Abstract 18: Fisetin effectively inhibits cancer stem cell properties in pancreatic ductal adenocarcinoma

Abstract Introduction: Cancer stem cells (CSCs) are crucial in the aggressiveness and chemoresistance of pancreatic ductal adenocarcinoma (PDAC). Targeting CSCs is essential for effective treatment and preventing relapse. Fisetin, a natural flavonoid, has demonstrated anticancer, anti-inflammatory, and antioxidant properties. This study investigates the anti-CSC effects of fisetin in PDAC. Methods: CSCs were isolated from Panc-1 cells and explant cultures of PDAC tissue using fluorescence-activated cell sorting (FACS) with specific surface markers (CD44, CD24, CD133, CD326/ESA). The isolated CSCs were treated with various doses of fisetin for 24 and 48 hours, and the half-maximal inhibitory concentration (IC50) was determined using the MTT assay. The effects of fisetin on colony formation, cell cycle, apoptosis, migration, invasion , and sphere formation were evaluated. Additionally, the expression of epithelial-mesenchymal transition (EMT) markers and stemness markers was assessed at both gene and protein levels. The molecular mechanisms underlying the anti-CSC effects of fisetin were investigated through mRNA sequencing, followed by validation using ELISA. Results: Fisetin significantly reduced viability and colony formation of CSCs in a dose- and time-dependent manner. The apoptotic cells increased from 2.06% in untreated to 50.9% in fisetin-treated CSCs. This was further supported by enhanced expression of apoptosis markers Bid and Bcl-2 in fisetin-treated CSCs. Migration capacity was significantly reduced, with the wound closure area increasing from 1.18% to 29.5%. Fisetin also induced cell cycle arrest, with a notable increase in the percentage of cells in the G2 and S phases. The size of tumor spheroids was significantly reduced after treatment. Additionally, fisetin elevated the expression of the epithelial marker E-cadherin and reduced the level of the mesenchymal marker N-cadherin. The expression of transcription factors SOX2 and OCT4, which are associated with the maintenance of CSCs, was decreased. Reactive oxygen species (ROS) levels in CSCs were significantly reduced. Fisetin treatment led to the downregulation of genes involved in inflammatory signalling pathways, including BCL2A1 (NF-κB target gene), CXCL1, CXCL8, CX3CL1, and CCL2. KEGG pathway analysis showed that the NF-κB and cytokine-cytokine receptor signalling pathways were significantly downregulated following fisetin treatment. ELISA further confirmed the significant downregulation of NF-κB p65 signaling (p = 0.013) in fisetin-treated CSCs. Conclusion: Fisetin effectively targets and inhibits CSCs in PDAC, modulating key markers of epithelial-mesenchymal transition and stemness, while downregulating NF-κB signaling. These results support the need for further validation in in vivo models. Citation Format: Anjali Aggarwal, Vinit Sharma, Rajesh Gupta, Mayank Sharma, Ankita Semwal. Fisetin effectively inhibits cancer stem cell properties in pancreatic ductal adenocarcinoma [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 18.