体外模型评估肿瘤细胞多药耐药性：生化、形态学技术及药理策略

ATP结合盒（ABC）转运蛋白的过表达是化疗失败的重要原因，代表肿瘤细胞适应抗癌药物的机制之一，即多药耐药（MDR）。本综述旨在介绍检测多药耐药（MDR）调节因子或抑制剂的体外模型的主要方法，包括用于化疗敏感性研究的生化及形态学技术。研究发现，化疗暴露的癌细胞中，MDR蛋白，尤其是糖蛋白-1（P-gp或ABCB1）、多药耐药相关蛋白1（MRP1或ABCC1）、多药耐药相关蛋白2（MRP2或ABCC2）及癌症耐药蛋白（ABCG2）表现出过表达，且通过多种技术手段进行检测。其中最常用的方法包括：(i) 比色/荧光间接生物测定法，(ii) 罗丹明及其外排分析，(iii) 利用荧光显微镜和流式细胞仪测定3,30-二乙氧基酰胺碳酰基吡咯啉碘化物的释放以检测P-gp功能及其他ABC转运蛋白，(iv) 钙青霉素乙酰氧基甲基酯的排除，(v) ATP酶分析以区分与ABC转运蛋白的不同相互作用类型，(vi) 形态学观察以详细描述转化细胞的表型特征，(vii) 分子检测耐药相关蛋白（RT-qPCR），(viii) 2D和3D模型，(ix) 类器官（organoids），以及(x) 微流控技术。随后，建立了用于检测化疗MDR细胞的体外模型，以评估新型疗法对调节或抑制肿瘤细胞生长、克服临床耐药的潜力。值得注意的是，包括抗miRNA、抗体药物偶联物（针对天然产物）及表观遗传修饰等多种疗法也被视为有前景的替代方案，目前尚无抗MDR疗法能显著改善患者生活质量。因此，亟需开发新的临床耐药性标志物，以更可靠反映新型抗肿瘤药物在体内的实际疗效。

The overexpression of ATP-binding cassette (ABC) transporters contributes to the failure of chemotherapies and symbolizes a great challenge in oncology, associated with the adaptation of tumor cells to anticancer drugs such that these transporters become less effective, a mechanism known as multidrug resistance (MDR). The aim of this review is to present the most widely used methodologies for induction and comprehension of in vitro models for detection of multidrug-resistant (MDR) modulators or inhibitors, including biochemical and morphological techniques for chemosensitivity studies. The overexpression of MDR proteins, predominantly, the subfamily glycoprotein-1 (P-gp or ABCB1) multidrug resistance, multidrug resistance-associated protein 1 (MRP1 or ABCCC1), multidrug resistance-associated protein 2 (MRP2 or ABCC2) and cancer resistance protein (ABCG2), in chemotherapy-exposed cancer lines have been established/investigated by several techniques. Amongst these techniques, the most used are (i) colorimetric/fluorescent indirect bioassays, (ii) rhodamine and efflux analysis, (iii) release of 3,30-diethyloxacarbocyanine iodide by fluorescence microscopy and flow cytometry to measure P-gp function and other ABC transporters, (iv) exclusion of calcein-acetoxymethylester, (v) ATPase assays to distinguish types of interaction with ABC transporters, (vi) morphology to detail phenotypic characteristics in transformed cells, (vii) molecular testing of resistance-related proteins (RT-qPCR) and (viii) 2D and 3D models, (ix) organoids, and (x) microfluidic technology. Then, in vitro models for detecting chemotherapy MDR cells to assess innovative therapies to modulate or inhibit tumor cell growth and overcome clinical resistance. It is noteworthy that different therapies including anti-miRNAs, antibody-drug conjugates (to natural products), and epigenetic modifications were also considered as promising alternatives, since currently no anti-MDR therapies are able to improve patient quality of life. Therefore, there is also urgency for new clinical markers of resistance to more reliably reflect in vivo effectiveness of novel antitumor drugs.