Fusobacterium Nucleatum Subspecies Animalis Influences Proinflammatory Cytokine Expression and Monocyte Activation in Human Colorectal Tumors

The gastrointestinal tract has a high density of commensal microbes that are not only important to digestive physiology but also critical to immune system development and function. However, an altered intestinal microbiota is believed to be a potential risk factor for colorectal carcinogenesis (1, 2). Early studies linked certain enteric bacterial infections with inflammatory bowel disease and colorectal cancer, particularly pathogenic bacterial species such as Streptococcus gallolyticus (3), Enterococcus faecalis (4), adherent Escherichia coli (5), and enterotoxigenic Bacteroides fragilis (6). However, the role of these species as causal factors for colorectal carcinogenesis remains to be determined.

Recent discoveries using next-generation sequencing and functional studies established an association between Fusobacterium spp. infection and colorectal cancer development (7, 8). Fusobacterium is a genus of anaerobic Gram-negative bacteria previously known to be oral pathogens. They are mainly associated with dental plaque biofilms and inflammatory periodontal diseases but are also found in individuals with extraoral infections (9). Fusobacterium encompasses at least 15 species (10, 11), of which Fusobacterium nucleatum was one of the most frequently identified species in periodontal and bowel diseases (9, 12). Moreover, the taxonomy of F. nucleatum consists of five subspecies—nucleatum, polymorphum, fusiforme, animalis, and vincentii—with fusiforme and vincentii indicated to be the same subspecies according to genomic analysis (13, 14). However, the etiological relationship between the F. nucleatum subspecies and colorectal cancer development remains to be determined.

It is known that colorectal mucosal immunity plays an important role in maintenance of mucosal symbiosis with the gut microbiota (15). Chronic relapse and remission of inflammation can result in repeated epithelial injury and DNA damage, leading to colorectal carcinogenesis (15). Inflamed mucosa and neoplasia secrete proinflammatory cytokines, including IL17A, IL1B (IL1β), IL6, and TNF, all of which are implicated to promote colorectal tumorigenesis (16, 17). The recruitment of myeloid-derived cells to the inflamed mucosa is partly mediated by chemokine signaling. Chemokine (C-C motif) ligand 20 (CCL20), also known as macrophage inflammatory protein-3α or liver activation-regulated chemokine, is one of upregulated chemokines in the inflammatory microenvironment (18, 19). CCL20 protein expression can be stimulated by lipopolysaccharide (LPS) and TNF via the NF-κB pathway (20). It is the sole high-affinity ligand for chemokine (C-C motif) receptor 6 (CCR6; ref. 21). The CCL20/CCR6 axis regulates recruitment of CCR6⁺ immune cells, including subsets of IL17-expressing T helper cells (Th17), regulatory T cells, and dendritic cells, to neoplastic lesions (22–25). Interestingly, colorectal cancer can hijack CCL20/CCR6 function to promote hepatic metastasis of colorectal cancers (19, 26–28). However, the mechanisms involving the tripartite relationships among specific bacteria, tumor cells, and immune cells in the tumor microenvironments have yet to be fully understood.

We hypothesized that specific Fusobacterium subspecies prevalent in colorectal cancer patients play critical roles in promoting proinflammatory response and neoplastic development. To test this hypothesis, we profiled the differential microbiomes in human colorectal cancers and adjacent mucosal tissue by high-throughput bacterial 16S rDNA sequencing. Then, using the Fusobacterium 16S gene-targeted sequencing, we identified the specific Fusobacterium spp. and subspecies associated with colorectal tumors. Moreover, we examined Th17 proinflammatory cytokine/chemokine expression in the paired colorectal tissue specimens using cytokine array analysis and specific ELISA. Finally, we assessed the phenotypes of colorectal cancer cells and monocytes responding to Fusobacterium infection in coculture systems. The results demonstrated that F. nucleatum ssp. animalis and dysregulated CCL20 protein expression are important factors associated with human colorectal cancer progression.

