肿瘤相关巨噬细胞通过激活IGF-1R信号通路诱导三阴性乳腺癌细胞对白蛋白紫杉醇耐药的研究

Investigation on the determination of tumor-associated macrophages inducing the drug resistance of albumin-bound paclitaxel in triple-negative breast cancer cells by activating IGF-1R signaling pathway

目的探讨肿瘤相关巨噬细胞(TAM)对三阴性乳腺癌(TNBC)细胞白蛋白紫杉醇(Nab-PTX)化疗敏感性的影响及作用机制。方法构建并鉴定TAM模型;通过Transwell小室共培养法建立TAM与TNBC细胞系MDAMB-231细胞共培养模式,分为对照组(MDA-MB-231细胞及空白小室)、Nab-PTX组(MDA-MB-231细胞、空白小室及0.5 nmol/L Nab-PTX)、TAM组(MDA-MB-231细胞、含M2型巨噬细胞的小室)、TAM+Nab-PTX组(MDA-MB-231细胞、含M2型巨噬细胞的小室及0.5 nmol/L Nab-PTX)、胰岛素样生长因子1受体(IGF-1R)抑制剂组(MDA-MB-231细胞、含M2型巨噬细胞的小室、0.5 nmol/L Nab-PTX及4 nmol/L IGF-1R抑制剂Linsitinib);CCK-8法检测各组细胞增殖情况;流式细胞术检测细胞凋亡;实时荧光定量PCR(q PCR)检测多药耐药蛋白(MDR)1和胱天蛋白酶(Caspase)-3mRNA水平;Western blot检测IGF-1R信号通路关键蛋白表达。结果THP-1细胞经诱导分化为M2型巨噬细胞;与Nab-PTX组相比,TAM+Nab-PTX组细胞增殖水平升高,凋亡率降低(P<0.01),MDR1 mRNA表达升高,Caspase-3mRNA表达降低(P<0.05),IGF-1R信号通路关键蛋白激活(P<0.01);与TAM+Nab-PTX组相比,IGF-1R抑制剂组细胞增殖水平降低,凋亡率升高,MDR1 mRNA表达降低,Caspase-3 mRNA的表达增高,IGF-1R信号通路关键蛋白的表达降低(P<0.01)。结论TAM可能通过激活IGF-1R信号通路诱导TNBC细胞对Nab-PTX耐药。

Objective To investigate the effect of tumor-associated macrophages(TAM)on the chemosensitivity of triple-negative breast cancer(TNBC)cells to albumin-bound paclitaxel(Nab-PTX).Methods TAM model was constructed and identified.TNBC cell line MDA-MB-231 was established by Transwell cell co-culture method.They were divided into the control group(MDA-MB-231 cells and blank chamber),the Nab-PTX group(MDA-MB-231 cells,blank chamber and 0.5 nmol/L Nab-PTX),the TAM group(MDA-MB-231 cells,containing M2-type macrophages),the TAM+Nab-PTX group(MDA-MB-231 cells,cells containing M2-type macrophages and 0.5 nmol/L Nab-PTX)and the insulin like growth factor 1 receptor(IGF-1R)inhibitor group(MD-MB-231 cells,macrophage compartment containing M2-type,0.5 nmol/L Nab-PTX and 4 nmol/L IGF-1R inhibitor Linsitinib).The cell survival rate of each group was determined by CCK-8 method.Flow cytometry was used to detect cell apoptosis.The mRNA levels of multidrug resistant protein(MDR)1 and Caspase-3 were detected by real-time quantitative PCR(qPCR).The key proteins of IGF-1R signaling pathway were detected by Western blot assay.Results THP-1 cells were induced to differentiate into M2 macrophages.Compared with the Nab-PTX group,the cell proliferation rate was increased and apoptosis rate was decreased in the TAM+Nab-PTX group(P<0.01).The mRNA expression of MDR1was increased and Caspase-3 mRNA was decreased(P<0.05).The key protein of IGF-1R signaling pathway was activated(P<0.01).Compared with the TAM+Nab-PTX group,the proliferation rate was decreased and apoptosis rate of cells was increased in the IGF-1R inhibitor group.The mRNA expression of MDR1 was decreased,the mRNA expression of Caspase3 was increased,and the expression of key proteins in IGF-1R signaling pathway was decreased(P<0.01).Conclusion TAM may induce resistance of TNBC cells to Nab-PTX by activating IGF 1R signaling pathway.