A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

Embracing cancer complexity: Hallmarks of systemic disease.

The last 50 years have witnessed extraordinary developments in understanding mechanisms of carcinogenesis, synthesized as the hallmarks of cancer. Despite this logical framework, our understanding of the molecular basis of systemic manifestations and the underlying causes of cancer-related death remains incomplete. Looking forward, elucidating how tumors interact with distant organs and how multifaceted environmental and physiological parameters impinge on tumors and their hosts will be crucial for advances in preventing and more effectively treating human cancers. In this perspective, we discuss complexities of cancer as a systemic disease, including tumor initiation and promotion, tumor micro- and immune macro-environments, aging, metabolism and obesity, cancer cachexia, circadian rhythms, nervous system interactions, tumor-related thrombosis, and the microbiome. Model systems incorporating human genetic variation will be essential to decipher the mechanistic basis of these phenomena and unravel gene-environment interactions, providing a modern synthesis of molecular oncology that is primed to prevent cancers and improve patient quality of life and cancer outcomes.

Bacteria-derived L-lactate fuels cervical cancer chemoradiotherapy resistance.

Accumulating studies have demonstrated the presence of viable and metabolically active bacterial communities within a range of solid tumor types. However, the precise mechanisms by which these microbes modulate their infected tumor niches or impact patient responses to cancer treatments remain to be elucidated. Recently, Colbert et al. revealed that L-lactate produced by intratumoral Lactobacillus iners reprograms metabolic capabilities of cervical tumors to support chemoradiotherapy resistance. This finding has implications for many solid cancer types.

INVADEseq to identify cell-adherent or invasive bacteria and the associated host transcriptome at single-cell-level resolution.

Single-cell RNA sequencing (scRNAseq) technologies have been beneficial in revealing and describing cellular heterogeneity within mammalian tissues, including solid tumors. However, many of these techniques apply poly(A) selection of RNA, and thus have primarily focused on determining the gene signatures of eukaryotic cellular components of the tumor microenvironment. Microbiome analysis has revealed the presence of microbial ecosystems, including bacteria and fungi, within human tumor tissues from major cancer types. Imaging data have revealed that intratumoral bacteria may be located within epithelial and immune cell types. However, as bacterial RNA typically lacks a poly(A) tail, standard scRNAseq approaches have limited ability to capture this microbial component of the tumor microenvironment. To overcome this, we describe the invasion-adhesion-directed expression sequencing (INVADEseq) approach, whereby we adapt 10x Genomics 5' scRNAseq protocol by introducing a primer that targets a conserved region of the bacterial 16S ribosomal RNA gene in addition to the standard primer for eukaryotic poly(A) RNA selection. This 'add-on' approach enables the generation of eukaryotic and bacterial DNA libraries at eukaryotic single-cell level resolution, utilizing the 10x barcode to identify single cells with intracellular bacteria. The INVADEseq method takes 30 h to complete, including tissue processing, sequencing and computational analysis. As an output, INVADEseq has shown to be a reliable tool in human cancer cell lines and patient tumor specimens by detecting the proportion of human cells that harbor bacteria and the identities of human cells and intracellular bacteria, along with identifying host transcriptional programs that are modulated on the basis of associated bacteria.

Fusobacterium sphaericum sp. nov. , isolated from a human colon tumor, is prevalent in various human body sites and induces IL-8 secretion from colorectal cancer cells.

