Society: AGA

BACKGROUND AND AIMS: Chemoresistance is a major cause of colorectal cancer (CRC) recurrence and death. The critical role of gut microbiome in the efficacy of CRC chemotherapy remains unclear. We aim to characterize the gut microbiota signatures in responders and non-responders to chemotherapy and identify chemoresistance-associated pathogens in CRC.

METHODS: Stool samples were collected from CRC patients before chemotherapy (n=64) and analyzed by shotgun metagenomic sequencing. The effect of enterotoxigenic Bacteroides fragilis (ETBF) on chemoresistance was assessed in CRC cells and CRC mouse models of xenografts, Apc^Min/+or azoxymethane (AOM)/dextran sodium sulfate (DSS) induced CRC tumorigenesis. Phage VA7 was used for targeting ETBF in vitro and in vivo. Bacterial adhesin-host receptor interaction was investigated by far western, biotin pull-down, and GST pull-down assays followed by mass spectrometry. RNA-seq was performed to evaluate involved pathways.
RESULTS: Bacteroides fragilis was identified as the top enriched bacterium in stool samples of CRC patients who were resistant to chemotherapy, and its abundance was associated with poor prognosis (P=0.019). Consistent with this observation, we demonstrated that ETBF co-culture with CRC cells (HT29 and HCT116) inhibited 5-FU and oxaliplatin (OXA)-induced cell apoptosis (both P<0.001). ETBF promoted chemoresistance in xenografts of nude mice (P<0.001). In particular, oral gavage of ETBF to Apc^Min/+mice significantly attenuated the antitumor efficacy of 5-FU and OXA. Such effect was further confirmed in the second CRC tumorigenesis mouse model induced by AOM/DSS. Mechanistically, we revealed that ETBF activated Notch signaling pathway genes (Notch1, NICD, Hes1, MAML1, and c-Myc) identified by RNA-seq and confirmed by Western blot. ETBF-mediated chemoresistance critically depends on its direct interaction with CRC cells, as neither ETBF supernatant nor non-contact transwell co-culture significantly affected the efficacy of 5-FU and OXA. Concordantly, fluorescence in situ hybridization (FISH) and qPCR demonstrated the significant enrichment of ETBF in colonic tumors compared to adjacent normal tissues (P = 0.002) in mice. Scanning electron microscopy (SEM) visualized the attachment of ETBF to colon epithelial cells. ETBF surface protein SusD/RagB was identified to mediate its attachment to CRC cells. Finally, we demonstrated that ETBF-targeting phage VA7 selectively inhibited ETBF in vitro and in mice (P<0.05), and that VA7 co-administration abolished ETBF-induced chemoresistance (P<0.001).
CONCLUSION: We identified for the first time that ETBF provokes chemoresistance in CRC cells through its direct interaction with colon epithelial cells and activating Notch1 signaling. Targeting ETBF by phage VA7 is a promising therapeutic strategy to overcome ETBF-mediated chemoresistance in CRC.

Background
Diet interacts intimately with the gut microbiome and is also a major risk factor for colorectal cancer (CRC). However, epidemiologic evidence on how diet may influence CRC through alteration of the gut microbiome remains limited.

Methods
We leveraged two cohorts of 307 healthy men and 212 healthy women with serial stool metagenomes and detailed dietary data. We empirically characterized a dietary pattern associated with the CRC gut microbiome signature (labeled CRC Microbial Dietary Score [CRC-MDS]), which sums the abundances of 30 species enriched and 13 depleted in CRC based on their loadings in a recent meta-analysis of metagenomic studies (Thomas et al. Nat Med. 2019). We then applied the CRC-MDS to three prospective cohorts – the Health Professionals Follow-up Study (1986-2014), the Nurses’ Health Study (1984-2014), and the Nurses’ Health Study II (1991-2015), in which we assessed diet by semi-quantitative food frequency questionnaires every four years, confirmed self-reported CRC cases through medical record review, and collected tumor tissues from a subset of CRC patients. We evaluated the CRC-MDS association with CRC risk using Cox proportional hazards model, and further assessed CRC risk according to subsites and tumor tissue presence of Fusobacterium nucleatum (F. nucleatum).

