胆囊癌相关成纤维细胞通过PDGF-BB促进胆囊癌淋巴转移机制研究

Lymphatic metastasis represents the main mode of tumor spread for gallbladder cancer(GBC) and is an important prognostic factor in patients with GBC, but the mechanisms of lymphatic metastasis are not fully understood. Cancer associated fibroblast(CAF) is an important component of the tumor microenvironment, it promotes tumor progression, invasion and metastasis through multiple mechanisms. Our preliminary study showed that the expression of CAF and Neuropilin-1(NRP-1) in gallbladder carcinoma was closely related to lymphatic metastasis; after we successfully separated and cultured CAF in gallbladder carcinoma, then we confirmed that the secretion of PDGF-BB in CAF was increased compared with normal fibroblast(NF). At the same time, based on that PDGF-BB is an important growth factor contributing to lymphangiogenesis, the existence of molecular Akt and STAT3 in PDGF-BB downstream signaling pathway may upregulate VEGF secretion, NRP-1 is a co-receptor of VEGF, we assume that,on the one hand, PDGF-BB secreted by CAF could promote the ability of invasion of cancer cells and up-regulate the expression of NRP-1 in cancer cells, on the other hand, it could promote the lymphangiogenesis of lymphatic endothelial cells (LEC) in tumor and promote the secretion of VEGF in LEC to recruit gallbladder cancer cells to invade into lymphatic vessels, and eventually promote lymph metastasis of gallbladder cancer. Several methods including histopathology, cytokine array, cell co-culture, in vitro culture of 3D model and experimental zoology will be applied in this study and the assumption will be confirmed from three aspects of histology, in vitro and in vivo experiments. This study will elucidate the mechanisms of lymphatic metastasis of gallbladder carcinoma from the point of view of tumor microenvironment, it has the important theory significance and scientific value.

淋巴转移是胆囊癌的主要转移方式，是影响预后的重要因素，但其机制不明。肿瘤相关成纤维细胞(CAF)是肿瘤微环境中重要的组成成分，通过多重机制促进肿瘤生长、侵袭和转移。我们前期研究表明胆囊癌CAF及神经菌毛素-1（NRP-1）的表达与淋巴转移相关；经分离培养胆囊癌CAF后，检测CAF分泌的PDGF-BB较正常成纤维细胞（NF）增高。同时基于PDGF-BB为淋巴管生成因子，其下游信号通路中存在调节VEGF的分子Akt及STAT3，NRP-1为VEGF的共受体，我们设想CAF通过分泌PDGF-BB一方面作用于癌细胞，促进其侵袭能力并上调NRP-1表达，另一方面作用于淋巴内皮细胞（LEC），促进肿瘤淋巴管形成，并促进LEC分泌VEGF招募胆囊癌细胞侵入淋巴管，最终促进胆囊癌淋巴转移。本研究拟通过细胞因子芯片、体外3D共培养及实验动物学等技术，从组织学、体外实验及体内实验三个层面证实上述设想。

胆囊癌是最常见的胆道恶性肿瘤，淋巴转移是胆囊癌的主要转移方式，是影响患者预后的重要因素，但其机制不明。肿瘤相关成纤维细胞(Cancer associated fibroblast, CAF)是肿瘤微环境中重要的组成成分，通过多重机制促进肿瘤生长、侵袭和转移。本课题研究针对胆囊癌肿瘤微环境中特异性的肿瘤相关成纤维细胞（CAF），探讨CAF通过分泌特定细胞因子分别作用于肿瘤细胞及淋巴内皮细胞，进而促进胆囊癌淋巴转移的作用机制。. 已完成的研究工作包括：1) 通过回顾性分析176例进展期胆囊癌患者资料，证实淋巴结转移是影响经根治性手术的患者预后最重要的因素，而阳性淋巴结数目为更为精准的淋巴结分期指标；2)建立了胆囊癌贝叶斯网络模型生存预测模型，结合重要度理论分析证实淋巴转移是影响手术治疗GBC患者预后最重要的因素之一；3) 通过组织学实验证实胆囊癌CAF的标志物与患者淋巴转移、淋巴管密度密切相关，影响患者预后；4) 通过组织块法和酶消化法成功地对人胆囊癌相关成纤维细胞与正常成纤维细胞进行了原代培养及鉴定；5) 通过体外实验证实CAF可促进胆囊癌细胞增殖、侵袭，促进淋巴内皮细胞的增殖、迁移及小管形成能力；6) 通过人半定量细胞因子芯片证实CAF通过旁分泌IL-8与IGFBP3引起胆囊癌细胞及淋巴内皮细胞生物学改变；7) 通过组织学、体外、体内实验证实NRP-1在胆囊癌中的表达与CAF相关，CAF分泌的IL-8可上调癌细胞的NRP-1表达，同时NRP-1对癌细胞有促增殖、侵袭的作用，使用表达谱芯片测序证实胆囊癌CAF通过分泌IL-8，上调了癌细胞NRP-1表达，并进一步通过上调BBC3、IGF1R、EGR1，下调CCNA2和MDM2促进肿瘤进展，进而诱发胆囊癌的淋巴转移。. CAF分泌IL-8、IGFBP3作用于LEC后细胞下游信号通路检测及CAF相关抗体治疗体内实验正在进行中。. 本研究截止目前已发表文章18篇，尚有3篇SCI文章及2篇中文文章处于审稿阶段，培养硕士研究生4名，均已毕业，培养博士研究生1名。. 综上，本研究从胆囊癌肿瘤微环境中肿瘤相关成纤维细胞的角度进一步阐明了胆囊癌淋巴转移的发生机制，基本达到预期目标。