The human colon epithelial cell line CCD841CoN, colorectal cancer cell lines (SW480, HT29, HCT116, and RKO), and monocyte THP-1 were obtained from the ATCC. The human primary colorectal cancer cell line HCP1 was generated in our laboratory under a protocol approved by the Institutional Review Board (IRB) of The University of Texas MD Anderson Cancer Center (UTMDACC; ref. 29). Cell lines were routinely cultured in minimum essential medium or RPMI 1640 complete medium containing 10% FBS under standard human cell culture conditions at 37°C. The normoxic culture was performed in an incubator with 5% CO₂ and 21% O₂, whereas the hypoxic culture was carried in 5% CO₂ and 2% O₂. Cells were free of mycoplasma as confirmed using a MycoAlert mycoplasma detection kit (Lonza Group).

The F. nucleatum ssp. animalis Gharbia and Shah (ATCC 51191) type strain NCTC 12276 was obtained from the ATCC. The strain was cultured in CDC anaerobe 5% sheep blood agar plates (Becton Dickinson and Company) under anaerobic conditions using an AnaeroGen Compact system (Oxoid) with a 37°C incubator.

Surgical residual colorectal adenoma or adenocarcinoma and adjacent normal tissue specimens were collected from patients by members of the Department of Pathology at UTMDACC. Informed consent for use of the paired frozen specimens was obtained from the patients according to an IRB-approved protocol. Clinical characteristics of the patients in this study are listed in Supplementary Table S1. Aliquots of each of the tissue specimens were utilized for protein extraction and cytokine panel analysis; the tissue aliquots were also used in DNA extraction and bacterial taxonomic analyses (assays described below).

Total DNA was extracted from the frozen tissue specimens using a QIAamp kit (QIAGEN) according to the manufacturer's instructions and stored at −20°C prior to sequencing analysis. Microbial 16S ribosomal RNA (rRNA) gene sequencing was performed as described previously (30) with minor modifications. Briefly, the V4 region of 16S rRNA gene was amplified using PCR and sequenced on the MiSeq platform (Illumina) using a 2 × 250-bp paired-end protocol yielding pair-end reads. The primers used for amplification contained adapters for MiSeq sequencing and single-end barcodes, allowing for pooling and direct sequencing of PCR products (30). The read pairs were de-multiplexed based on the unique molecular barcodes, and reads were merged using the USEARCH software program (version 7.0.1090; ref. 31) with no mismatches and a minimum overlap of 50 bases. In addition, a quality filter was applied to the resulting merged reads, and reads with more than 0.05 expected errors were discarded. Custom analytic software packages and pipelines developed at the Alkek Center for Metagenomics and Microbiome Research (CMMR) at Baylor College of Medicine were used with the 16S rRNA gene profiling pipeline to provide summary statistics and quality control measurements for the sequencing run. 16S rRNA gene sequences were clustered into operational taxonomic units (OTU) at a similarity cutoff value of 97% using the UPARSE algorithm (32). OTUs were mapped to classification data in an optimized version of the SILVA database (33) containing only the 16S V4 region to determine taxonomies of bacteria. Abundances of OTUs were recovered by mapping the de-multiplexed reads to the UPARSE OTUs. A custom script was used to construct an OTU table from the output files for downstream analyses of alpha-diversity, beta-diversity, and phylogenetic trends in bacteria.

To further examine the 16S V4 reads corresponding to the Fusobacterium genus, we used a Fusobacterium 16S V4 consensus sequence as a query sequence for the data search. The reads containing the matched sequence were mapped to the nonredundant nucleotide collection (nr/nt) database from NCBI to determine the closest species identities. In addition, we performed Fusobacterium genus 16S rDNA–targeted PCR and Sanger sequencing to confirm the Fusobacterium spp. and subspecies in the specimens. Briefly, the Fusobacterium 16S rDNA was first amplified using PrimeSTAR high-fidelity DNA polymerase (Clontech) using outer primers (Univ-8F′ 5′-AGAGTTTGATCHTGGCTCAG-3′; FUSO-v5r 5′-GACCCCCAACACCTAGTAAT-3′) and then followed with inner primers for a nest PCR (FUSO-v2df 5′-GGGACAACATYTRGAAAYGRATGC-3′; FUSO-v4r 5′-GCYYACCTCTCCAGTACTCTAG-3′). The resulting 16S V2–V4 region amplicons (508 nucleotides length) were sequenced bidirectionally at CMMR. The entire sequences were used for mapping in the SILVA database to determine species identities. The PCR amplicons were also cloned and sequenced individually in a confirmatory study. In addition, we used a different set of inner primers (FUSO-v2f 5′-GCCTCACAGNTAGGGACAACAT-3′; FUSO-v4r 5′-GCYYACCTCTCCAGTACTCTAG-3′) to produce 16S V2–V4 amplicons in an independent study. The PCR products were gel-purified and sequenced directly using a V4 internal consensus primer (FUSO-cv4r: 5′-CCRCCTAGACGCGCTTTAC-3′) at the UTMDACC Sequencing and Microarray facility. This method can identify approximate 437 nucleotides length sequence of V2–V3 regions. The PCR amplicons were also cloned and sequenced individually for confirmation. The sequence data were analyzed using the Basic Local Assignment Search Tool, and sequence alignment was performed using the Clustal Omega software program to compare with the reference sequences in the Ribosomal Database Project database (http://rdp.cme.msu.edu/index.jsp).

