Abstract
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.
GRAPHICAL ABSTRACT
IMPACT AND IMPLICATIONS
PCTS are a potentially valuable preclinical cancer model, retaining the native TME, while offering a 3Rs approach to translational research, yet their use in CCA remains limited and the model has not been comprehensively characterised. This study establishes patient-derived CCA PCTS as a viable ex-vivo platform and provides the first detailed proteomic characterisation of how this model behaves during prolonged culture. Our proteomic findings are informative more broadly for researchers developing PCTS as a platform in other cancer types, underscoring the impact of ex-vivo culture and the need to consider whether the proteomic changes described here may influence model behaviour and potential drug sensitivity.