli for performing the bioinformatics analyses; C. Faccani for technical support for the in vitro experiments; and E. Mileti, C. Burrello and M. R. Giuffré for their technical support for the in vivo experiments. This work has been supported by grants from the Italian Association for Cancer Research (grant no. AIRC IG 17628) and the European Research council (grant no. 615735, HOMEOGUT ERC) to M.R. E.Z., I.S. and A.B. were/are recipients of a FIRC fellowship. T.S. is a recipient of a fellowship from Fondazione Veronesi. G.F. is a recipient of a grant from the Italian Ministry of Health (grant no. GR-2013-02359806).

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Authors

Contributions

E.Z. and C.P. ideated, performed and analysed all of the experiments. T.S. and F.S. helped with the execution of experiments. A.B., I.S. and S.Guglietta helped with the execution of the mouse experiments. B.F., M.M. and G.Pesole performed the 16S rRNA metagenomic analysis. L.M. and W.V. designed and carried out the histological analyses. G.N. performed the ex vivo stimulation of human colonic mucosa experiments. A.B. performed the confocal analyses. J.T. executed the metabolomic analyses. B.O. assisted in the execution of the in vitro experiments. K.A. and K.H. isolated F. PB1 and carried out the germ-free experiments. S.A. and S.Guglielmetti set up F. PB1 growth and supernatant production. S.C. set up H. biformis and L. lactis growth and supernatant production. G.F. performed the FACS analyses. F.A. and N.S. performed the phylogenetic analysis and human CRC dataset interrogation. G.Penna contributed with ideas and interpretation of results. M.R. ideated the study, coordinated the work and wrote the manuscript.

Corresponding author

Correspondence to Maria Rescigno.

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Extended data

Extended Data Fig. 1 Faecal microbiota diversity upon tumour progression in ApcMin/+ mice.

a, Chao1 and Simpson indexes of the faecal microbiota of WT and ApcMin/+ mice; n = 8 mice/group. Box plots show the interquartile range, median value and whiskers min to max. Data from two independent experiments. b, Tumour multiplicity in the small intestine of ApcMin/+ mice assessed at different time points; weeks 6, 28 n = 5; weeks 8, 26 n = 8; weeks 10 n = 9; weeks 12, 16, 24 n = 3; weeks 14 n = 7; weeks 18 n = 10; weeks 20 n = 33; weeks 22, 30 n = 4 mice/group. c, Normalized read counts for differentially represented species in WT and ApcMin/+ mice shown as heatmaps. The counts were log transformed and used to define the colour gradient of the heatmap; n = 8 mice/group. Data from two independent experiments. a-c, Data are represented as means ± s.e.m. Source data

Extended Data Fig. 2 Mucin production is altered in the ileum of ApcMin/+ mice.

a, Representative Western blots of two independent experiments with consistent results showing the abundance of Muc1, Muc13 and Muc20 in ileal extracts from WT (healthy tissues, H) and healthy (H) and tumour (T) tissues of ApcMin/+ mice analysed at 11 weeks of age; Right: densitometric quantification (WT n = 3; ApcMin/+ n = 4 biologically independent samples). b, Representative confocal images of WT and ApcMin/+ mice ileum intestinal tissues stained with Muc1 (red) and DAPI (blue); scale bars, 50 μm. Mean fluorescence intensity of MUC1 was measured to quantify the expression of the protein in the intestinal of ApcMin/+ and WT mice (WT n = 7, ApcMin/+ n = 5 biologically independent samples). c, ApcMin/+ mice were treated with vehicle (n = 10 mice/group) or F. PB1 (n = 11 mice/group) from week 8 to 10. Number of microscopic lesions, shown as % relative to vehicle, was evaluated on H&E slides by an expert pathologist. a-c, Data from two independent experiments and represented as means ± s.e.m. P values were determined by one-way ANOVA using Tukey post-test (a), two-tailed unpaired Mann Whitney U test (b), or two-tailed unpaired Student’s t-test (c). Source data

Extended Data Fig. 3 F. PB1 does not have a major impact on immune cells.

