Fusobacterium nucleatum and cancer

Abstract Accumulating evidence demonstrates that the oral pathobiont Fusobacterium nucleatum is involved in the progression of an increasing number of tumors types. Thus far, the mechanisms underlying tumor exacerbation by F. nucleatum include the enhancement of proliferation, establishment of a tumor‐promoting immune environment, induction of chemoresistance, and the activation of immune checkpoints. This review focuses on the mechanisms that mediate tumor‐specific colonization by fusobacteria. Elucidating the mechanisms mediating fusobacterial tumor tropism and promotion might provide new insights for the development of novel approaches for tumor detection and treatment.


| INFEC TIVE AG ENTS AND C AN CER
In 1911, a causal role of microbes in cancer was first revealed by Peyton Rous who demonstrated that sarcoma can be induced in chickens by a virus. 1 The link between a virus and human cancer was demonstrated 53 years later by Epstein, Achong and Barr as evidenced by the presence of Epstein-Barr virus in Burkitt lymphoma cells visualized by electron microscopy. 2 This was followed with the association of hepatitis B and C viruses with liver cancer, papillomavirus with cervical cancer and herpesviruses with Kaposi sarcoma. 3 In contrast to viruses, which play critical roles in cancer, bacteria were first considered as anti-cancer agents (reviewed in reference 4 ).
In 1813, Vautier reported that patients with cancer who developed gas gangrene showed tumor regression. 5 German physicians Busch and Fehleisen independently observed the regression of tumors in patients with cancer suffering from erysipelas infection. In 1868, Busch infected a cancer patient with erysipelas and noted tumor shrinkage. In 1882, Fehleisen repeated this treatment and identified Streptococcus pyogenes as the causative agent of erysipelas. 4 Furthermore, in the United States in the early 1890s, a surgeon named William Coley pioneered the use of bacteria and their extracts (Coley's toxins) to evoke anti-tumor immunity and successfully treat cancer patients. 6 However, the high-degree of success of newly developed radiation therapy led to a decline in the application of Coley's toxins as cancer treatment (reviewed in reference 7 ).
Bacterial-based anticancer treatment reemerged in 1990, when the FDA approved the Bacillus Calmette-Guérin (BCG) vaccine, a live attenuated form of Mycobacterium bovis that is used against tuberculosis, for treating noninvasive bladder cancer. 8,9 Currently, BCG is the only anti-cancer bacterial agent approved for routine clinical use. 4 BCG, and fungal-derived polysaccharide β-glucan, can promote a sustained enhanced response of myeloid and natural killer (NK) cells to secondary infectious, inflammatory challenges, and tumors. This process of non-specific memory of innate immune cells, facilitates the heightened response of these cells, as well as that of their progeny, to future challenges, and has been termed ''trained innate immunity'' or ''innate immune memory''. 10,11 Trained immunity is mediated via transcriptomic, epigenetic, and metabolic reprogramming. 11 NK cells, 12 and the induction trained immunity, 13 are hypothesized to play important roles in BCG immunotherapy for noninvasive bladder cancer. 14 The realization that Helicobacter pylori is a causative agent of gastric cancers in the 1990s indicated that bacteria are involved in tumor promotion. [15][16][17][18] Furthermore, mice that were genetically susceptible to cancer developed significantly fewer tumors under germ-free advanced genomic sequencing and microbiome characterization methods indicate the association of bacterial species with specific cancers. 21,22 Multiple features of tumor, including proliferation, survival, progression, immunogenicity, sensitivity, and resistance to therapy, are affected by their interaction with the components of their microbial environment. 22,23 Although some bacterial species can promote cancer, those found to have reduced abundance in cancers might have cancer-inhibitory actions or antagonistic interactions with tumor-promoting bacteria. 24,25 Among the first bacteria suggested as potential cancer drivers are Escherichia coli strains that generate a mutagenic toxin called colibactin, which can induce single-strand DNA breaks, and fragilysin-expressing Bacteroides fragilis, which is genotoxic and can cleave the tumor suppressor protein E-cadherin. 19 Streptococcus gallolyticus (former Streptococcus bovis) bacteremia is an indicator of colorectal cancer since 1951 26 ; however, the specific bacteriacancer interaction is not understood. Overall, approximately 16% to 20% of cancer incidence can be linked to infectious agents. [27][28][29] A recent report comprehensively characterized the microbiome of seven solid tumors. 21 Cancer is among the comorbidities affected by periodontal pathobionts. [30][31][32] Fusobacterium nucleatum the focus of the review, is an oral oncobiont mostly associated with the development of periodontitis. Highly abundant F. nucleatum has been detected in various types of cancer, including colorectal (CRC), 33,34 pancreatic, 35,36 esophageal, 37,38 and breast cancers, 39,40 and associated with shorter survival in patients with CRC, pancreatic, and esophageal cancers. 35,37,38,41,42 Accumulating evidence indicating that F. nucleatum accelerates tumorigenesis 40,43,44,45,46,47,48 and induces resistance to chemotherapy 49-52 may provide rational for the association of high amounts of F. nucleatum with poor disease outcome.

