Initial associations with human tissue-associated microbes.

GF 129/SvEv Apc^MinΔ850/+;Il10^−/− mice were inoculated with pooled tissue-derived microbes from biofilm-negative tissues collected from healthy patients via colonoscopy biopsy (BF-bx) or biofilm-positive tumor tissues collected from CRC patients (BF+T) via surgical resection. Patient tissues were collected and screened for biofilm status via fluorescence in situ hybridization (FISH) (biofilm-positive criteria, polymicrobial, within the mucus layer, spanning 200 μm, and >10⁹ bacteria/ml) as described previously (12). Tissues were analyzed with the universal bacterial probe (EUB338), and a nonsense probe (non338) was used as a negative control (12, 13). Additional FISH analysis was conducted on biofilm-positive tumor tissues with probes to detect Bacteroidetes, Lachnospiraceae, Fusobacteria, and Proteobacteria (12, 13). The probe sequences are listed in Table S4 in reference 12. Each inoculum was prepared anaerobically by homogenizing tissue (tissue pooled from five different patients) in phosphate-buffered saline (PBS), and FISH images for these tissues can be found in Fig. S1 in reference 13. Each mouse received 100 to 200 μl of inoculum, and associations were carried out for 12 to 20 weeks (Fig. 1AI). Tissues and/or stools from mice collected 12 weeks after association were used for transcriptome sequencing (RNA-seq), microRNA sequencing (miRNA-seq), and 16S rRNA gene sequencing analyses (Fig. 1AII).