建立和表征三个抗阿法替尼的肺腺癌PC-9细胞系与增加剂量的阿法替尼开发
Summary
开发了一种从肺腺癌PC-9细胞中建立抗阿法替尼细胞系的方法,并对抗性细胞进行了特征描述。该耐药细胞可用于研究表皮生长因子受体酪氨酸激酶抑制剂抑制剂机制,适用于非小细胞肺癌患者。
Abstract
分子靶向抑制剂的习得抗药性是癌症治疗中的一个严重问题。在大多数国家,肺癌仍然是导致癌症相关死亡的主要原因。发现”致癌驱动因素突变”,如表皮生长因子受体(EGFR)-激活突变,以及随后开发EGFR酪氨酸激酶抑制剂(TKIs)(gefitinib,erlotinib,近几十年来,阿法替尼、达科米尼布和奥西米替尼的肺癌治疗发生了戏剧性的变化。然而,这些药物仍然不能有效在非小细胞肺癌(NSCLC)携带EGFR激活突变的患者。在获得抗药性后,NSCLC的系统性进展仍然是治疗EGFR突变阳性NSCLC患者的重大障碍。在这里,我们提出了一个逐步剂量升级方法,用于从NSCLC PC-9细胞中建立三个独立的获得性阿法替尼耐药细胞系,在EGFR exon 19中携带EGFR-激活突变的15碱基对。简要介绍了三个独立的阿法替尼-抗细胞系的特征化方法。EGFR TKIs 的后天电阻机制是异构的。因此,必须检查对EGFR-TKIs具有获得性强的多个细胞系。使用这种循序渐进的剂量升级方法,需要10到12个月才能获得具有获得性耐药性的细胞系。发现新的后天抵抗机制将有助于制定更有效和安全的治疗策略。
Introduction
五种酪氨酸激酶抑制剂,靶向表皮生长因子受体(EGFR),包括格菲替尼、埃尔洛替尼、阿法替尼、达科米替尼和奥西米替尼,目前可用于治疗EGFR突变阳性非小细胞肺患者癌症(NSCLC)。在过去的十年中,随着新的潜在EGFR-TKIs的发现,针对此类患者的疗法得到了巨大的发展。在肺腺癌患者中,大约50%的亚洲人和15%的白种人患者中,在EGFR中发现细胞突变。EGFR中最常见的突变是EGFR外电21和15碱基对(bp)缺失中的EGFR外电192中的L858R点突变。在NSCLC的EGFR突变阳性患者中,EGFR-TKIs比以前的铂双子化疗3标准提高了反应率和临床结果。
格菲替尼和埃尔洛替尼是第一批经批准的小分子抑制剂,通常被称为第一代EGFR TKIs。这些EGFR TKI通过与ATP竞争和逆向结合到ATP结合位点4来阻止酪氨酸激酶活性。阿法替尼是第二代EGFR TKI,不可逆转和共价结合于EGFR的酪氨酸激酶域,并被定性为泛人EGFR家族抑制剂5。
尽管这些疗法在NSCLC患者中具有显著的好处,但获得抗药性是不可避免的。对第一代和第二代EGFRTKIs最常见的抗药性机制是EGFR外大20中T790M突变的出现,该突变存在于肿瘤样本6、7、8的50-70%中。其他抵抗机制包括旁路信号(对MET,IGF1R和HER2),转化为小细胞肺癌,和诱导上皮到中位体过渡,这发生在临床前和临床9。EGFR TKI 的电阻机制是异构的。通过在临床前研究中识别新的抗药性机制,有可能开发新的治疗方法来克服耐药性。最佳序列疗法,使患者的临床效益最大化,必须考虑抵抗机制和治疗目标。
必须选择正确的亲细胞系,因为它是所有后续实验的基础。选择策略从临床相关性开始;有必要选择化疗和放疗幼稚的细胞系。以前的化疗和/或放射治疗可能诱发耐药性途径的改变和耐药性标记物表达的变化。在这项研究中,PC-9细胞,携带15 bp缺失在EGFR exon 19,用于建立对阿法替尼的后天抵抗。该细胞系来自一名日本NSCLC患者,他之前没有接受化疗和放疗。
由于阿法替尼是每天口服的,持续的体外治疗,其中细胞在阿法替尼的存在下不断培养,在临床上是相关的。实验各步骤中使用的药物剂量必须针对所选的亲子细胞系进行优化。细胞毒性测定可用于确定合适的药物范围,该范围应与药物的药代动力学信息相媲美。
在整个选择过程中,整个细胞群作为一个群体保持;不使用克隆或其他分离方法。细胞首先持续暴露于低水平的药物。随后,在细胞适应在药物存在的情况下生长后,药物的剂量缓慢地增加到药物10、11的最终最佳剂量。或者,脉冲药物管理或诱变可用于选择抗药性细胞,这些细胞也在药物治疗12、13之前进行。不幸的是,一般不报告耐药性没有发展的情况。制定选择策略的目的是试图模拟癌症患者的条件,以重建临床相关的抗药性。有时,为了识别与耐药性机制相关的分子变化,使用高药物浓度。这种模式在临床上变得不那么相关。
在这里,我们描述了一种从PC-9细胞中建立三个独立的抗阿法替尼细胞系的方法,在EGFR exon 19中含有15 bp缺失,以及抗阿法替尼细胞系的初始表征。