| FUSOBAC TERIUM NUCLE ATUM IN THE OR AL C AVIT Y
Fusobacterium nucleatum is a gram-negative, spindle-shaped, nonspore forming, oral anaerobe and is one of the most abundant gram-negative species residing in the human oral cavity. 68,69 It is one of the pathobionts that outgrow during dysbiosis that precedes periodontal disease 68,69 and assist keystone species such as Porphyromonas gingivalis 70 in disrupting host-microbial homeostasis and inducing periodontitis. 71,72 It can be found on the dorsal surface of the tongue 73,74 and in multispecies biofilms at the gingival margin of the tooth, where it is hypothesized to play an important role in the development of the subgingival dental plaque. Owing to its abundant adhesion mechanisms, F. nucleatum can bind many oral bacterial species. Attachment between different oral colonizers is termed coaggregation or coadherence. [75][76][77] By coaggregation with early oral colonizers capable of attaching to oral surfaces, such as Streptococcus species (via the RadD adhesin), 78 and the largely anaerobic secondary colonizers that are associated with periodontal disease, including Porphyromonas gingivalis (via Fap2 as will be discussed below), Treponema denticola, and Aggregatibacter actinomycetemcomitans, and bridging them, F. nucleatum play a scuffle-like, structurally supportive role in the oral biofilm that can resist washing by the saliva and gingival crevicular fluid. Multispecies bridging also facilitates multi-species community existence, including communication, cross-feeding, and metabolic interactions ( Figure 1). 55,75,76,79

| FUSOBAC TERIUM NUCLE ATUM I S OVER ABUNDANT IN COLOREC TAL C AN CER
CRC is the second most common cause of cancer deaths in the United States 80 and the fourth leading cause of cancer-related deaths worldwide. 81 The burden of CRC is rapidly increasing in developing countries as they adopt western lifestyles. 81 In 2012, two studies employing applied computational approaches found F I G U R E 1 F. nucleatum acts as a bridging organism in dental plaques. A. Scanning electron microscopic image of a multispecies human oral biofilm. B. Schematic representation showing the ability of F. nucleatum to function as a "bridging" organism connecting the early colonizers, such as Streptococcus species via the RadD adhesin, and the largely anaerobic secondary colonizers, including Porphyromonas gingivalis via Fap2, Treponema denticola, and Aggregatibacter actinomycetemcomitans increased fusobacteria (particularly F. nucleatum) DNA or RNA levels in colorectal cancer tissues compared to adjacent normal tissues. 33,34 This discovery was unexpected as fusobacteria are the core resident members of the human oral microbiome and infrequently found in the gut. 82,83 Live F. nucleatum directly isolated from biopsy samples 34,84,85 and patient-derived xenografts in mice 46 confirmed these metagenomic results. Interestingly, the proportion of F. nucleatum-high colorectal cancers gradually increased from rectal cancers to the cecal cancers. 86 Remarkably, a stronger association between F. nucleatum and CRC patients was found in Asiatic populations than in European and American populations (for a recent systematic review and meta-analysis, please see references 42,87 ). In addition, F. nucleatum in CRC patients was frequently detected with other oral anaerobic species including Peptostreptococcus spp. 46,88 Leptotrichia and Campylobacter. 89 Increasing evidence indicates that the presence of F. nucleatum in colon cancer is associated with resistance to chemotherapy, disease recurrence, and poor prognosis, which will be discussed in detail in section 9 below.