qPCR analysis of 16S rRNA gene was carried out using HotStart-IT SYBR Green qPCR master mix (Affymetrix) with Fusobacterium-specific quantitative PCR primers (FUSO-v2f 5′-GCCTCACAGNTAGGGACAACAT-3′; FUSO-v4r 5′-GCYYACCTCTCCAGTACTCTAG-3′). Also, analysis of an internal control gene HNRNPK was performed using specific primers (HNRNPK-f2: 5′-GTCTCCCATCAAAGGACGTG-3′; HNRNPK-r1: 5′-GTCTCCAGGTCTCCCTCTTC-3′). The reactions and data processing were performed using a StepOnePlus Real-Time PCR System (Applied Biosystems).

Soluble proteins were extracted from frozen tissue specimens in cold PBS (pH 7.4) containing a protease inhibitor cocktail (Roche) using a Polytron homogenizer protocol (Kinematica). Quantitative Luminex cytokine assays were performed using a MILLIPLEX Human Th17 cytokine panel kit (HT17MG-14K-PX25; EMD Millipore) according to the manufacturer's instructions at the Functional Genomics and Microbiome Core in the Texas Medical Center-Digestive Diseases Center. Human CCL20 protein ELISA was performed using the extracted tissue protein solution with a Quantikine ELISA kit (R&D Systems) according to the manufacturer's protocol.

A Boyden chamber assay was used to assess cell migration in vitro. Briefly, human colorectal cancer cells and monocytes were prepared and suspended in antibiotic-free RPMI 1640 medium containing 1% FBS. Cells (2 × 10⁵) were then seeded in the upper chamber (filter pore size, 8 μm; Corning). A CCL20-neutralizing antibody (AF360; R&D Systems) or control IgG was added to the upper chamber at a final concentration of 0.4 μg/mL in the medium. F. nucleatum ssp. animalis (ATCC 51191) suspension was prepared by washing and diluting of the bacterium in PBS. An aliquot of bacterial suspension (1 × 10⁴ cells) or PBS (control) was added to the bottom chamber containing antibiotic-free RPMI 1640 medium with 1% FBS. After incubation under standard mammalian cell culture conditions for 24 hours, the upper chamber filters were fixed and stained using the PROTOCOL Hema 3 stain set (Fisher Scientific). Migrated cells on the bottom side of filter were counted in five random fields per filter. The ImageJ software program (National Institutes of Health, Bethesda, MD) was used for cell quantification.

Differences in values for the paired colorectal tumor and adjacent normal tissue specimens were assessed using the Wilcoxon matched-pairs signed rank test. Differences in the means of the cell line in vitro assays were assessed using a two-tailed unpaired Student t test. A P value < 0.05 was considered significant.

