TLR3-Dependent Polarization of Adipose-Derived Mesenchymal Stem Cells Regulates Pancreatic Cancer Cell Behavior in an Indirect Co-culture Model

Purpose Toll-like receptor 3 (TLR3) signaling is known to regulate the interactions between mesenchymal stem cells (MSCs) and tumor cells; however, its functional impact in the context of pancreatic cancer remains insufficiently defined. This study investigated how pharmacological activation or inhibition of TLR3 in adipose-derived MSCs (ADMSCs) modulates cellular inflammatory responses and alters the paracrine effects exerted on Panc-1 cells. Methods ADMSCs were phenotypically validated by flow cytometry and subsequently exposed to graded doses of the TLR3 agonist Poly(A:U) and the antagonist CU-CPT4a. Optimal doses were identified through cell viability (MTT) assays and expression analyses of IL6, TNFA, IL10, and TGFB1. These doses were then applied in indirect transwell co-culture models at MSC:Panc-1 ratios of 10:1, 1:1, and 1:10 to determine the optimal co-culture condition. At the selected MSC:Panc-1 = 10:1 ratio, Panc-1 cell proliferation (Calcein AM), apoptosis (Annexin V/PI), cell cycle distribution, colony-forming capacity, EMT-related genes (CD44, ZEB1, VIM, CDH1, CLDN1), and metastasis-associated genes (MMP2, MMP9, TIMP1, VEGFR2, PLAU) were evaluated. Results TLR3 activation induced a dose-dependent, biphasic pro-inflammatory response in ADMSCs while maintaining viability at 1 µg/mL. In contrast, TLR3 inhibition generated a strong anti-inflammatory phenotype, with maximal induction of TGFB1 and IL10 at 0.5 µg/mL. In co-culture, MSCs -particularly T3 + MSCs -significantly reduced Panc-1 viability, proliferation, and colony formation; increased G1/S retention; and enhanced apoptotic death. At the gene level, TLR3 activation promoted epithelial stabilization and reduced extracellular matrix degradation, whereas TLR3 inhibition favored a more mesenchymal and invasive phenotype. Conclusion Overall, the findings identify TLR3 signaling in ADMSCs as a critical regulator of tumor-stroma interactions and demonstrate that controlled TLR3 activation may enhance the antitumor potential of MSC-based therapeutic strategies in pancreatic cancer.