胆囊癌肝浸润发生率高且预后差，但胆囊癌肝浸润过程受免疫微环境调控的机制目前仍未知。我们前期在组织样本中发现胆囊癌肝浸润灶周围存在大量肿瘤相关巨噬细胞（TAM）和活化的肝星状细胞（aHSC），但其作用未明。我们进一步研究发现胆囊癌细胞可能通过VEGFA和GM-CSF募集并激活巨噬细胞为TAM，TAM随后可能通过分泌Angiogenin活化HSC，aHSC可释放外泌体促进胆囊癌细胞迁移侵袭。据此我们提出假说：近肝侧胆囊癌细胞可与TAM、aHSC形成正反馈环路，最终促进胆囊癌肝浸润。本项目拟通过3D培养技术、RIP等细胞与分子生物学实验，裸鼠胆囊原位成瘤模型，及临床样本研究进一步探明：胆囊癌细胞通过TAM活化HSC的确切机制，以及aHSC通过外泌体促进胆囊癌细胞肝浸润的具体分子机制。项目研究成果将从免疫微环境角度揭示胆囊癌肝浸润的分子机制，为TAM和aHSC作为胆囊癌的免疫治疗靶点提供科学依据。

Gallbladder cancer (GBC) hepatic invasion is of high morbidity and mortality. However, the role of immune microenvironment on GBC hepatic invasion remains unknown. Our preliminary data showed that around the GBC hepatic invasion lesions, there existed a large number of tumor-associated macrophages (TAMs) and activated hepatic stellate cells (aHSCs). Nevertheless, how TAMs and aHSCs affect the hepatic invasion of GBC cells is unclear. Our further preliminary results showed that GBC cells could recruit and activate macrophages to a TAM-like phenotype probably via VEGFA and GM-CSF, respectively. TAMs subsequently activated HSCs probably through secretion of Angiogenin. Furthermore, aHSCs could release exosomes to promote GBC cells migration and invasion. We thus hypothesize that GBC cells on the hepatic side could form a positive feedback loop with TAM and aHSC, which finally promotes GBC cells hepatic invasion. Our project will include cellular and molecular biology experiments such as 3D cell culture technology and RNA immunoprecipitation (RIP) assay, as well as gallbladder orthotopic xenograft model and plenty of clinical tissue samples to clarify the following scientific questions: 1) The mechanism of how GBC cells activate HSCs through TAMs. 2) The molecular mechanism of how aHSCs promote GBC cells invasion and metastasis through exosomes. Our research will uncover the molecular mechanism of GBC hepatic invasion from the perspective of immune microenvironment, and provide scientific evidences in developing effective immunotherapy against GBC by targeting TAMs and aHSCs.