To determine profiles of the microbiome associated with human colorectal tumors and normal colorectal mucosa, we conducted Illumina MiSeq analysis of bacterial 16S rDNA in the frozen tissue specimens obtained from patients with colorectal cancer. This analysis yielded a total of 638,542 high-quality sequence reads, with an average of 15,963 reads per sample. Comparison of the microbial taxonomic compositions at the phylum level demonstrated that Fusobacteria was one of the most abundant phyla in the colorectal tissue specimens. The distribution between the tumor and normal mucosa was approximately an average of 15.3% Fusobacteria OTUs in the tumors versus 7.7% Fusobacteria OTUs in the normal tissue. We observed a similar result at the genus level of increased Fusobacterium in the tumor compared with mucosa (P < 0.05, Fig. 1). In addition, Gemella as a low abundant genus in the tissues was also increased in the tumor (P < 0.05). In contrast, Bacteroides, Lachnospiraceae, and Blautia were relatively overrepresented in the adjacent normal mucosa compared with tumor (P < 0.05).

In order to determine the Fusobacterium spp. and subspecies (ssp.) composition in the colorectal tumor and normal mucosa specimens, we first analyzed the Illumina MiSeq 16S sequencing data that mapped the Fusobacteria OTUs in the specimens. Using a Fusobacterium 16S V4 consensus sequence as a query sequence for the data search, we found reads mapping to F. nucleatum with higher hits in the tumor specimens (Supplementary Fig. S1). We then performed an independent experiment using the Fusobacterium genus–specific PCR and deep sequencing of 16S rDNA V2–V4 regions to determine Fusobacterium spp. and ssp. in the specimens. The results demonstrated that the Fusobacterium population in the specimens was composed of five species: Fusobacterium periodonticum, Fusobacterium canifelinum, Fusobacterium varium, Fusobacterium simiae, and F. nucleatum. Of these species, F. nucleatum was the most abundant (Fig. 2A). The sequence analysis also demonstrated reads matching four F. nucleatum subspecies—F. nucleatum ssp. nucleatum, F. nucleatum ssp. polymorphum, F. nucleatum ssp. fusiforme/vincentii, and F. nucleatum ssp. animalis—of which F. nucleatum ssp. animalis was unambiguously predominant (Fig. 2B).

To further validate the presence of F. nucleatum ssp. animalis in colorectal tumors, we performed additional confirmatory sequencing of the Fusobacterium 16S V2-V4 at a different sequencing core facility with cloning or without cloning of PCR amplicons. We found the cloned amplicons were all F. nucleatum ssp. animalis sequences (Supplementary Fig. S2A) but could not exhaust the possibility of finding other Fusobacterium species or subspecies in clone pools. We then used a Fusobacterium genus 16S consensus primer to sequence the amplicons without cloning. Results demonstrated that all sequences in four colorectal cancer specimens were again identical to that of F. nucleatum ssp. animalis (Fig. 3A; Supplementary Fig. S2A) except for one that contained a minor overlapping sequence aligned to potentially F. nucleatum ssp. nucleatum or F. nucleatum ssp. fusiforme/vincentii (Supplementary Fig. S2B). In a parallel experiment, we also sequenced the F. nucleatum ssp. animalis Gharbia and Shah (ATCC 51191) type strain as a positive control. Figure 3A shows the representative sequence alignments of the V2 hypervariable region of 16S rRNA gene. These sequences were derived from four specimens and compared with the type strain sequence and the Fusobacterium spp. reference sequences from the Ribosomal Database Project database. Taken together, these results confirmed that F. nucleatum ssp. animalis was the predominant Fusobacterium subspecies associated with colorectal cancer in the samples evaluated.

We then established a qPCR assay based on the confirmed 16S rDNA sequences of Fusobacterium spp., including F. nucleatum, F. simiae, F. periodonticum, and F. canifelinum, in the colorectal tumors. However, the qPCR primer specificity excludes F. varium. In addition, the qPCR also detects four F. nucleatum subspecies: animalis, fusiforme/vincentii, nucleatum, and polymorphum. Analysis of this subpopulation of Fusobacterium spp. and ssp. in the colorectal specimens showed that their relative abundance was significantly higher in the tumor than in the normal mucosa in the paired analysis (P < 0.05; Fig. 3B).

