M2型肿瘤相关巨噬细胞通过IL-10介导胰腺癌上皮间质转化的作用及机制

Epithelial-mesenchymal transition(EMT) is an early step during tumor process and is regulated by the inflammatory microenvironment.M2 tumor-associated macrophage(TAM) is a major component and increased TAM infiltration often correlates with poor prognosis，but the regulation mechanism of EMT is unavailable. In the preliminary experiments, we found that M2-polarized TAMs enhance EMT and migratory capability of pancreatic cancer cells partially through the activation of TLR4 signaling. Importantly, IL-10 neutralizing antibody in the co-culture medium significantly reversed EMT. Therefore, IL-10 might be the mediator responsible for TLR4 signaling.The following question is : is IL-10 the key mediator responsible for EMT? what is the sorce of IL-10? what is the roles of IL-10 in vitro or in vivo? what is the mechanism of IL-10 effects? The study will answer the above questions. In the preliminary experiments, the levels of phosphorylated STAT3 and mRNA and protein of snail were significantly elevated when co-culutured with M2 TAM. a study in the jounal of Nature reported that LIV-1, a breast-cancer-associated zinc transporter protein, is essential for the nuclear localization of snail during zebrafish gastrulation,at the same time, LIV-1 is a downstream target of STAT3. Base on the above, we have the hypothesis that IL-10 secreted from M2 TAM induced EMT in pancreatic cancer, partially through the IL-10R/JAK/STAT3/LIV-1/Snail signaling pathway, Futhermore, STAT3,as a transcription factor, might regulate snail both on mRNA and protein levels. We will address the issues by the advantage of co-culture, RNA interference, gene over expression, luciferase reporter system, and the model of orthotopic pancreatic tumor and so on. The study will provide a new understanding of the mechanism of EMT regulated by M2 TAM and a new target in the tumor therapy.

上皮间质转化（EMT）是肿瘤进展中的早期事件，并受炎性微环境的影响，M2型肿瘤相关巨噬细胞（tumor-associated macrophage, TAM）是炎性微环境中的主要组分,但对EMT的作用靶点不详。我们前期研究提示M2 TAM通过自身TLR4信号活化促进了胰腺癌细胞EMT（snail mRNA和核内蛋白升高，E-cadherin下降），而且TLR4下游靶分子IL-10可能是主要因子，并发现癌细胞内P-STAT3活性增强，结合文献报道锌转运蛋白LIV-1可促进snail核内移位，而LIV-1是STAT3的下游基因。我们推测M2 TAM可能通过分泌IL-10，经IL-10R/JAK/STAT3/LIV-1/snail通路导致了EMT。本课题拟采用细胞共培养、RNAI、过表达、磷酸化抑制剂、体内原位成瘤等手段验证假说。研究结果将为揭示M2 TAM对EMT的调控关系提供新的视角和靶点。

M2型肿瘤相关巨噬细胞（tumor-associated macrophage, TAM）在胰腺癌中广泛浸润并提示预后不良。上皮间质转化（Epithelial-mesenchymal transition(EMT) ）是肿瘤进展中的重要生物学过程。本项目研究M2 TAM在胰腺癌EMT中的作用及分子机制。 主要研究内容：采用细胞共培养、RNAI、过表达、磷酸化抑制剂、体内原位成瘤等手段研究： 1). 体外实验研究M2型TAM在胰腺癌EMT中的作用: M2型TAM与胰腺癌细胞株共培养，观察癌细胞形态的变化以及EMT分子标志E-cadherin / Vimentin和EMT关键转录因子Snail表达的影响。2）体外实验研究M2型TAM通过TLR4/IL-10介导了胰腺癌细胞的EMT: 通过对M2型TAM上TLR4 RNAI及加入TLR4/IL-10中和型抗体研究TLR4/IL-10对胰腺癌细胞株 EMT及增殖、迁移和侵袭的影响。3)体外实验研究胰腺癌细胞内IL-10R/ JAK/STAT3/ LIV-1/Snail通路对EMT的作用：M2型TAM与胰腺癌细胞株共培养，检测癌细胞内IL-10R、JAK、STAT3、LIV-1及snail的表达和活性变化；对关键分子STAT3进行阻断及激活，研究对EMT的影响。4）体内实验研究M2型TAM通过分泌IL-10导致了胰腺癌EMT。重要结果和关键数据：1）M2 TAM促进了胰腺癌EMT，同时促进了癌细胞的恶性生物学行为：E-cadherin mRNA及蛋白表达下降，Vimentin及Snail mRNA及蛋白表达下降，同时胰腺癌细胞增殖、迁移、侵袭能力增强；2）对M2 TAM上TLR4进行RNA干扰，显著逆转了EMT发生；3）在共培养液分别加入TLR4、IL-10中和性抗体，显著逆转了EMT发生；4）共培养后，癌细胞内IL-10R、JAK、STAT3、LIV-1及snail表达及活性增强；5）对癌细胞内STAT3进行阻断及激活后，分别抑制和增强了EMT发生；6）皮下移植瘤和原位瘤实验显示M2 TAM通过IL-10促进了胰腺癌的生长和EMT。以上结果提示M2型TAM通过IL-10R/JAK/STAT3/LIV-1/snail通路促进了胰腺癌的EMT，为干预胰腺癌进展提供了新靶点和新思路。