A Patient-Derived Ex Vivo Tissue Model of Cholangiocarcinoma Using Precision-Cut Tissue Slices

Abstract

Background & Aims: Cholangiocarcinoma (CCA) is an aggressive malignancy with poor five-year survival. Improving outcomes requires preclinical models that faithfully recapitulate the native tumour microenvironment. Precision-Cut Tissue Slices (PCTS) retain in-vivo architecture and represent a promising candidate. We aimed to establish a patient-derived PCTS model of CCA, characterise its response to ex-vivo culture, and assess its use as a platform for therapeutic testing. Methods: PCTS were generated from 25 patients undergoing curative-intent resection for presumed CCA (2022-2024) and cultured for up to 15 days. Viability and histological architecture were assessed serially. Quantitative proteomics (SWATH-DIA) was performed at Days 0, 3, 7 and 15. A subset were treated with staurosporine or clinically relevant chemotherapy (5'-deoxy-5-fluorouridine, gemcitabine {+/-} cisplatin). Results: PCTS viability was maintained to Day 15, with no significant reduction from baseline. PCTS retain tumour architecture, cytokeratin-19-positivity and resident CD3+/CD68+ immune cells during culture. Proteomic profiling quantified 4,578 proteins and identified a staged response, with differentially abundant proteins increasing from 8 (Day 3) to 127 (Day 7) and 180 (Day 15), stabilising thereafter. This comprised of an early loss of inflammatory and stromal proteins, sustained cellular stress responses and metabolic reprogramming. CCA subtype-specific proteomic signatures were preserved throughout culture. Staurosporine produced dose-dependent cytotoxicity whilst chemotherapy responses were variable. Conclusions: Patient-derived CCA PCTS maintain viability, tumour architecture and subtype identity for 15 days while undergoing a defined proteomic response to culture, and retain pharmacological responsiveness,