a-c, Flow cytometry analyses of the small intestinal and colonic lamina propria of germ-free ICR mice monocolonized with F. PB1 (GF + F. PB1 n = 10 mice/group) for the presence of T reg (a), Th17 (b) and Th1 (c) cells. Germ-free (GF, n = 9 mice/group) and SPF (n = 7 mice/group) mice were used as controls. d-g WT and ApcMin/+ mice treated with vehicle (Veh) or F. PB1 from week 8 to 12. d,e, Flow cytometric analysis of T reg, Th1 and Th17 cell populations in the small intestinal lamina propria. FoxP3+CD25+ are gated on the live CD45+ CD3+ CD4+ cells; Helios+ is gated on the FoxP3+ CD25+ cells (WT Veh, ApcMin/+ F. PB1 n = 12; WT F. PB1 n = 14; ApcMin/+ Veh n = 11 mice/group). IL17+, IFNγ+ and IL17+ IFNγ+ cells are gated on the live CD45+ CD3+ CD4+ cells (WT Veh, ApcMin/+ Veh n = 9; WT F. PB1 n = 11; ApcMin/+ F. PB1 n = 10 mice/group). Data shown as % of CD45+ cells (d) or as absolute number / whole tissue (e). f,g, Flow cytometric analysis of mononuclear phagocytes (CD11b+F4/80+ macrophages, CD11c+CD11b+ dendritic cells and Ly6ChiCD11b+ inflammatory monocytes) and neutrophils (Ly6G+CD11b+) in the small intestinal lamina propria (WT Veh n = 5; WT F. PB1, ApcMin/+ F. PB1 n = 6; ApcMin/+ Veh n = 4). Data shown as percentages relative to the CD45+ CD3- population (f) or as absolute number / whole tissue (g). h, Flow cytometric analysis of peripheral blood cells. Data shown as absolute number / ml blood (WT Veh, ApcMin/+ F. PB1 n = 13; WT F. PB1 n = 15; ApcMin/+ Veh n = 12 mice/group) P values were determined by one-way ANOVA with Bonferroni post-test (a-d), Kruskal-Wallis with Dunn post test (e), two-tailed unpaired Mann-Whitney U test (f,g,h Ly6G+CD11b+) or two-tailed unpaired Student’s t-test (h, Ly6ChiCD11b+). a-h, Data are represented as means ± s.e.m. Source data

Extended Data Fig. 4 F. PB1 administration alters microbiota and its metabolic profile.

a, LEfSe comparison results between the microbiota of Vehicle and F. PB1 treated mice at 12 weeks with the highest linear discriminant analysis LDA score (log10 ≥ 2.0); Veh, n = 8; F. PB1 n = 14 mice/group. b, Faecal concentrations of succinate, isovalerate and valerate in WT and ApcMin/+ mice treated with vehicle (Veh) or F. PB1 as detected by UPLC–HR–MS; WT Veh, WT F. PB1 n = 6; ApcMin/+ Veh n = 11; ApcMin/+ F. PB1 n = 10 mice/group. Data from two independent experiments and represented as means ± s.e.m. P values were determined by two-way ANOVA with Bonferroni post-test. Source data

Extended Data Fig. 5 SCFAs and F. PB1 spent medium (SUP) increase histone H3 acetylation and reduce NFATc3-calcineurin pathway in vitro on mouse intestinal tumour cell lines.

a, Representative WB showing H3K27 acetylation, PP2B-A and NFATc3 in cell lines treated (+) or not (-) with a mix of SCFAs. Three independent experiments were performed with consistent results. b, Densitometric quantification of WB in Fig. 4d showing NFATc3 and PP2B-A (normalized to actin) and H3K27 acetylation (normalized to vinculin). Two or three independent experiments were performed with consistent results (n = 2 or n = 3 biologically independent experiments). Data are represented as means ± s.e.m. and P values were determined by two-tailed unpaired Student’s t-test. c, Densitometric quantification of WB in Fig. 4e showing NFATc3 (normalized to vinculin) and H3K27 acetylation (normalized to H3 tot). To calculate the protein expression induced by SUP evap as a percentage, the densitometric value of SUP was assumed to be 100%. Data from two independent WB (n = 2 biologically independent experiments). Source data

Extended Data Fig. 6 Effect of F. PB1 spent medium (SUP) in tumourigenesis.

a-c, Representative histologies from ileal and colon sections of ApcMin/+mice (a,Veh n = 13; SUP n =11 mice/group; b,Veh n = 6; SUP n =7 mice/group) and AOM and DSS treated WT mice (c,Veh n = 5; SUP n = 6 mice/group) treated with vehicle or F. PB1 SUP. Sections stained for H&E from FFPE blocks of swiss roll of small (a, 5X magnification, scale bars 500 μm; b, 3X magnification, scale bars 750 μm) and large (c, 2X magnification, scale bars 900 μm) bowel and treated as indicated by labels. Slides were scanned by Aperio Scanscope and digital images were obtained. Dysplastic lesions were selected and measured. In the lower right high power insert, a detail of dysplastic glands (200X magnification, blue scale bars 100 μm).