Cancerous tissue is a largely unexplored microbial niche that provides a unique environment for the colonization and growth of specific bacterial communities, and with it, the opportunity to identify novel bacterial species. Here, we report distinct features of a novel Fusobacterium species, F. sphaericum sp. nov. ( Fs ), isolated from primary colon adenocarcinoma tissue. We acquire the complete, closed genome of this organism and phylogenetically confirm its classification into the Fusobacterium genus. Phenotypic and genomic analysis of Fs reveal that this novel organism is of coccoid shape, rare for Fusobacterium members, and has species-distinct gene content. Fs displays a metabolic profile and antibiotic resistance repertoire consistent with other Fusobacterium species. In vitro, Fs has adherent and immunomodulatory capabilities, as it intimately associates with human colon cancer epithelial cells and promotes IL-8 secretion. Analysis of the prevalence and abundance of Fs in ∼1,750 human metagenomic samples shows that it is a moderately prevalent member of the human oral cavity and stool. Intriguingly, analysis of ∼1,270 specimens from patients with colorectal cancer demonstrate that Fs is significantly enriched in colonic and tumor tissue as compared to mucosa or feces. Our study sheds light on a novel bacterial species that is prevalent within the human intestinal microbiota and whose role in human health and disease requires further investigation.

The intratumoral microbiota: From microniches to single cells.

Solid tumors are composed of a complex and dynamic collection of cell types. Here I discuss the important relationships between cancer cells and bacterial members of the intratumoral microbiota that may provide a fitness advantage within the tumor ecological niche.

The cancer chemotherapeutic 5-fluorouracil is a potent Fusobacterium nucleatum inhibitor and its activity is modified by intratumoral microbiota.

Fusobacterium nucleatum (Fn) is a dominant bacterial species in colorectal cancer (CRC) tissue that is associated with cancer progression and poorer patient prognosis. Following a small-molecule inhibitor screen of 1,846 bioactive compounds against a Fn CRC isolate, we find that 15% of inhibitors are antineoplastic agents including fluoropyrimidines. Validation of these findings reveals that 5-fluorouracil (5-FU), a first-line CRC chemotherapeutic, is a potent inhibitor of Fn CRC isolates. We also identify members of the intratumoral microbiota, including Escherichia coli, that are resistant to 5-FU. Further, CRC E. coli isolates can modify 5-FU and relieve 5-FU toxicity toward otherwise-sensitive Fn and human CRC epithelial cells. Lastly, we demonstrate that ex vivo patient CRC tumor microbiota undergo community disruption after 5-FU exposure and have the potential to deplete 5-FU levels, reducing local drug efficacy. Together, these observations argue for further investigation into the role of the CRC intratumoral microbiota in patient response to chemotherapy.

Effect of the intratumoral microbiota on spatial and cellular heterogeneity in cancer.

The tumour-associated microbiota is an intrinsic component of the tumour microenvironment across human cancer types1,2. Intratumoral host-microbiota studies have so far largely relied on bulk tissue analysis1-3, which obscures the spatial distribution and localized effect of the microbiota within tumours. Here, by applying in situ spatial-profiling technologies4 and single-cell RNA sequencing5 to oral squamous cell carcinoma and colorectal cancer, we reveal spatial, cellular and molecular host-microbe interactions. We adapted 10x Visium spatial transcriptomics to determine the identity and in situ location of intratumoral microbial communities within patient tissues. Using GeoMx digital spatial profiling6, we show that bacterial communities populate microniches that are less vascularized, highly immuno­suppressive and associated with malignant cells with lower levels of Ki-67 as compared to bacteria-negative tumour regions. We developed a single-cell RNA-sequencing method that we name INVADEseq (invasion-adhesion-directed expression sequencing) and, by applying this to patient tumours, identify cell-associated bacteria and the host cells with which they interact, as well as uncovering alterations in transcriptional pathways that are involved in inflammation, metastasis, cell dormancy and DNA repair. Through functional studies, we show that cancer cells that are infected with bacteria invade their surrounding environment as single cells and recruit myeloid cells to bacterial regions. Collectively, our data reveal that the distribution of the microbiota within a tumour is not random; instead, it is highly organized in microniches with immune and epithelial cell functions that promote cancer progression.

Cancer as microenvironmental, systemic and environmental diseases: opportunity for transdisciplinary microbiomics science.

