VEGFR2/PKD1信号通路对NSCLC来源外泌体分泌的影响及其机制研究

Tumor-derived exosomes play a key role in immunosuppressive microenvironment. Targeted antiangiogenesis against vascular endothelial growth factor receptor 2 (VEGFR2) has been demonstrated to enhance the anti-tumor effect of DC-T cellular therapy on non-small cell lung cancer(NSCLC). Our primary results showed that the inhibition of protein kinase D1 (PKD1) reduced the secretion of exosome from NSCLC cells, which might be related to Vps34. Moreover, VEGFR2 regulates the activity of PKD1 by direct phosphorylation. Based on these findings, we hypothesized that VEGFR2 could activate Vps34 through phosphorylating PKD1,which further initiates the formulation and secretion of exosome. To verify this hypothesis, we will first demonstrate that VEGFR2 activates the PKD1/Vps34 pathway and induces NSCLC derived exosome. Then, the molecular mechanism of VEGFR2/PKD1 signaling network on regulating exosome secretion will be studied. Finally, SCID mice transplantation tumor model will be utilized to verify the effect of anti-angiogenesis therapy on the secretion of exosome in vivo. Together, our research will provide a novel information for the combination of antiangiogenesis and immune therapy in the treatment of NSCLC.

肿瘤细胞来源的外泌体在免疫抑制性微环境中起关键作用，而靶向VEGFR2的抗血管生成治疗能够增强NSCLC免疫治疗的效果。申请者前期发现，NSCLC细胞中PKD1活性的抑制能够减少肿瘤来源外泌体的分泌，文献提示这个过程可能与Vps34有关。进一步探索显示，细胞表面VEGFR2参与细胞内PKD1磷酸化的调控。基于此，本研究中提出以下假说：NSCLC细胞表面VEGFR2可通过对PKD1相关通路的调控，影响胞内Vps34介导的外泌体形成及分泌。为验证上述假说，本研究将首先明确VEGFR2对PKD1/Vps34通路及NSCLC来源外泌体分泌的影响。进而，在分子及细胞水平对VEGFR2/PKD1通路调控肿瘤来源外泌体分泌的分子机制进行研究。最后，建立SCID鼠异植瘤模型，在体内验证VEGFR2对NSCLC来源外泌体分泌的影响，为肿瘤抗血管靶向疗法和免疫疗法的联合应用提供新的思路及实验基础。

本研究证实了NSCLC细胞中VEGFR2活性对PKD1相关通路及外泌体分泌的影响，而且证实VEGFR2对外泌体分泌的影响依赖其激酶活性；进一步研究发现，VEGFR2靶向药物Apatinib不仅降低NSCLC来源外泌体的分泌，还改变了外泌体中内含物的成分，进一步抑制微环境中Treg增殖。本研究证实了VEGFR2活性的抑制能够通过减少NSCLC来源外泌体分泌，也初步探索了VEGFR2活性的抑制通过影响外泌体成分对免疫微环境的影响，将为抗肿瘤血管生成靶向疗法和免疫疗法的联合应用提供新的思路和实验基础。同时，本研究也为探索外泌体相关免疫调节的机理提供新思路。

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