Yamaoka Toshimitsu, Ohba Motoi, Arata Satoru, Ohmori Tohru
Institute of Molecular Oncology, Showa University;
Institute of Molecular Oncology, Showa University.
J Vis Exp. 2017 Aug 11(126):55967. doi: 10.3791/55967.
Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.
耐药性是癌症治疗中的一项重大挑战。在体外生成耐药亚系对于发现克服这一挑战的新机制而言是必要的。在此，描述了一种用于建立对表皮生长因子受体（EGFR）-酪氨酸激酶抑制剂（TKI）吉非替尼和一种MET-TKI PHA665752双重耐药的两步剂量递增法。该方法基于抑制剂的简单逐步剂量递增以在细胞系中诱导获得性耐药。生成耐药亚系的另一种方法是一步将细胞暴露于高浓度抑制剂。与这种方法相比，逐步剂量递增法成功诱导获得性耐药的可能性更高。激活型EGFR突变是非小细胞肺癌（NSCLC）中存在这些突变时EGFR-TKI治疗反应的生物标志物，EGFR-TKI是针对此类NSCLC的一线应用治疗。然而，尽管有有效反应的报道，但EGFR-TKI的使用仍受到限制，因为肿瘤不可避免地会产生耐药性。EGFR-TKI耐药背后的主要机制包括EGFR第20外显子守门位点的二次突变T790M以及MET的旁路信号。因此，针对这一问题的一个潜在解决方案是EGFR-TKI与MET-TKI联合使用。这种联合治疗在体外研究模型中已显示出有效性。通过对吉非替尼进行12个月的逐步剂量递增，经MET扩增建立了获得性吉非替尼耐药，并在先前的研究中生成了一个名为PC-9MET1000的细胞系。为了进一步研究获得性MET-TKI和EGFR-TKI耐药的机制，在吉非替尼存在的情况下，对这些细胞逐步递增剂量给予MET-TKI PHA665752，持续12个月。该方案也已成功应用于多种联合疗法以建立对其他抑制剂分子的获得性耐药。