Cancer is generally regarded as a localised disease, with the well-established role of the tumour microenvironment. However, the realm of cancer goes beyond the tumour microenvironment, and cancer should also be regarded as a systemic and environmental disease. The exposome (ie, the totality of exposures), which encompasses diets, supplements, smoking, alcohol, other lifestyle factors, medications, etc, likely alters the microbiome (inclusive of bacteria, viruses, archaea, fungi, parasites, etc) and immune system in various body sites and influences tumour phenotypes. The systemic metabolic/inflammatory status, which is likely influenced by exposures and intestinal physiological changes, may affect tissue microenvironment of colorectum and any other organs. Germline genomic factors can modify disease phenotypes via gene-by-environment interactions. Although challenges exist, it is crucial to advance not only basic experimental research that can analyse the effects of exposures, microorganisms and microenvironmental components on tumour evolution but also interdisciplinary human population research that can dissect the complex pathogenic roles of the exposome, microbiome and immunome. Metagenomic, metatranscriptomic and metabolomic analyses should be integrated into well-designed population research combined with advanced methodologies of artificial intelligence and molecular pathological epidemiology. Ideally, a prospective cohort study design that enables biospecimen (such as stool) collection before disease detection should be considered to address reverse causation and recall biases. Robust experimental and observational research together can provide insights into dynamic interactions between environmental exposures, microbiota, tumour and immunity during carcinogenesis processes, thereby helping us develop precision prevention and therapeutic strategies to ultimately reduce the cancer burden.

An international report on bacterial communities in esophageal squamous cell carcinoma.

The incidence of esophageal squamous cell carcinoma (ESCC) is disproportionately high in the eastern corridor of Africa and parts of Asia. Emerging research has identified a potential association between poor oral health and ESCC. One possible link between poor oral health and ESCC involves the alteration of the microbiome. We performed an integrated analysis of four independent sequencing efforts of ESCC tumors from patients from high- and low-incidence regions of the world. Using whole genome sequencing (WGS) and RNA sequencing (RNAseq) of ESCC tumors from 61 patients in Tanzania, we identified a community of bacteria, including members of the genera Fusobacterium, Selenomonas, Prevotella, Streptococcus, Porphyromonas, Veillonella and Campylobacter, present at high abundance in ESCC tumors. We then characterized the microbiome of 238 ESCC tumor specimens collected in two additional independent sequencing efforts consisting of patients from other high-ESCC incidence regions (Tanzania, Malawi, Kenya, Iran, China). This analysis revealed similar ESCC-associated bacterial communities in these cancers. Because these genera are traditionally considered members of the oral microbiota, we next explored whether there was a relationship between the synchronous saliva and tumor microbiomes of ESCC patients in Tanzania. Comparative analyses revealed that paired saliva and tumor microbiomes were significantly similar with a specific enrichment of Fusobacterium and Prevotella in the tumor microbiome. Together, these data indicate that cancer-associated oral bacteria are associated with ESCC tumors at the time of diagnosis and support a model in which oral bacteria are present in high abundance in both saliva and tumors of some ESCC patients.

Complete Genome Sequence of Morganella morganii CTX51T, Isolated from a Human Cecal Adenocarcinoma.

We report the complete genome sequence of Morganella morganii CTX51T, a strain isolated from the resected tumor of a patient with cecal colorectal adenocarcinoma of the cecum. The genome comprises a circular chromosome of 4.19 Mbp, with an overall GC content of 50.4% and one circular plasmid of 8.48 kbp.

Complete Genome Sequence of Clostridium cadaveris IFB3C5, Isolated from a Human Colonic Adenocarcinoma.

We report the complete genome sequence of Clostridium cadaveris IFB3C5, a strain isolated from the resected tumor of a treatment naive colorectal cancer patient. This genome is comprised of a singular chromosome of approximately 3.63 Mbp in length, contains two plasmids, and has an overall mean GC content of 31.7%.