Protocol
Representative Results
Discussion
在这里,我们描述了一种建立三个独立的抗阿法替尼细胞系的方法,并通过与亲的PC-9细胞进行比较来对这些细胞进行特征化。通过逐步剂量升级暴露,父母PC-9细胞在10-12个月内获得对阿法替尼的抵抗力。临床上,EGFR TKIs的抗性机制是异质的,因此,在使用阿法替尼进行初步治疗后,PC-9细胞被分成三个独立的p100碟,并进一步暴露在阿法替尼。最初,细胞生长没有显著减缓,但随着药物浓度接近IC50值,细?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢高级癌症转化研究所的成员,感谢他们在英语编辑方面给予的周到评论和编辑。这项工作得到了JSPS KAKENHI(赠款编号:16K09590到T.Y.)的支持。
Materials
| afatinib | Selleck | S1011 | |
| anti-EGFR monoclonal antibody | cell signaling technology | 4267S | |
| bicinchoninc acid assay | sigma | B9643 | |
| cell-culture treated 10cm dish | Violamo | 2-8590-03 | |
| CELL BANKER1 | TakaRa | CB011 | cryopreservation media |
| CellTiter 96 | Promega | G4100 | Non-Radioactive Cell Proliferation Assay; Dye solution and Solubilization/Stop solution |
| DMSO | Wako | 043-07216 | |
| ECL solution | Perkin Elmer | NEL105001EA | |
| FBS | gibco | 26140-079 | |
| GeneAmp 5700 | Applied Biosystems | fluorescence-based RT-PCR-detection system | |
| GraphPad Prism v.7 software | GraphPad, Inc. | a statistical software | |
| NanoDrop Lite spectrophotometer | Thermo | spectrophotometer | |
| Nonfat dry milk | cell signaling technology | 9999S | |
| Pen Strep | gibco | 15140-163 | |
| phosphatase inhibitor cocktail 2 | sigma | P5726 | |
| phosphatase inhibitor cocktail 3 | sigma | P0044 | |
| Powerscan HT microplate reader | BioTek | ||
| Power SYBR Green master mix | Applied Biosystems | SYBR Green master mix | |
| protease inhibitor cocktail | sigma | P8340 | |
| QIAamp DNA Mini kit | Qiagen | 51306 | DNA purification kit |
| QIAquick PCR Purification Kit | QIAGEN | PCR purification kit | |
| RPMI-1640 | Wako | 189-02025 | with L-Glutamine and Phenol Red |
| TBST powder | sigma | T9039 | |
| Trans-Blot SD Semi-Dry Electrophoretic Transfer cell | Bio-Rad | semi-dry t4ransfer apparatus | |
| 96 well microplate | Thermo | 130188 |
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