Establishment of extrahepatic bile duct cancer organoids using suspension culture and their application in clinical drug screening

The present study aims to explore the feasibility of establishing human extrahepatic cholangiocarcinoma (eCCA) organoids via suspension culture, to biologically characterize their biological features and to evaluate their drug sensitivity, thereby providing a preclinical model for personalized precision therapy. Surgically resected tumor tissues were obtained from patients with eCCA. Tumor cells were isolated by mechanical dissociation and enzymatic digestion, and were cultured in a matrix‑free suspension system for three‑dimensional organoid formation and continuous passaging. Histomorphological features of the organoids and the corresponding parental tumor tissues were compared by H&E staining. Immunohistochemistry was used to detect the expression of the cholangiocyte markers cytokeratin (CK)7 and CK19, as well as Ki‑67, to confirm tissue origin and proliferative activity. In addition, the organoid model was used for in vitro sensitivity testing of conventional chemotherapy regimens. The organoids exhibited morphological features of moderately to poorly differentiated adenocarcinoma, consistent with those of the parental tumors, including tubular structures, solid nests and nuclear atypia. Immunohistochemical analysis showed high expression of CK7 and CK19, confirming cholangiocytic origin, along with high Ki‑67 expression, indicating active proliferative capacity. Drug sensitivity testing revealed substantial heterogeneity in treatment responses among organoid lines derived from different patients, indicating variable sensitivity to chemotherapeutic agents. In conclusion, an eCCA organoid model was successfully established using a suspension culture method, and it largely preserved the histopathological and cellular phenotypic features of the parental tumor. This model functions as an in vitro ‘patient surrogate’ for drug‑sensitivity testing, with potential for evaluating chemotherapy and targeted‑drug efficacy. However, the observed clinical concordance in a single patient is preliminary and its definitive predictive value requires validation in larger prospective cohorts. Theoretically, this system offers advantages in scalability and standardization for clinical applications, although direct comparisons with traditional matrices remain to be conducted.