Extended Data Fig. 7 L. lactis does not colonize the mouse gut but its spent medium (SUP) has anti-tumourigenic effect in vivo.

a, Quantification of SCFAs in SUP of F. PB1 and L. lactis by UPLC–HR–MS. Data from 2 or 6 independent experiments (L.lactis SUP n = 2; F. PB1 SUP n = 6 biologically independent experiments). b,c, ApcMin/+ mice received vehicle (Veh, n = 7 mice/group), F. PB1 or L. lactis (n = 8 mice/group) from week 8 to 10. b, Tumour multiplicity in the small intestine normalized to vehicle-treated ApcMin/+ mice. c, Area and maximum diameter (axis lenght) of ileal dysplastic lesions normalized to the total number of lesions per mouse. Box plots show the interquartile range, median value and whiskers min to max. d, qPCR of L. lactis abundance normalized to panbacterial primers targeting the 16S rRNA gene (UNI 16S) in bacterial DNA extracted from faeces (both at time 0 and 48h after last gavage) and mucus from the ileum and colon of WT and ApcMin/+ mice pretreated or not with antibiotics (ABX) and then monocolonized with L. lactis (n = 4 mice/group). e,f, 11 weeks old ApcMin/+ mice treated with broths not fermented (Veh) or fermented by L. lactis (SUP) in the presence of ABX (n = 6 mice/group). e, tumour multiplicity in the small intestine normalized to vehicle-treated ApcMin/+ mice. f, Area and maximum diameter of ileal dysplastic lesions normalized to the total number lesions per mouse. Box plots show the interquartile range, median value and whiskers min to max. g, Cell proliferation assay on mouse CRC cell lines treated or not with sodium lactate at different concentrations for 48h. t0 is the signal from cells at the time of stimulation. Two independent experiments were performed with consistent results. Data presented as means ± s.d. of a representative experiment (n = 6 biologically independent samples). P values were determined by two-tailed unpaired Mann-Whitney U test (e,f), one-way ANOVA with Bonferroni post-test (b,g) or Kruskal-Wallis test with Dunn post-test (c,d). Data are presented as means ± s.e.m. in a-f. Source data

Extended Data Fig. 8 H. biformis does not colonize the mouse gut but its spent medium (SUP) increases histone H3 acetylation, reduces NFATc3 activation and proliferation in vitro on human colorectal cancer cell lines.

a, Cell proliferation assay on human CRC cell lines treated or not (NT) with broth fermented by F. PB1 (F. PB1 SUP) or by H. biformis (H. biformis SUP) and with a mix of SCFAs as a control. t0 is the signal from cells at the time of stimulation. Two independent experiments were performed with consistent results. Data are presented as means ± s.d. of a representative experiment (n = 6 biologically independent samples). b, Densitometric quantification of WB in Fig. 6e showing NFATc3 and H3K27 acetylation (normalized to vinculin). Two or three independent experiments were performed with consistent results (n = 2 or n = 3 biologically independent experiments). c-e, ApcMin/+ mice received vehicle (Veh) or H. biformis from week 8 to 10 (n = 5 mice/group). c, tumour multiplicity in the small intestine normalized to vehicle-treated ApcMin/+ mice. d, Area and maximum diameter (axis lenght) of ileal dysplastic lesions normalized to the total number of lesions per mouse. Box plots show the interquartile range, median value and whiskers min to max. e, qPCR of H. biformis abundance normalized to panbacterial primers targeting the 16S rRNA gene (UNI 16S) in bacterial DNA extracted from faeces and mucus from the ileum. P values were determined by one-way ANOVA using Bonferroni (a) or Tukey (b, NFATc3 HT29 and H3K27 Caco2) post-test, by two-tailed unpaired Student’s t-test (b, H3K27 HT29) or by two-tailed Mann-Whitney U test (c-e). Data are represented as means ± s.e.m. in b-e. Source data

Extended Data Fig. 9 Treatments with SCFAs or F. PB1 spent medium (SUP) increase histone H3 acetylation, reduce NFATc3 activation and proliferation in human CRC samples.

a-c, Human colon tumour samples (hCRC) incubated ex vivo with broth fermented with F. PB1 (SUP) or non-fermented (Veh); with medium alone (NT) or with SCFAs MIX. a, Representative immunohistochemistry of one out of three independent experiments of H3K27 acetylation and NFATc3 expression. Scale bars, 50 μm. b,c, Representative images of ileal dysplastic lesions stained with Ki67. Scale bars, 50 μm. Three independent experiments were performed with consistent results. Dot plots show the percentage of Ki67 positive cells relative to vehicle (b) or to NT (c). Data from three independent experiments (b, n = 7; c, n = 5 biologically independent samples), are represented as means ± s.e.m. and P values were determined by two-tailed unpaired Mann-Whitney U test. Source data

Extended Data Fig. 10 Model.

Upon intestinal tumourigenesis selectively some bacterial species do not expand, due to different mucus composition or to competition with other bacteria and their corresponding metabolites. If these bacterial species are reintroduced, they induce the release of SCFAs. F. PB1 or H. biformis metabolic products act as HDAC inhibitors in the adenomas inducing an increase in acetylation and downmodulation of the calcineurin-NFATc3 pathway, which is involved in cell proliferation.

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Supplementary Tables 1–3 and flow cytometry gating strategy.

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Zagato, E., Pozzi, C., Bertocchi, A. et al. Endogenous murine microbiota member Faecalibaculum rodentium and its human homologue protect from intestinal tumour growth. Nat Microbiol 5, 511–524 (2020). https://doi.org/10.1038/s41564-019-0649-5

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