免疫载药纳米微粒靶向阻断SDF-1/CXCR4/IL-6环路及调控TAMs治疗胰腺癌的研究

Previous studies have found that there are a large number of M2 type of tumor associated macrophage cells (TAMs, CD163 +) in the pancreatic cancer (PC) stroma, which may induce SDF-1 cytokine secretion through IL-6 in vitro. What is more, SDF-1 cytokine can further promote the invasion of PC cells. Consequently, we hypothesis that there may be a SDF-1/ CXCR4 / IL-6 positive feedback loop between M2-TAMs and PC cells. In addition, it is reported that overexpressing HRG can induce transformation of M2 type of TAMs to M1 type. In this study, we will further study the function and molecular mechanisms of this loop; using the nanotechnology we have already established build a nanoparticle delivery system for PC targeted therapy: ① including human single-chain antibody CD163; ② nanoparticles loading drug and histidine glycoprotein (HRG) gene. Cracking peptide can connect AMD3100 with the nanoparticles; ③ the increasing of HRG gene can induce M2-TAMs transformation to M1 type cells which act as tumor-suppressor. This new nanoparticles can target HRG gene to M2-TAMs, and induce M2-TMA2 cells to transform to M1 type; At the same time, this nanoparticle system can also release AMD3100, as a result blocking the reciprocal loop between PC and M2-TAM2, and finally double targeted control the micro-environment of PC tumor in humanized mouse model, providing a new way for the treatment of PC.

我们前期研究发现胰腺癌间质富含促癌M2型肿瘤相关巨噬细胞（TAMs，CD163+），其在体外被胰腺癌细胞（可能经IL-6）诱导分泌SDF-1，SDF-1又促进胰腺癌细胞侵袭，该作用可被SDF-1受体CXCR4阻断剂AMD3100阻断，因此，M2型TAMs与癌细胞间可能存在SDF-1/CXCR4/IL-6正反馈环路。此外，上调富含组氨酸糖蛋白（HRG）基因可诱导M2型TAMs向抑癌M1型转化。本研究将深入探讨和验证上述环路作用机制；并用我们已建立纳米技术针对此新靶点及M2型TAMs构建胰腺癌靶向治疗纳米载体：①含人源CD163单链抗体；②载HRG基因，同时用可裂解肽将AMD3100与载体相连；③此新型纳米载体可将HRG基因靶向导入M2型TAMs，诱使其向M1型转化；并局部释放AMD3100，阻断M2型TAMs与癌细胞互惠作用，在人源化鼠原位癌模型双靶点调控肿瘤微环境，为治疗胰腺癌提供新思路。

本课题在前期工作基础上分三部分完成。第一部分阐明TAMs与胰腺癌细胞相互作用的细胞因子环路，包括SDF-1/CXCR4-IL-6和CCL1/CCL7IL-10。我们的研究发现TAMs与胰腺癌细胞存在SDF-1/CXCR4/IL-6相互作用环路：①SDF-1通过诱导胰腺癌细胞发生上皮-间质转化（EMT），促进肿瘤细胞迁移和侵袭；②通过经尾静脉注射裸鼠转移瘤模型，发现SDF-1促进胰腺癌肝转移；③SDF-1通过激活NF-κB通路促进胰腺癌细胞分泌IL-6；④IL-6通过激活STAT3通路促进TAMs分泌SDF-1。另外，我们研究还发现肿瘤相关巨噬细胞和胰腺癌细胞存在另一个细胞因子环路，即CCL1/CCL7-IL-10相互作用环路：①IL-10可激活胰腺癌细胞ERK通路上调CCL1；②CCL1可激活TAMs细胞AKT通路上调IL-10；③IL-10和CCL7皆能促进胰腺癌细胞迁移、侵袭能力。第二部分主要研究如何诱导M2型TAMs转型为M1型TAMs：①利用慢病毒转染M2型TAMs建立稳定过表达HRG细胞株；②M2型TAMs稳定过表达HRG后可发生M1型转换；③HRG诱导的M1型TAMs抑制胰腺癌细胞恶性生物学行为。第三部分体内研究利用载药纳米工具双靶点调控肿瘤微环境的抗胰腺癌效应：①SDF-1/IL-6和TAMs可作为调控靶点抑制胰腺癌生长及转移；②成功制备scFv-NPs用于体内靶向投递纳米微粒。本研究结果提示TAMs和胰腺癌细胞间存在复杂的炎症因子调控网络，而SDF-1/CXCR4-IL-6和CCL1/CCL7-IL-10可能是这个复杂网络的两个重要调控环路，后期有望能利用构建成功的纳米微粒实现双靶点调控肿瘤微环境发挥胰腺癌免疫靶向治疗的作用。