顺铂耐药肺腺癌A549/DDP细胞释放富含IGFBP3的外泌体诱导巨噬细胞M2型极化并促进A549细胞继发耐药  

The secondary drug resistance of lung adenocarcinoma A549 cells pomoted by IGFBP3-rich exosome released from A549/DDP cells through M2 polarization of macrophages

作　　者：张征峥[1] 王晓峰 吕品[1] 钱乾 张玲[1] 崔玲[1] 宋淑霞[1] ZHANG Zhengzheng;WANG Xiaofeng;LÜPin;QIAN Qian;ZHANG Ling;CUI Ling;SONG Shuxia(Department of Immunology,Hebei Medical University,Hebei Key Laboratory of Immune Mechanism and Intervention for Serious Diseases,Shijiazhuang 050017,Hebei Province,China;Department of Oncology,The Fourth Hospital of Shijiazhuang,Shijiazhuang 050000,Hebei Province,China)

机构地区：[1]河北医科大学免疫学教研室,河北省重大疾病免疫机制及干预重点实验室,河北石家庄050017 [2]石家庄市第四医院肿瘤科,河北石家庄050000

出　　处：《肿瘤》2024年第4期346-357,共12页Tumor

基　　金：河北省自然科学基金资助项目(H2020206424);河北省高等学校科学技术研究项目(ZD2022137)

摘　　要：目的:探讨顺铂(cisplatin,DDP)耐药型人肺腺癌(lung adenocarcinoma,LUAD)细胞株A549/DDP释放的富含胰岛素样生长因子结合蛋白3(insulin-like growth factorbinding protein 3,IGFBP3)的外泌体对诱导巨噬细胞分化及其对DDP耐药的影响。方法:体外培养亲本A549及A549/DDP细胞,用不同浓度的DDP处理48 h后计算IC_(50)值。收集A549或A549/DDP细胞培养上清液,利用超速离心法分离外泌体,分别命名为A-exo或A/D-exo。用终浓度为15μg/m L的PMA诱导THP-1细胞分化为M0型巨噬细胞,与A549细胞按1∶1比例混合后接种于裸鼠腋下;在肿瘤细胞接种当天,在肿瘤细胞接种部位分别注射PBS、A-exo和A/D-exo,同时每4 d 1次腹腔注射DDP进行治疗;于小鼠荷瘤的第35天,经流式细胞法检测移植瘤组织中人源CD11b^(+)CD206^(+)或CD11b^(+)CD86^(+)巨噬细胞的募集情况。用抗体芯片筛选A-exo或A/D-exo中携带的蛋白,并用ELISA法检测A-exo和A/D-exo中IGFBP3蛋白的含量进行验证。用不同浓度的rh IGFBP3处理A549或A549/DDP细胞,分别采用MTS法和Transwell小室实验检测rh IGFBP3对细胞增殖或迁移能力的影响。用rh IGFBP3处理M0巨噬细胞4 d,收集培养上清液;采用ELISA法检测不同浓度的rh IGFBP3对M0巨噬细胞产生TGF-β及TNF-α含量的影响;另外,用rh IGFBP3或rh IGFBP3预处理的M0巨噬细胞培养上清液处理A549细胞,并再次检测DDP对A549细胞的IC_(50)值。结果:DDP对A549/DDP细胞的IC_(50)值较A549细胞显著提高(P<0.01);与PBS及A-exo组比较,A/D-exo可明显促进A549细胞移植瘤的生长(P<0.05),并促进CD11b^(+)CD206^(+)巨噬细胞募集到肿瘤组织中(P<0.05)。成功收集获得外泌体A-exo和A/D-exo;与A-exo比较,A/D-exo中携带高水平的IGFBP3;分析显示,LUAD患者中IGFBP3的表达水平明显上调,且IGFBP3高表达患者的总生存率较IGFBP3低表达者有所降低。高浓度rh IGFBP3(100 ng/m L)对A549或A549/DDP细胞均有明显的促增殖作用(P均<0.05),但对A549或A549/DDP细�Objective:To investigate the effects of insulin-like growth factor-binding protein 3(IGFBP3),which is carried in exosomes released by cisplatin(DDP)-tolerant human lung adenocarcinoma(LUAD)A549/DDP cells,on differentiation of macrophages and its effect on DDP resistance of A549 cells.Methods:The parental A549 and A549/DDP cells were cultured in vitro,and the IC_(50) values were calculated after treatment with different concentrations of DDP for 48 h.The supernatants of A549 or A549/DDP cells culture were collected,and the exosomes were isolated using ultracentrifugation and named A-exo or A/D-exo,respectively.THP-1 cells were induced to differentiate into M0-type macrophages with PMA(15μg/mL),mixed with A549 cells at a ratio of 1∶1,and then inoculated in the axillae of nude mice;on the day of tumor cell inoculation,the tumor cells were injected with PBS,A-exo,and A/D-exo at the inoculation site of the tumor cells,respectively,and at the same time,the treatment was carried out by intraperitoneal injection of DDP 1 time every 4 d.On the 35th day of the tumor loading in mice,the recruitment of human CD11b^(+)CD206^(+)or CD11b^(+)CD86^(+)macrophages in transplanted tumor tissues was detected by flow cytometry(FCM).Antibody microarrays were used to screen for proteins carried by A-exo or A/Dexo and validated by detecting the amount of IGFBP3 protein in A-exo and A/D-exo by ELISA method.A549 or A549/DDP cells were treated with different concentrations of rhIGFBP3,and the effects of rhIGFBP3 on the proliferation or migration ability of the cells were detected by MTS assay and Transwell assay,respectively.M0-type macrophages were treated with rhIGFBP3 for 4 d,and the culture supernatant was collected;the effects of different concentrations of rhIGFBP3 on the production of TGF-βand TNF-αcontent by M0-type macrophages were detected by ELISA;in addition,A549 cells were treated with rhIGFBP3 or culture supernatant of M0-type macrophages pretreated with rhIGFBP3,and again detected the IC_(50) value of DDP on A549 cells.R

关 键 词：肺腺癌 外泌体 IGFBP3 巨噬细胞分化 耐药性 

分 类 号：R734.2[医药卫生—肿瘤]