F. nucleatum is known for its invasive and inflammatory pathogenicity in cases of periodontal disease. We hypothesized that its prevalence with colorectal tumors likely induces a similar inflammatory response via activation of colorectal mucosal immune cells. To examine the cytokine expression in colorectal tissue obtained from our study patients, we performed two independent Luminex multiplex analyses of Th17 cytokines to compare the expression in colorectal tumor and adjacent mucosal tissue specimens. The analyses of first 16 pairs of specimens (Exploratory Set) and then additional 12 pairs of specimens (Confirmatory Set) demonstrated the significant increase of cytokines CCL20, IL17A, and TNF, but decrease of IL21, in the tumor (Table 1). The finding of differential expression of CCL20, IL17A, and TNF proteins supports the suspected inflammatory statuses of the human colorectal tumors. More importantly, the differential expression of CCL20 protein was not only significantly higher but also present at all stages of tumors (Fig. 4), suggesting that CCL20 protein is probably involved in the tumor initiation and progression throughout their entire courses. In addition to the Luminex assay, we also used a human CCL20-specific ELISA that confirmed the differential expression of CCL20 protein levels in the colorectal tumor specimens (Supplementary Fig. S3).

It is unclear whether Fusobacterium infection directly contributes to CCL20 protein upregulation in colorectal tumors. To determine the effect of Fusobacterium on CCL20 protein expression, we set up a human cell–bacterium coculture system under normoxic (optimal for human cell growth) and hypoxic (favorable for anaerobic Fusobacterium proliferation) conditions. F. nucleatum ssp. animalis (ATCC 51191 strain) was used to represent the predominant subspecies found in the colorectal tumors. Conditioned media were collected from cocultures and measured for the CCL20 protein concentrations by ELISA, which demonstrated that the CCL20 protein expression in HCP1 cells was markedly induced in the presence of F. nucleatum ssp. animalis under normoxic conditions (Fig. 5A). Note that HCP1 is a low-passage colorectal cancer cell line (29). However, we observed a very subtle increase of CCL20 protein expression in high-passaged colorectal cancer cell lines (SW480, HT29, HCT116, and RKO) but no induction in a noncancerous colonic cell line CCD841 after coculture with F. nucleatum ssp. animalis (Supplementary Fig. S4A). In contrast, THP-1 monocytes were highly responsive to presence of F. nucleatum ssp. animalis, which not only markedly induced CCL20 protein expression (Fig. 5B; Supplementary Fig. S4B) but also apparently induced cellular morphologic changes from unanchored round cells to anchored spiky cells (Supplementary Fig. S5). This phenotypic change suggests that the monocytes were activated by interaction with F. nucleatum ssp. animalis. This is similar to a previous finding that LPS isolated from F. nucleatum or Porphyromonas gingivalis can stimulate the THP-1 cells to express activation antigens and macrophage phenotype (34). Furthermore, we observed that hypoxic stress conditions effectively increased basal expression of CCL20 protein in HCP1 and THP-1 cells, with an additive effect on the CCL20 protein expression in THP-1 cells by F. nucleatum ssp. animalis (Fig. 5B). These results suggested that both Fusobacterium infection and a hypoxic microenvironment can induce upregulation of CCL20 protein expression in cancerous epithelial cells and immune cells and may together contribute to aberrant CCL20 protein expression in colorectal tumors.

On the basis of the observations described above, we hypothesized that F. nucleatum ssp. animalis infection-triggered cellular responses in both colorectal cancer cells and monocytes/macrophages can lead to downstream effects, such as increased chemokine-mediated cell migration. To test this hypothesis, we performed a cell migration assay using a Boyden chamber system in which we seeded HCP1 or THP-1 cells in the top chambers and placed F. nucleatum ssp. animalis suspension or control medium in the bottom chambers. In addition, to assess the effect of CCL20 protein expression on cell motility, we used a CCL20-neutralizing antibody to block the chemokine function in the Boyden chamber system. The results demonstrated that the THP-1–monocyte migration was markedly enhanced by incubation with F. nucleatum ssp. animalis (Fig. 5C). We also observed that migration of THP-1 cells was partially inhibited by the CCL20-neutralizing antibody, suggesting that CCL20 signaling is indeed involved in regulation of monocyte/macrophage motility. However, the HCP1 colorectal cancer cell migration was not affected by the presence of F. nucleatum ssp. animalis or CCL20-neutralizing antibody (Supplementary Fig. S6). These observations warrant further investigation to better understand the molecular mechanisms that regulate the interactions between F. nucleatum subspecies and human cells.

