Colorectal cancer-associated microbiota contributes to oncogenic epigenetic signatures

Human Fecal Microbiota Transfer to Germ-Free Mice: Clinical and Routine Biochemical Parameters Among Groups.

We performed human fecal microbiota transfer (FMT) to germ-free mice utilizing material from patient donors with CRC (n = 9) and healthy control donors (n = 9) as illustrated in SI Appendix, Fig. S1. In addition, mice received the carcinogen azoxymethane (AOM) or saline control for 10 wk in order to assess potential effects of chemically induced stress. As shown in SI Appendix, Tables S1 A and B, FMT had no effect on the general blood parameters of the recipient mice as no significant differences were observed among any of the groups (a total of 185 mice) at 7 and 14 wk. At 14 wk, mice that received both AOM and CRC microbiota (CRC-μ) showed a lower mean body weight compared to the normal microbiota (N-μ) controls. This was accounted to reduced food intake observed in the CRC-μ group when compared to the N-μ controls (1.5 ± 0.1 g/100 g food intake vs. 2.0 ± 0.1 g/100 g food intake, respectively; P < 0.01). Interestingly, this weight effect disappeared when mice were given PEG in their drinking water, which is known to prevent bacterial colonization of the gut (SI Appendix, Fig. S2). In line with this, the luminal bacterial load, adherence of luminal bacteria to the colonic mucosa, and short-chain fatty acid (SCFA) concentrations in the cecum were significantly lower in mice that received PEG as compared to the controls (SI Appendix, Fig. S2 A and B). These results suggest that human CRC FMT may induce systemic changes in mice mediated by the interaction of bacteria with the colonic mucosa.

Dysbiosis-Related Histological Alterations in Colonic Tissue After Human FMT in Mice.

In order to delineate the predominant modifications occurring in murine colonic tissues following FMT, the number of aberrant crypt foci (ACF), i.e., clusters of enlarged thick crypts visualized by methylene blue at low magnification, were enumerated. Whereas histological features appeared normal in the group of mice receiving only N-μ, mild inflammation and ACF were seen at significant levels in the groups given CRC-μ, AOM, or both (Fig. 1 A–D, H, and I). After 7 or 14 wk, ACF numbers were highest in the group given CRC-μ + AOM, compared to all other groups, and were also significantly higher in the groups given CRC-μ alone or AOM alone, compared to the N-μ only group (Fig. 1 H and I). No ACFs were seen in mice given PEG, in which the microbiota was efficiently reduced in the mucus layer proximal to the mucosa. These latter samples were therefore not further examined. According to the colon length, numbers of ACFs became significantly higher in groups given CRC-μ versus N-μ, when only 20 mm of the left colonic mucosa was examined (13 ± 3 versus 5 ± 1; P < 0.05; Fig. 1I).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Histological patterns of murine colonic mucosa following FMT. After the intestine was removed from cecum to anus, mucosa was carefully pinned flat, without folds, to examine the totality of the colonic mucosa which were stained with 0.2% methylene blue (left slides of coupled slides A to D) or HES (right slides of coupled slides A to D). Numbers of mice were as follows: n = 53 in 7-wk study (CRC-μ transfer, n = 30, and N-μ transfer, n = 23) and n = 132 in 14-wk study (CRC-μ transfer, n = 66, and N-μ transfer, n = 66). (Scale bar: 50 μm.) (A) After FMT from healthy human controls (N-μ), no ACF were visible in the colonic mucosa (here after 14 wk in Left), and pattern of crypts was normal by HES staining (Right). (B) Elevated numbers of ACF were observed after FMT from patients with CRC (CRC-µ) as compared to N-μ with multiple ACF (arrow in Left) as verified by HES (Right). (C) ACF counts were higher in the animals given AOM as compared to N-μ. Arrows indicate double ACF under blue coloration (Left) and illustration on HES slide of colonic mucosa. (D) The combination of CRC-µ and AOM increased the ACF count with dysplasia in rare cases (arrow). (E) No ACF nor inflammatory cell infiltrate was visible in mice given N-μ under NaCl (HES), although FISH staining showed density of bacteria trapped in the mucus layer (arrow, Left). (F) No ACF and no inflammatory cell infiltrate nor injury were noticed when mice received polyethylene glycol (PEG) in their drinking water as shown by H&S staining (HES) when FISH staining shows clear decrease in density of bacteria trapped in the proximal mucus layer (arrow). (G) Representative pictures of KI67 staining after human FMT from patients with CRC (CRC-µ) or from N-μ recipients. (H) Cell proliferation assessed by Ki67 staining and ACF quantification 7 and 14 wk after FMT in mice in the intestinal mucosa were higher in CRC-µ than in N-μ recipients. (I) The number of ACF counts were enhanced depending on the length of mucosa examined and the mice subgroups. (J) Comparative transcriptional levels of a set of inflammatory cytokines in the colonic mucosa assessed by murine cytokine qPCR quantification showing a trend to higher IL1, IL6, MIP2, and IL17 and lower IFNγ, IL10, and IL23 in mice given CRC-μ alone (fold vs. N-μ given mice) with AOM boosting this effect that reached significance for IL6, TNFα, and IL10 in mice given CRC-μ + AOM (vs. N-μ + AOM). (K) Inflammatory cell infiltrate in the colonic mucosa as assessed by semiquantification on HES stained slides (10 consecutive fields) under optic microscope magnification 20: a pathologist blinded to animal groups used a semiquantitative score to evaluate myeloid cell infiltrate in the colonic mucosa as 0, 1, and 2 indicating absence, scarce, and numerous inflammatory cells, respectively. The groups were compared by 1-way ANOVA followed by the Tukey–Kramer multiple comparisons post hoc test. No significant difference in between mice groups was observed. *P < 0.05; NS, not significant.

