基于微流控芯片技术巨噬细胞介导的CD47促进非小细胞肺癌侵袭转移的机制研究

In the tumor microenvironment, macrophage promotes lung cancer cells metastasis and invasion by enhancing the expression of integrin-associated protein-CD47, however, the CD47-dependent molecular mechanism involved in tumor metastasis remains further to be elucidated. By using the RNA-sequencing analysis, we compared the transcriptome profiling of lung cancer cells co-cultured with macrophage or not and found that CD47 positively regulates Wnt/PCP pathway. Moreover, our previous data reveal that Cdc42 belonging to the Wnt/PCP pathway was the downstream target of CD47. Thus, we hypothesize that the macrophage could up-regulate the expression of CD47 in the non-small cell lung cancer (NSCLC), which contributed to activate the Wnt/PCP pathway and promote the metastasis and invasion. In this project, we aim to construct a metastatic model using the microfluidic chip technology. Using this model, we can not only investigate the metastatic and invasive ability of cancer cell co-cultured with macrophage in a real-time and dynamic manner, but also clarify the relationship of CD47 with the key molecules of Wnt/PCP pathway in the cancer cell. Then, the transwell platform will assist to verify the results of the metastatic model. Finally, we will further confirm the correlation of CD47 and Wnt/PCP pathway with the ability of metastasis and invasion in the tissue specimen of NSCLC. Collectively, our project aims to clarify that CD47 down regulated the Wnt/PCP pathway to promote the invasion and metastasis of NSCLC, mediated by macrophage. These data may provide more effective targets for the prevention and treatment of metastasis and invasion in NSCLC.

肿瘤微环境的巨噬细胞使肿瘤细胞表面整合素相关蛋白-CD47高表达且与肿瘤侵袭转移密切相关，但具体分子机制不明。我们通过RNA-Seq技术对巨噬细胞共培养高表达CD47的非小细胞肺癌(NSCLC)细胞株转录组测序分析，发现Wnt/PCP通路分子显著富集。通过细胞功能实验证实该通路关键分子Cdc42受CD47调控，为其下游分子。由此推测巨噬细胞介导NSCLC的CD47-Wnt/PCP通路促进肺癌侵袭转移。本课题拟利用微流控芯片技术构建模拟肿瘤微环境，实时动态观察细胞侵袭转移的体外模型，比较有无巨噬细胞共培养时肺癌细胞CD47、Wnt/PCP关键节点表达和侵袭变化，从功能和形态上明确巨噬细胞介导NSCLC的CD47-Wnt/PCP通路促进侵袭转移。最后，transwell平台和肺癌标本验证芯片模型结果。本研究将揭示巨噬细胞介导CD47促进NSCLC转移的分子机制，为NSCLC转移防治提供新思路。

肺癌是全球最常见的恶性肿瘤之一，近年来NSCLC的发病率和死亡率呈持续上升的趋势，主要原因是肿瘤细胞具有较强的侵袭性及转移能力。既往研究表明CD47与NSCLC的侵袭转移密切相关，然而，具体分子机制目前仍无报道，确切的分子通路有待进一步的探索。本课题组利用RNA-seq技术初步筛选Wnt/PCP通路与CD47关系最为显著。Wnt/PCP通路Wnt家族Wnt5a和Wnt11等配体与细胞表面的Frizzled受体（FZD）结合，使胞内的Dishevelled蛋白（Dvl）激活，进一步活化Rho家族的RhoA、Rac1和Cdc42等，通过调节细胞形态影响细胞的迁移。因此我们首先针对CD47通过Wnt/PCP通路下游的Rho/ROCK信号影响NSCLC的侵袭转移进行研究。并进一步证实了c-myc作为调控因子，作用于CD47的启动子区域，调控CD47的作用。除以上研究工作之外，我们在分析Wnt通路的同时关注到了Wnt6分子。Wnt6的高表达与食管鳞状细胞癌、肠癌肝转移术后患者和脑胶质母细胞瘤患者的不良预后密切相关；其高表达也增加了结直肠腺瘤的发病风险。然而，迄今为止尚无针对Wnt6与NSCLC的相关研究。因此我们首次验证了Wnt6在NSCLC的高表达，其表达水平与NSCLC临床分期明确相关。进一步评估Wnt6在NSCLC中的诊断效能高于传统的血清学肿瘤标志物。为肺癌的早期诊断、预后判断以及个体化治疗提供新思路。另外，本项目研究工作中，利用微流控芯片技术低消耗、高通量、模拟体内微环境等优势，构建了微流控芯片肺癌转移模型，可以对肿瘤细胞的侵袭转移过程动态实时观察，利用这一技术手段较好的模拟了肿瘤侵袭转移的空间过程，广泛使用于本课题的研究工作中，为课题研究提供了有效技术手段。