Abstract 122: Fusobacterium nucleatum in saliva of oral cancer patients induces tumor stemness defense phenotype in cancer stem cells

Abstract Background: Oral squamous cell carcinoma (OSCC) is a highly aggressive cancer. Cancer Stem Cells (CSCs) having cell surface markers CD44, ALDH, and EpCAM+ have been identified in OSCC. However, the host pathogen interaction between CSCs and oral microbiome has not yet been studied. A number of studies have recently demonstrated the presence of specific oral bacteria populations and their lipopolysaccharides (LPS) in the tumor microenvironment (1). We previously reported pathogenic bacterial internalization in the CSCs (2). Based on the findings, we have developed an in-vitro model to investigate how oral microbiota may integrate into the tumor microenvironment's CSC population and control its activity. Here, we investigated the host/pathogen interaction between CSCs and FN. Methods: Oral bacteria FN was selectively isolated using FEA agar plates from oral saliva of oral cancer patients (n=10) at stage 3 or 4. The isolated bacteria was co cultured with SCC 25 cells at MOI of 1: 50. After co culturing the stemness genes such as SOX 2, OCT 4, Nanog, LPS and ABCG2+ cancer stem cell markers were analysed. ABCG2+ CSCs and ABCG2- cells were immunomagnetically sorted and lysed followed by RNA extraction and RT QPCR was performed to observe if FN has selectively internalised in the CSCs. Also ABCG2+ and ABCG 2- cells were used to perform clonogenic assay. Results: Coculture of FN with SCC 25 cell line induced high expression of stemness genes SOX, OCT 4 and Nanog along with CSC markers ABCG2+. Moreover high expression of LPS was observed indicating the presence of FN in the coculture. Importantly live bacteria was found internalized in ABCG2+ cells but not in ABCG2- cells. High clonogenicity was observed in ABCG2+ CSCs compared to ABCG2- cells. Notably, we found that live bacteria and their LPS, mostly Fusobacterium nucleatum isolated (FN) from clinical subjects, were capable of invading CSCs in the in-vitro setting. Post the host-pathogen interaction; it enabled the activation of a niche modulatory tumor stemness defense (TSD) phenotype in the CSCs. These aggressive CSCs with the TSD phenotype have been found to have a critical role in the progression and relapse of oral cancer. Conclusion: Expression of stemness genes infers the niche defence mechanism of the CSCs of the tumour microenvironment due to infection of FN. Importantly presence of live bacteria in ABCG2+ CSCs indicates that FN selectively infects ABCG2+ CSCs but not ABCG2- cells. High clonogenicity of ABCG2+ CSCs confirms the TSD phenotypic attributes of the CSCs. References: 1. doi: 10. 3389/fimmu. 2023. 1198269. 2. doi: 10. 3389/fimmu. 2022. 933329. Citation Format: Partha Jyoti Saikia, Bidisha Pal, Lekhika Pathak, Shirsajit Mitra, Tutumoni Baishya, Rupam Das, Bikul Das. Fusobacterium nucleatum in saliva of oral cancer patients induces tumor stemness defense phenotype in cancer stem cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 122.