Wnt Stimulation and Inhibition in the Development and Phenotype of Patient‐Derived Gallbladder Organoids

ABSTRACT Background Gallbladder cancer (GBC) is a highly lethal malignancy with limited experimental models to study disease biology or evaluate therapeutic responses. Although canonical Wnt activation is commonly used for patient‐derived organoid (PDO) development and expansion, gallbladder PDOs have also been generated under Wnt‐inhibitory conditions. No comparative assessments have determined how Wnt pathway modulation influences gallbladder PDO development, phenotype, or drug response. Methods This study systematically compared the impact of canonical Wnt activation (WNT Act medium containing CHIR99021) versus inhibition (WNT Inh medium containing DKK1) on the establishment, propagation, molecular features, and therapeutic responses of PDOs generated from malignant or non‐malignant gallbladder tissues derived from the same patient. Results Both media supported successful PDO generation with comparable efficiency, preserving biliary epithelial functions and marker expression. Transcriptomic profiling confirmed selective enrichment of canonical Wnt target genes in PDOs generated in WNT Act cultures. WNT Act conditions enabled markedly superior long‐term propagation, whereas WNT Inh cultures more consistently retained the dysplastic features in malignant samples. Gemcitabine response assays demonstrated significantly greater drug sensitivity in PDOs grown in WNT Act medium, a phenotype reversible upon media switching but requiring extended adaptation, indicating a dynamic and context‐dependent influence of Wnt‐signaling on chemotherapeutic vulnerability. Conclusion Collectively, the findings reveal a trade‐off between long‐term propagation and histological fidelity in gallbladder PDOs and show that Wnt‐signaling modulates gemcitabine sensitivity in a reversible manner. This comparative framework provides practical guidance for selecting culture conditions for gallbladder PDO‐based disease modeling, and precision oncology applications.