Yekula Anudeep, Minciacchi Valentina R, Morello Matteo, Shao Huilin, Park Yongil, Zhang Xuan, Muralidharan Koushik, Freeman Michael R, Weissleder Ralph, Lee Hakho, Carter Bob, Breakefield Xandra O, Di Vizio Dolores, Balaj Leonora
Department of Neurosurgery, Massachusetts General Hospital, Boston, MA, USA.
Department of Surgery, Pathology & Laboratory Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
J Extracell Vesicles. 2019 Nov 27;9(1):1689784. doi: 10.1080/20013078.2019.1689784. eCollection 2020.
Tumour cells release diverse populations of extracellular vesicles (EVs) ranging in size, molecular cargo, and function. We sought to characterize mRNA and protein content of EV subpopulations released by human glioblastoma (GBM) cells expressing a mutant form of epidermal growth factor receptor (U87) and with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87 cancer cells, non-cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87 glioma-bearing mice using differential centrifugation (EVs that sediment at 10,000 × or 100,000 × are termed large EVs and small EVs, respectively) were characterized using transmission electron microscopy (TEM), confocal microscopy, nanoparticle tracking analysis (NTA), flow cytometry, immunofluorescence (IF), quantitative-polymerase chain reaction (qPCR), droplet digital polymerase chain reaction (ddPCR) and micro-nuclear magnetic resonance (μNMR). We report that both U87 and HUVEC release a similar number of small EVs, but U87 glioma cells alone release a higher number of large EVs compared to non-cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87 glioma cells was comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Similarly, EGFRvIII mRNA in large and small EVs isolated from the serum of U87 glioma-bearing mice is comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Here we report for the first time a direct comparison of large and small EVs released by glioma U87 cells and from serum of U87 glioma-bearing mice. Both large and small EVs contain tumour-specific EGFRvIII mRNA and proteins and combining these platforms may be beneficial in detecting rare mutant events in circulating biofluids.
肿瘤细胞释放出大小、分子载荷和功能各异的细胞外囊泡（EVs）群体。我们试图表征由表达表皮生长因子受体（EGFR）突变形式的人胶质母细胞瘤（GBM）细胞释放的EV亚群的mRNA和蛋白质含量，并研究其大小、形态以及肿瘤相关物质的存在情况。使用差速离心法从GBM U87癌细胞、非癌性人脐静脉内皮细胞（HUVEC；对照）和U87荷瘤小鼠血清中纯化出两种EV亚群（分别沉降在10,000×或100,000×的EVs被称为大EVs和小EVs），并通过透射电子显微镜（TEM）、共聚焦显微镜、纳米颗粒跟踪分析（NTA）、流式细胞术、免疫荧光（IF）、定量聚合酶链反应（qPCR）、液滴数字聚合酶链反应（ddPCR）和微核磁共振（μNMR）对其进行表征。我们发现，U87细胞和HUVEC释放的小EVs数量相似，但与非癌性HUVEC相比，单独的U87胶质瘤细胞释放的大EVs数量更多。来自U87胶质瘤细胞的两种EV亚群中的EGFRvIII mRNA相当，但大EVs中的EGFR蛋白（野生型+vIII）水平显著更高。同样，从U87荷瘤小鼠血清中分离出的大、小EVs中的EGFRvIII mRNA相当，但大EVs中的EGFR蛋白（野生型+vIII）水平显著更高。在此，我们首次对胶质瘤U87细胞和U87荷瘤小鼠血清释放的大、小EVs进行了直接比较。大、小EVs均含有肿瘤特异性的EGFRvIII mRNA和蛋白质，结合这些平台可能有助于检测循环生物流体中罕见的突变事件。