Molecular and Pathology Features of Colorectal Tumors and Patient Outcomes Are Associated with Fusobacterium nucleatum and Its Subspecies animalis.

BACKGROUND: Fusobacterium nucleatum (F. nucleatum) activates oncogenic signaling pathways and induces inflammation to promote colorectal carcinogenesis. METHODS: We characterized F. nucleatum and its subspecies in colorectal tumors and examined associations with tumor characteristics and colorectal cancer-specific survival. We conducted deep sequencing of nusA, nusG, and bacterial 16s rRNA genes in tumors from 1,994 patients with colorectal cancer and assessed associations between F. nucleatum presence and clinical characteristics, colorectal cancer-specific mortality, and somatic mutations. RESULTS: F. nucleatum, which was present in 10.3% of tumors, was detected in a higher proportion of right-sided and advanced-stage tumors, particularly subspecies animalis. Presence of F. nucleatum was associated with higher colorectal cancer-specific mortality (HR, 1.97; P = 0.0004). This association was restricted to nonhypermutated, microsatellite-stable tumors (HR, 2.13; P = 0.0002) and those who received chemotherapy [HR, 1.92; confidence interval (CI), 1.07-3.45; P = 0.029). Only F. nucleatum subspecies animalis, the main subspecies detected (65.8%), was associated with colorectal cancer-specific mortality (HR, 2.16; P = 0.0016), subspecies vincentii and nucleatum were not (HR, 1.07; P = 0.86). Additional adjustment for tumor stage suggests that the effect of F. nucleatum on mortality is partly driven by a stage shift. Presence of F. nucleatum was associated with microsatellite instable tumors, tumors with POLE exonuclease domain mutations, and ERBB3 mutations, and suggestively associated with TP53 mutations. CONCLUSIONS: F. nucleatum, and particularly subspecies animalis, was associated with a higher colorectal cancer-specific mortality and specific somatic mutated genes. IMPACT: Our findings identify the F. nucleatum subspecies animalis as negatively impacting colorectal cancer mortality, which may occur through a stage shift and its effect on chemoresistance.

Patients with mesenchymal tumours and high Fusobacteriales prevalence have worse prognosis in colorectal cancer (CRC).

OBJECTIVES: Transcriptomic-based subtyping, consensus molecular subtyping (CMS) and colorectal cancer intrinsic subtyping (CRIS) identify a patient subpopulation with mesenchymal traits (CMS4/CRIS-B) and poorer outcome. Here, we investigated the relationship between prevalence of Fusobacterium nucleatum (Fn) and Fusobacteriales, CMS/CRIS subtyping, cell type composition, immune infiltrates and host contexture to refine patient stratification and to identify druggable context-specific vulnerabilities. DESIGN: We coupled cell culture experiments with characterisation of Fn/Fusobacteriales prevalence and host biology/microenviroment in tumours from two independent colorectal cancer patient cohorts (Taxonomy: n=140, colon and rectal cases of The Cancer Genome Atlas (TCGA-COAD-READ) cohort: n=605). RESULTS: In vitro, Fn infection induced inflammation via nuclear factor kappa-light-chain-enhancer of activated B cells/tumour necrosis factor alpha in HCT116 and HT29 cancer cell lines. In patients, high Fn/Fusobacteriales were found in CMS1, microsatellite unstable () tumours, with infiltration of M1 macrophages, reduced M2 macrophages, and high interleukin (IL)-6/IL-8/IL-1ß signalling. Analysis of the Taxonomy cohort suggested that Fn was prognostic for CMS4/CRIS-B patients, despite having lower Fn load than CMS1 patients. In the TCGA-COAD-READ cohort, we likewise identified a differential association between Fusobacteriales relative abundance and outcome when stratifying patients in mesenchymal (either CMS4 and/or CRIS-B) versus non-mesenchymal (neither CMS4 nor CRIS-B). Patients with mesenchymal tumours and high Fusobacteriales had approximately twofold higher risk of worse outcome. These associations were null in non-mesenchymal patients. Modelling the three-way association between Fusobacteriales prevalence, molecular subtyping and host contexture with logistic models with an interaction term disentangled the pathogen-host signalling relationship and identified aberrations (including NOTCH, CSF1-3 and IL-6/IL-8) as candidate targets. CONCLUSION: This study identifies CMS4/CRIS-B patients with high Fn/Fusobacteriales prevalence as a high-risk subpopulation that may benefit from therapeutics targeting mesenchymal biology.