Fusobacterium infection has been implicated as one of possible etiological factors in human inflammatory bowel disease (35, 36) and colorectal adenoma and carcinoma (1, 7, 8, 37–39). However, the specific pathogenic/oncogenic species of Fusobacterium and mechanism are largely unclear. In this study, we used 16S rDNA sequencing analysis to profile microbiota in the paired colorectal tumor and mucosal tissue specimens and found that Fusobacterium OTUs were relatively enriched with colorectal tumor, a result that is in line with previous reports (7, 8, 40). However, the relative abundance of Fusobacterium in the specimens was quite variable as also observed by others (7), suggesting many other factors (e.g., antibiotic therapy and bowel preparation) potentially affecting bacterial dynamics in the specimens. In addition to the sequencing analysis, we established a qPCR assay and demonstrated differential distribution of Fusobacterium in the colorectal specimens. Further improvement of the assay specificity for detecting the disease-relevant species will help resolve some inconsistencies regarding the role of Fusobacterium in colorectal cancer (41–43).

Fusobacterium spp. and ssp. are very heterogeneous (10, 11). In our in-depth taxonomic analysis, we found that F. nucleatum was the prevalent species present in the tumor specimens we profiled. Our analysis further revealed that F. nucleatum ssp. animalis was the most dominant subspecies associated with colorectal cancer in the same specimens. Interestingly, a recent study of the fecal samples from colorectal cancer patients showed that F. nucleatum ssp. vincentii and F. nucleatum ssp. animalis were the top-ranked gut microbial species associated with colorectal cancer (44). Although we agree that the predominance of F. nucleatum sp. animalis was associated with colorectal cancer, our data did not support F. nucleatum ssp. vincentii as an abundant species in the tissue specimens of colorectal tumors (Fig. 3A; Supplementary Fig. S2). Further validation is critical for the identification of the specific Fusobacterium pathogen and its oncogenic mechanism(s) that, in turn, may theoretically be targets for prevention and possibly even treatment of colorectal cancer.

F. nucleatum ssp. animalis infections associated with human diseases have been documented in the literature. Studies have demonstrated an association of F. nucleatum ssp. animalis virulence with inflammatory periodontal diseases and adverse pregnancy outcomes (45, 46). Interestingly, F. nucleatum ssp. animalis was found more frequently in the isolates from gastrointestinal tract than in the isolates from oral sites (47). A draft genome sequence of F. nucleatum ssp. animalis was reported (48), but information of its unique genetic features is lacking. In addition, F. nucleatum ssp. animalis has two bacteriophage variants (49). Because bacteriophages are known to contribute to the host strain's virulent phenotype, the F. nucleatum ssp. animalis bacteriophage may be another layer of the complexity of this subspecies' virulence.

Investigators have explored the function of Fusobacterium spp. in carcinogenesis. Early studies demonstrated Fusobacterium strains that are invasive and proinflammatory in the oral mucosa (50). The strains can also invade colorectal cancer cells and induce cytokine secretion (51–53). In colorectal adenomas and carcinomas, invasive Fusobacterium spp. can be visualized using FISH (8, 54). In an Apc^Min/+ mouse model of colorectal cancer, F. nucleatum promoted intestinal carcinogenesis via the bacterial virulence factor FadA, which binds to E-cadherin on the host cell surface and activates β-catenin signaling and inflammatory response (52). On the other hand, F. nucleatum increased tumor multiplicity and selectively recruited tumor-infiltrating myeloid cells, predominantly myeloid-derived suppressor cells, with potent immunosuppressive activity (40). F. nucleatum infection has also been associated with NF-κB activation and proinflammatory gene expression (40, 53). Similarly, in the present study, we found that expression/secretion of proinflammatory cytokines IL17A and TNF was markedly higher in colorectal tumors than in adjacent mucosal tissue. In addition, we found that CCL20 protein was increased in the colorectal adenomas and carcinomas compared with mucosa.

