胃癌腹膜转移中肿瘤转移干细胞的捕获和"上皮-间质转化"的作用机制

The prognosis of gastric cancer patients is often poor due to fatal peritoneal metastases. The mechanism of peritoneal metastases in gastric cancer is complicated and remains one of the hot research topics. Recently, the conception of metastatic cancer stem cell (MCSC), which is the metastasis-related subpopulation of cancer stem cell, has been raised but still lack of deeply research, including in gastric cancer. Metastasis of MCSC is a multistep "homing" process, in which MCSC is able to migrate directionally and colonize in specific target tissue. Peritoneal metastases in gastric cancer is a simplified "homing" pattern, and we hyptheize its important steps should be the adhesion, migration and niche construction of MCSC in peritoneum. The theory "epithelial-mesenchymal transition" might probably play important role in MCSC peritoneal metastasis of gastric cancer. Our research team has already captured the primary and circulatory cancer stem cell of human gastric cancer before, and preserves them by subculture till now. Based on these previous achievements, we aim to capture the MCSC of peritoneal metastases in human gastric cancer, and identify its stemness feature, high tumorigenicity and high invasive ability. Then, we will investigate whether MCSC is able to gain the invasive and migratory ability by "epithelial-mesenchymal transition", when exposed to the physical or chemical factors simulated in peritoneal cavity.

胃癌腹膜转移机制是当前研究热点之一。近年提出了"肿瘤转移干细胞"（MCSC）的概念，其为具有转移特性的肿瘤干细胞亚群，但目前尚鲜有研究。MCSC转移是其趋向性迁移并定植到特定靶组织的多步骤"归巢"过程。胃癌腹膜转移是简化的"归巢"模式，我们认为其关键环节是MCSC在腹膜的粘附、迁移。本团队前期已捕获人胃癌原代和循环肿瘤干细胞并传代培养保存。在此基础上，本项目拟进一步捕获人胃癌腹膜转移灶的MCSC，并鉴定其干细胞性、高成瘤性、高侵袭性。然后研究MCSC在腹腔理化因素暴露下，是否会出现"上皮-间质转化"的特性和能力，使MCSC获得侵袭和迁移的转移能力？

胃癌腹膜转移缺少有效的治疗手段，预后差。探索胃癌肿瘤干细胞（CSC-hGC）及其上皮-间质转化（EMT）在腹膜转移过程中的作用有实际的科学价值。本组在2014期间外科胃癌病例中腹膜转移率为5.2%（10/192）。收集8例粟粒样P2/P3病例的腹膜种植瘤组织进行无血清培养，以期细胞成球生长捕获胃癌腹膜转移肿瘤干细胞（pMCSC-hGC）。但均无成球生长趋势，可能的影响因素为（1）腹膜转移瘤的组织量少或/和（2）培养条件与原发肿瘤组织培养有所不同。因此调整方案采用已捕获传代的CSC-hGC[Cell Res. 2012;22:248-58]建立裸鼠腹腔种植模型来捕获胃癌腹腔移植瘤肿瘤干细胞（pMCSC-tGC）。将经慢病毒转染稳定表达绿色荧光蛋白和荧光素酶的CSC-hGC进行裸鼠腹腔注射构建腹膜转移动物模型。活体小动物成像证实腹膜移植瘤模型成瘤率可达100%。用CSC-hGC腹腔注射成瘤后收集腹腔移植瘤行无血清培养捕获了第一代pMCSC-tGC(G1)，然后将其再次注射连续成瘤同法捕获第二代pMCSC-tGC(G2)。Transwell实验提示pMCSC-tGC(G2)较CSC-hGC具有更强的迁移能力和侵袭能力。免疫组织化学检测提示第二代移植瘤较第一代的E-cad、Snail表达增强，而α-SMA表达降低。免疫细胞荧光检测提示与CSC-hGC相比pMCSC-tGC(G2)的E-cad表达上调，而α-SMA、Vimentin和MMP2表达下调。定量PCR检测提示与CSC-hGC相比pMCSC-tGC(G2)的E-cad、α-SMA、MMP9和Vimentin表达均下调。本项目发现，CSC-hGC可以在裸鼠腹腔连续成瘤保持干细胞特性，经裸鼠腹腔移植富集后pMCSC-tGC(G2)的迁移和侵袭能力更强。此现象提示（1）CSC-hGC在转移过程中获得更强的迁移和侵袭能力；或者（2）具有更强迁移和侵袭能力的CSC-hGC亚群成为pMCSC-tGC。即CSC-hGC的侵袭转移能力是获得性或为固有异质性尚不明确。此外，pMCSC-tGC倾向间质性表型下调而上皮性表型不稳定。此现象提示间质-上皮转化（MET）可能在胃癌腹膜种植转移的肿瘤干细胞“归巢”过程中发挥作用。本研究仅为围绕肿瘤干细胞在腹膜种植转移中的作用开展的初步探索性研究，实验发现为进一步探索提出了新的理论假设和研究方向。