Results
The CRC-MDS was characterized by high consumption of foods with greater industrial processing (e.g., processed meat, butter, and low/high-energy drinks) and low consumption of foods rich in fiber (e.g., fruits, vegetables, nuts, and whole grains) (Figure). The score was positively correlated with Western diet (Spearman ρ =0.44) and empirical dietary index for hyperinsulinemia score (ρ =0.54), and negatively correlated with the prudent dietary pattern score (ρ =-0.53). In the three cohorts of 259,190 participants, we documented 3,819 incident cases of CRC over 3,223,696 person-years of follow-up. Compared with participants in the lowest quintile of the CRC-MDS, those in the highest quintile had an increased risk of CRC (hazard ratio [HR], 1.25; 95% confidence interval [CI], 1.12-1.39; P_trend <0.001) after adjusting for putative CRC risk factors. The association was attenuated but remained statistically significant after adjusting for other dietary patterns (see details in Table). The association was similar across CRC subsites but differed according to the presence of F. nucleatum in tumor tissue. The CRC-MDS was strongly associated with a higher risk of F. nucleatum-positive CRC (HR_{Q5 vs Q1}, 2.52; 95% CI, 1.68-3.76; P_trend <0.001) but not F. nucleatum-negative CRC (HR_{Q5 vs Q1}, 1.09; 95% CI, 0.95-1.25; P_trend =0.19) (P_{heterogeneity} = 0.03).

Conclusions
The CRC-MDS was positively associated with CRC risk, especially F. nucleatum-positive CRC. Our findings support the gut microbiome as a mediator of diet in CRC development.

<b>Figure. Weights for food groups comprising the CRC-MDS in men (A) and women (B)</b>

Figure. Weights for food groups comprising the CRC-MDS in men (A) and women (B)

<b>Table. Hazard ratios (95% CIs) for colorectal cancer according to the CRC-MDS</b>

Table. Hazard ratios (95% CIs) for colorectal cancer according to the CRC-MDS

Background: Invasive bacterial biofilms are associated with irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and colorectal cancer (CRC) particularly in the proximal colon, but limited data is available on their prevalence on histologically normal colonic tissues or polyps. The present study examined biofilms in a healthy screening colonoscopy cohort to determine the variability of mucosal biofilms along the colonic axis in normal tissues and in early colonic lesions.

Methods: Biofilms were examined in a screening colonoscopy cohort of 2,091 individuals from three sites in Maryland and/or Pennsylvania, USA. Six methacarn-fixed, histologically normal colonic biopsies per patient (3 left, 3 right colon) were evaluated by fluorescence in situ hybridization (FISH) with the EUB338 probe on a scale from 0-3 (3 being a biofilm, with >20 adherent bacteria within 1 mm of the epithelium along a 200 mm stretch). Patients with IBD and patients on blood thinners were ineligible for the study. Inclusion criteria for the present analysis included patients without a history of CRC or new-onset CRC, and optimally acquired biopsy samples as previously reported (large capacity biopsy forceps and >7 hours time from bowel prep completion to the start of colonoscopy) (N = 1,226). Forty-one paired, formalin-fixed polyp tissues from 21 individuals (7 biofilm positive, 14 biofilm negative on normal mucosa) were also screened for biofilms.

Results: A total of 11.3% (138/1,226) of patients were scored as biofilm positive on their histologically normal tissue. A high degree of intra-individual variability was observed, with only 29/138 biofilm-positive patients having biofilms on both right and left colonic biopsies. Biofilms on histologically normal tissues were not associated with polyp status using linear regression modeling (see Rifkin et al, DDW 2023 abstract). While a similar prevalence and distribution of biofilms was observed on polyps vs. histologically normal tissues (10.5% vs. 11.3%, respectively), the polyp bacterial scores overall were nearly double that of the paired, normal tissues (p = 0.0005, Mann-Whitney, median score of 2 on polyps vs. 1.15 on normal tissues). This difference was weighted strongly by biofilm-negative individuals (Figure 1A, p < 0.0001). Polyp bacterial scores significantly correlated with polyp size (p = 0.014, Spearman’s rank correlation). Furthermore, several large polyps displayed dramatic bacterial crypt invasion that, while still separated from the epithelium by mucus (Figure 1B), were reminiscent of the prolific invasion seen in colorectal cancer specimens.

