M2型巨噬细胞来源外泌体对乳腺癌细胞系4T1干性特征的影响

目的探讨M2型巨噬细胞来源的外泌体对乳腺癌细胞系4T1细胞肿瘤干性特征的影响。方法用超速离心法分离M2型巨噬细胞来源的外泌体;用透射电子显微镜、纳米粒子追踪技术,Westernblot等对其进行鉴定;然后以乳腺癌细胞系4T1为肿瘤模型,使用Dil染料标记M2型巨噬细胞来源的外泌体,通过免疫荧光技术观察其能否进入4T1细胞。此外,用流式细胞计量术、共培养体系和Transwell实验探究在M2型巨噬细胞来源的外泌体影响下的4T1细胞的干性特征。结果M2型巨噬细胞在体外被成功诱导,分离并鉴定了M2型巨噬细胞来源的外泌体。通过免疫荧光,看到Dil标记的外泌体可成功进入4T1细胞。M2型巨噬细胞来源的外泌体与4T1细胞共孵育后,可明显增强4T1细胞的迁移能力(P<0.001)和成球能力(P<0.05);外泌体抑制剂GW4869可明显抑制4T1细胞的成球能力(P<0.05)。同时,M2型巨噬细胞来源的外泌体可明显增加4T1细胞中CD44highCD24low细胞的比例(P<0.001)。结论M2型巨噬细胞来源的外泌体能够促进4T1肿瘤细胞的迁移和成球能力,并明显增加4T1细胞中肿瘤干细胞的比例。

Effect of M2 macrophage-derived exosomes on stemness of breast cancer cell line 4T1

Objective To explore the effect of M2 macrophage-derived exosomes on stemness of 4 T1 cells. MethodsM2 macrophage-derived exosomes were isolated by ultracentrifugation. And transmission electron microscopy, nanoparticle tracking technology and Western blot were performed to identified the exosomes. Then, the M2 macrophage-derived exosomes were labeled with Dil which makes it available for us to observe their transfer to breast cancer cell line 4 T1 by immunofluorescence. After that, flow cytometry, co-culture system and transwell chamber were used to investigate the tumor phenotype and stemness of 4 T1 under the influence of M2 macrophage-derived exosomes. Results First, the M2 macrophages were successfully induced in vitro. In addition, the M2 macrophage-derived exosomes were successfully isolated and verified. M2 macrophage-derived exosomes labeled with Dil could be transferred into 4 T1 cells. After co-incubation with M2 macrophage-derived exosomes, the migration(P<0.001) and sphere formation(P<0.05) of 4 T1 cells were significantly enhanced. While after using exosome inhibitor GW4869,the number and volume of spheres formed by 4 T1 stem cells were reduced(P<0.05). The proportion of CD44highCD24low cells in 4 T1 cells(P<0.001) was significantly increased after incubating with M2 macrophage-derived exosomes. Conclusions M2 macrophage-derived exosomes increases the migration and sphere formation ability of 4 T1 tumor cells, so is the proportion of tumor stem cells in 4 T1 cells.