Multiphoton Microscopy of FITC-labelled Fusobacterium nucleatum in a Mouse in vivo Model of Breast Cancer.

Over the past decades, the main techniques used to visualize bacteria in tissue have improved but are still mainly based on indirect recognition of bacteria. Both microscopy and molecular recognition are being improved, but most procedures for bacteria detection in tissue involve extensive damage. Here, we describe a method to visualize bacteria in tissue slices from an in vivo model of breast cancer. This method allows examining trafficking and colonization of fluorescein-5-isothiocyanate (FITC)-stained bacteria in various tissues. The protocol provides direct visualization of fusobacterial colonization in breast cancer tissue. Rather than processing the tissue or confirming bacterial colonization by PCR or culture, the tissue is directly imaged using multiphoton microscopy. This direct visualization protocol causes no damage to the tissue; therefore, all structures can be identified. This method can be combined with others to co-visualize bacteria, types of cells, or protein expression in cells.

High mannose level in bladder cancer enhances type 1 fimbria-mediated attachment of uropathogenic E. coli.

Bladder cancer is the 4th leading cancer in men. Tumor resection followed by bladder instillation of Bacillus Calmette-Guérin (BCG) is the primary treatment for high-risk patients with Non-Muscle Invasive Bladder Cancer (NMIBC) to prevent recurrence and progression to muscle-invasive disease. This treatment, however, lacks efficiency and causes severe adverse effects. Mannose residues are expressed on bladder surfaces and their levels were indicated to be higher in bladder cancer. Intravesical instillations of a recombinant Pseudomonas aeruginosa (PA) overexpressing the mannose-sensitive hemagglutination fimbriae (PA-MSHA), and of a mannose-specific lectin-drug conjugate showed efficiency against NMIBC in murine models of bladder cancer. Urothelial mannosylation facilitates bladder colonization by Uropathogenic E. coli (UPEC) via the interaction with the FimH mannose lectin, positioned at the tip of type 1 fimbria. A recombinant BCG strain overexpressing FimH on its outer surface, exhibited higher attachment and internalization to bladder cancer cells and increased effectivity in treating bladder cancer in mice. Investigating the pattern of mannose expression in NMIBC is important for improving treatment. Here, using tissue microarrays containing multiple normal and cancerous bladder samples, and lectins, we confirm that human bladder cancer cells express high mannose levels. Using UPEC mutants lacking or overexpressing type 1 fimbria, we also demonstrate that tumor-induced hypermannosylation increases type 1 fimbria mediated UPEC attachment to human and mouse bladder cancer. Our results provide an explanation for the effectiveness of PA-MSHA and the FimH-overexpressing BCG and support the hypothesis that mannose-targeted therapy holds potential for improving bladder cancer treatment.

Colon Cancer-Associated Fusobacterium nucleatum May Originate From the Oral Cavity and Reach Colon Tumors via the Circulatory System.

Fusobacterium nucleatum is a common oral bacterium that is enriched in colorectal adenomas and adenocarcinomas (CRC). In humans, high fusobacterial CRC abundance is associated with chemoresistance and poor prognosis. In animal models, fusobacteria accelerate CRC progression. Targeting F. nucleatum may reduce fusobacteria cancer progression and therefore determining the origin of CRC F. nucleatum and the route by which it reaches colon tumors is of biologic and therapeutic importance. Arbitrarily primed PCR performed previously on matched same-patients CRC and saliva F. nucleatum isolates, suggested that CRC F. nucleatum may originate from the oral cavity. However, the origin of CRC fusobacteria as well as the route of their arrival to the tumor have not been well-established. Herein, we performed and analyzed whole genome sequencing of paired, same-patient oral, and CRC F. nucleatum isolates and confirmed that CRC-fusobacteria originate from the oral microbial reservoir. Oral fusobacteria may translocate to CRC by descending via the digestive tract or using the hematogenous route during frequent transient bacteremia caused by chewing, daily hygiene activities, or dental procedures. Using the orthotropic CT26 mouse model we previously showed that IV injected F. nucleatum colonize CRC. Here, we compared CRC colonization by gavage vs. intravenous inoculated F. nucleatum in the MC38 and CT26 mouse orthotropic CRC models. Under the tested conditions, hematogenous fusobacteria were more successful in CRC colonization than gavaged ones. Our results therefore provide evidence that the hematogenous route may be the preferred way by which oral fusobacteria reach colon tumors.

Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression.

Fusobacterium nucleatum is an oral anaerobe recently found to be prevalent in human colorectal cancer (CRC) where it is associated with poor treatment outcome. In mice, hematogenous F. nucleatum can colonize CRC tissue using its lectin Fap2, which attaches to tumor-displayed Gal-GalNAc. Here, we show that Gal-GalNAc levels increase as human breast cancer progresses, and that occurrence of F. nucleatum gDNA in breast cancer samples correlates with high Gal-GalNAc levels. We demonstrate Fap2-dependent binding of the bacterium to breast cancer samples, which is inhibited by GalNAc. Intravascularly inoculated Fap2-expressing F. nucleatum ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria are impaired in tumor colonization. Inoculation with F. nucleatum suppresses accumulation of tumor infiltrating T cells and promotes tumor growth and metastatic progression, the latter two of which can be counteracted by antibiotic treatment. Thus, targeting F. nucleatum or Fap2 might be beneficial during treatment of breast cancer.

Fusobacterium nucleatum supresses anti-tumor immunity by activating CEACAM1.

Fusobacterium nucleatum (F. nucleatum) is an oral anaerobe found to be enriched in colorectal cancer (CRC). Presence of F. nucleatum in CRC has been correlated with resistance to chemotherapy and poor prognosis. We previously demonstrated that the Fap2 outer-surface protein of F. nucleatum binds and activates the human inhibitory receptor TIGIT which is expressed by T and Natural Killer (NK) cells, and inhibits anti-tumor immunity. Here we show that F. nucleatum also binds and activates the human inhibitory receptor CEACAM1 leading to inhibition of T and NK cells activities. Our results suggest that using CEACAM1 and TIGIT inhibitors and specific targeting of fusobacteria should be considered for treating fusobacteria-colonized tumors.

The Helicobacter pylori HopQ outermembrane protein inhibits immune cell activities.

We previously showed that the colorectal cancer colonizing bacterium Fusobacterium nucleatum protects tumors from immune cell attack via binding of the fusbacterial Fap2 outer-membrane protein to TIGIT, a checkpoint inhibitory receptor expressed on T cells and NK cells. Helicobacter pylori, the causative agent for peptic ulcer disease, is associated with the development of gastric adenocarcinoma and MALT lymphoma. The HopQ outer-membrane adhesin of H. pylori was recently shown to bind carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) including CEACAM1, an inhibitory receptor expressed mainly by activated T and NK cells. Here we investigated the possibility that similar to Fap2, HopQ can also inhibit immune cell activities by interacting with CEACAM1. We used several approaches to confirm that HopQ indeed interacts with CEACAM1, and show that CEACAM1-mediated activation by HopQ, may inhibit NK and T cell functions.

Tumor Targeting by Fusobacterium nucleatum: A Pilot Study and Future Perspectives.

Colorectal adenocarcinoma (CRC) is a common tumor with high mortality rates. Interestingly, CRC was found to be colonized by the oral anaerobic bacteria Fusobacterium nucleatum, which accelerates tumor progression and enables immune evasion. The CRC-specific colonization by fusobacteria is mediated through the recognition of tumor displayed Gal-GalNAc moieties by the fusobacterial Fap2 Gal-GalNAc lectin. Here, we show high Gal-GalNAc levels in additional adenocarcinomas including those found in the stomach, prostate, ovary, colon, uterus, pancreas, breast, lung, and esophagus. This observation coincides with recent reports that found fusobacterial DNA in some of these tumors. Given the tumorigenic role of fusobacteria and its immune evasion properties, we suggest that fusobacterial elimination might improve treatment outcome of the above tumors. Furthermore, as fusobacteria appears to specifically home-in to Gal-GalNAc-displaying tumors, it might be engineered as a platform for treating CRC and the above common, lethal, adenocarcinomas.