Conclusions: Colonic biofilms are dynamic ecosystems that may be transient and focal in healthy mucosa but appear to co-evolve with early colonic lesions along the adenoma-carcinoma sequence and may facilitate progression to colorectal cancer.

<b>Figure 1. Polyps display higher bacterial scores and more pronounced bacterial invasion than paired normal tissues</b>. Forty-one polyps from 21 individuals (range: 1-4 polyps per patient) and their paired normal tissues were screened for bacterial invasion on a scale from 0-3 (3 = biofilm positive). <b>(A) </b>Polyps from biofilm-negative patients, as determined by biofilm status on their normal tissue, displayed higher bacterial scores than their paired normal tissues. Polyps from biofilm-positive patients were equivalent to their biofilm-positive normal tissues but were still significantly higher than polyps from biofilm-negative patients. Mann-Whitney p-values are shown. <b>(B) </b>A formalin-fixed, transverse tubular adenoma from a biofilm-positive patient presented with prolific crypt infiltration throughout the tissue (white arrows). Blue = DAPI, yellow = EUB338, green = autofluorescence.

Figure 1. Polyps display higher bacterial scores and more pronounced bacterial invasion than paired normal tissues. Forty-one polyps from 21 individuals (range: 1-4 polyps per patient) and their paired normal tissues were screened for bacterial invasion on a scale from 0-3 (3 = biofilm positive). (A) Polyps from biofilm-negative patients, as determined by biofilm status on their normal tissue, displayed higher bacterial scores than their paired normal tissues. Polyps from biofilm-positive patients were equivalent to their biofilm-positive normal tissues but were still significantly higher than polyps from biofilm-negative patients. Mann-Whitney p-values are shown. (B) A formalin-fixed, transverse tubular adenoma from a biofilm-positive patient presented with prolific crypt infiltration throughout the tissue (white arrows). Blue = DAPI, yellow = EUB338, green = autofluorescence.

Introduction: Chronic inflammation in the colon of patients with inflammatory bowel diseases (IBD) represents a risk factor for developing colitis-associated cancer (CAC). Activating transcription factor 6 (ATF6) signaling provides a novel link between the endoplasmic reticulum unfolded protein response (UPR^ER) of the epithelium and the development of spontaneous colorectal cancer (CRC). In this study, we characterize the impact of IBD-related risk factors on ATF6-mediated tumorigenesis in gnotobiotic mouse models and patients.
Methods: We generated a novel transgenic mouse model for spontaneous CAC by crossing mice expressing the active form of ATF6 (nATF6) in intestinal epithelial cells (IEC) with Interleukin-10 deficient mice (nATF6^IEC,Il10^-/-). Specific pathogen-free (SPF) and germ-free (GF) mice were characterized for tumor formation (incidence, frequency and histology), inflammation (gene expression analysis, immunostaining and immune cell infiltration) and mucus properties (mucus staining and FISH). Spatial microbial signatures of tumor and tumor-associated tissue regions were analyzed by 16S rRNA amplicon sequencing under SPF conditions as well as fecal microbial transfer (FMT) of IBD patient stool, and IBD-relevant simplified human microbiota (SIHUMI). ATF6 expression patterns were quantified in IBD patient tissue.
Results: Constitutive expression of nATF6 drives CAC in Il10^-/- mice. Strikingly, tumor-free monoallelic nATF6^IEC mice (tg/wt;+/+) develop colonic tumors with an incidence of 70% in the absence of Il10 (tg/wt;-/-). tg/tg;-/- mice never exceed a lifespan of 10 weeks. FACS and CHIP cytometry analyses show neutrophil and monocyte infiltrates together with a decrease in NK cells in the tumor niche. tg/wt;-/- mice display a loss of mucus and increased bacterial penetration of the mucus layer. Spatial analyses show that the mucosa-associated microbiota predicts the tumorigenic phenotype, with alterations restricted to tumor sites. GF mice remain tumor-free. IBD patient FMT into GF mice restores the tumorigenic phenotype in susceptible mice (75% tumor incidence). Interestingly, even an IBD-relevant minimal consortium (SIHUMI) induces colonic tumors with an incidence of 64%. Staining in IBD patients reveals higher ATF6 expression in active disease compared to remission.
Conclusion: Our novel CAC mouse model shows that IBD susceptibility increases the risk to develop ATF6-driven tumors. Tumorigenesis is microbiota-dependent, with mucosa-associated microbiota predicting tumorigenic phenotype. Both IBD patient FMT and SIHUMI successfully transfer the disease phenotype and validate tumor risk. Moreover, IBD patients with active disease show increased ATF6 expression, confirming its clinical relevance. Our findings clearly identify ATF6 as a driver of CAC, suggesting its suitability as a biomarker for cancer development in IBD patients.