| CRC-A SSO CIATED F. NUCLE ATUM ORIG INATE S FROM THE OR AL MICROB IOTA
Although F. nucleatum is a common oral isolate, it is not abundantly isolate, thereby supporting fusobacteria from the oral cavity may seed and become enriched in colorectal cancers. 85 The frequent cooccurrence of F. nucleatum in tumors with potential oral coaggregation partners, including Peptostreptococcus spp. 46,88 Leptotrichia and Campylobacter spp., 89 also substantiate the oral origin of colorectal cancer-colonizing fusobacteria.

| OR AL F. NUCLE ATUM C A N TR ANS LOC ATE TO COLOREC TAL TUMOR S VIA THE HEMATOG ENOUS ROUTE
Considering the oral origin of colon cancer-associated fusobacteria, the route of their oral to tumor transmission remained to be resolved.
Kostic et al 45 demonstrated that oral fusobacteria can reach colon tumors by descending via the digestive tract. 45 However, hematogenous translocation that can occur during frequent gingival bleeding 91 is also possible. Such hematogenous transfer of oral fusobacteria to the placenta was previously observed, thus explaining its high occurrence in preterm births. 92 (Reviewed in this volume by Y. W. Han).
Abed et al 85 studied the preferred oral tumor route by employing two orthotropic mouse colon cancer models, namely MC38 in C57BL/6 mice and CT26 in BALB/C mice. They compared colon tumor colonization by F. nucleatum that was intravascularly injected via the tail vein or administered via oral gavage. Under the tested conditions, tumor colonization by the intravascularly injected fusobacteria is more efficient than that of the gavage-inoculated ones in both mouse models. 85 Intravenously injected fusobacteria were detected in mouse CT26 colon tumors at 2 h post-delivery, and their levels remained stable at 6 h post-infection. Fusobacterial proliferation in the tumor was observed at 24 h and 72 h post-infection. 85 The magnitude of bacteremia resulting after a dental procedure and routine daily activities is significantly lower (<10 4 CFU/ml) 93 than that tested in the experiments described above (1 × 10 7 -1 × 10 8 F. nucleatum per mouse). However, when fusobacteria were inoculated in physiological doses in the orthotropic MC38 CRC model, tumor-associated fusobacteria were also detected in mice inoculated with the more physiologic dose range (1 × 10 4 F. nucleatum 93 ).
Increased doses resulted in increased proportion of mice-bearing tumors with intertumoral fusobacteria. In detail, fusobacteria were detected in the tumors of 45% of mice-bearing tumors inoculated with 5 × 10 3 to 1 × 10 4 F. nucleatum; 60%, 5 × 10 4 to 1 × 10 5 ; and 100%, 5 × 10 6 to 1 × 10 7 . Thus, lowering the fusobacterial inoculation dose did not suppress colon tumor colonization but rather reduced its efficiency. These results may explain the heterogeneity observed in fusobacterial occurrence in 3% to 56% of human colorectal cancer. 55 The above results do not rule out that oral fusobacteria, which are constantly swallowed, may colonize colon tumors through the digestive tract. However, the hematogenous dissemination of oral fusobacteria to CRC is biologically conceivable as bloodstream travel circumvents the toxicity of low gastric pH and bile acids encountered upon descent to the gastrointestinal tract. Furthermore, bloodstream travel affords fusobacteria an escape from competition with the endogenous colonic microbiota. 85

