Animals. All experiments with 129/SvEv GF ApcMinΔ850/+ and ApcMinΔ850/+;Il10–/– multiple intestinal neoplasia (Min) mice were conducted at the University of Florida. GF ApcMinΔ850/+ and ApcMinΔ850/+;Il10–/– mice were previously described (20). Experiments using GF C57BL/6 and GF or SPF C57BL/6 ApcMinΔ716/+ mice were conducted at Johns Hopkins University.

Biofilm evaluation and microscopy. Patient tissues were collected and screened for biofilms, as described previously (11, 15, 22). In brief, patient tumor (T) and normal flanking (NF, collected from the distal margin of the surgical resection) tissues were collected after surgical resection, and healthy control patient tissues were collected from the right and left colon by colonoscopy biopsy (bx). Part of the patient tissue was fixed in Carnoy’s solution (60% methanol, 30% acetic acid, and 10% chloroform) and biofilm status assessed by FISH with a universal bacterial probe (EUB338); bacterial density was quantified with ImageJ (NIH). Sections were stained with periodic acid–Schiff (PAS) to confirm mucus presence and preservation, and successive sections were hybridized with the EUB338 universal bacterial probe. Carnoy’s fixed tissues were defined as biofilm positive if there were more than 109 bacteria/ml for CRC or tumor patients or more than 108 bacteria/ml for colonoscopy biopsies invading the mucus layer (within 1 μm of the epithelium) for at least 200 μm of the epithelial surface. All human colon samples were screened twice in a randomized, blinded manner.

Because in mice, only the distal 3 cm of the colon contains a striated, nonpermissive mucus layer, at 1 week after infection, the distal 3 cm of each GF C57BL/6 mouse colon was separated from the remainder of the colon and fixed using Carnoy’s solution (38), with residual stool intact to preserve the mucus layer. This distal 3 cm region was screened by FISH with the EUB338 all-bacterial probe and DAPI for the relative percentage of the entire distal colonic epithelium that was in direct contact (within 1 μm) with bacteria. Each mouse colon was scored twice in a blinded, randomized manner, with the 2 scores averaged to obtain a final percentage for each mouse. Data presented are representative of 2 separate experiments, one with shams, BF-Bx, and BF+T, and a second with shams, BF-Bx, and BF+Bx. PAS stains of a sequential slide section were used to confirm the presence of mucus.

Human colonic biofilms were imaged as previously described (15). Confocal images of FISH-stained mouse colons were taken on a Zeiss LSM 780 META laser scanning microscope at ×400 with LSM Zen imaging software with linear unmixing in the Johns Hopkins University School of Medicine Microscope Core Facility, unmixed using Zen software, and finally merged in ImageJ. PAS-stained mouse colons were imaged on a Nikon Eclipse E800 microscope with a Nikon DXM1200 camera.

Human tissue inocula preparation. The study design was developed to give priority to reproducibly testing using multiple murine models the hypothesis that human mucosal microbiota populations could induce colon carcinogenesis. Thus, to allow for limitations in available human tissues and GF mice, we used inocula composed of tissue from 5 different patients (Supplemental Table 1). Biofilm negative (BF–) and positive (BF+) inocula were prepared from 3 mm diameter tissue pieces that were collected from healthy or CRC patients, snap-frozen, and stored at -80°C. The BF-bx and BF+bx inocula were pooled from separate groups of healthy patients, while the BF+NF and BF+T inocula were from the same set of CRC patients except in one instance (Supplemental Table 1). All inocula were prepared anaerobically by mincing and homogenizing tissue pieces in PBS in an anaerobic hood to a final dilution of 1:20 (weight/volume).

