Mestivier, Denis

Abstract Oral commensals such as Parvimonas micra are increasingly implicated in sporadic colorectal cancer (CRC). Several findings support an emerging notion that alterations in host DNA methylation constitute a mechanism by which P. micra adapts to the host microenvironment and contributes to pathogenic processes. However, multiple limitations hinder definitive conclusions, including the correlative nature of the human studies and the absence of human microbiota in mouse models. Here, we investigated how a complex human microbiome influences the adaptation and oncogenic potential of P. micra in a spontaneous mouse model of polyposis. Adult mice, following antibiotic conditioning, received fecal microbiota transplants (FMT) from a healthy human donor, with or without supplementation of P. micra. P. micra caused significant reshaping of the gut microbial community and altered co-occurrence network structures, which however largely recovered within 21 days post FMT. Despite the transient nature of these changes, P. micra caused a marked increase in both the number and invasive potential of intestinal polyps. Epigenomic profiling of polyp crypts revealed global DNA hypomethylation alongside gene-specific hypermethylation, accompanied by transcriptional changes in key oncogenic pathways. Affected pathways included Wnt signaling, epithelial-to-mesenchymal transition, circadian rhythm regulation, and host–microbe interactions. These findings demonstrate that P. micra exerts a profound influence on microbial ecology, host epigenetic programming, and tumorigenesis in a genetically predisposed host, highlighting its role as a microbial driver of intestinal carcinogenesis.

Objective How tumour-adherent microbiome can influence outcomes in patients with colorectal cancer (CRC). Methods and analysis CRC tissues (n=97) were analysed using RNAseq, whole exome sequencing (WES) and 16 r sRNA sequencing. Inflammatory/immune cells were quantified by immunohistochemistry in mice and human tissues. Patient outcomes were analysed according to virulent bacteria and gene RNA levels in tumour tissues. We performed faecal microbiota transfer (FMT) in germ-free mice using presurgery stool samples from nine patients with sporadic CRC. Changes in gene mutations and DNA methylation in the colonic mucosa were measured and compared with those in mice receiving FMT from nine healthy individuals. Immune cells in the tumour tissues of donor and mouse recipients were compared. Results FMT from CRC donors led to preferential DNA demethylation, but specific host genes, including pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), were hypermethylated compared with FMT from healthy donors. CRC faecal samples showed elevated levels of virulent bacteria, reduced levels of commensals and triggered colon inflammation in mice. Notably, PHLPP1 was downregulated in CRC tumours, and its expression varied in accordance with other CRC cardinal genes (n=137, as identified in the scientific literature to be involved in colon carcinogenesis), and was most pronounced in inflammatory bowel disease associated CRC. Tumours with reduced PHLPP1 expression are more inflammatory and correspond to patients with a higher relative abundance of adherent virulent bacteria and poorer outcomes. Conclusion The composition of CRC tumour-adherent bacteria predicts epigenetic modifications and altered expression of cardinal CRC genes such as PHLPP1. PHLPP1 downregulation characterises patients with CRC with inflammatory tumours and poor survival outcomes.

Summary Background Colorectal cancer (CRC) results from the accumulation of mutations and epigenetic changes in gut epithelial cells likely due to gut microbiota dysbiosis. However, limited research has been done to explore the link between host tumour dysbiosis and disease outcome. Methods The mechanisms influencing outcomes of 97 colorectal cancer (CRC) patients, including 13 with Lynch syndrome, 20 with inflammatory bowel disease (IBD), and 64 sporadic cases, were analyzed using a multiomics approach. These patients were categorized into two groups: “disease-free/stable disease” and “progression disease” survival outcomes. The analysis included tumor adherent microbiota composition (16S rRNA), somatic gene mutations (WES), gene expression (RNAseq), immune markers (RNAscope), and immune infiltrate cells (immunohistochemistry). Results IBD-CRC patients had worse outcomes than those with Lynch or sporadic CRC, regardless of TNM staging or treatment. Symbiotic bacteria like Lactococcus lactis were significantly reduced in IBD-CRC tissues. Patient outcomes were influenced by the abundance of virulent ( Escherichia coli ) relative to beneficial bacteria ( Lactococcus lactis ). Although no significant increase in deleterious somatic mutations was found in IBD-CRC. 16sRNA revealed increased virulent- and decreased anti-inflammatory symbiotic-bacteria correlating with the upregulation of oncogenes and downregulation of anti-oncogenes like PHLPP1. The multiplex in situ hybridization of CD8, IFNγ and PHLPP1 an anti-oncogene revealed significant decrease of immune cells with detectable PHLPP1 expression in IBD-CRC tumour tissues as compared to sporadic CRCs. Conclusion The poor outcomes in IBD-CRC patients are likely due to gut dysbiosis and immune cell alterations, possibly triggered by microbiota-related epigenetic pathways. What You Need to Know BACKGROUND AND CONTEXT Colorectal cancer (CRC) is associated with gut microbiota dysbiosis. Inflammatory bowel disease-related CRC (IBD-CRC) is classified as an environment-related condition. NEW FINDINGS In relation with patient outcomes, tumour tissues from three types of CRC (Sporadic-, IBD-, and Lynch syndrome-CRC) were analyzed using a multiomic approach. This included examining tissue adherent virulent bacteria, gene analyses, and quantifying immune cell infiltration in the mucosa. IBD-CRC patients had the worst outcomes, associated with the down regulation of PHLPP1 gene, virulent/symbiotic imbalance, and immune response failure. LIMITATIONS Lack of animal experiments using FMT of fresh stool from IBD-CRC patients. CLINICAL AND TRANSLATIONAL RESEARCH RELEVANCE Among the different types of CRC, IBD-CRC patients showed a greater imbalance between harmful and beneficial bacteria, along with immune response failure. Lay summary This study compares the pathological and clinical characteristics of patients with colorectal cancer (CRC) across three distinct etiologies: sporadic CRC, inflammatory bowel disease (IBD)-associated CRC, and Lynch syndrome-associated CRC (LS-CRC). Distinct differences in tumor-adherent microbiota, gene expression and immune response profiles were observed. Notably, IBD-CRC patients demonstrated the poorest prognosis depending on microbe-host gene interaction highlighting potential biomarkers for disease prognosis and treatment strategies.

e16062 Background: Lynch syndrome (Ly) but not Sporadic (Sp) CRC patients are responsive to immunotherapies. We have shown micorbiota from CRC patients induces precancerous colonic lesions in germ-free mice. The aim was to investigate microbiota linked compromised anti tumor immune response. Methods: We compared 1) Whole metagenomic fecal microbiotas of CRC patients [Sp, n = 53+Ly, n = 19] to controls (n = 90) or being first degree relatives of Ly patients (n = 14), respectively; 2) Tumoral to normal mucosa-adherent microbiota (16sRNA sequences); 3) Normal to tumoral tissues’ mRNA RT-PCRs of IL1b, IL4, IL6, IL8, IL17, TNFa, TGFb, FasL, Granzyme A&B, Thy1; 4) CD3, CD4, CD8, Granzym, KL1, FoxP3, RORgT, MastCell, cells in tumor tissues quantified by immunohistochemistry (IHC), between subgroups. Associations between bacteria, tissue immune cell infiltrates and clinical data (3-yrs follow up or death) were established by using unsupervised Systems Biology and Bioinformatics analyses (Institut Pasteur Shaman platform and Qlucor program). Anti tumoral immune response links with bacteria were based on differences between Ly and Sp CRC patients. Tumor tolerance links with bacteria were identified using germ-free mice (n = 185) who orally received microbiota (9 Sp CRC patients vs 9 controls) with colonic mucosa examined 3 months after human fecal transfer (whole exome sequencing, methylchips, IHC for CD3, CD11b, FoxP3, RORgT, ILC3). Results: Enrichment in Peptostreptococcus, Thermodesulfovibrio and diminution in Ruminococcus, Eubacterium, Coprococcus were linked with FoxP3 and IL-17 over expressions in tumor tissues. Enrichment in Escherichia, Enterobacter, Thioalkalimicrobium, and diminution in Eubacterium, Faecalibacterium and Butyrovibrio were associated with higher FoxP3, FasL, Granzyms A&B, Perforin, Thy1 mRNAs/mRNACD3. Diminution of Eubacterium, Dorea, Faecalibacterium genera and enrichment in Fusobacterium, E. Coli/Shigella were associated with higher CD3, FoxP3, GranzymA and B and FOXP3-RORgT cells in tumor tissues. Higher fecal enrichments in oral bacteria (parvimonas micra) and lack of Thermodesulfovibrio enrichment were associated with higher FoxP3-RORgt cells and short survival; in mice ILC3 and FoxP3-RORgT cells were linked with Sp CRC dysbiosis. Species linked with immunotolerance are identified. Conclusions: Fecal and tissue microbiota can impact failure of anti tumoral immune response in sporadic CRC patients by enhancing overexpression of IL-17 and reducing cytotoxic T-cell infiltration in tumors. Clinical trial information: NCT01270360 .

Sporadic colorectal cancer (CRC) is a result of complex interactions between the host and its environment. Environmental stressors act by causing host cell DNA alterations implicated in the onset of cancer. Here we investigate the stressor ability of CRC-associated gut dysbiosis as causal agent of host DNA alterations. The epigenetic nature of these alterations was investigated in humans and in mice. Germ-free mice receiving fecal samples from subjects with normal colonoscopy or from CRC patients were monitored for 7 or 14 wk. Aberrant crypt foci, luminal microbiota, and DNA alterations (colonic exome sequencing and methylation patterns) were monitored following human feces transfer. CRC-associated microbiota induced higher numbers of hypermethylated genes in murine colonic mucosa (vs. healthy controls’ microbiota recipients). Several gene promoters including SFRP1,2,3, PENK, NPY, ALX4, SEPT9, and WIF1 promoters were found hypermethylated in CRC but not in normal tissues or effluents from fecal donors. In a pilot study ( n = 266), the blood methylation levels of 3 genes ( Wif1 , PENK , and NPY ) were shown closely associated with CRC dysbiosis. In a validation study ( n = 1,000), the cumulative methylation index (CMI) of these genes was significantly higher in CRCs than in controls. Further, CMI appeared as an independent risk factor for CRC diagnosis as shown by multivariate analysis that included fecal immunochemical blood test. Consequently, fecal bacterial species in individuals with higher CMI in blood were identified by whole metagenomic analysis. Thus, CRC-related dysbiosis induces methylation of host genes, and corresponding CMIs together with associated bacteria are potential biomarkers for CRC.