NSCLC^CXCR4+外泌体介导非小细胞肺癌干性转化对放射敏感性的影响及机制研究

Radiosensitivity of non-small cell lung cancer (NSCLC) is closely related to tumor microenvironment, activation of Cancer Stem Cells (CSCs) and epithelial-mesenchymal transition (EMT) pathway genes. We previously demonstrated that, X-ray irradiation enhanced the expression of CXCR4, a molecular marker of CSCs, through the regulation of tumor hypoxia microenvironment, and finally, promoted the metastasis and invasiveness of NSCLC cells. In our preliminary experiment, after X-ray irradiation, we obtained the CXCR4 overexpressed NSCLC cells (NSCLC^CXCR4+) by sorting with flow cytometry. The exosomes released from the NSCLC^CXCR4+ cells (Exo^CXCR4+) were extracted. We have found that NSCLC^CXCR4+ cells may induce cancer stem cell-like properties of adjacent NSCLC cells via exosomes-mediated pathways, and then the transformed CSCs regulate the radiosensitivity of NSCLC cells via targeting EMT. We will use various molecular biology methods to 1) investigate the cancer stem cell-like transformation of adjacent NSCLC cells mediated by the Exo^CXCR4+; 2) illustrate the mechaniam how the transformed CSCs regulate the radiosensitivity of NSCLC via targeting EMT; 3) explore the mechanism of cancer stem cell-like transformation by identifing the ingredients of Exo^CXCR4+.

非小细胞肺癌(NSCLC)的放射敏感性与肿瘤微环境的改变、肿瘤干细胞（CSCs）的活化及肿瘤细胞上皮间质转化（EMT）等密切相关。我们前期研究结果证实，X射线能够通过上调干细胞分子标志物CXCR4促进NSCLC细胞的侵袭迁移能力。本课题预实验通过流式分选法得到了X射线诱导的CXCR4高表达的NSCLC（NSCLC^CXCR4+）细胞，并提取了其释放的外泌体Exo^CXCR4+。本项目拟通过干细胞成球实验、克隆存活实验、凋亡检测、小动物IGRT精确放疗及高通量二代测序等方法在细胞水平及裸鼠原位移植瘤模型上进一步揭示NSCLC^CXCR4+外泌体对邻近肿瘤细胞干性转化的影响；明确NSCLC干性转化通过EMT影响放射敏感性的机制；同时通过鉴定NSCLC^CXCR4+外泌体的成份，揭示外泌体关键成分对肿瘤细胞干性转化的机制。

电离辐射杀灭恶性肿瘤细胞的同时，通过微环境改变，可以影响恶性肿瘤的诸多生物学行为。本研究前期分离得到电离辐射诱导的CXCR4高表达的NSCLC(NSCLC^CXCR4+)细胞，并提取了其释放的外泌体Exo^CXCR4+。Exo^CXCR4+处理后观察细胞形态以及分子标志物免疫荧光检测均显示NSCLC细胞发生上皮间质转化（EMT）。细胞凋亡实验、克隆形成实验以及γH2AX foci免疫荧光结果显示，Exo^CXCR4+可以导致NSCLC细胞细胞凋亡减少、克隆存活率增加以及DNA双链断裂减少。Western Blot实验结果显示相关的分子机制主要涉及PI3K/ pAkt /mTOR及MAPK / pERK1/2信号通路。对43例肺腺癌组织样本进行免疫组化染色，结果提示CXCR4高表达与肺腺癌患者分期晚及淋巴结转移的临床特征相符合。外泌体Exo^CXCR4+在动物水平也可导致肿瘤细胞对辐射抵抗。本研究的完成提示电离辐射照射后的NSCLC细胞可以通过产生外泌体，调控未受照射细胞发生干性转化及EMT，最终导致对电离辐射抵抗。