GF and SPF mouse colonization. Six- to fourteen-week-old GF 129/SvEv ApcMinΔ850/+ and ApcMinΔ850/+;Il10–/– mice (males and females) (University of Florida) were transferred to gnotobiotic isolators (separate isolator for each experimental group) and gavaged with 100–200 μl of inoculum. Mice were euthanized at indicated time points, and colonoscopy was performed as described previously (39) before sacrifice. The small intestine, cecum, and colon were cut open longitudinally, and macroscopic tumors were counted. About 1 to 2 × 0.5 cm snips were taken from the proximal and distal colon, flash-frozen in liquid nitrogen, and stored at –80°C until analysis. The rest of the colon was Swiss-rolled and fixed in 10% neutral buffered formalin solution. Swiss rolls were processed, paraffin-embedded, sectioned, and stained with H&E by the Molecular Pathology Core at the University of Florida. Histological scoring of inflammation was performed blindly by 2 investigators, as described previously (5). Whole colon inflammation scores were calculated as the average between the proximal and distal colon region scores, while distal colon inflammation scores represent the inflammation score in the distal colon region.

Murine reassociation experiments utilized 6- to 13-week-old GF 129/SvEv ApcMinΔ850/+;Il10–/– mice. For these mice, the inocula were anaerobically homogenized murine colon tissues of 4 mice originally inoculated with human BF-bx (for 12 weeks) or BF+T (for 16–20 weeks) inocula. Flash-frozen murine tissues were stored at –80°C until anaerobic inocula preparation. Once inocula were prepared, they were stored at –80°C until administration into mice as described above.

The 1-week association experiment for flow cytometry analysis with GF ApcMinΔ850/+ mice was completed in 2 mini-isolators (1 per inoculation group; see Results). For this experiment, the BF-bx inoculum was diluted 1:2, while the BF+T inoculum was diluted 1:4. Each mouse received 100 μl of the diluted inoculum, and mice were euthanized 1 week after inoculation. Colons were shipped overnight on ice to Johns Hopkins University, where flow cytometry analysis (below) was performed.

GF C57BL/6 mice (Johns Hopkins University) were similarly housed and inoculated, as indicated in Results, using individual isolators (1 isolator per condition), followed by mouse colon harvest after 1 week for biofilm analysis or flow cytometry, as described below. Prior to inoculation of approximately 6-week-old male and female SPF ApcMinΔ716/+ mice (Johns Hopkins University), mice were given water containing 500 mg/l cefoxitin for 48 hours. After removal of antibiotic water for 24 hours, mice were inoculated by oral gavage with human colon mucosal homogenates as indicated.

Lamina propria lymphocyte isolation. Colon lamina propria lymphocyte (LPL) isolation has been previously described (21). Briefly, colons were flushed, minced, and digested using 400 U/ml liberase and 0.1 mg/ml DNAase1 (Roche Diagnostics). Leukocytes were isolated by 60%/80% Percoll gradient separation (GE Healthcare Life Sciences).

Flow cytometry. Staining and flow cytometry analysis have been previously detailed (22). Briefly, LPL were stained for 30 minutes with the LIVE/DEAD Fixable Aqua Viability Stain (Thermo Fisher Scientific) and then washed. Fluorochrome-conjugated antibodies were added for cell-surface staining of lymphoid and myeloid cell subsets and incubated 30 minutes on ice. Stained cells were then washed and analyzed using an LSRFortessa flow cytometer (BD Bioscience) and BD DIVA software (TriStar Inc.). In some experiments, analysis of IFN-γ– and IL-17–producing cells was performed using intracellular cytokine staining (ICS), following a 3.5-hour in vitro stimulation of LPL in the presence of eBioscience Stimulation Plus Protein Transport Inhibitor Cocktail (PMA, ionomycin, Brefeldin A, Monensin; Thermo Fisher Scientific). IFN-γ, IL-17, and Foxp3 staining were performed after fixation of cell surface–stained cells using the Foxp3 Fixation/Permeabilization Kit (Thermo Fisher Scientific), as previously described (21). Mucosal dendritic cells were defined as CD11chiMHC IIhiCD103+, macrophages as CD11b+Ly-6C–Ly-6G–F4/80+I-E/Ahi (Mɸ-I-E/Ahi) or I-E/Alo (Mɸ-I-E/Alo), inflammatory MO as CD11bH\hiLy6ChiLy-6G–, PMN neutrophils as CD11bhiLy6G+Ly-6GLo, Tregs as CD3+CD4+Foxp3+, CD4 non-Tregs as CD3+CD4+Foxp3–, CD8+ T cells as CD3+CD4+Foxp3–CD8+, γδ T cells as CD3+CD4+Foxp3–CD8–γδTCR+, and NKT cells as CD3+CD4+Foxp3–CD8–γδTCR–. ILCs were defined as CD3–Thy1.2hi. The gating strategies have been previously detailed (22).