| FAP2-G LYC ANS INTER AC TIONS G U IDE F. NUCLE ATUM COLONIZ ATI ON IN COLOREC TAL C AN CER
Whether oral fusobacteria translocate to colon tumors via the blood circulation or descending through the digestive tract, mechanisms that home and localize fusobacteria to colorectal tumors must exist.
Tumor-induced conditions, including increased blood supply, blood vessel leakiness, hypoxia, and immunosuppressive microenvironment, are non-specific factors that might contribute to a niche that promotes fusobacterial survival. However, these local environmental conditions are apparently not sufficient to enable the localization of other abundant oral anaerobic bacteria, such as Porphyromonas gingivalis, to colon cancers. 94 Therefore, specific factors and mechanisms might be required for CRC colonization by fusobacteria. Current evidence suggests that tumor localization by F. nucleatum is dictated by glycan-lectin interactions.
an unknown structural-related sugar moiety is hypothesized as a tumor ligand for fusobacterial attachment. Gal-GalNAc was found to be over-displayed in sections of colorectal adenocarcinoma and has been suggested as a biomarker for colon cancer. 95  O-Ser/Thr), which is also called core 1 glycan, T-antigen, or Thomsen-Friedenreich antigen. 96 In normal cells, N-acetylneuraminic acid, the predominant sialic acid in human and many mammalian cells, is frequently added to cap and mask the GalNAc and Gal-GalNAc residues. 96,97 However, in many carcinomas (such as CRC), truncated O-GalNAc glycans are formed, and sialic acid is not added to the exposed GalNAc and Gal-GalNAc. 97,98 As a result, high levels of GalNAc (Tn antigen) and Gal-GalNAc (T antigen) have been detected in colon cancer and additional human tumors including lung, breast and liver carcinoma. 96,99,100 Such high levels of unmasked Tn-and Tantigens are associated with tumor invasion and metastasis. 99 In the dental plaque, the coaggregation of F. nucleatum with many gram-negative species can be inhibited by galactose and GalNAc indicating that F. nucleatum expresses a lectin (previously termed adhesin) that binds these sugar molecules present on the receptor of these coaggregation-partner bacteria. 75,101 Transposon mutagenesis and mutant screening results identified the outer-membrane Fap2 protein as the fusobacterial lectin that mediates GalNAc-inhibited coaggregation. 102 Interestingly, in previous studies, Fap2 was found to enable the ability of F. nucleatum to induce apoptosis in lymphocytes. 103,104 Therefore, it is plausible that Fap2 mediates the binding of F. nucleatum to lymphocytes, and enable additional fusobacterial factors to induce this apoptosis-mediated immune evading mechanism.
As Gal-GalNAc is over-displayed by colon tumors, it has potential as an oncotarget for fusobacterial Fap2. In agreement with this, the attachment of F. nucleatum to colon cancer cell lines and colon cancer sections correlated with the amounts of Gal-GalNAc detected on the target cells. In addition, its attachment was reduced upon O-glycanase treatment and inhibited by soluble GalNAc in a dose-dependent manner. 85

| G AL-G ALNAC IS OVER-DIS PL AYED IN MANY ADENOC ARCINOMA S
Evidence suggests that oral F. nucleatum can hematogenously translocate to and specifically colonize colon cancer tumors 85 via recognition and attachment to Gal-GalNAc (or related sugars), which is highly displayed in colon cancer. 85,94 This indicates that F. nucleatum can reach other Gal-GalNAc-displaying tumors through the same mechanism.
A screen for tumors that display high Gal-GalNAc levels and might be targeted by fusobacteria was conducted, and Gal-GalNAc levels of 20 different types of tumors were determined based on fluorescently labeled peanut agglutinin (PNA), a Gal-GalNAc-specific lectin. 105 In agreement with previous reports, 99 high Gal-GalNAc levels were detected in 10 tumors types of epithelial tissues with glandular origin or/and characteristics (Figure 2A). 105 Of which, nine were adenocarcinomas, namely that of the stomach, prostate, ovary, colon, uterus, pancreas, breast, lung, and esophagus. The remaining one was a squamous cell carcinoma of the cervix. In addition, Gal-GalNAc levels were significantly higher in seven of these adenocarcinomas than in the matched normal control tissues ( Figure 2B), whereas those in the stomach, lung, and cervix of the normal control samples were high and similar to those of their respective adenocarcinomas. 105 Concurring with the speculation that fusobacteria can home-in and accumulate in cancers that display high Gal-GalNAc levels, fusobacterial DNA levels were reported to be overabundant in the pancreas, 35,36 esophagus, 37 gastric, 106,107 cervical, 108 and breast 39 adenocarcinomas. Importantly, similar to its prevalence in colorectal cancer, 41,109 fusobacterial occurrence in pancreatic tumors was associated with shorter survival. 35 High levels of F. nucleatum nucleic acids in esophageal cancer was also associated with shorter survival 37 and poor response to neoadjuvant chemotherapy. 38 Interestingly, high levels of Gal-GalNAc are also found in the placenta, [110][111][112] another extraoral niche, in which F. nucleatum is associated with pathology (Reviewed in this volume by Y. W. Han).
Fap2-inactivated mutants were deficient in placental colonization, 102 suggesting that, Fap2-Gal-GalNAc interaction might be involved in placental colonization by F. nucleatum, similar to tumor colonization.

