TAM通过PLGF/Flt-1和TGF-β1通路促进非小细胞肺癌生长和肿瘤血管异生的研究

Tumor-associated macrophages promotes non-small cell lung cancer growth and tumor vascularization via PLGF/Flt-1 and TGF-β1 signal pathway

目的探讨胎盘生长因子(PLGF)介导非小细胞肺癌(NSCLC)细胞和肿瘤相关巨噬细胞(TAM)之间相互作用的具体机制。方法采用尾静脉注射法将转染腺病毒(AAV)的NSCLC A549细胞注入10周龄的雄性NOD/SCID小鼠体内,构建小鼠肺癌动物模型。流式细胞技术检测瘤体内各种巨噬细胞(MΦ)亚型,RT-qPCR检测肿瘤细胞和TAM中PLGF、血管内皮生长因子受体(Flt-1)和转化生长因子β1(TGF-β1)表达水平。根据A549细胞和MΦ细胞的培养条件不同,分为单纯A549组,单纯MΦ组,A549+MΦ组,A549+MΦ+sFlt-1组(加入10μg/L sFlt-1),PLGF+MΦ组(加入100 ng PLGF)和PLGF+MΦ+sFlt-1组(加入100 ng PLGF和10μg/L sFlt-1)。Transwell、MTT法分别检测不同共培养条件下A549细胞的增殖和迁移能力。根据HUVEC细胞的培养条件,分为对照组、条件培养基(CM)组、CM+SB431542组、TGF-β1组和TGF-β1+SB431542组,HUVEC细胞胶原凝胶测定法检测肿瘤血管异生情况。结果与RFP-细胞相比,PLGF主要由肿瘤细胞表达(31.72±4.69 vs.2.31±0.06,P<0.001),而Flt-1主要由CD163+的巨噬细胞表达(P<0.001)。A549+MΦ组A549细胞增殖能力显著强于单纯A549组和A549+MΦ+sFlt-1组(OD值:3.62±0.23 vs.4.53±0.34 vs.3.71±0.37,P<0.05)。TAM/M2巨噬细胞中TGF-β1的表达量高于M1巨噬细胞(0.99±0.23 vs.0.07±0.02,P<0.05)。CM组和TGF-β1组中HUVEC细胞管状结构的形成最多(P<0.05),TGF-β1抑制剂SB431542可抑制这一现象。结论TAM和NSCLC细胞间通过PLGF/Flt-1和TGF-β1信号通路间的交互作用,促进NSCLC的生长和血管异生。

Objective The aim of this study was to investigate the mechanism of placental growth factor(PLGF)mediated crosstalk between non-small cell lung cancer(NSCLC)cells and tumor-associated macrophages(TAM).Methods A NSCLC model in mice was established by tail vein injecting NSCLC A549 cells into 10-weeks-old male NOD/SCID mice,which were transfected by adeno-associated virus(AAV).A flow cytometry was used to detect various macrophage(MΦ)subtypes.Levels of PLGF,vascular endothelial growth factor receptor-1(Flt-1)and transforming growth factorβ1(TGF-β1)were examined in tumor cells and TAM by RT-qPCR.With the different cultured media of A549 cells and MΦcells,it could divide into A549 group and MΦgroup,A549+MΦgroup,A549+MΦ+sFlt-1 group(with 10μg/L sFlt-1),PLGF+MΦgroup(with 100 ng PLGF)and PLGF+MΦ+sFlt-1 group(with 100 ng PLGF and 10μg/L sFlt-1).Transwell assay and MTT assay were used to detect the proliferation and migration of A549 cells with different co-culture conditioned media.With the different cultured media of HUVEC cell,it could divide into control group,conditioned media(CM)group,CM+SB431542 group,TGF-β1 group and TGF-β1+SB431542 group.HUVEC cells collagen gel assay was used to detect the tumor vascularization.Results Compared with RFP-cells,PLGF was predominantly expressed by tumor cells(31.72±4.69 vs.2.31±0.06,P<0.001),while Flt-1 was predominantly expressed by CD163+macrophages(P<0.001).The proliferation ability of A549 cells in the A549+MΦgroup was significantly higher than in A549 group and A549+MΦ+sFlt-1 group(OD value:3.62±0.23 vs.4.53±0.34 vs.3.71±0.37,P<0.05).TGF-β1 in TAM/M2 macrophages was higher than that in M1 macrophages(0.99±0.23 vs.0.07±0.02,P<0.05).The formation of tubular structures in HUVEC cells were the most in the CM group and TGF-β1 group(P<0.05),which was inhibited by TGF-β1 inhibitor SB431542.Conclusion The crosstalk between TAM and NSCLC cells via PLGF/Flt-1 and TGF-β1 signal pathways promotes the growth and vascularization of NSCLC.