Molecular and phenotypic characterization of 5-FU resistant colorectal cancer cells: toward enrichment of cancer stem cells

Cancer stem cells (CSCs) as a subgroup of cells within a tumor capable of self-renewal, thereby driving tumor initiation and spread. Addressing treatment failures in cancer, linked to CSCs and their resistance mechanisms, requires effective preclinical models for testing targeted therapies. Caco2- and HT-29-resistant cells were generated by repeated treatment of cells with growing concentrations of 5-fluorouracil (5-FU) anticancer drug for an extended time. The sensitivity of 5-FU-resistant cells was evaluated by cytotoxicity assay. Stemness, epithelial-mesenchymal transition (EMT), migration and drug resistance characteristics were assessed through gene expression investigation by real-time PCR. The expression of CD44, CD133, and CD66 were evaluated by flow cytometry. To end, the bioinformatic analysis estimated the molecular function and biological pathways considering the differential expression of selected genes and proteins. 5-FU-exposed cells displayed increased resistance to 5-FU. The gene expression analysis showed an upregulation of stemness genes (KLF4, SOX2, OCT4, C-MYC), enhanced scavenging system, and elevated expression of CSC surface markers (CD44 and CD133) compared to parental cells. Additionally, pro-EMT genes (TWIST1, SNAIL1, ZEB1, Vimentin, and N-cadherin) were significantly upregulated compared to parental cells, with the downregulation of E-cadherin as an EMT suppressor gene reflected in increased migration capacity. Moreover, increased expression of ABC transporter genes (ABCB1, ABCC1) was observed, correlating with enhanced drug resistance. The bioinformatic analysis highlighted pathways related to microRNAs in cancer, cells pluripotency, and proteoglycans. Methods of drug exposure take priority over spheroid formation, particularly due to their enhanced efficacy in stemness, EMT, and surface markers. This positions them as a promising protocol for establishing experimental models of CSCs. © The Author(s) 2025.