Genomic analysis identifies association of Fusobacterium with colorectal carcinoma

Abstract

The tumor microenvironment of colorectal carcinoma is a complex community of genomically altered cancer cells, nonneoplastic cells, and a diverse collection of microorganisms. Each of these components may contribute to carcinogenesis; however, the role of the microbiota is the least well understood. We have characterized the composition of the microbiota in colorectal carcinoma using whole genome sequences from nine tumor/normal pairs. Fusobacterium sequences were enriched in carcinomas, confirmed by quantitative PCR and 16S rDNA sequence analysis of 95 carcinoma/normal DNA pairs, while the Bacteroidetes and Firmicutes phyla were depleted in tumors. Fusobacteria were also visualized within colorectal tumors using FISH. These findings reveal alterations in the colorectal cancer microbiota; however, the precise role of Fusobacteria in colorectal carcinoma pathogenesis requires further investigation.

Figure 1.

Whole-genome sequencing analysis of the colorectal cancer microbiome. (A) Schematic of experimental and computational whole-genome sequencing analysis workflow. (B) Hierarchical clustering of phylotype relative abundance measurements demonstrates that microbial composition of tumor/normal pairs within individuals is more highly correlated than tumor/tumor pairs or normal/normal pairs from different individuals. (Green) Normal samples; (purple) tumors. (C) Linear discriminant analysis (LDA) coupled with effect size measurements identifies Fusobacterium as the most differentially abundant taxon in colon tumor versus normal specimens by whole-genome sequencing in nine individuals. Tumor-enriched taxa are indicated with a positive LDA score (black), and taxa enriched in normal tissue have a negative score (gray). Only taxa meeting an LDA significant threshold of 1.8 are shown. (D) Percent relative abundance for the genus Fusobacterium is depicted across all samples in the order of the labels in B, demonstrating a tumor-enrichment in most individuals.

Figure 2.

16S rDNA sequencing analysis of the colorectal cancer microbiome. (A) Schematic of experimental and computational 16S rDNA sequencing analysis workflow. (B) Beta-diversity distances calculated using phylotype relative abundance measurements between all pairs of samples demonstrate that the microbial composition of tumor/normal pairs within individuals is more highly correlated than tumor/tumor pairs, normal/normal pairs, or tumor/normal pairs from different individuals. (C) Linear discriminant analysis (LDA) coupled with effect size measurements identifies Fusobacterium as the most differentially abundant taxon in colon tumor versus normal specimens by 16S rDNA sequencing in 95 individuals. Tumor-enriched taxa are indicated with a positive LDA score (black), and taxa enriched in normal tissue have a negative score (gray). Only taxa meeting an LDA significant threshold of 4.2 are shown. (D) A cladogram representation of data in C. (Red) Tumor-enriched taxa; (blue) taxa enriched in normal tissue. The brightness of each dot is proportional to its effect size.

Figure 3.

Fluorescence in situ hybridization (FISH) detects enrichment of Fusobacteria in colorectal tumors. (A) FISH using an Oregon-Green 488-conjugated ‘‘universal bacterial'' 16S rDNA-directed oligonucleotide probe (EUB338, green) (top left); and Cy3-conjugated Fusobacterium (FUSO, red) (top right and bottom center) 16S rDNA-direct oligonucleotide probe demonstrates the presence of bacteria and Fusobacterium within the colonic mucosa of colorectal tumor samples. Representative images are shown with a 10-μm scale bar in the lower corner of each panel; white arrowheads mark bacteria. Epithelial cell nuclei were stained with DAPI. (B) To determine whether Fusobacterium was enriched in tumor versus normal pairs, three random 40× fields were chosen for scoring by an observer blind to tumor/normal status, using selection criteria of mucosal tissue depth and a minimum of five bacteria visualized by the EUB338 probe per field. Each dot represents data from either a tumor or normal sample from nine tumor/normal paired cases. The mean, SEM, and P-values (calculated by a Wilcoxon matched-pairs signed rank test) are shown.

Figure 4.

Phylogenetic analysis identifies several Fusobacterium species in human colon cancer tissues. (A) Approximately-maximum-likelihood phylogenetic trees were constructed on the V3–V5 region of the 16S rDNA gene using 31 reference Fusobacterium species along with the five most prominent OTUs identified in colon cancer specimens (indicated in red). Nodes that have bootstrap support above 50% and 75% are indicated with a white and black dot, respectively. The mean percent relative abundance in tumor (T) and normal (N) of each OTU is indicated in parentheses. The full names of the reference strains appear in Supplemental Table S6. (B) The abundance of the indicated OTU relative to all other phylotypes in a given specimen is shown for the two most abundant Fusobacterium OTUs in tumors (x-axis) and normal colon tissue (y-axis); each point represents tumor and normal abundance data for a different individual. The lower-right quadrant of the graph highlights the substantial proportion of patients for whom the Fusobacterium abundance is >10% in tumors but <10% in the matched normal.