CCL20 and its receptor CCR6 are known for their important roles in the recruitment of immune cells and their paradoxical functions in regulation of both immunological tolerance and inflammation (55). Clinical studies have established that overexpression of CCL20 and CCR6 is associated with colorectal cancer progression (19, 26, 27, 56). In preclinical studies, the CCL20/CCR6 axis has proven to be critical for intestinal tumorigenesis in mice. For example, using the mutagen MNU plus Helicobacter pylori to induce colorectal tumorigenesis in wild-type C57BL mice, investigators showed that tumor-associated macrophages recruited CCR6⁺ regulatory T cells to the tumor microenvironment via CCL20 signaling and promoted tumor growth (24). However, in Apc^Min/+ mice, knockout of CCR6 decreased spontaneous intestinal tumorigenesis via reduction of macrophage recruitment to the inflamed intestinal mucosa (57). Taking our own data into consideration for the F. nucleatum ssp. animalis interaction with colorectal cancer cells and monocytes, the response was stronger in the monocytes and showed a macrophage activation phenotype with increased migration and CCL20 protein expression, particularly under a hypoxic condition mimicking the tumor microenvironment. In a recent study of Fusobacterium and T-cell density in colorectal cancer, investigators showed that Fusobacterium-high cases were inversely associated with the density of CD3⁺ pan-T cells (58). Future studies need to address whether the Fusobacterium-induced cytokine/chemokine signaling, including CCL20/CCR6 axis, selectively regulates the inflammation and immune suppression in tumor microenvironment, thus promoting the colorectal tumor growth and progression.

In summary, we identified five species of Fusobacterium in clinical colorectal cancer specimens, of which F. nucleatum was the most predominant. We also identified and confirmed that F. nucleatum ssp. animalis was the most prevalent subspecies of F. nucleatum in colorectal tumors. Moreover, we found that colorectal tumors differentially expressed the proinflammatory cytokines IL17A, IL21, TNF, and CCL20, with CCL20 being highly expressed at every stage of CRC. In in vitro assays, coculture with F. nucleatum ssp. animalis markedly induced CCL20 protein expression in certain colorectal cancer cells. Similarly, the coculture strongly stimulated CCL20 protein expression in monocytes and induced monocyte/macrophage activation and migration, suggesting that F. nucleatum ssp. animalis interacts directly with monocytes, which may recruit other immunoregulatory cells via CCL20 signaling and subsequently promote colorectal cancer progression. These findings provide unique insight into the tumorigenic mechanism mediated by Fusobacterium spp. and mucosal immunity. More importantly, we identified a specific subspecies of tumor-associated F. nucleatum ssp. animalis that may be useful for early-stage detection of colorectal cancer and for antibiotic therapy or vaccination against for colorectal cancer.

No potential conflicts of interest were disclosed.

Conception and design: X. Ye, R. Bhattacharya, L.M. Ellis

Development of methodology: X. Ye, L. Xia, J.F. Petrosino

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Ye, R. Wang, F. Fan, L. Xia, N.J. Ajami, J.F. Petrosino, S. Venable, D. Maru, L.M. Ellis

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Ye, R. Bhattacharya, N.J. Ajami, M.C. Wong, D.P. Smith, J.F. Petrosino, W. Qiao, V. Baladandayuthapani

Writing, review, and/or revision of the manuscript: X. Ye, D.R. Boulbes, N.J. Ajami, S. Venable, L.M. Ellis

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.R. Boulbes, D.P. Smith, D. Maru, L.M. Ellis

Study supervision: L.M. Ellis

We thank Drs. James Versalovic and Jennifer K. Spinler of the Department of Pathology and Texas Children's Microbiome Center at Baylor College of Medicine, and Drs. Diane L.S. Hutchinson and Suzanne Kennedy of the Alkek Center for Metagenomics and Microbiome Research at Baylor College of Medicine for their help and discussion during this study. The authors also thank Donald R. Norwood of the UTMDACC Department of Scientific Publications for editorial assistance.

This work was supported in part by NIH Cancer Center Support Grant CA016672, NIH grant R01CA157880 (to L.M. Ellis), Department of Defense grant CA100879 (to L.M. Ellis), the William C. Liedtke, Jr., Chair in Cancer Research (to L.M. Ellis), and a Texas Medical Center-Digestive Disease Center (TMC-DDC) Pilot Award (to X. Ye). In addition, this work used the TMC-DDC core facility that was supported in part by PHS grant P30 DK56338 (to principal investigators including J.F. Petrosino).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.