| B RE A S T C AN CER COLONIZ ATI ON BY F. NUCLE ATUM
Fusobacterium nucleatum is enriched in the breast cancer microbiome, 21,39,40 which supports the hypothesis that fusobacteria can reach tumors via the circulatory system. A study focusing on breast cancer 40 revealed that Gal-GalNAc levels increase along with the progression of human breast cancer, similar to colon cancer ie, transition from adenoma to adenocarcinoma. 94 The most dramatic rise in Gal-GalNAc levels occurs in the transition from hyperplasia to atypical hyperplasia. 40 Breast cancer, which develops in a sequence of events, begins with non-neoplastic epithelial cells undergoing hyperplasia, atypical hyperplasia, carcinoma in situ, and eventually invasive adenocarcinoma. The conversion from benign hyperplasia to carcinoma in situ (the stage preceding invasive carcinoma) is speculated to occur at the transition from hyperplasia to atypical ductal hyperplasia. 113 Importantly, the presence of F. nucleatum gDNA in breast cancer samples was correlated with high Gal-GalNAc levels. 40 In mouse models of breast cancer, when fap2expressing F. nucleatum ATCC 23726 was intravascularly inoculated, specific colonization of mammary tumors was observed ( Figure 3).
In contrast, fap2-inactivated F. nucleatum mutants showed impaired tumor colonization. 40 The inoculation of F. nucleatum into C57BL/6 mice orthotopically implanted with AT3 breast cancer cells resulted in the impaired accumulation of tumor-infiltrating CD4+ and CD8+ T cells. Tumors obtained from F. nucleatum-inoculated mice were significantly larger in volume than those from non-inoculated ones. The progression of lung metastasis was also significantly enhanced in the F I G U R E 2 Gal-GalNAclevelsare increased in human adenocarcinomas. (A) Tumors are arranged according to increasing Gal-GalNAc levels. Examined adenocarcinomas that displayed high levels of Gal-GalNAc are marked with dark gray (right). (B) Gal-GalNAc levels in the tumors (shaded dots) described in (A) were compared to those in matched normal tissue controls (hollow dots). Of the nine examined adenocarcinomas, seven showed significantly higher Gal-GalNAc levels than the matched control tissues. The normal tissue controls for the esophagus, lung, and skin were used twice for the respective esophagus adenocarcinoma and esophagus squamous cell carcinoma (Esophagus SCC), the respective lung adenocarcinoma and lung SCC, and for the melanoma and SCC. Each symbol represents the fluorescent intensity of a sample from different patient. Data are presented as the mean ± SEM (*P < .05, **P < .01, ***P = .0001 analyzed by two-tailed Mann-Whitney test; ****P < .0001 analyzed by two-tailed t-test). This figure is from reference 105 F I G U R E 3 BreastcancercolonizationbyF. nucleatum. Schematic representation depicting the mechanism of the translocation of oral F. nucleatum to breast tumor via blood circulation. The bacterial lectin Fap2 enables the specific binding of F. nucleatum to cancerous cells that over-display Gal-GalNAc  35,36 and breast cancer. 40 Fusobacterial presence has been associated with poor prognosis in colon, rectal, pancreatic, and esophageal cancers 35,37,41,109,114 and with treatment failure in colorectal and esophageal cancers. 38,49 In an animal model of colon and breast cancer, F. nucleatum accelerated tumor growth and metastatic progression. 40,[44][45][46] Tumor acceleration by F. nucleatum involves the promotion of proliferation, 43,44 generation of a pro-tumorigenic immune microenvironment, 45  In human CRC tissues, high levels of F. nucleatum correlated with high levels of BIRC3. Moreover, high levels of F. nucleatum, TLR4, and BIRC3 were more likely to be detected in CRC patients with recurrence than in those without. 50 In another study, the incubation of HCT116 and HT29 cells with F. nucleatum significantly upregu-

| Fusobacterium nucleatum inhibits the recruitment of anti-cancer tumor-infiltrating T cells
Accumulated evidence indicates that tumor-colonized F. nucleatum can also interfere with the recruitment of TILs. In colorectal carcinoma tissues, the abundance of F. nucleatum was inversely correlated with T-cell density. 115,131,132 In post-neoadjuvant locally treated advanced rectal cancer, fusobacterial persistence was associated with a lack of CD8 + T cells. 109 In an AT3 orthotropic mouse model of breast cancer, F. nucleatum accelerated cancer progression by inhibiting the recruitment of TILs.  103 Importantly, the immunomodulated pro-tumorigenic effect of F. nucleatum is expected to be more significant in humans because the activity of NK and some T cells in tumors can be further weakened by the inhibitory interactions between Fap2 and TIGIT 116

and between
CbpF and CEACAM 117 checkpoints (as discussed below).