Human FMT to Germ-Free Mice: Effect on Epithelial Proliferation and Gene Expression.

Epithelial cell proliferation in the colonic mucosa was visualized by KI67 staining (as illustrated in Fig. 1G). At 7 wk following FMT, a significant increase of proliferation was observed in the groups given AOM as compared to all other groups (Fig. 1H). However, at week 14, cell proliferation was significantly greater in the CRC-μ, as compared to N-μ recipients with AOM exerting an additional effect (Fig. 1 H and I), and ACFs increased with the rate of epithelial cell proliferation. These results are consistent with those reported after FMT from only 5 CRC patients as compared to 5 control individuals’ microbiota (13). Further, to investigate the involvement of Wnt and Notch pathways that control intestinal cell fate, mRNA of key mediators was quantified. qPCR analysis of mRNA from the colonic mucosa of recipient mice revealed an increase of 1.7-fold, 1.9-fold, and 1.9-fold expression levels of the transcription factors HES1, KLF4, and ELF3, respectively, but not of MATH1 in CRC-μ compared to N-μ recipients (SI Appendix, Table S2).

Human FMT to Germ-Free Mice: Mucosal Inflammation.

Early after FMT (7 wk), a mild inflammatory response reflected by higher IL1β, IL6, and MIP2α levels and lower IL10, IL23, and INFγ levels was detected in CRC-μ recipient animals as compared to N-μ controls. AOM treatment tended to to amplify this stimulatory effect as assessed by mRNA cytokine quantification (Fig. 1J). In addition, semiquantitative evaluation of mucosal myeloid cells along the entire histologically examined intestine (10 fields per sample, 3 samples per mouse) showed a trend toward increased numbers in human CRC-μ as compared to N-μ recipients but without reaching statistical significance at any time point (Fig. 1K).

Microbiota Characterization Following Human FMT in Mice.

To assess for the impact of the microbial communities on the observed differences in the mucosal phenotypes, murine fecal samples were analyzed by 16S rRNA gene sequencing. At baseline, the fecal microbiota of the CRC-µ (n = 9 donors) group contained a higher proportion of Fusobacteria, Parvimonas, Butyrivibrio, Gemella, and Akkermansia and lower proportions of Ruminococcus, Bifidobacterium, Eubacteria, and Lachnospira, as compared to stools from the N-μ (n = 9 donors) group (SI Appendix, Table S3 and Fig. S3).

During the follow-up, all groups (n = 6 per group and at each time point) showed a moderate decrease in Coccoides, Clostridium leptum, and Bifidobacterium on day 7 and stability in the community of bacteria afterward, as estimated by qPCR (SI Appendix, Table S4). Based on 16S rRNA gene sequencing of mouse stools (n = 6 in each group at each time point of the follow-up), 85% of OTUs remained unchanged over time. Overall, the bacterial species most associated with significant histopathological alterations, i.e., precancerous lesions (e.g., ACF), were Firmicutes, Clostridia, and Clostridiales. These histological changes were also associated with lower counts of genera with an antiinflammatory effect (i.e., Faecalibaterium and Eubacterium) and of butyrate-producing bacteria (Firmicutes species) (SI Appendix, Fig. S4). Interestingly, the abundance of Coprococcus was lower and of Bacteroides was higher in CRC-μ vs. N-μ. Covariations of these 2 genera with histological changes characterized by ACF numbers and with mRNA levels of the previously quantified transcription factors in mice colonic parameters were analyzed. Coinertia analysis revealed that these parameters were associated with variation of the fecal bacteria. The first component of the analysis showed a significant separation between N-μ and CRC-μ recipients. This first component was significantly associated linearly with Bacteroides and Coprococcus genera abundances in mice stools (SI Appendix, Fig. S5). These allowed a significant separation between N-μ and CRC-μ recipients regarding bacteria and mRNA levels of key transcription factors involved in Wnt and Notch pathways.

Human FMT to Germ-Free Mice Alters DNA.

DNA mutations.

In order to analyze the potential of FMT to induce DNA modifications in the host, whole-genome sequencing of murine colonic mucosal tissues (n = 12, 3 in each group of recipients) was performed, covering 220,000 exons within 24,000 genes. Overall, the incidence of mutations at global exon/intron level was significantly higher in intestinal vs. spleen tissues. Results of the colonic mucosa showed a trend toward increased DNA alterations in exons or introns of CRC-μ compared to N-μ recipients, but this did not reach significance (Fig. 2A). In addition, mice given CRC-μ and AOM showed the highest levels of DNA alterations in exons or introns showing a trend to an additive effect of CRC-μ as compared to N-μ. Interestingly, Principal Component Analysis (PcA) scatter diagrams separated animal subgroups according to the total DNA changes in single nucleotides in the colonic mucosa or spleen samples suggesting a link between gene mutation and type of microbiota (N or CRC) or treatment (saline or AOM) given to mice (Fig. 2 B and C).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

DNA changes in mice after human FMT. Whole genome sequencing of total DNA extracted from colonic mucosa (n = 12) and spleen samples (n = 6) was performed. (A) All single-nucleotide polymorphisms (SNPs) in the colon and spleen samples are compared to the reference mouse genome (GRCm38), and mutation levels within gene segments are indicated. Mice given AOM showed the highest levels of DNA alterations in exons or introns with a trend of additive effect of CRC-μ as compared to N-μ. (B) Distribution of animal subgroups according to the total gene mutations. DNA changes in single nucleotides with PcA scatter diagrams for colonic or spleen samples in the groups of mice. The groups are identified by the type of human microbiota received (CRC-μ or N-μ for CRC patients’ or controls’ stool, respectively) and type of treatment (AOM or NaCl for azoxymethane or saline, respectively). (C) Correlation circle of targeted gene mutations in the colonic mucosa and spleen tissues according to PcA. Vector length reflects targeted gene mutation weight in the first 2 component analyses; targeted mutated genes are indicated (those of Wnt pathway in red color). (D) When mutations in all Wnt genes were pooled together, rates of mutations were significantly higher in mice given AOM with an additive effect of CRC-μ. The total number of mutations in Wnt pathway genes in both colonic mucosa and spleen (Sp) was the highest in the animals given the CRC-μ and AOM combination (see also SI Appendix, Fig. S6). The number of mutations was greater in colonic mucosa (but not in spleen tissues) with AOM combined with CRC microbiota compared to AOM combined with control microbiota. There was no significant effect in between colonic mucosa due to CRC-μ alone as compared to N-μ alone. Col, colonic mucosa; Sp, spleen. (E) DNA epigenetic changes were investigated by using mEPIC array (39). The methylation level of probes (n = 63,987) were estimated after bisulfite modification of DNAs. Changes based on the methylation of probes were investigated on DNAs from colon samples (n = 16; 4 mice from each experimental group): the level of methylated probes was quantified as reported (39) and ranged from 0 (not methylated) to 1 (fully methylated). DNAs were classified as unmethylated if the methylation value was <0.2 and as hypermethylated if the methylation value was >0.799. Overall, mean and median values of all probes pooled (n = 63,987) in each group of mice showed lowest values in the group of CRC-μ + AOM recipients and highest in control microbiota recipients. (F) Most of the probes were unmethylated in all animal groups; elevated numbers of both hypomethylated and hypermethylated probes were observed in the mice given CRC-μ + AOM. (G) Probes whose methylation level changed or remained unchanged under AOM in mice given N-μ or CRC-μ. Mice receiving CRC-μ had a greater number of genes with changed methylation levels. (H) Number of hypermethylated probes in each group showing highest level in mice given CRC-μ and AOM combination compared to all other groups. **P < 0.01, *P < 0.05; NS, not significant.

In order to investigate the local oncogenic potential of CRC-μ, we went on to perform in-depth analysis of the following selected gene pathways (number of genes analyzed): Wnt and β–catenin (19 genes), Notch (4 genes), PPAR (3 genes), SMAD (2 genes), TGF-β (2 genes), ACRV (2 genes), DKK (4 genes), TCF (2 genes), MYC (1 gene), and SOCS (1 gene). Changes were most prominent in pivotal Wnt pathway genes (with indel and single nucleotide polymorphisms in 11 Wnt genes), with no significant differences between the CRC-μ and N-μ groups. When all DNA mutations were pooled, however, the number of DNA changes was highest in the CRC-μ + AOM recipients vs. the N-μ recipients (Fig. 2D). Again, DNA changes were significantly more numerous in colonic than in spleen tissues. Interestingly, in the spleen, DNA changes were similar among AOM-exposed animals that received CRC-μ or N-μ. Finally, an unsupervised analysis between mutated genes (including only selected genes as indicated above) and recipient status showed that changes in Wnt, PPARγ, and Notch pathway genes were associated with CRC-μ recipient, with an additive effect of AOM treatment (Fig. 2C and SI Appendix, Fig. S6 and Table S2).

Gene Methylation in Human Colonic Samples: From Bench to Bed.

To investigate whether the gene methylation patterns observed in the mouse were also associated with CRC dysbiosis in humans, we first investigated methylated genes in CRC tissues and effluents such as blood and stool (n = 9) and in normal tissue effluent controls (n = 9), both obtained from the same individuals as for the FMT in mice (Fig. 3A). In order to develop a quick and easy methylation test, panels of genes were selected, based on the difference between normal and CRC in various effluents and tissues. Among the various genes classified as hypermethylated in the individual samples (tissue, stool, and blood) from the same individuals, only 8 (Wif1-regulating gene and SEPT9, SFRP1,2,3, PENK, NPY, and ALX4 genes) of these were common to all samples and were therefore subjected to further characterization (Fig. 3B and SI Appendix, Figs. S8–S11). We selected Wif1 to represent the Wnt pathway and NPY and PENK, 2 other tissue CRC-associated hypermethylated genes, for methylation testing, as at least either one was found hypermethylated in all tumor tissues. The sum of the methylation levels of the 3 selected genes was found to serve best when discriminating between effluents from normal and cancer individuals (details in SI Appendix). Finally, to conceive easy and reproducible biological testing for large cohort screening, a housekeeping gene plus selected genes (i.e., ALB, WIF1, NPY, and PENK) were combined in a multiplex procedure for qPCR measurement in blood. A cumulative methyl index (CMI) was determined in blood samples from participants with normal colonoscopy or with a cancer diagnosed (32 patients with CRC and 46 with extra colonic cancer; SI Appendix). To validate its performance, CMI assessment was performed in a pilot study including 266 individuals (SI Appendix, Table S5) from a well-characterized cohort designed as CCR1 (SI Appendix, Table S6) and in a final validation study including 999 individuals (Table 1) from a second cohort designed as CCR2 (SI Appendix, Table S7). The specificity and the sensitivity of CMI > 2 in blood was 95 and 59%, respectively, in the pilot study when those with positive fecal occult blood test (FOBT) in stool were 97 and 43%, respectively. In the validation study, half of individuals were enrolled through mass CRC-screening programs and had a new fecal immunochemical test (FIT) to directly measure human hemoglobin in stools, and all had a blood with a threshold of CMI > 2 for positivity.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

Identification of hypermethylated genes related to fecal microbiota in human. Overview on the strategy from experimental approach for the validation of gene methylated targets in human based on microbiota donors (CRC patients or controls) in germ-free mice experiments. Methylated genes in CRC-associated tissues and fluids were identified based on their power for showing differences between normal colonoscopy individuals and CRC patients. (A) Human tissues and effluents were submitted to methylation gene array. Based on significant differences of methylation values in CpG probes between control (n = 9) and CRC patient (n = 9) donors, genes were selected according to the promoter segments hypermethylated in CRC patients. (B) Bidimensional (Right) and tridimensional (Left) distribution of genes regarding the difference in methylation values are indicated; in red color are indicated 7 selected more discriminant genes regarding CRC patients and controls. D, difference.

Validation of a CMI.

Demographic, clinical characteristics, and results of CMI were compared between CRC patients and those with normal colonoscopy in the validation study; in addition, FIT was performed in those asymptomatic individuals who underwent colonoscopy, and we investigated whether positive FIT and CMI > 2 could be independent parameters for diagnosis of CRC (Table 1 and SI Appendix, Table S7).

A CMI > 2 correlated significantly with CRC in 999 individuals in the validation study. Interestingly, a tendency of CMI increase with age was noted; however, this observation did not reach significance. By keeping specificity of both tests superior to 95%, the negative predictive value (mean, extremes) of CMI (set at >2) test was better than FIT (set at >150 ng/mL): 84.09 [81.5 to 86.4] and 81.64 [77.5 to 85.3], respectively (SI Appendix, Table S8). A multivariate model including all individuals with and without neoplasia adjusted for age, gender, and FIT results showed CMI > 2 was independently associated with CRC (Table 1). Regarding CMI test and keeping with threshold for positivity set at ≥2, the sensitivity for the detection of CRC could reach 37% in the validation study.

After stratification on Tumor-Nodes-Metastasis classification from the American Committee on Cancer (SI Appendix, Table S9), the CMI value levels increased significantly and linearly with increasing tumor stages.

Dysbiosis and Associations with Methylation of Genes.

The 16S rRNA gene sequencing on stool samples (n = 513) indicated a great β-diversity in patients with CRC and in those with a CMI > 2 (P < 0.05). Furthermore, CMI levels were significantly correlated with dysbiosis. In the validation cohort, we hierarchically clustered bacterial phylotypes on the genus level based on the similarity of their dynamics in patients with CRC and in those with CMI > 2 (Fig. 4).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Distribution of bacteria in fecal microbiota from patients with CRC and controls with normal colonoscopy findings. Overall, 348 individuals from CCR2 cohort (173 asymptomatic individuals enrolled via a mass CRC-screening program and 165 patients from Vatnimad and symptomatic subcohorts, respectively) enrolled. Invasive carcinoma, carcinoma in situ, or specific carcinoma either on flat mucosa or within a polyp in the rectum or colon were defined as CRC (n = 177); controls had no malignancy or significant polyp visible by full colonoscopy (n = 171). (A) Pattern of microbiota clustering according to the diagnosis as assessed by principal coordinate analysis. The genus-level analysis based on distance matrix variances showed significant differences between CRC patients and controls. Fecal DNA was subjected to metagenomic sequencing of the conserved V3 to V4 region of the 16S rRNA gene. The amplicons were purified, quantified, and pooled and then sequenced on an Illumina MiSeq platform. For the analysis of 16S rRNA gene sequences, raw MiSeq FASTQ files were demultiplexed, quality-filtered using Trimmomatic, and merged. Taxonomic assignations were performed using Qiime2 (default parameters) with the SILVA-123 database. The statistical analysis was done with MetagenomeSeq (36). (B) Pattern of microbiota clustering according to the blood methylation test as assessed by principal coordinate analysis. Analysis of variance using distance matrices on 789 OTUs (metagenomeSeq_1.16.0) from 362 individuals (175 with normal colonoscopy findings and 187 with advanced neoplasia) demonstrated a significant difference between the groups with positive and negative blood CMI values (>2 and ≤2, respectively). (C) Distribution of genera in fecal microbiota in the groups with positive and negative blood CMI values (>2 and ≤2, respectively) (Shaman c3bi platform; Institut Pasteur, http://shaman.c3bi.pasteur.fr/). A maximum likelihood phylogenetic tree was tested (Individuals, Materials, and Methods). Brown to red colors indicate negative CMI results (≤2), and blue colors indicate positive CMI results (>2). Note that diversity was less in the group with a positive CMI compared to the group with a negative CMI.

Interestingly, principal coordinate analysis on the microbiota when incorporating the diagnosis and the level of blood methylation test (Fig. 4A), separated into 2 main clusters which differed significantly between participants with versus without a CMI > 2, regardless of whether they did or did not have CRC (Fig. 4B). A single cluster differed significantly in abundance between controls with and without a CMI > 2 (P < 0.05, Wilcoxon signed-rank test; Fig. 4C).

Although microbiota composition varied across cohorts, several genera such as Parvimonas and Parasutterella were more abundant in CRC patients in both cohorts, whereas Eubacterium was more abundant in controls (Table 2 and SI Appendix, Table S10). Parvimonas genus was also more abundant in individuals with blood CMI > 2 vs. ≤2 in the current as well as in our pilot study. The whole metagenomic analysis showed 20 bacteria species, including several Parvimonas species, differed in abundance in patients with CMI > 2 (n = 53) and in those with CMI ≤ 2 (n = 90) (Table 3).