肿瘤细胞微粒介导血管内皮屏障损伤在胃癌血道转移中的作用及机制

The integrity of vascular endothelial barrier is critical for transmigrating through the endothelium and promoting distant metastasis for tumor cells. Studies reported that tumor-derived microparticles (TMPs) and platelet-derived microparticles (PMPs) were increased with tumor progression, but whether they could promote tumor metastasis by influencing the permeability of vascular endothelial had not been reported. Our previous data showed that the hematogenous metastasis of hepatic carcinoma was promoted by mice hepatocellular carcinoma cells derived microparticles, which was rich in vascular permeability factor VEGFA, it induced high capillary permeability of capillaries in mice. In addition, the gastric cells derived microparticles in vitro increased the permeability of vascular endothelial. These data and the published reports have led us to hypothesize that TMPs may promote hematogenous metastasis by activating vascular endothelial, promoting cancer cells to adhere and transmigrate through the endothelium and increaseing the permeability of vascular endothelial. We will test these hypotheses by achieving three specific aims. First, we chose to study gastric cancer because it has a higher incidence the western region, including Gansu than other parts of China. Second, we will use Nanodiscs, microfluidic technology, measurement of electrical resistance across endothelial cell monolayers, Transwell migration model, mass spectrometry, xenograft and small animal imaging in vivo to explore the effects of TMPs on vascular endothelial barrier. Third, we will define that the role of the structural integrity of TMPs, VEGFA-VEGFR2-VE-cadherin signaling pathways and synergy with PMPs in endothelial barrier disruption that is induced by TMPs. Together, these studies are designed to identify the mechanisms in promoting cancer hematogenous metastasis, new diagnostic/prognostic biomarkers, and novel therapeutics.

血管内皮屏障完整性是影响肿瘤细胞跨内皮迁移、导致肿瘤血道转移的关键。研究发现外周血肿瘤细胞微粒（TMPs）和血小板微粒（PMPs）水平随肿瘤进展增高，但其能否通过影响血管内皮通透性促进肿瘤转移，目前尚未见报道。我们前期研究发现小鼠肝癌细胞微粒能促进肿瘤转移、体内诱导血管高通透性；胃癌细胞微粒体外可诱导内皮通透性增加。我们推测：肿瘤细胞微粒可能通过激活血管内皮，介导肿瘤细胞与内皮粘附；增加内皮通透性，促进肿瘤细胞跨内皮迁移，导致肿瘤血道转移。据此本项目以我国西部高发胃癌为研究对象，应用微流体技术、跨内皮电阻测定、Transwell迁移模型、Nanodiscs、质谱分析、移植瘤和小动物成像等技术，进一步探讨TMPs对血管内皮屏障的影响；阐明TMPs结构完整性、VEGFA-VEGFR2-VE-cadherin信号通路及血小板微粒与TMPs介导内皮屏障损伤的相关性，为肿瘤转移机制研究提供新思路。

血管内皮屏障完整性是影响肿瘤细胞跨内皮迁移、导致肿瘤血道转移的关键。研究发现外周血肿瘤细胞微粒TMPs水平随肿瘤进展增高，但其能否通过影响血管内皮通透性促进肿瘤转移，目前尚未见报道。本项目以我国西部高发胃癌为研究对象，对胃癌患者血浆TMPs水平与临床病理因素相关性的研究发现胃癌患者外周血TMPs及PMPs水平均较健康受试者显著增高，其血浆水平与肿瘤组织分化程度和远处转移具有相关性，证实TMPs和PMPs协同参与胃癌进展。体外跨内皮电阻测定、Transwell迁移等实验结果表明肿瘤细胞微粒通过激活血管内皮细胞，刺激VWF释放增加，增强肿瘤细胞对内皮的粘附；TMPs通过影响细胞骨架蛋白及细胞表面粘附分子的表达，增加血管内皮通透性，促进肿瘤细胞跨内皮迁移；小鼠移植瘤实验发现通过尾静脉注射不同剂量TMPs增加其血浆含量，能够促进胃癌细胞的肺转移，应用Lactadherin降低荷瘤小鼠外周血TMPs水平，可减少由TMPs介导的胃癌细胞肺转移增加，进一步证实TMPs通过增加内皮屏障的通透性，促进肿瘤肺转移。机制探讨表明TMPs介导内皮屏障损伤的作用与激活VEGFA-VEGFR2-Src-VE-cadherin通路，降低VE-cadherin表达密切相关；此外，研究发现TMPs结构完整性不同，对内皮屏障的影响也有差异，主要是微粒表面膜蛋白在发挥作用，蛋白组学分析筛选出肿瘤细胞微粒特异性富集的CD147蛋白可能是TMPs的主要效应蛋白。本项目首次从肿瘤细胞微粒对内皮屏障完整性的作用展开研究，经过四年的工作，已经基本完成项目计划内容，达到预期研究目标，对拟解决的关键问题做出了合理、科学的解答，阐明VEGFA-VEGFR2通路及CD147蛋白的可能机制，为进一步的胃癌血道转移机制研究及防治提供新思路和新靶点。