| Fusobacterium nucleatum activates immune checkpoints
While the presence of F. nucleatum in human colorectal cancer 115,131,132 and in a mouse model of breast cancer 40 116 More recently, the suppression of immune cell anti-tumor activity by F. nucleatum through the activation of an additional immune cell suppressing receptor CEAMAM1, was reported. 117,119 Thus, in addition to reducing the number of immune cells infiltrating the fusobacterial-colonized tumor microenvironment, fusobacteria can further protect tumors by activating checkpoints to suppress immune-cell anti-tumor activity.

| Fusobacterium nucleatum promotes metastasis
Fusobacterium nucleatum has been detected in CRC metastases to the liver and lymph nodes 33,34,46,94 and is associated with increased number of liver metastases in colorectal cancer. 46,133 In a mouse model of breast cancer, F. nucleatum promoted lung metastasis. 40 The presence of F. nucleatum was also shown to promote the successful establishment of CRC patient-derived xenografts in mice. 46 The proposed mechanism by which F. nucleatum promotes metastasis involves the induction of proinflammatory cytokines that stimulate tumor cell migration and invasion. F. nucleatum-infected CRC cells secrete cytokines IL-8 and CXCL1, which promote the invasive motility of infected and non-infected cells. 134 Upon incubation with F. nucleatum, human and mouse breast cancer cells also induced the overexpression and increased secretion of the matrix metalloproteinase 9 (MMP-9). 40 Proteases of the MMP family play vital roles in many biological processes that involve matrix remodeling. In particular, MMP-9 activity has been related to cancer pathology, including invasion, angiogenesis, and metastasis. 135 Therefore, in addition to immune modulation, which is the putative major mechanism of F. nucleatum action in AT3 breast cancer model in C57BL/6 mice, the induction of MMP might be another mechanism by which F. nucleatum accelerates breast tumor progression.
Generally, metastasis is responsible for more than 90% of cancerassociated mortality and is the main cause of breast cancer-related deaths. Patients with localized breast cancer have a 5-year survival rate of 98%, which dramatically decreases to 26% in patients with metastatic breast cancer. 136 More studies are required to completely understand the pro-metastasis mechanisms of F. nucleatum.

| FUSOBAC TERIUM NUCLE ATUM A S A P OTENTIAL D IAG NOS TIC B I OMARKER
Microbiome-based oncology diagnostics are promising novel approaches for tumor detection. A recent report demonstrated the potential of plasma-derived, cell-free microbial nucleic acids for tumor screening. Good discrimination was achieved between samples from donors with tumors and those from healthy ones and among 32 different cancer types. 137 Therefore, the overabundance of F. nucleatum in tumors can be utilized as a strategy for tumor detection. Although a number of approaches have been explored, adequate screening capabilities have not yet been achieved.

| Stool screening for CRC detection
The early detection of cancers is important to reduce CRC mortality. 138 Fecal occult blood testing (FOBT) is a common non-invasive cost-efficient method to screen for CRC 138 ; However, FOBT has moderate sensitivity. [138][139][140] Almost a decade ago, F. nucleatum was reported to be enriched in stool samples from colorectal adenoma and carcinoma patients compared to healthy subjects. 45 Many reports have since corroborated this finding, particularly those involving Asian cohorts. 141 A recent review and meta-analysis demonstrated the potential of a fecal F. nucleatum -based test for detecting colorectal cancer; however, additional clinical trials should be performed to verify this. 141 The combination of fecal quantification of F. nucleatum and FOBT was shown to increase the specificity and sensitivity of the latter, 142,143 indicating the applicability of this combination method as a large population-based screening strategy employing large noninvasive samples for colorectal cancer. To date, the quantification of F. nucleatum has been performed using quantitative PCR. 141 It is expected that future developments of novel antibody-or enzymaticbased assays might enable the combination of FOBT with fecal fusobacterial testing (FFT) in a single test.