DNA extractions and 16S rRNA qPCR assay. DNA was extracted from human tissue inocula and biofilm-positive or biofilm-negative inoculated GF ApcMinΔ850/+ and ApcMinΔ850/+;Il10–/– stools (collected at 1 week and end point) and distal colon tissues (end point) using phenol/chloroform separation followed by DNeasy Blood & Tissue Kit (QIAGEN). 16S rRNA qPCR was performed on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad) using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). The following universal bacteria 16S rRNA gene primers were used: forward, AGAGTTTGATCCTGGCTCAG; and reverse, ACTGCTGCCTCCCGTAGGAG.

16S rRNA amplicon sequencing. The V1–V3 hypervariable region of the 16S rRNA gene was amplified using primer pairs 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 534R (5′-ATTACCGCGGCTGCTGG-3′). Both the forward and the reverse primers contained universal Illumina paired-end adapter sequences as well as unique individual 4- to 6-nucleotide barcodes between the PCR primer sequence and the Illumina adapter sequence to allow multiplex sequencing (Supplemental Table 4). PCR products were visualized on an agarose gel before samples were purified using the Agencourt AMPure XP Kit (Beckman Coulter) and quantified by qPCR with the KAPA Library Quantification Kit (KAPA Biosystems). Equimolar amounts of samples were then pooled and sequenced with an Illumina MiSeq.

16S rRNA-Seq analysis. Taxonomic ranks were assigned for the forward reads using the ribosomal database project (RDP) classifier (40) done with confidence set to 80%. Reads were grouped by genera, and the counts were normalized and log 10 transformed (41) using the following formula:

(Equation 1)

where RC is the read count for a particular genus in a particular sample, n is the total number of reads in that sample, the sum of x is the total number of reads in all samples, and N is the total number of samples. This equation is used to standardize the effect of adding the pseudocount to samples of different sequencing depth. The PCoA was generated from the Bray-Curtis distance of the normalized and log 10 -transformed counts using the capscale function in the vegan R package (42, 43).

Genera significant for biofilm group (BF-bx, BF+bx, BF+NF, or BF+T) were detected using the lme function in the R nlme package, with the REML method (44) to fit a generalized mixed linear model of the following form: genera ~ group + 1|cage + ɛ, where genera indicates the log 10 -normalized abundance of a particular genera, group indicates the biofilm group, and 1|cage indicates that we used the cage as a random effect. We then ran an ANOVA analysis on the above model to generate P values for the biofilm group. We filtered genera absent in more than a quarter of the samples. The P values for cage were calculated using an ANOVA of this model and a model with the cage removed (genera ~ group + ɛ). All P values were then adjusted for multiple hypothesis testing using the method of Benjamini and Hochberg (45). The heatmap was generated using the R function ggplot2 (46). The code and tables used to generate the 16S rRNA-Seq figures can be found at: https://github.com/afodor/biofilm

We performed 2 additional analyses on the 16S rRNA data, the first utilizing QIIME (47), version 1.9.1, closed reference at 97% similarity level using the Greengenes reference dataset release 13_8, and the second employing Deblur (48) workflow, version 1.0.3, with the default parameters (using Deblur’s default positive and negative reference filtering) and trim length set to 100 bases. Both pipelines showed similar broad separation between the BF+ and BF-bx samples (Supplemental Figure 6, C and D).

