Functional and molecular profiling of primary intrahepatic cholangiocarcinoma cell lines along its tissue histo-morphological spectrum

Background and Aim Cholangiocarcinoma (CCA) is a biliary cancer with an insidious onset and non-specific symptoms, often leading to late diagnosis and limited curative options. Intrahepatic CCA (iCCA) is not a single patho-bio-molecular entity but an umbrella term encompassing at least two distinct histo-morphological subtypes according to International Classification of Diseases for Oncology (version 3.2, 2019): small bile duct (SBD) and large bile duct (LBD) type iCCA. The main aim of this work is to standardize a reliable and reproducible protocol for isolating and culturing in vitro primary cell lines from SBD and LBD type iCCA. Materials and Methods iCCA tissues were collected from N=23 patients (average 72 year; SD 9.51) who underwent surgical resection, after obtaining informed consent. Tissue of origin at the diagnosis were characterized by histo-morphology and Next-Generation Sequencing (NGS; OCAplus panel®). Following our cell isolation protocol, the obtained primary cells were characterized through various functional assays assessing their ability to maintain viability (Trypan Blue Exclusion), proliferative potential (MTS), and clonogenic capacity (Colony Formation), along with the molecular profiling. Finally, selected cell lines (N=2) were challenged in 3D culture conditions and global transcriptional programs were assessed in paired samples by RNAseq. Results A high success rate of cell isolation (~80%) was achieved in 23 iCCA samples. Functional characterization in N=4 SBD and N=4 LBD type iCCA cell lines revealed high cell viability with increasing proliferation at the later time points (p≤0.05). Data were supported by Colony Formation Assay, with LBD type forming higher number of self-renewing colonies than the SBD type (p≤0.05). The molecular characterization of the mutational profile in tumour tissues and paired primary cell lines, revealed strong concordance in several cases between cells and tissue, identifying shared genetic alterations (amplifications, deletions, indels, copy number variations, and single-nucleotide variants) in key genes involved in cellular signaling, tumor growth, DNA repair, and drug resistance. Compared with paired 3D cultures, 2D cell lines upregulate extracellular matrix remodelling, stromal activation and immune-associated signatures. Discussion In this study, we present an efficient and reproducible model for establishing patient-derived cell lines, which preserve most of the original phenotypic and genotypic features. This use represents a valuable approach for testing multiple therapeutic agents in patients who exhibit resistance to conventional chemotherapy, immunotherapy, or targeted therapies ("Try-it-all" strategy). Despite the lack of in vivo tumorigenicity assessment, the overall prospects remain highly innovative and ready to clinical translation.