van der Vos Kristan E, Abels Erik R, Zhang Xuan, Lai Charles, Carrizosa Esteban, Oakley Derek, Prabhakar Shilpa, Mardini Osama, Crommentuijn Matheus H W, Skog Johan, Krichevsky Anna M, Stemmer-Rachamimov Anat, Mempel Thorsten R, El Khoury Joseph, Hickman Suzanne E, Breakefield Xandra O
Departments of Neurology and Radiology, Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, Massachusetts (K.E.v.d.V., E.R.A., X.Z., C.L., S.P., O.M., M.H.W.C., J.S., X.O.B.); Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Boston, Massachusetts (E.C., T.R.M., J.E.K., S.E.H.); Neuropathology Service, Massachusetts General Hospital and Department of Pathology, Harvard Medical School, Boston, Massachusetts (D.O., A.S-R.); Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts (A.M.K.); Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, the Netherlands (K.E.v.d.V.).
Neuro Oncol. 2016 Jan;18(1):58-69. doi: 10.1093/neuonc/nov244. Epub 2015 Oct 3.
To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo.
Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels.
Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA.
Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs.
为了解神经胶质瘤操纵其微环境的能力，我们在体外培养和体内观察了囊泡的转移以及肿瘤释放的细胞外RNA对小胶质细胞表型的影响。
分离原发性人胶质母细胞瘤（GBM）细胞释放的细胞外囊泡（EVs）并分析其中的微小RNA（miRNAs）。将原代小鼠小胶质细胞暴露于GBM-EVs中，分析其摄取情况以及对增殖和特定miRNAs、mRNAs及蛋白质水平的影响。对于体内分析，将小鼠胶质瘤细胞植入小鼠脑内，通过活体双光子显微镜、免疫组织化学、荧光激活细胞分选分析以及RNA和蛋白质水平监测小胶质细胞和单核细胞/巨噬细胞对EVs的释放和摄取。
小胶质细胞大量摄取GBM-EVs，导致其增殖增加且细胞因子谱向免疫抑制方向转变。GBM-EVs中高水平的miR-451/miR-21转移至小胶质细胞，同时miR-451/miR-21的靶标c-Myc mRNA水平降低。在体内分析中，我们直接观察到胶质瘤细胞释放的EVs及其被脑内小胶质细胞和单核细胞/巨噬细胞摄取的过程。来自荷瘤脑的解离小胶质细胞和单核细胞/巨噬细胞显示miR-21水平升高而c-Myc mRNA水平降低。
活体显微镜检查证实了神经胶质瘤释放EVs并被脑内小胶质细胞和单核细胞/巨噬细胞摄取。我们的研究还支持GBM释放的EVs被小胶质细胞摄取后的功能效应，这部分与miRNA水平升高、靶标mRNAs及编码蛋白质水平降低有关，推测这是肿瘤操纵其周围环境的一种方式。