| Tumor detection based on antibody responses
Immune assays based on the serum, salivary, or fecal anti-F. nucleatum antibodies may also offer new opportunities for CRC screening.
Thus far, serum anti-F. nucleatum antibodies could not discriminate between CRC patients and the controls with sufficient specificity and sensitivity. [144][145][146] One study used multiplex serology assay to simultaneously measure antibody responses to 11 F. nucleatum recombinant antigens in prediagnostic serum samples from colorectal cancer patients and matched controls (n = 485 each). However, colorectal cancer risk was not significantly associated with antibody response to each F. nucleatum protein or combined positivity to any of the 11 proteins. 145 In a subsequent study, ELISA-based testing found that the levels of F. nucleatum IgA and IgG antibodies in the CRC group were higher than those in the healthy controls. However, the discriminative ability of the ELISA test was not adequate for diagnosis. 146 Notably, plasma anti-F. nucleatum IgG level and salivary IgA level against F. nucleatum and specifically against Fap2, has been recently reported to be associated with pancreatic malignancy. 147 However, the diagnostic potential of these findings should be confirmed by future studies.

| Elimination of tumor-colonized F. nucleatum
As mentioned above (section 9), high fusobacterium load in tumors has been associated with poor disease outcomes in humans. 35,37,38,41,42,49,51,52 In animal models, systemic antibiotic treatment eliminated tumor-colonized fusobacteria and subsequently suppressed fusobacterial-induced tumor exacerbation, suggesting the effectivity of antibiotic treatment for cancer patients. 40,46 Unfortunately, in some cases, antibiotics might interfere with anti-tumor treatment. Gut microbiota can influence anti-tumor chemotherapy, 148,149 immunotherapy, [150][151][152][153][154][155][156] radiotherapy, 157 and allogeneic bone marrow transplantation 158 via various proposed mechanisms. 23 Fecal transplantation to restore the gut microbiota following antibiotic treatment might address this issue, especially if in the future, fecal transplant will be considered to aid anti-cancer (chemotherapeutic, immunotherapeutic) treatments. 25 Bacteriophages are viruses that prey and replicate in bacteria. The use of bacteriophages for targeting specific oncobacteria, including tumor-colonized F. nucleatum, has been recently suggested. 25 Importantly, a fusobacteria bacteriophage with a potential to eradicate tumor-colonized F. nucleatum has been recently identified. 159

| Tumor targeting strategies using F. nucleatum and Fap2
Due to their specific homing to glycan-overdisplaying tumors, F. nucleatum or Fap2 could potentially be used as a platform for targeting tumors and metastases that display high levels of Gal-GalNAc (or related sugars). Recent advances in the genetic manipulation of F. nucleatum 134,160 have facilitated the ability to weaken fusobacterial tumor-enhancing actions in the future by for example mutating FadA and/or nullifying TIGIT and CEACAM1 activation. Such enfeebled strains can then be engineered to express anti-cancer payloads. Possible anti-tumor agents might include antigens that induce trained innate immunity, or antigens that induce innate and adaptive anti-tumor immune responses, and/or enzymes that locally convert a nontoxic prodrug to a cytolytic drug. Such strategies are currently being tested with several tumor-colonizing bacteria including Salmonella and Listeria (reviewed in 5 ).
Importantly, live bacteria are currently used for cancer treatment. 161 In case of adverse effects, this treatment can be terminated using antibiotics. For over three decades, the intravesical administration of live bacillus Calmette-Guérin, a vaccine against tuberculosis, has been used to treat bladder cancer. 161 Anecdotally, bladder cancer patients treated with BCG have significantly reduced risk of Alzheimer's disease and Parkinson's disease compared to those not treated with BCG. The beneficial effect of BCG on neurodegenerative diseases has been attributed to the possible activation of longterm nonspecific immune effects. 162 A more advanced version of this tumor-targeting approach might be targeting tumor-colonized fusobacteria with bacteriophages engineered to express anti-cancer payloads such as described above. Gal-GalNAc, chondroitin sulfate is an oncofetal antigen shared between placental trophoblasts and cancer cells. 164,165 Recombinant VAR2CSA (rVAR2) coupled to magnetic beads can capture circulating tumor cells in a blood sample, thus serving as a potential tool for novel cancer diagnostics. 166 The conjugation of a toxin to rVAR2 can also direct anti-tumor therapeutics. 165

