Akkermansia muciniphila inhibits tryptophan metabolism via the AhR/β-catenin signaling pathway to counter the progression of colorectal cancer

Akkermansia muciniphila (A. muciniphila), a gram-negative anaerobic bacterium, is selectively decreased in the fecal microbiota of patients with colorectal cancer (CRC), but its molecular mechanism in CRC development remains inconclusive. In this study, we first confirmed the inhibitory effect of A. muciniphila on CRC formation and analyzed the metabolic role of intestinal flora in human Polyps, A-CRA (advanced colorectal adenoma) and CRC samples. To better clarify the role of A. muciniphila in CRC development, a pseudo-germ-free (GF) azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model was established, followed by infection with or without A. muciniphila. Metabolomic analysis and RNA-seq analysis showed tryptophan-mediated aryl hydrocarbon receptor (AhR) was significantly down-regulated in A. muciniphila-infected CRC mice. Then, mice with intestinal specific AhR deficiency (AhRfl/fl Cre) were generated and were used in 2 murine models: AOM/DSS treatment as a model of carcinogen-induced colon cancer and a genetically induced model using ApcMin/+ mice. Notably, AhR deficiency inhibited CRC growth in the AOM/DSS and ApcMin/+ mouse model. Moreover, AhR deficiency inhibited, rather than enhanced, tumor formation and tumor-derived organoids in Apc-deficient cells both in vivo and in vitro by activating Wnt/β-catenin signaling and TCF4/LEF1-dependent transcription. Furthermore, the antitumor effectiveness of A. muciniphila was abolished either in a human colon cancer tumor model induced by subcutaneous transplantation of AhR-silenced CRC cells, or AhR-deficienty spontaneous colorectal cancer model. In conclusion, supplementation with A. muciniphila. protected mice from CRC development by specifically inhibiting tryptophan-mediated AhR/β-catenin signaling.


Electron microscopic
The intestinal tissue of AOM/DSS mice treatment with A. muciniphila (1×10 8 colony forming units) or Vehicle (E. coli MG1655 or the same volume of phosphate buffer saline) were excised and fixed in 0.1M phosphate buffer containing 2.5% glutaraldehyde and 2.0% paraformaldehyde (pH 7.4).Then the tissues were fixed, dehydrated, polymerized and then examined using the transmission electron microscope as previously described [1].

CRISPR/Cas9-mediated knockout of AhR
The genomic sequence of AhR was located at ensemble.org.Locate the exon or any exon of our interest (that may contain a functional domain, hot spot, etc.), copy and paste 23-500 nt (~200 nt optimal) onto crispr.mit.edu to design CRISPR sgRNA strands for nuckase.Pick the top-ranked strands (with predicted faithfulness scores close to 100), to ligate into CRISPR constructs, at least two for nuclease.For every sgRNA strand, two oligos were designed and ordered, forward and reverse, complimentary to each other, with 5' overhang CACC for one, AAAC for the other sticky ends for the BbsI/BsmBI site (Figure 2E).Perform the sub-cloning process: oligo insert annealing to form the oligo duplex, dilute the phosphorylated and annealed oligo duplexes 1:100 in H2O, followed by the process of LentiCRISPR v2 plasmid digestion and oligo insert ligation.Each construct was quickly transformed into Stbl3-competent cells, and at least six clones of each construct were selected for Sanger sequencing to validate insert ligation using the U6-Forward primer.
Puromycin was used to weed out the CRISPR negative cells and the isogenic single-cell clones were obtained in a 96-well plate using serial dilution method.Single clones of transduced cells were screened for indels in AhR coding sequence by locus PCR/Sanger sequencing, and RT-PCR.Those without full-length AhR expression were used to perform further experiments.

Transfection of plasmids, siRNAs, and lentivirus production
Specific siRNAs were used to knock down AhR (Sequences of all the primers are shown in Supplementary Table 1; Qiagen).Transfection procedures were performed according to manufacturers' instructions, with Lipofectamin 2000 as transfection reagent (Invitrogen).Briefly, 2×10 4 cells were plated in each well of a 6-well plate and incubated overnight.A mixture of Lipofectamine 2000 (10 nM) with siRNA (50 nM) was added, followed by a 48 h incubation in regular medium.sh-AhR and sh-control lentiviral particles used to transfect DLD-1 cells were generated by cotransfection of 293T cells.The GFP positive cells, transfected with sh-AhR-GFP-Lentivirus, were sorted and the stable clones were cultured as previously described [2].

Animals and Xenograft Models
We have summarized all animal models in Supplementary Table 7 and represented them with representative molding method etc. Male athymic nude mice (NCr-nu), 8-12 weeks old, were purchased from Sino-British SIPPR/BK Lab Animal Co., Ltd (Shanghai, China, license No. SCXK 2008-0016), and maintained under pathogen-free conditions.When the xenograft tumors reached an average size of 100 mm 3 (almost 8 days), all the animals were injected through the vena caudalis every 2 days as the above four groups mentioned.After treatment for 28 days, luciferase intensity was detected by bioluminescence.At the end point (when tumor volume reached ~2000 mm 3 ), the animals were euthanized; their tumor was excised, cleaned, and imaged; and their tumor mass was excised.
Tumor volume and two perpendicular diameters (A and B) were recorded every 3 days.The average tumor radius was calculated as (A+B)/4.Tumor volume (V) was estimated (assuming a spherical shape) using the formula V = (4/3) π r 3 .

In vivo BrdU assay
According to 5-bromo-2-deoxyuridine (BrdU) in vivo kit's instructions (BrdU; Sigma-Aldrich), prepare a fresh BrdU solution at 10 mg/ml in saline every time and keep it refrigerated in the dark.The solution is sonicated in an ultrasound water bath for a few minutes immediately before injection.It was injected intraperitoneally into the mice once daily for 5 days as described previously [3], then intestine tissues were harvested after cleanout with PBS.The tissues were fixed on glass slides with 2.5% paraformaldehyde, Epitopes were retrieved by heat induction with Antigen Decloaker 10X (Biocare Medical, Concord, CA) in a rice cooker for 10 minutes at 120°C.After blocking non-specific binding (Protein Block, 30 minutes, room temperature), tissues were incubated for 2 hrs at RT (room temperature) with mouse anti-BrdU (1:100, BD Biosciences, San Jose, CA).Then the tissues were labeled for 1 hr at RT with AlexaFluor-488 goat anti-mouse IgG (1:200), AlexaFluor-647 goat anti-chicken IgG (1:200).The cell nuclei were stained twice for 10 minutes at RT with DAPI as a counter stain.Images were takenby the Leica DMi8 Laser Scanning Confocal.

weight loss score+ stool consistency score+ occult blood score)/3 Table 6. Histopathologic analysis of neoplastic lesions and the degree of dysplasia Group carcinoma/tumor number malignant degree of carcinoma
[5] -/-+ A. muciniphila 5/20 25% Histopathologic analysis of neoplastic lesions and the degree of dysplasia were assessed according to standard criteria and classification of adenomas of the colon.tubularadenomawith high-grade dysplasia characterized; low grade adenocarcinomas with focal submucosal invasion[5].