Metagenomic analysis to identify novel infectious agents in systemic anaplastic large cell lymphoma.

Systemic anaplastic large cell lymphoma (ALCL) is a rare CD30-expressing T-cell non-Hodgkin lymphoma. Risk of systemic ALCL is highly increased among immunosuppressed individuals. Because risk of cancers associated with viruses is increased with immunosuppression, we conducted a metagenomic analysis of systemic ALCL to determine whether a known or novel pathogen is associated with this malignancy. Total RNA was extracted and sequenced from formalin-fixed paraffin-embedded tumor specimens from 19 systemic ALCL cases (including one case from an immunosuppressed individual with human immunodeficiency virus infection), 3 Epstein-Barr virus positive diffuse large B-cell lymphomas (DLBCLs) occurring in solid organ transplant recipients (positive controls), and 3 breast cancers (negative controls). We used a pipeline based on the Genome Analysis Toolkit (GATK)-PathSeq algorithm to subtract out human RNA reads and map the remaining RNA reads to microbes. No microbial association with ALCL was identified, but we found Epstein-Barr virus in the DLBCL positive controls and determined the breast cancers to be negative. In conclusion, we did not find a pathogen associated with systemic ALCL, but because we analyzed only one ALCL tumor from an immunosuppressed person, we cannot exclude the possibility that a pathogen is associated with some cases that arise in the setting of immunosuppression.

The relationship between gastrointestinal cancers and the microbiota.

The contribution of the microbiota to disease progression and treatment efficacy is often neglected when determining who is at the highest risk of developing gastrointestinal cancers or designing treatment strategies for patients. We reviewed the current literature on the effect of the human microbiota on cancer risk, prognosis, and treatment efficacy. We highlight emerging research that seeks to identify microbial signatures as biomarkers for various gastrointestinal cancers, and discuss how we could harness knowledge of the microbiome to detect, prevent, and treat these cancers. Finally, we outline further research needed in the field of gastrointestinal cancers and the microbiota, and describe the efforts required to increase the accuracy and reproducibility of data linking the microbiome to cancer.

Association of Fusobacterium nucleatum with Specific T-cell Subsets in the Colorectal Carcinoma Microenvironment.

PURPOSE: While evidence indicates that Fusobacterium nucleatum (F. nucleatum) may promote colorectal carcinogenesis through its suppressive effect on T-cell-mediated antitumor immunity, the specific T-cell subsets involved remain uncertain. EXPERIMENTAL DESIGN: We measured F. nucleatum DNA within tumor tissue by quantitative PCR on 933 cases (including 128 F. nucleatum-positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on Bifidobacterium, microsatellite instability (MSI), tumor whole-exome sequencing, and M1/M2-type tumor-associated macrophages [TAM; by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay]. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between F. nucleatum and T-cell subsets. RESULTS: The amount of F. nucleatum was inversely associated with tumor stromal CD3+ lymphocytes [multivariable OR, 0.47; 95% confidence interval (CI), 0.28-0.79, for F. nucleatum-high vs. -negative category; P trend = 0.0004] and specifically stromal CD3+CD4+CD45RO+ cells (corresponding multivariable OR, 0.52; 95% CI, 0.32-0.85; P trend = 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden. F. nucleatum was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs. CONCLUSIONS: The amount of tissue F. nucleatum is associated with lower density of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells.