A Novel Multidrug-Resistant Cell Line from an Italian Intrahepatic Cholangiocarcinoma Patient

SIMPLE SUMMARY: Intrahepatic cholangiocarcinoma (ICC) has limited prognosis and therapies. The first-line gemcitabine-based therapy provided poor benefits in terms of survival due to the development of resistance. Gemcitabine-resistance mechanisms were studied on pancreatic cancer models or cell lines derived from ICC patients of Eastern countries. Since ICC has different etiology and genetic/molecular characteristics depending on the ethnicity, appropriate preclinical models that recapitulate their biology are required. Thus, we aimed to establish and characterize an Italian ICC cell line, named 82.3. Cells were isolated from a patient-derived xenograft. After one year, immunophenotypical, biological, genetic, molecular features, and in vivo tumorigenicity in NOD/SCID mice were investigated. Furthermore, 82.3 cells displayed resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin. This model could be exploited either to investigate drug resistance mechanisms or to test alternative drugs through the identification of suitable targets to overcome drug resistance. ABSTRACT: Chemotherapy resistance is a relevant clinical issue in tumor treatment, in particular in biliary tract carcinoma (BTC), for which there are no effective therapies, neither in the first nor in the second line. The development of chemoresistant cell lines as experimental models to investigate the mechanisms of resistance and identify alternative druggable pathways is mandatory. In BTC, in which genetics and biological behavior depend on the etiology, ethnicity, and anatomical site of origin, the creation of models that better recapitulate these characteristics is even more crucial. Here we have established and characterized an intrahepatic cholangiocarcinoma (iCCA) cell line derived from an Italian patient, called 82.3. Cells were isolated from a patient-derived xenograft (PDX) and, after establishment, immunophenotypic, biological, genetic, molecular characteristics, and tumorigenicity in vivo in NOD/SCID mice were investigated. 82.3 cells exhibited epithelial morphology and cell markers (EPCAM, CK7, and CK19); they also expressed different cancer stem markers (CD44, CD133, CD49b, CD24, Stro1, PAX6, FOXA2, OCT3/4), α–fetoprotein and under anchorage-independent and serum-free conditions were capable of originating cholangiospheres. The population doubling time was approximately 53 h. In vitro, they demonstrated a poor ability to migrate; in vivo, 82.3 cells retained their tumorigenicity, with a long latency period (16 weeks). Genetic identity using DNA fingerprinting analysis revealed 16 different loci, and the cell line was characterized by a complex hyperdiploid karyotype. Furthermore, 82.3 cells showed cross-resistance to gemcitabine, 5-fluorouracil, carboplatin, and oxaliplatin; in fact, their genetic profile showed that 60% of genes (n = 168), specific for drug resistance and related to the epithelial-mesenchymal transition, were deregulated in 82.3 cells compared to a control iCCA cell line sensitive to chemotherapeutics. RNA sequencing analysis revealed the enrichment for genes associated with epithelial to mesenchymal transition (EMT), vasculature development, and extracellular matrix (ECM) remodeling, underlining an aggressive phenotype. In conclusion, we have created a new iCCA cell line of Caucasian origin: this could be exploited as a preclinical model to study drug resistance mechanisms and to identify alternative therapies to improve the prognosis of this tumor type.