背景:肿瘤细胞释放细胞外囊泡(EV),这有助于巨噬细胞向肿瘤相关巨噬细胞(TAM)的极化。RNA结合蛋白IGF2BP2/IMP2的高表达水平与肿瘤细胞增殖增加相关。入侵,临床预后不良。然而,缺乏对IMP2是否会影响癌细胞衍生电动汽车的货物的理解,从而调节巨噬细胞极化。
方法:从表达IMP2的HCT116亲本细胞(WT)和CRISPR/Cas9IMP2敲除(KO)细胞中分离EV。电动汽车根据MISEV指南进行了表征,通过microRNA-Seq评估microRNA货物,并通过蛋白质组学分析了蛋白质货物。原代人单核细胞衍生的巨噬细胞(HMDM)被EV极化,使用qPCR和流式细胞术评估基因和表面标志物的表达,分别。巨噬细胞的形态变化,以及癌细胞的迁移潜力,通过Incucyte®系统和通过酶谱法评估巨噬细胞基质降解潜力。使用Seahorse®分析仪定量巨噬细胞代谢活性的变化。对于体内研究,将电动汽车注射到斑马鱼幼虫的卵黄囊中,和巨噬细胞通过荧光激活细胞分选分离。
结果:来自WT和KO细胞的EV具有相似的大小和浓度,并且对25个囊泡标志物呈阳性。与KOEV相比,WTEV极化的巨噬细胞中促肿瘤基因的表达更高,TNF和IL6的表达降低。在体内处理的斑马鱼幼虫的巨噬细胞中观察到类似的模式。WTEV极化的巨噬细胞显示出较高丰度的TAM样表面标志物,较高的基质降解活性,以及对癌细胞迁移有更高的促进作用。MicroRNA-Seq显示WT和KOEV的microRNA组成存在显着差异,特别是WT电动汽车中miR-181a-5p的丰度很高,这在KO电动汽车中是不存在的。巨噬细胞吞噬和吞噬作用的抑制剂拮抗miR-181a-5p进入巨噬细胞的递送和miR-181a-5p靶标DUSP6的下调。蛋白质组学数据显示KO与KO中蛋白质货物的差异WT电动汽车,差异丰富的蛋白质主要参与代谢途径。WTEV处理的巨噬细胞比KOEV处理的细胞表现出更高的基础耗氧率和更低的细胞外酸化率。
结论:我们的结果表明IMP2决定了癌细胞释放的电动汽车货物,从而调节电动汽车对巨噬细胞的作用。IMP2的表达与使巨噬细胞向肿瘤促进表型极化的EV的分泌有关。
BACKGROUND: Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization.
METHODS: EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting.
RESULTS: EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells.
CONCLUSIONS: Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs\' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.