Saikosaponin D Induced Apoptosis and Anti-Tumor Effect Enhancing Chemotherapy Sensitivity of Gemcitabine in Patient-Derived Cholangiocarcinoma Organoids.

BACKGROUND: Cholangiocarcinoma (CCA), an aggressive malignancy with limited therapeutic options and poor survival rates, poses an urgent clinical challenge necessitating innovative therapeutic strategies. Saikosaponin D (SSd), a bioactive compound derived from Bupleurum, has demonstrated anticancer potential in various malignancies. However, its role in CCA remains unexplored. This study investigated the antitumor effects and potential mechanisms of SSd, both alone and in combination with gemcitabine, utilizing patient-derived cholangiocarcinoma organoids (PDCOs). METHODS: Four PDCO models were established from surgical tumor tissues and metastatic ascites of CCA patients. Histological, immunohistochemical (Cytokeratin 7, Cytokeratin 19 and Ki-67), and immunofluorescence analyses validated the fidelity of organoids to the primary tumors. Drug sensitivity testing evaluated SSd (0.001-50µM) and gemcitabine alone or in combination (ratios 1:1, 1:2, 1:4) using dose-response curves and combination index (CI) analysis. Apoptosis mechanisms were assessed via TUNEL staining, caspase-3 activity assays, and the JC-1 assay for the mitochondrial membrane potential. RESULTS: SSd exhibited dose-dependent growth inhibition across PDCOs, with half-maximal inhibitory concentration (IC50) values ranging from 0.9µM to 13µM. Gemcitabine showed IC50 values spanning 0.03µM to 11.1µM. Notably, the SSd-gemcitabine combination at 1:4 ratio demonstrated synergistic effects (CI=0.0005), significantly reducing organoid viability. Ascites-derived PDCOs (eg, CCA182) displayed lower sensitivity to SSd, correlating with prior chemotherapy exposure. Apoptosis induction by SSd was confirmed through increased TUNEL-positive cells, elevated caspase-3 activity, and mitochondrial depolarization. Histological and molecular profiles of PDCOs closely mirrored those of the primary tumors, preserving spatial heterogeneity (eg, glandular structures in tissue-derived PDCOs vs compact morphology in ascites-derived models). CONCLUSION: The combination of SSd and gemcitabine exerts a synergistic antitumor effect in patient-derived cholangiocarcinoma organoids through activating caspase-3 and triggering mitochondrial dysfunction-mediated apoptosis.