| CON CLUDING REMARK S
The terms alpha-bugs 168 also referred to as bacterial drivers 19 have been proposed to describe certain members of the microbiome that possess direct pro-oncogenic features or the ability to shift the local bacterial community to one that promotes mucosal immune responses and epithelial cell changes, consequently resulting in the development of colorectal cancer. Alpha-bugs have been also suggested to enhance carcinogenesis by selectively "crowding out" cancer-protective microbial species. 168 "Classical" bacterial drivers possess virulence factors that might initiate cancer formation. These factors include the colibactin genotoxin of several E. coli strains that can induce single-strand DNA breaks 169 and the B. fragilis toxin fragilysin (BFT). BFT, a metalloproteinase, is genotoxic to colonic epithelial cells, upregulates spermine oxidase, a polyamine catabolic enzyme that contributes to increased production of reactive oxygen species and DNA damage. 170 Fragilysin also promotes the proliferation of intestinal epithelial cells in a mechanism involving cleavage of the tumor suppressor protein E-cadherin. 171,172 Currently, H. pylori is the only bacterium that is classified as a direct carcinogen. Epidemiological evidence and experimental data indicate that prevalence of H. pylori is associated with the development of gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. 173 H. pylori in the stomach mucosa is crucial in the chronic inflammatory process, which leads to gastric cancer development. 173 Thus, the cytotoxin-associated gene A (CagA) protein of H. pylori, which is delivered to gastric epithelial cells via bacterial type IV-secretion, is an oncoprotein that can induce malignant neoplasms in mammals. 174,175 Unlike the cancer drivers mentioned above, based on the current evidence, F. nucleatum is a "passenger" 19 bacteria that colonizes an already formed tumor and accelerates its progression through manipulation of β-catenin signaling, 43,44 host cytokine production (IL-8 and CXCL1), 134 anti-tumor immunity, and chemoresistance. These mechanisms are illustrated in Figure 4.
Occurrence of F. nucleatum is found to be associated with poor disease outcome in an increasing number of tumor types suggesting that targeting intratumor fusobacteria will improve prognosis.
High Gal-GalNAc level is found in all tumor-types colonized by fusobacteria indicating that it is an oncoantigen that plays a role in the specificity of tumor colonization by fusobacteria by serving as a ligand to Fap2. It is therefore tempting to assume that fusobacterial overabundance will be found in all Gal-GalNAc overdisplaying tumors. Due to the tumor specificity, fusobacteria and Fap2 hold potential for use for tumor screening and treatment.
The fusobacterial adhesin FadA binds to E-cadherin and activates the β-catenin/WNT signaling pathway, thus promoting cell proliferation. 43 The bacterial endotoxin lipopolysaccharide (LPS) activates the Toll-like receptor 4 (TLR4) to trigger the upregulation of miR21. This decreases the levels of RAS GTPase RASA1 and activates the RASmitogen-activated protein kinase (MAPK) cascade to enhance cell proliferation. 120,121 Fusobacterium nucleatum LPS interactions with TLR4 can also upregulate BIRC3, which inhibits apoptosis by directly inhibiting the caspase cascade, thereby increasing cell resistance against cytotoxic drugs. 50 In addition, LPS/TLR4 interactions downregulate the expression of miR18a and miR4802, which is associated with that of autophagy elements ULK1 and ATG7, resulting in increased autophagy and subsequently enhancing cell resistance to therapy. 49,125 Lastly, F. nucleatum inhibits apoptosis by upregulating the expression anoctamin-1 (ANO1) in a TLR4-dependent manner to contribute to chemoresistance. 51 The non-lectin domain of Fap2 inhibits the anti-tumor activity of TILs and NK cells at the tumor site by activating the human TIGIT checkpoint. 116 Fusobacterial CbpF further suppresses the anti-tumor activity of TILs and NK cells by activating the human CEACAM1 checkpoint. 117,118 F I G U R E 4 VariousmechanismsutilizedbyF. nucleatum to accelerate tumor progression. The fusobacterial Fap2 domain binds tumordisplayed Gal-GalNAc to enable tumor colonization. 40,94 Tumor acceleration is then mediated with the following mechanisms