Babajnai Amirhesam, Rahmani Saeed, Asadi Mohammad Jamal, Gheytanchi Elmira, Adibhesami Glavizh, Vakhshiteh Faezeh, Madjd Zahra
Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran.
Department of Computer Engineering, Sharif University of Technology, Tehran, Iran.
Cancer Cell Int. 2025 Apr 18;25(1):154. doi: 10.1186/s12935-025-03758-2.
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.
癌症干细胞（CSCs）作为肿瘤内能够自我更新的细胞亚群，从而驱动肿瘤的起始和扩散。解决与CSCs及其耐药机制相关的癌症治疗失败问题，需要有效的临床前模型来测试靶向治疗。通过用浓度不断增加的5-氟尿嘧啶（5-FU）抗癌药物长时间反复处理细胞，产生了对Caco2和HT-29耐药的细胞。通过细胞毒性试验评估5-FU耐药细胞的敏感性。通过实时PCR进行基因表达研究，评估干性、上皮-间质转化（EMT）、迁移和耐药特性。通过流式细胞术评估CD44、CD133和CD66的表达。最后，生物信息学分析考虑所选基因和蛋白质的差异表达，估计分子功能和生物学途径。暴露于5-FU的细胞对5-FU的耐药性增加。基因表达分析显示，与亲代细胞相比，干性基因（KLF4、SOX2、OCT4、C-MYC）上调，清除系统增强，CSC表面标志物（CD44和CD133）表达升高。此外，与亲代细胞相比，促EMT基因（TWIST1、SNAIL1、ZEB1、波形蛋白和N-钙黏蛋白）显著上调，作为EMT抑制基因的E-钙黏蛋白下调反映在迁移能力增强上。此外，观察到ABC转运蛋白基因（ABCB1、ABCC1）表达增加，与耐药性增强相关。生物信息学分析突出了与癌症中的微小RNA、细胞多能性和蛋白聚糖相关的途径。药物暴露方法优先于球体形成，特别是由于它们在干性、EMT和表面标志物方面具有更高的功效。这使它们成为建立CSCs实验模型的有前景的方案。
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