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.

Fusobacterium nucleatum and cancer.

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.

Placental colonization by Fusobacterium nucleatum is mediated by binding of the Fap2 lectin to placentally displayed Gal-GalNAc.

While the existence of an indigenous placental microbiota remains controversial, several pathogens are known to be involved in adverse pregnancy outcomes. Fusobacterium nucleatum is an oral bacterium that is one of several bacteria associated with preterm birth. Oral fusobacteria translocate to the placenta hematogenously; however, the mechanisms localizing them to the placenta remain unclear. Here, using peanut agglutinin, we demonstrate that the level of Gal-GalNAc (Galß1-3GalNAc; Thomsen Friedenreich antigen) found on trophoblasts facing entering maternal blood rises during gestation and is recognized by the fusobacterial Fap2 Gal-GalNAc lectin. F. nucleatum binding to human and mouse placenta correlates with Gal-GalNAc levels and is reduced upon O-glycanase treatment or with soluble Gal-GalNAc. Fap2-inactivated F. nucleatum shows reduced binding to Gal-GalNAc-displaying placental sections. In a mouse model, intravenously injected Fap2-expressing F. nucleatum, but not a Fap2 mutant, reduces mouse fetal survival by 70%.

CEACAM1 Activation by CbpF-Expressing E. coli.

Recent studies on the oral, anaerobic, gram-negative bacterium Fusobacterium nucleatum revealed its presence and involvement in colorectal, esophageal and breast cancer. We previously demonstrated that F. nucleatum binds and activates the human inhibitory receptors TIGIT and CEACAM1 leading to inhibition of T and NK cell anti-tumor immunity. CEACAM1 was found to be bound and activated by the fusobacterial trimeric autotransporter adhesin CbpF. Here we report the generation of a recombinant E. coli expressing full-length CbpF that efficiently binds and activates CEACAM1.

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.