Oral microbiome reprograms oral cancer stem cells to the highly immunosuppressive tumor stemness defense (TSD) phenotype

Abstract Oral squamous cell carcinoma (OSCC) is a global health disease affecting developing countries, mainly the South and South East Asian countries. This cancer type is mostly HPV-16 negative and exhibits aggressiveness and resistance to therapies. Our previous studies have shown an oral microbiota (Fusobacterium nucleatum a gram negative anaerobic bacillus) associated with tumour progression in oral squamous cell carcinoma (1). The enhanced degree of stemness involved may confer the CSCs to be highly aggressive, self-sufficient, transient and niche modulatory, the tumour stemness defense (TSD) phenotype (2). The TSD characteristics are expressed as a part of “stem cell niche defence mechanism” (2). The TSD phenotype in ABCG2+ CSCs are characterized by the activation of the MYC-HIF-2alpha stemness pathway (3), as well as altruistic defense mechanism (4). Here we further studied the role of F.N. in reprograming of oral CSCs to TSD phenotype. Methods: SCC-25 cell line containing EpCAM+/ABCG2+ CSCs were treated with saliva samples of oral cancer patients (n=15). The cells were grown in antibiotic-treated media for 72 hours and then grown in serum-free conditioned media obtained from naïve MSC culture (2). The treated cells were then subjected to immunomagnetic sorting to obtain EPCAM+/ABCG2+ CSCs; TSD phenotype of the sorted CSCs were evaluated as described (2). Some cells were lysed and subjected to bacterial culture to detect F.nucleatum. Next, we evaluated the therapy resistance of the saliva-treated CSCs with chemotherapy, targeted therapy, immunotherapy and small molecular inhibitors of stemness pathways. We also obtained primary oral cancer EpCAM+/ABCG2+ of platinum treated patients (n=12), and lysed these cells for bacterial culture to detect F.nucleatum phenotype. Results: We confirmed the previous findings that F.nucleatum can be recovered from the EpCAM+/ABCG2+ of SCC-25 cells treated with patient derived saliva (processed saliva samples from relapsed cases; n=6/16 live bacteria recovered). These EpCAM+/ABCG2+ CSCs exhibited TSD phenotype, including the activation of MYC-HIF-2alpha stemness pathway, as well as TLR2/4/NFkB pathway. Importantly, the intracellular F.nucleatum exhibited an increase in surface proteins Fap 2, FadA, the two proteins involved in oncogenesis. We confirmed the FN induced TSD phenotype by treating SCC-25 cells with laboratory derived FN (ATCC 25586). Importantly, the primary tumor derived EpCAM+/ABCG2+ CSCs of 6/18 subjects exhibited the presence of F.N. Conclusion: Our findings indicate the potential role of oral microbiome especially the F. nulceatum in oral cancer stem cell reprograming to the highly aggressive TSD phenotype. 1. Cancer research. 2018;78(13 Supplement):3064-. 2. PMID: 362488583. 3. PMID: 33887214.4. PMID: 31266772 Citation Format: Partha Jyoti Saikia, Lekhika Pathak, Shirsajit Mitra, Bikul Das. Oral microbiome reprograms oral cancer stem cells to the highly immunosuppressive tumor stemness defense (TSD) phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 648.