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Improving our understanding of this issue is crucial for developing strategies that can overcome drug refractoriness. We have established and characterized two novel human cell sublines derived from extrahepatic CCA EGI-1 cells that are resistant to cisplatin and 5-fluorouracil (5-FU). Migration and proliferation were analyzed using holographic microscopy. The expression of genes involved in drug uptake and efflux was determined by RT-qPCR. Cross-resistance to commonly used antitumor drugs was assayed using the MTT test. EGI-1 sublines resistant to cisplatin (CR) or 5-FU (FR) exhibited more than a three-fold increase in resistance to cisplatin and 5-FU, respectively, and showed reduced proliferation, migration, and colony-formation rates, along with an altered cell cycle compared to wild-type cells, while retaining tumorigenic capacity. The analysis of the transportome showed downregulation of uptake transporters and upregulation of the export pumps MRP3\/4. EGI-1 cells with acquired resistance to 5-FU demonstrated cross-resistance to irinotecan and gemcitabine, while cisplatin-resistant cells showed decreased sensitivity to 5-FU and platinum derivatives. These resistant cell lines offer valuable models for investigating the molecular basis of chemoresistance in CCA, providing a robust platform for the development and evaluation of novel therapeutic strategies.<\/jats:p>","DOI":"10.3390\/molecules30143053","type":"journal-article","created":{"date-parts":[[2025,7,21]],"date-time":"2025-07-21T17:27:36Z","timestamp":1753118856000},"page":"3053","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Drug-Resistant Cholangiocarcinoma Cell Lines for Therapeutic Evaluation of Novel Drugs"],"prefix":"10.3390","volume":"30","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-5134-1475","authenticated-orcid":false,"given":"Kevin","family":"Delgado-Calvo","sequence":"first","affiliation":[{"name":"Experimental Hepatology and Drug Targeting (HEVEPHARM) Group, Institute of Biomedical Research of Salamanca (IBSAL), Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), University of Salamanca, Campus M. Unamuno s\/n, 37007 Salamanca, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7714-1691","authenticated-orcid":false,"given":"Elisa","family":"Lozano","sequence":"additional","affiliation":[{"name":"Experimental Hepatology and Drug Targeting (HEVEPHARM) Group, Institute of Biomedical Research of Salamanca (IBSAL), Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), University of Salamanca, Campus M. Unamuno s\/n, 37007 Salamanca, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6891-1554","authenticated-orcid":false,"given":"Oscar","family":"Briz","sequence":"additional","affiliation":[{"name":"Experimental Hepatology and Drug Targeting (HEVEPHARM) Group, Institute of Biomedical Research of Salamanca (IBSAL), Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), University of Salamanca, Campus M. 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