Background: Colorectal cancers (CRC) commonly arise from premalignant tubular adenomas (TA) or sessile serrated adenomas (SSA). Although the molecular genetics underlying these lesions are distinct, it is unknown whether they are associated with distinct gut microbiomes.

Objective: To study the microbiome taxonomic and functional features associated with premalignant colonic adenoma subtype.

Methods: 1197 consecutive participants 18 years or older, referred for an outpatient colonoscopy were enrolled from the GI Disease and Endoscopy Registry (GIDER), Massachusetts General Hospital, Boston between 2015 and 2022. Stool samples were collected prior to colonoscopy. DNA was extracted using Allprep DNA/RNA KIT (Qiagen). Metagenomic libraries were constructed using Nextera XT DNA Library Preparation Kit (Illumina) and sequenced on HiSeq2500 2x101PE platform, targeting ~2.5Gb of sequence per aliquot. Metagenomic taxonomic and functional profiles were generated using the bioBakery meta’omics workflow. Only 971 participants were included in the analysis, after non-mutualistic exclusion for not providing stool samples (n=111), being on concomitant antibiotics or probiotics (n=44), presence of colitis (n=4), a personal history of a hereditary cancer syndrome (n=29), and suboptimal metagenomic read counts (n=30). Participants also completed a brief food frequency questionnaire. Age, gender, family history of CRC, body mass index, and smoking status, as well as concomitant medications at baseline were all recorded.

Results: Within GIDER, we identified 19 and 8 significant microbial species, as well as 270 and 140 unique microbial enzymes associated with either TA or SSA, respectively. 14 and 12 microbial species were differentially abundant in proximal and distal TAs. TAs were associated with diminished methane metabolism whereas SSAs were enriched in sulfate reduction and secondary bile acid metabolism. We further predicted 51 and 39 significant microbial metabolites associated with either TA or SSA, which allowed us to prioritize key microbial pathways; microbes associated with SSA were heavily involved in maintenance of antioxidant mechanisms, mainly through NAD-NADPH metabolism, and microbes associated with TA were enriched for amino-acid driven lipogenesis. Finally, through mediation analysis, we identified Flavonifractor plautii as a key microbial mediator of TA, via both diet (vegetables) and oral medication (aspirin).

Conclusion: Our findings suggest different adenoma subtypes may be influenced by distinct microbiome signatures. The microbial metabolic diversity of subjects with either TA or SSA reveals opportunities to exploit these unique dependencies through both pharmacological inhibition of metabolic targets and dietary intervention as a potential strategy for lowering the incidence of early CRC and CRC-related deaths.

