Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment

Abstract

Increasing evidence links the gut microbiota with colorectal cancer. Metagenomic analyses indicate that symbiotic Fusobacterium spp. are associated with human colorectal carcinoma, but whether this is an indirect or causal link remains unclear. We find that Fusobacterium spp. are enriched in human colonic adenomas relative to surrounding tissues and in stool samples from colorectal adenoma and carcinoma patients compared to healthy subjects. Additionally, in the Apc(Min/+) mouse model of intestinal tumorigenesis, Fusobacterium nucleatum increases tumor multiplicity and selectively recruits tumor-infiltrating myeloid cells, which can promote tumor progression. Tumors from Apc(Min/+) mice exposed to F. nucleatum exhibit a proinflammatory expression signature that is shared with human fusobacteria-positive colorectal carcinomas. However, unlike other bacteria linked to colorectal carcinoma, F. nucleatum does not exacerbate colitis, enteritis, or inflammation-associated intestinal carcinogenesis. Collectively, these data suggest that, through recruitment of tumor-infiltrating immune cells, fusobacteria generate a proinflammatory microenvironment that is conducive for colorectal neoplasia progression.

Figure 1. Fusobacterium is enriched in adenoma versus adjacent normal tissue and detected at a higher abundance in stool from CRC and adenoma cases than from healthy controls

(A)Fusobacterium abundance for normal tissue (x-axis) vs adenoma (y-axis) is plotted. 29 matched adenoma normal tissues pairs were tested. Each symbol represents data from one patient (adenoma and normal tissue). (B) Fecal Fusobacterium abundance from healthy subjects (n=30), subjects with colorectal adenomas (n=29), and colorectal cancer (n = 27). See also Table S1.

Figure 2. Fusobacterium nucleatum promotes small and large intestinal tumorigenesis and is enriched in tumor tissues of Apc^Min/+ mice

(A) Colon tumor counts and histologic colitis scores from Apc^Min/+, Il10^–/– and T-bet^–/– X Rag2^–/– mice fed F. nucleatum (F. nuc), Streptococcus spp., or TSB control. Mice were started on the 8-week feeding regimen at 6 weeks of age. (B) Representative images of colons (ruler numbers in cm) and (C) colonic histological analysis of Apc^Min/+ mice (100 icrometer scale bar). (D) Fusobacterium abundance in matched tumor (T) versus normal (N) tissues from colons of Apc^Min/+ mice fed F. nucleatum measured by quantitative PCR. (E) Representative FISH images of colonic tumor and matched normal colonic tissue from an Apc^Min/+ mouse fed F. nucleatum using a Fusobacterium 16S rDNA-directed probe (50 icrometer scale bar). (F) Small intestinal aberrant crypt foci, adenoma, and adenocarcinoma counts and (G) histologic enteritis scores in Apc^Min/+ mice fed F. nucleatum, Streptococcus species, or TSB control. (H) Representative sections of small intestines from Apc^Min/+ mice (ruler numbers in cm). *** P < 0.0001, ** P < 0.001, * P < 0.01. See also Fig. S1.

Figure 3. F. nucleatum selectively expands myeloid-derived immune cells, but not lymphoid immune cells in the intestinal tumor microenvironment

Multicolor flow cytometric analyses: (A) Percentage (left panel) with representative density plots and the number (right panel) of intratumoral myeloid cells and lymphoid cells. Data for CD11b⁺ myeloid cells, CD3⁺CD4⁺ T cells, or CD3⁺CD8⁺ T cells are shown. Mean percentages ± s.e.m are shown within each plot. n= 6, 4, or 15 for not treated, S. sanguinis, or F. nucleatum. *** P < 0.001, ** P < 0.01, * P < 0.05 (B) A schematic of the gating strategy for subset identification of intratumoral myeloid cells. Representative density plots of myeloid cells are shown. The initial analysis region used CD11b vs. Gr-1 to identify CD11b⁺Gr-1⁺ MDSC population (violet boxes) and CD11b⁺Gr-1macrophage population (light green boxes). Arrows show the gating progression used to discriminate the 9 subsets. Each number on the density plot represents the corresponding subset of myeloid cells. (C) Cell number/gram tumor for myeloid cells from the treatment groups. Each symbol represents data from an individual mouse. See also Fig. S2.

Figure 4. A Fusobacterium-associated human colorectal cancer gene signature shared and validated in mice

(A) Immune cell types enriched in Fusobacterium-associated mouse tumors are shown with the human marker gene utilized to determine abundance in the TCGA CRC RNA-seq data set. (B) Ingenuity Pathway Analysis Biological Function Gene Ontology categories enriched for Fusobacterium abundance-correlating gene sets are shown. (C) Fusobacterium spp. transcript relative abundance are plotted for the 133 TCGA colon tumors (upper panel and lower panel x-axis) and scaled expression values for the top 50 ranked genes denoted as the row Z-score (y-axis) are shown in a heat map (lower panel) with a purple (low expression)–yellow (high expression) color scale. (D) Western blot of nuclear extracts from human colon cancer with a high or low Fusobacterium relative abundance. Fusobacterium relative abundance and western blot densitometry are shown in the lower panel. (E) qPCR analysis of a selection of the top 50 ranked genes in (B) in colon and small intestinal tumors from F. nucleatum vs TSB fed Apc^Min/+ mice. Data are represented as mean +/− SEM. Tumors from 6–9 mice per group were used. See also Fig. S3.