RIP1依赖的线性程序性坏死促进乳腺癌血管生成拟态的机制研究

Vasculogenic mimicry (VM), identified in 1999, is a new tumor blood supply pattern. However, the space basement of VM channel in the tumor microentertainment with a high pressure is not clear. Previously, we have found that there were some line pattern programmed necrosis cells (LPPCN) surrounding to VM channels. There is association in distribution and time-line between VM and LPPCN. In this study, we will investigate the expression of RIP1 signaling pathway, which is essential in necroptosis, in 102 breast cancers analyze their clinical significance and the correlation between them, LPPCN and VM. Furthermore, we will establish a LPPCN model in vitro and a coculture system to observe the effect of LPPCNs on breast cancer cells and HUVCs. nanoLC-MS/MS will be used to identify the shedding proteins from cell membrane of LPPCN cells through RIP1-activaed ADAMs. The proteins associated with VM formation will be identified. Moreover, 3D cell culture, multiple immunofluorescent in 3D cell culture system, transfection, IP, EGFP transgenic SCID mice, whole mount, confocal and three dimensional image reconstruction will be performed to validate the molecular mechanisms which LPPCN induce breast cancer cells EMT, necroptosis and endothelial cells migration to promote VM formation and connection with host microcirculation. The necroptosis inhibitors will be administrated in breast cancer-bearing mice to assess the efficiency of them in treating tumors with VM. The results will reveal the origin and space of tumor cells lining VM channels. It will deep insight of LPPCN cells inducing VM through regulating other cells’ biological behaviors , and it will provide more new molecular targets for treatment of tumor with VM.

血管生成拟态(VM)是一种全新的肿瘤血供模式，前期发现VM周围线性排列的程序性坏死细胞（LPPCN）可能是VM形成的空间基础，但其影响VM的机制尚不清楚。本研究拟在102例人乳腺癌中观察程序性坏死关键RIP1通路成员与LPPCN、VM和内皮依赖性血管的空间分布关系及病理意义；构建LPPCN体外研究模型和细胞共培养体系观察其对周围细胞的影响；蛋白质谱鉴定由LPPCN细胞膜上解离的蛋白，筛选影响VM形成和血管内皮迁移的分子；运用多重荧光、细胞转染、IP和转基因小鼠、whole mount染色、共聚焦三维图像重建等验证LPPCN通过诱导乳腺癌细胞EMT和程序性坏死，促进VM形成并与宿主血管连通的分子机制；在动物模型中评估程序性坏死抑制剂治疗具有VM乳腺癌的可行性。结果有望明确VM形成的空间基础，阐明LPPCN调控周围细胞生物学行为主动促进VM的机制，并为临床治疗有VM的肿瘤提供新靶点和实验依据。

血管生成拟态是三阴乳腺癌重要的微循环模式，与宿主血管连通成为肿瘤功能性微循环，前期研究观察到细胞程序性坏死与其在空间分布上一致，但其具体过程与机制尚不明确。本项目在人乳腺癌样本、体外实验和动物模型中，探讨了RIP1依赖的细胞程序性坏死促进乳腺癌血管生成拟态形成的分子机制。在108例乳腺癌组织标本中，通过免疫组化和PAS/CD31双染等检测了肿瘤微循环模式、程序性坏死及RIP1通路分子表达和空间分布特征，观察到程序性坏死与VM、EVD等空间分布相近且表达RIP1等分子，提示程序性坏死细胞与乳腺癌肿瘤微循环构筑密切相关。通过氯化钴诱导三阴乳腺癌MDA-MB-231细胞成功构建了体外程序性坏死模型，质粒转染调控癌细胞RIP1表达，发现RIP1下调后MDA-MB-231细胞三维成管能力和侵袭转移能力减弱，VM相关蛋白表达降低。MDA-MB-231细胞程序性坏死条件培养基可增强人静脉内皮细胞HUVEC增殖能力、三维成管能力和迁移能力，加入RIP1抑制剂Necrostatin-1后上述现象均被抑制，提示RIP1依赖的程序性坏死参与乳腺癌VM和EDV形成。进而采用Label-free蛋白质谱分析了TSZ诱导的MDA-MB-231程序性坏死细胞培养基，筛选出程序性坏死细胞的差异蛋白，上调的差异蛋白包括ADAM10，ADAMTS13，CDH5等，下调的差异蛋白包括EIF4E, OTUB1和ITGA3等。KEGG分析显示上调差异蛋白参与了necroptosis和HIF-signalling pathway等信号转导通路。GO Slim分析显示上调蛋白可影响血管生成、炎症反应等。网上乳腺癌数据库分析显示ADAM10、EIF4E和ITGA3等差异蛋白与人三阴乳腺癌预后相关，它们的表达与VM调控分子表达密切相关，进而在体外细胞培养中证实ADAM10抑制剂GI254023x 可阻断RIP1依赖的程序性坏死对VM形成和EVD的促进作用。在TA2小鼠三阴乳腺癌移植瘤模型中，证实RIP1抑制剂Necrostatin-1可通过抑制VM和EVD抑制移植瘤生长。研究结果说明程序性坏死可调控周围细胞生物学行为，主动促进肿瘤微循环形成的机制，丰富了肿瘤血管生成理论，并为临床治疗有VM的肿瘤提供新靶点和实验依据。研究结果发表于SCI论文5篇，其余4篇正在投稿中，培养研究生6名。

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