Background: The gastric microbiome is considered one of the key factors contributing to the gastric cancer environment and progression. Enterococcus hirae (Eh) is a specific pathogen that has repeatedly emerged in gastric cancer environments based on microbiome analysis. We identified a high abundance of Eh in patients with gastric cancer. Hence, we investigated the impact of Eh infection on gastric cancer development by using gastric epithelial cells (GECs).
Methods: Eh strains were isolated from human gastric cancer biopsy samples, and one strain has been chosen for this study. The effect of Eh infection on the GECs (AGS, MKN-74) was investigated by using cytotoxicity assay and immunoblotting. Comet assay was used to confirm DNA double-strand breaks (DSBs). Gastroids model was used to evaluate the pro-inflammation cytokine expression using real-time-PCR.
Results: In vitro infection of current Eh strain 106 induced cell cytotoxicity more than H. pylori (Hp) strain on GECs (Fig 1A, B). However, Eh infection did not increase IL-8 mRNA expression compared to the Hp and control (Fig 1C). Interestingly, Eh significantly stimulated DNA DSBs (gH2AX expression and comet formations) on GECs, even without their adhesion to cells (Fig 1D-F). We found that Eh-conditioned medium (CM) has the capability to promote DNA damage and a significant reduction of GECs cell viability (Fig 2A, B). Further investigation revealed that the CM contains an extracellular superoxide compound which is secreted by Eh (Fig 2C). Next, we investigated the co-infections of Hp and Eh on GECs. Even though Eh suppressed H. pylori-stimulated IL-8 expression in co-infection on the GECs (Fig 3), their co-infection induced synergistic effect as shown by the highest among DNA damages.
Conclusion: Taken together, Eh might promote gastric cancer via genomic instability induction of host cells. Eh remain sustainable since they have the capability to suppress the pro-inflammatory cytokine expression. These suppressive effects may partially explain the mechanism of Eh’s defense system against the host immune system. Because of these occurrences, Eh has been suggested as a potential pathogen of gastric cancer.

<b>Figure 1. Virulence characteristics of Eh. (A)</b> In 24hpi, Eh induced morphological changes in AGS cells. <b>(B)</b> Eh induced AGS cell death than control and Hp. Error bars indicate mean ± SD. <b>(C)</b> IL-8 mRNA expressions of gastroids by Hp and Eh infections<b> (D)</b> Eh-associated DSBs arise independently of the cell-cycle phase which started to appear in 12hpi as same as Hp infection. <b>(E)</b> Characterization of comets in Eh and Hp infections. <b>(F)</b> The tail moments levels of DNA fragmentation following Eh and Hp infections.<br /> <b>Figure 2. CM effects of Eh on GECs (A) </b>gH2AX expression of GECs infected with live or CMs of Eh and Hp for 24hrs. <b>(B) </b>Cell viability of GECs treated with CMs of Eh at different concentrations.<b> (C) </b>Extracellular superoxide measurement of Eh and Hp at different concentrations.<br /> <b>Figure 3.</b> <b>Co-infection of Hp and Eh.</b> IL-8 protein expressions of GECs by Hp, Eh, and co-infections at 6hrs and 12hrs.

Figure 1. Virulence characteristics of Eh. (A) In 24hpi, Eh induced morphological changes in AGS cells. (B) Eh induced AGS cell death than control and Hp. Error bars indicate mean ± SD. (C) IL-8 mRNA expressions of gastroids by Hp and Eh infections (D) Eh-associated DSBs arise independently of the cell-cycle phase which started to appear in 12hpi as same as Hp infection. (E) Characterization of comets in Eh and Hp infections. (F) The tail moments levels of DNA fragmentation following Eh and Hp infections.
Figure 2. CM effects of Eh on GECs (A) gH2AX expression of GECs infected with live or CMs of Eh and Hp for 24hrs. (B) Cell viability of GECs treated with CMs of Eh at different concentrations. (C) Extracellular superoxide measurement of Eh and Hp at different concentrations.
Figure 3. Co-infection of Hp and Eh. IL-8 protein expressions of GECs by Hp, Eh, and co-infections at 6hrs and 12hrs.

Yoshiyuki Tsukamoto

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