Identification of Ankrd35 as a mediator of cisplatin response in non-small cell lung cancer by in vivo screening of murine tumor derived xenografts. Identification of Ankrd35 as a mediator of cisplatin response in non-small cell lung cancer by in vivo screening of murine tumor derived xenografts

Using a pooled shRNA library, we performed a focused screen of 81 shRNAs selected for involvement in response to chemotherapy in an ex vivo 3D primary tumor cell culture system. Our results establish Ankrd35, a previously uncharacterized ankyrin repeat domain protein, as an important mediator of chemoresistance in vivo. Overall design: We analyze shRNA abundance at two different timepoints using 4 biological replicates for genes predicted to play a functional role in chemoresistance and re-growth of the tumor after chemotherapy and compare these results to LKR10 cells (a similar murine NSCLC cell line grown in 2D culture) treated in parallel with cisplatin. The first analysis compares untreated groups T1 v. T0 for any in vivo shRNA vulnerabilities in mouse-derived xenografts (MDXs) and LKR10 cells; the second analysis compares cisplatin treated T1 v. untreated T1 groups for chemosensitizers in MDXs and LKR10 cells..

本研究采用混合式短发夹RNA（short hairpin RNA, shRNA）文库，针对参与化疗应答的81条筛选得到的shRNA，在离体原代肿瘤细胞三维培养体系中开展靶向筛选。本研究结果证实，Ankrd35——一种此前尚未被功能表征的锚蛋白重复结构域蛋白——是体内化疗耐药的重要介导因子。实验整体设计：本研究针对被预测在化疗耐药及化疗后肿瘤再生中发挥功能作用的基因，设置两个不同时间节点，通过4组生物学重复检测shRNA丰度，并将结果与经顺铂平行处理的LKR10细胞（一种采用二维（2D）培养的相似小鼠非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞系）进行对比。首次分析对比未处理组T1与T0，以筛选小鼠来源异种移植瘤（mouse-derived xenografts, MDXs）及LKR10细胞中的体内shRNA易感靶点；第二次分析则对比顺铂处理组T1与未处理组T1，以筛选MDXs及LKR10细胞中的化疗增敏剂。