Fusobacterium nucleatum is enriched in invasive biofilms in colorectal cancer

Abstract

Fusobacterium nucleatum is an oral bacterium known to colonize colorectal tumors, where it is thought to play an important role in cancer progression. Recent advances in sequencing and phenotyping of F. nucleatum have revealed important differences at the subspecies level, but whether these differences impact the overall tumor ecology, and tumorigenesis itself, remain poorly understood. In this study, we sought to characterize Fusobacteria in the tumor microbiome of a cohort of individuals with CRC through a combination of molecular, spatial, and microbiologic analyses. We assessed for relative abundance of F. nucleatum in tumors compared to paired normal tissue, and correlated abundance with clinical and pathological features. We demonstrate striking enrichment of F. nucleatum and the recently discovered subspecies animalis clade 2 (Fna C2) specifically in colon tumors that have biofilms, highlighting the importance of complex community partnerships in the pathogenesis of this important organism.

Fig. 1. The CRC cohort.

A Flow diagram of study participants and downstream analysis of samples. B Demographic characteristics of the cohort (n = 116), including age, sex, ethnicity, stage, and tumor location. EO-CRC early-onset colorectal cancer, defined as age under 50. LO-CRC late-onset colorectal cancer, defined as age 50 or above.

Fig. 2. Biofilm analysis of the CRC cohort.

A The location and biofilm status of CRC tumors overlaid on a diagram of a colon. One tumor marked as unknown had no fixed tissue suitable for screening. B Biofilm status of CRC tumors classified as either positive or negative by FISH. C Percent of left and right-sided tumors positive for biofilms. D–F Representative 40X images of multi-probe FISH of each biofilm subtype stained for DAPI (blue), Bacteroidetes (green), Fusobacterium (yellow), Lachnospiraceae (red), and Proteobacteria (pink). G Percent of tumors positive for biofilms stratified by tumor location and stage. C, G analyzed by Fisher’s exact test, with p < 0.05 considered significant. H Stacked bar graph depicting percent of tumor biofilms of each subtype screened by multi-probe FISH (n = 70) and stratified by tumor stage. Analyzed by nonparametric Kruskal-Wallis one-way ANOVA, with p < 0.05 considered significant.

Fig. 3. Abundance of Fusobacterium species in tumor/normal pairs.

Relative abundance on a log scale by 16S rRNA amplicon sequencing of each depicted Fusobacterium species in pairs of tumor (blue circle) and normal (clear circle) tissues. Bars indicate median. Of 112 individuals with CRC in the study, 108 tumor and normal pairs were available for 16S rRNA sequencing analysis. Numbers below each graph depict the number of samples out of the total in which each species was not detected by sequencing (i.e. 0% abundance). Analyzed by two-tailed Wilcoxon matched-pairs signed rank test, with p < 0.05 considered significant. See Fig. S7 for abundance of F. nucleatum as a sum of confident and ambiguous species assignments, and for F. varium abundance stratified by ethnicity.

Fig. 4. Fusobacterium nucleatum abundance by individual and tumor characteristics.

Relative abundance by 16S rRNA amplicon sequencing of Fusobacterium nucleatum in pairs of tumor (blue circle) and normal (clear circle), stratified by A sex, B age, C ethnicity, D tumor location, E tumor stage, F biofilm (BF) status, and G both biofilm status and tumor stage. Bars indicated median. Of 112 individuals with CRC in the study, 108 tumor and normal pairs were available for 16S rRNA sequencing analysis. Numbers below each graph depict the number of samples out of the total in which F. nucleatum was not detected by sequencing. Analyzed by two-tailed Wilcoxon matched-pairs signed rank test, with p < 0.05 considered significant. EO-CRC early-onset colorectal cancer. LO-CRC late-onset colorectal cancer. See Fig. S7 for abundance of F. nucleatum as a sum of confident and ambiguous species assignments, stratified by biofilm status. H Matrix of F. nucleatum and other oral biofilm organism abundances in tumor samples, analyzed by nonparametric Spearman correlation coefficient. See Fig. S8 for relative abundance of each organism in the matrix in tumor/normal pairs.

Fig. 5. Fusobacterium nucleatum subspecies animalis abundance by clade and correlated with FISH.

A Relative abundance by 16S rRNA amplicon sequencing of Fusobacterium nucleatum subspecies animalis clade 1 (Fna C1) and clade 2 (Fna C2) in pairs of tumor (blue circle) and normal (clear circle). Tumor/normal pairs analyzed by two-tailed Wilcoxon matched-pairs signed rank test, and tumor/tumor or normal/normal comparisons analyzed by two-tailed Mann Whitney test, with p < 0.05 considered significant. B Relative abundance of Fna C1 and C2 stratified by biofilm (BF) status and C, D by both biofilm status and tumor stage. Bars indicated median. Of 112 individuals with CRC in the study, 108 tumor and normal pairs were available for 16S rRNA sequencing analysis. Numbers below each graph depict the number of samples out of the total in which each clade was not detected by sequencing. E Heatmap depicting relative abundance by 16S rRNA amplicon sequencing for the Fusobacterium genus and each labeled Fusobacterium species or clade for n = 29 tumors where dense blooms of Fusobacteria were visualized by FISH.

Fig. 6. PICRUSt2 pathways associated with tumors, biofilms, and Fna C2 abundance.

A Heatmap of all statistically significant PICRUSt2 pathways with differences in gene content between tumor (n = 112) and normal (n = 112) samples for either biofilm-positive tissues (left) or biofilm-negative tissues (right), as analyzed by Mann Whitney test, with p < 0.05 considered significant. Data depicted as Log₂ Fold Change between tumor and normal samples (>0 indicates higher in tumor). B For each indicated comparison, the listed pathways are the 4 PICRUSt2 pathways with the greatest Log₂ Fold Change between groups. For each pathway, the genera listed are the top 3 taxa contributing to enrichment of that pathway. See Figs. S12, S13, and S14A for the full display of significantly enriched or depleted pathways for each comparison. See Table S4 for all taxa significantly contributing to each enriched pathway.