Abstract 6531: High resolution analysis of Fusobacterium infection in colorectal cancer

Previous research provided evidence pointing towards an important role for bacterial infection with Fusobacterium nucleatum (F. nucleatum) and F. animalis (previuosly F. nucleatum subsp. animalis) in colorectal cancer (CRC) progression. Both are oral commensal Gram negative anaerobes with recent evidence suggesting F. animalis predominates in CRC (Zepeda-Rivera et al., 2024). We recently applied spatial transcriptomics to demonstrate local effects of Fusobacterium infection on cancer and immune cell gene expression in CRC (Duggan et al, Gut Microbes, 2024). While Fusobacterium infection was observed in specific patches in the tumors, the precise cell types infected with bacteria and the precise localization of bacteria in cells still requires further elucidation. Commercially available strains of F. nucleatum (ATCC 25586) and F. animalis (ATCC 51191) were purchased and separately co-cultured with MDA-MB-468 breast cancer cells. Immuno cytochemistry using a commercial Fusobacteria antibody (ANT0084) and a Fusobacteria antiserum (Prof Slade group), along with pancytokeratin and Hoechst staining was undertaken. High resolution confocal microscopy using an LSM 980 Airyscan 2 microscope (Carl Zeiss, Jena, Germany) was performed to create image stacks of optical sections. CRC tissue sections were subsequently prepared and stained by immunohistochemistry (IHC) for pancytokeratin, CD68, and Fusobacteria (Slade antisera). Imaging of the full tumor face was undertaken at a lower resolution using Cell DIVE (Leica Microsystems, Wetzlar, Germany) to select regions rich in Fusobacteria for further study. Selected regions were then imaged using high resolution confocal microscopy. Fusobacteria antisera stained both F. nucleatum and F. animalis while the commercially produced antibody stained only F. nucleatum. Both bacteria can populate the cell surroundings, cell surface, cytoplasm as well as nucleus. The typical morphology of both F. nucleatum and F. animalis is seen, allowing antibody validation. In CRC tissue intracellular bacteria are seen in both pancytokeratin positive tumor epithelial cells as well as in CD68 (Cluster of Differentiation 68) positive cells (tumor associated macrophages). Tumor associated macrophages (TAMs) have an important role in the tumor immmune microenvironment. In particular, studies have highlighted the importance of M1/M2 macrophage polarization on immune-evasion and prognosis. Our spatial transcriptomic study highlighted reduced M2 polarization as associated with increased Fusobacterial load. The intracellular location of Fusobacteria may suggest a mechanism for altered macrophage polarization. References: Zepeda-Rivera et al, Nature Vol 628, pages 424-432 (2024)Flanagan et al, Eur J Clin Microbiol Infect Dis. 2014 Aug;33(8):1381-90 Duggan et al, Gut Microbes. 2024 Jan-Dec;16(1):2350149. Citation Format: Heiko Dussmann, Barry Maguire, Caoimbhe Burke, Arman Raman, John Burke, William M. Gallagher, Jochen H. Prehn. High resolution analysis of Fusobacterium infection in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6531.