Functional prediction. Sequences were also analyzed by PICRUSt to infer functional content as previously described (11, 25). PICRUSt counts associated with functional categories were also normalized to an even total per sample within each data set. Relative contributions of higher-level taxa to functional categories were determined using the metagenome_contributions.py in the PICRUSt package.

RNA extraction, rRNA depletion, and RNA-Seq. Total RNA was extracted from frozen ApcMinΔ850/+ proximal colon tissue snips using the mirVana miRNA Isolation Kit with phenol (Thermo Fisher Scientific, catalog AM1560), according to the manufacturer’s instructions, with the addition of an approximately 1:1 mix of 1 mm acid-washed glass beads and 0.1 mm zirconia beads and a Precellys 24 (Bertin Instruments, catalog EQ03119-200-RD000.0) bead beater for tissue disruption and lysis. Extracted RNA was treated with the Turbo DNA-free Kit (Thermo Fisher Scientific, catalog AM1907) to remove DNA. Quality control, rRNA depletion, and cDNA library preparation were performed by the University of Florida’s Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression and Genotyping core using the Agilent 2100 Bioanalyzer (Agilent Genomics, catalog G2939BA), Ribo-Zero Gold rRNA Removal Kit (Epidemiology) (Illumina, catalog MRZE724), and the ScriptSeq, version 2, RNASeq Library Preparation Kit (Illumina, catalog SSV21124) starting with 1 μg total RNA. Samples were sequenced by the University of Florida ICBR NextGen DNA Sequencing core on the Illumina HiSeq 3000 (2 × 100 run), multiplexing each sample into 3 lanes to avoid lane effect.

Metatranscriptome analysis. Trimmomatic was used to quality filter and trim the RNA-Seq reads, followed by alignment to the iGenome Mus musculus Ensembl GRCm38 reference genome using BWA, version 0.7.16a; reads with alignments to the host genome were excluded from further evaluation. The remaining set of unaligned reads was next assessed for rRNA and tRNA transcripts by aligning (BWA) to a collection of NCBI rRNA and tRNA sequences and the SILVA rRNA database; reads with rRNA and tRNA alignments were also excluded from further evaluation. Protein-coding gene-expression quantification and annotation were performed by aligning the remaining reads to the human gut microbiome integrated gene catalog (IGC) (49) using Bowtie2 (version 2.3.4.2) followed by quantification using featureCounts from the subread package, version 1.5.3. Differential expression analysis was performed using edgeR; a gene was considered significant if its FDR-adjusted P value was less than 0.05.

Data availability. 16S rRNA sequences were uploaded to the NCBI’s Sequence Read Archive (SRA PRJNA423288). RNA-Seq data were deposited in the NCBI’s SRA (PRJNA510126).

Statistics. Statistics for 16S rRNA and RNA-Seq data are described in the above analysis sections. Flow cytometry data were analyzed by 2-way ANOVA with correction for multiple comparisons (Tukey’s test). Fisher’s exact test was used to analyze proportions and Spearman’s rank correlation to evaluate the statistical dependence between the rankings of 2 variables. The log-rank (Mantel-Cox) test and the log-rank test for trend were used for survival analyses. All other data were analyzed using the nonparametric unpaired, 2-tailed Mann-Whitney U test for 2-group comparisons or the Kruskal-Wallis test for 3 or more group comparisons as labeled for each figure. Data are presented as mean ± SEM unless otherwise stated. P < 0.05 was considered statistically significant except, as indicated in the text or figure legends, when Bonferroni’s correction was applied for multiple comparisons. Within figures, the NS label designates P > 0.05.

Study approval. Animal experiments performed at the University of Florida were approved by the University of Florida Institutional Animal Care and Use Committee, and animal experiments performed at Johns Hopkins University were approved by the Johns Hopkins University Institutional Animal Care and Use Committee. Patient tissues were collected as described previously (11, 15, 22). This study was approved by the Johns Hopkins Institutional Review Board. All samples were obtained in accordance with the Health Insurance Portability and Accountability Act (HIPAA).