Abstract 4902: Gut bacteria as hidden architects of stemness maintenance in the progression of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, defined by an aggressive tumor microenvironment (TME) and a small pool of cancer stem cells (CSCs) that fuel recurrence and therapy resistance. Evidence suggests that bacteria migrating from the gut to the pancreas reshape the TME to sustain these CSCs. Although microbial imbalance is linked to other cancers, its role in PDAC stemness remains unclear. This study investigates how gut-derived microbes and their metabolites reprogram the tumor niche to enrich CSCs and accelerate PDAC progression. Methodology: Clinical profiling of PDAC patient samples was performed to identify diverse microbial species that contribute to dysbiosis. We performed tissue validation using 16S rRNA FISH (fluorescent In-situ hybridization) and lipopolysaccharide (LPS) staining to localize these bacteria in human and murine PDAC tissues. To define microbial effects, we exposed PDAC and normal pancreatic cell lines to bacterial supernatants, live bacteria, or purified LPS, followed by functional validation through tumor sphere, serial dilution, and colony assays with CSC markers assessed by immunofluorescence, western blotting, and qPCR. We next applied Omics Integration using LC-MS of bacterial supernatants and RNA sequencing of Antibiotics (Abx) treated mice to identify microbial metabolites and signaling pathways linked to CSC enrichment. In our In-Vivo studies using KrasLSL-G12D/+;Pdx1-Cre (KC), KrasLSL-G12D/+;Trp53R172H/+;Pdx1-Cre (KPC), and C57BL/6J (C57) mice treated with Abx, we evaluated how microbiome depletion reshapes CSC dynamics and PDAC progression. Results: Clinical Profiling of 62 resected PDAC samples showed that the basal-like subtype is enriched with Pseudomonas aeruginosa, Acinetobacter baumannii, and Sphingopyxis macrogoltabida, linking microbial dysbiosis to tumor aggressiveness. In-situ hybridization and LPS staining revealed stage-dependent bacterial accumulation in human PDAC and in KC and KPC tumors. LPS or bacterial supernatants elevated CSC and proliferation markers including SOX2, SOX9, NANOG, Cyclin A, and p-ERK, while normal pancreatic cells remained unchanged. Co-culture with P.aeruginosa and S.macrogoltabida produced the strongest stemness induction with LPS-CD44 co-localization. Functional assays and KPC organoids confirmed enhanced self-renewal and growth, most pronounced with S.macrogoltabida supernatant. Abx treatment in xenograft tumors altered tumor growth, and KC and KPC mice showed reduced PanIN lesions with decreased CD44 and Ki67 expression, consistent with loss of CSC maintenance. Conclusion: A dysbiotic microbiome within the PDAC microenvironment sustains CSC-driven aggressiveness through metabolite-mediated signaling. Targeting this microbiome-stemness axis may offer a therapeutic approach to limit PDAC progression and improve patient outcomes. Citation Format: Kirtana Arikath, Seoung Ryoung Choi, Annant Bir Kaur, Nivedeta Krishna Kumar, Venkatesh Varadharaj, Zahraa Wajih Alsafwani, Neelanjana Gayen, Poompozhil Mathivanan, Wyatt Petersen, Prabagaran Narayanasamy, Surinder K. Batra, Moorthy P. Ponnusamy. Gut bacteria as hidden architects of stemness maintenance in the progression of pancreatic cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4902.