区域淋巴结转移胃癌exosome重塑骨髓MSC作用及其分子机制

Regional lymph node metastasis is common in early gastric cancer. Previously, we successfully isolated and obtained mesenchymal stem cell-like cells from the regional metastatic lymph node tissues of gastric cancer,which is called GLN-MSC (Regional lymph node metastasis associated mesenchymal stem cells of gastric cancer). Exosomes both from lymph node metastasis derived-gastric cancer cell lines and serum of gastric cancer patients with regional lymph node metastasis could obviously reprogram bone marrow-derived MSC (BM-MSC) into GLN-MSC-like cells, which in turn promoted lymph node metastasis derived-gastric cancer cells metastasis in vitro and in vivo. The results of proteomics detection and analysis showed that the metastasis and microenvironment reprogramming related proteins were highly enriched in these exosomes, such as Wnt5a. However, the role and mechanism of BM-MSC reprogramming by the exosomes in the formation of regional lymph node metastatic gastric cancer microenviroment are not clear. Based on these, in the project, we will use the techniques including antibody chip and gene manipulation to perfect and confirm the role of regional lymph node metastatic gastric cancer derived exosomes in BM-MSC reprogramming and identify the key protein in the exosomes and elucidate its molecular mechanism in this process. Finally, we will establish the method for detecting the key protein levels in the serum exosome and determine their values in the diagnostic and therapeutic application. The project completion will be helpful for us to further understand the molecular mechanism of the malignant progression for the regional lymph node metastatic gastric cancer and find potential therapeutic targets and noninvasive diagnostic markers for gastric cancer patients with lymph node metastasis, which exhibit scientific significance and prospect of clinical application.

早期胃癌以区域淋巴结转移多见，前期我们发现区域淋巴结转移灶存在着胃癌淋巴结转移相关间充质干细胞GLN-MSC，淋巴结转移灶来源胃癌细胞和区域淋巴结转移胃癌血清exosome可显著重塑骨髓MSC向GLN-MSC样细胞转分化，促进转移灶来源胃癌细胞体内外转移。蛋白组学检测显示exosome高度富集转移、微环境重塑相关蛋白如Wnt5a等，但其重塑骨髓MSC参与区域淋巴结转移性胃癌微环境形成作用及其分子机制尚不明确。基于此，本项目拟采用抗体芯片、基因操纵等技术，完善并明确淋巴结转移性胃癌exosome重塑骨髓MSC作用，鉴定并阐明exosome重塑骨髓MSC关键蛋白及其分子机制，建立血清exosome关键蛋白检测技术并评估其诊疗应用价值。项目的完成将有助于深入理解区域淋巴结转移胃癌恶性进展分子机制，有望寻找到区域淋巴结转移胃癌治疗潜在靶点和非创伤性诊断指标，具有重要的科学意义和临床应用前景。

区域淋巴结转移是胃癌常见转移方式，也是影响胃癌预后重要因素，但其恶性进展分子机制尚待阐明。前期胃癌细胞诱导骨髓MSCs转分化的研究工作中意外发现：区域淋巴结转移胃癌细胞exosomes可通过特定驯化骨髓MSCs促进转移胃癌细胞进一步转移；蛋白组学检测显示淋巴结转移胃癌细胞exosomes高度富集转移相关蛋白等；区域淋巴结转移灶存在着MSC样基质细胞即GLN-MSCs。由此推测，区域淋巴转移胃癌细胞exosomes驯化骨髓MSCs参与转移淋巴结胃癌微环境形成可能是淋巴结转移胃癌恶性进展的关键。本项目以此为突破口，明确淋巴结转移胃癌来源exosome对骨髓MSCs的重塑作用，鉴定exosome重塑骨髓MSCs关键蛋白并阐明其调控分子机制，评估exosome中关键蛋白在区域淋巴结转移胃癌诊疗中的应用价值。研究发现：①淋巴结转移胃癌细胞和患者血清exosomes均较原发灶胃癌细胞和未转移患者血清exosomes具有更强驯化骨髓MSCs获得促胃癌淋巴结转移能力；②Wnt5a和CD44是淋巴结转移胃癌细胞exosomes驯化骨髓MSCs关键蛋白，可被直接递送至骨髓MSCs内，分别通过激活YAP信号和上调CPT1A表达促进脂肪酸氧化（FAO）代谢重编程驯化骨髓MSCs；③淋巴结转移胃癌细胞exosomes-CD44促进FAO代谢重编程直接赋予原发灶胃癌细胞淋巴结转移能力；④成功建立血清exosomes内Wnt5a和CD44 ELISA检测方法，淋巴结转移患者血清exosomes富集关键蛋白，其含量与淋巴结转移负荷高度相关；CD44、YAP和CPT1A表达与胃癌患者转移性病理特征和预后不良有关。系列研究揭示了淋巴结转移胃癌细胞exosomes不仅递送关键蛋白驯化骨髓MSCs构建有利于转移的新生环境，还直接递送至原发灶胃癌细胞传递恶性表型，从新的层面剖析淋巴结转移胃癌细胞exosomes推动淋巴结转移胃癌循环恶性进展的多向调节作用及分子机制，也从新的视角为阻断淋巴结转移胃癌恶性进展提供潜在治疗策略，为淋巴结转移胃癌诊断和负荷评估提供潜在检测生物标志物。