巨噬细胞对非小细胞肺癌表皮生长因子受体酪氨酸激酶抑制剂获得性耐药产生的促进作用研究

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been used successfully in the treatment of non-small cell lung cancer (NSCLC). NSCLC patients with EGFR mutations have shown good response to EGFR-TKI. However, the acquired resistance of NSCLC to EGFR-TKI has emerged and become a major clinical problem to be resolved. Current researches about acquired resistance to EGFR-TKI mostly focused on abnormal activation of key components in EGFR signal transduction pathway and other pathways as well as epithelial mesenchymal transduction (EMT). It is noteworthy that all of these are final changes of cell reconstruction, but the promoting agents of cell reconstruction in the microenvironment where cancer cells grow with stromal cells such as macrophage (MΦ) were ignored. In previous study, we found that EMT was often accompanied with the acquired resistance of NSCLC cells to EGFR-TKI in vitro, while the reactivations of key components in EGFR signal transduction pathway or bypass were not found. More interestingly, MΦ could help NSCLC cells escape from the inhibition of EGFR-TKI and accelerate the development of EMT. In this study, we aim to explore whether MΦ plays a key role in the development of acquired resistance to EGFR-TKI and further investigate whether some cytokines including TGF-β secreted by MΦ could confer NSCLC cells to develop EMT and then obtain the acquired resistance to EGFR-TKI. We followed more than 300 NSCLC patients treated with EGFR-TKI post of surgical operation and collected their cancer tissues. The relationship of MΦ function in cancer tissues and the patient response to EGFR-TKI will be analyzed to make sure the MΦ function in the acquired resistance in vivo. The co-culture system of MΦ and NSCLC cells established ourselves would be used to study the function of MΦ helping the cancer cells survive and obtain new features in the process of EGFR-TKI stimulation. High resolution melting analysis would be used to monitor the rare gene mutation (known or unknown) of cancer cells during adaptation to EGFR-TKI stimulation. RNA interference, protein chip, real-time PCR, and other common bio-molecular techniques would be also used to detect the gene expression at mRNA level and protein level to elucidate the mechanism of MΦ on promoting the acquired resistance of NSCLC cells to EGFR-TKI. We hope to disclose the molecular mechanism that MΦ could help the cancer cells develop acquired resistance by producing some cytokines which promote EMT of the cancer cells, and to find the cytokines secreted by MΦ, which could be used as targets to inhibit and even reverse the acquired resistance in the tailor treatment of future.

获得性耐药是EGFR酪氨酸激酶抑制剂（TKI）治疗非小细胞肺癌（NSCLC）亟待解决的问题。目前研究多着眼于T790M等EGFR及下游基因突变、上皮间质转化（EMT）、其他信号通路激活等癌细胞自身重塑的结果，却忽视了体内微环境中细胞重塑的始因。我们发现：①有的获得性耐药癌细胞株的EGFR通路并没重新激活；②耐药瘤株多有EMT；③巨噬细胞（MΦ）能增强癌细胞TKI耐受性。本申请将分析肿瘤内MΦ状态与NSCLC患者获得性耐药发生的关系，用自建高分辨熔解曲线分析基因突变监测技术、MΦ-NSCLC细胞可分离共培养等细胞分子生物学方法，探讨MΦ对癌细胞重塑及TKI获得性耐药发生的影响，拟证明MΦ通过诱导癌细胞EMT帮助NSCLC发生TKI获得性耐药，期待找到在TKI获得性耐药发生中起关键作用并可用作治疗分子靶标的MΦ分泌因子，为遏制EGFR-TKI获得性耐药开辟新思路。

获得性耐药是阻碍分子靶向药表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）治疗非小细胞肺癌（NSCLC）成功的瓶颈。目前认为NSCLC发生获得性耐药的机制主要是癌细胞的重塑。细胞重塑的基础是癌细胞能够在EGFR-TKI存在的微环境中生存。本课题拟证明微环境中的巨噬细胞（MΦ）将可能帮助癌细胞在EGFR-TKI微环境中生存并产生获得性耐药。本研究在完善自建的NSCLC-MΦ共培养体系的同时，又建成了基于微流控芯片的NSCLC细胞、MΦ及成纤维细胞共存且微环境可调的培养方法，诱导建成了包括含有或不含有继发性T790M突变的多个EGFR-TKI耐药细胞亚株。总结出了EGFR-TKI类药物影响细胞增殖和耐药产生的评价方法（包括检测细胞耐药指数、用药后细胞增殖回复时间等）和以高效分辨溶解曲线（HRM）技术为核心的亚细胞克隆多种类型基因变异检测技术。研究发现MΦ可以促进PC-9等不同遗传背景的NSCLC发生EGFR-TKI获得性耐药。活化的MΦ能够通过外泌体分泌等方式促进EGFR-TKI微环境中癌细胞自分泌胰岛素生长因子（IGF）及稳定表达胰岛素生长因子受体（IGFR），从而替代被TKI封闭的EGFR，活化下游增殖信号传导通路，维持癌细胞生存，致使癌细胞有机会发生重塑产生TKI耐药。MΦ亦可在该药物存在微环境中促进癌细胞发生上皮-间质转化，进一步对MΦ分型发现，在EGFR-TKI存在微环境中对癌细胞起保护作用的主要是Ⅰ型MΦ（M1）。成纤维细胞亦能对癌细胞有保护作用，且发挥作用时间早于MΦ。应用HRM技术检测了临床肺癌、肾癌、胃癌组织标本的EGFR通路关键基因热点突变，发现肉瘤属性是透明细胞肾癌原发耐受EGFR-TKI遗传学原因（已发表）等。该课题成果及科学发现将对深入研究EGFR-TKI获得性耐药机制，研发新型靶向治疗药物及临床EGFR-TKI靶向药物治疗决策制定有着重要意义。