Hypoxic Cancer-Secreted Exosomal miR-182-5p Promotes Glioblastoma Angiogenesis by Targeting Kruppel-Like Factor 2 and 4

Background: Glioblastoma (GBM) is the most lethal primary brain tumor and has a complex molecular profile. Hypoxia plays a critical role during tumor progression and in the tumor microenvironment (TME). Exosomes released by tumor cells contain informative nucleic acids, proteins and lipids involved in the interaction between cancer and stromal cells, thus leading to TME remodeling. Accumulating evidence indicates that exosomes play a pivotal role in cell-to-cell communication. The aim of this study was to clarify how hypoxia affects tumor angiogenesis via exosomes derived from GBM cells. Methods: Exosomes were isolated by total exosome isolation reagents from the conditioned medium of glioma cells and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting analysis. Human umbilical vein endothelial cells (HUVECs) were incubated with exosomes derived from different conditions, and the subsequent impacts were measured by tube formation assay, Western blotting, real-time quantitative PCR (RT-PCR), cell migration/transendothelial migration/endothelial permeability, fluorescent imaging of exosome uptake, luciferase reporter assay, brain orthotopic xenografts, fluorescence in situ hybridization (FISH), immunofluorescence (IF) and immunohistochemical (IHC) staining. Findings: Compared with the parental cells under normoxic conditions, the GBM cells produced more exosomes, and miR-182-5p was significantly up-regulated in the exosomes from GBM cells under hypoxic conditions. Exosomal miR-182-5p directly suppressed its targets Kruppel-like factor 2 and 4 (KLF2 and KLF4), leading to the accumulation of vascular endothelial growth factor receptor (VEGFR) in endothelial cells. Consequently, hypoxic GBM cells enhanced the angiogenesis caused by exosomes from hypoxic tumors under both normoxic and hypoxic conditions. In addition, exosomal miR-182-5p also inhibited tight junction related proteins (such as ZO-1, Occludin, and Claudin-5), thus enhancing vascular permeability and tumor transendothelial migration. The inhibition of miR-182-5p by inhibitor administration reduced angiogenesis and tumor growth in vivo. Furthermore, elevated levels of circulating miR-182-5p were found in the blood serum and cerebrospinal fluid (CSF) of glioma patients, and miR-182-5p levels were positively correlated with poor prognosis. Interpretation: Taken together, our study clarifies the clinical relevance and prognostic value of GBM-derived exosomal miR-182-5p under hypoxic conditions and reveals the unique intercellular crosstalk between tumor cells and HUVECs mediated by tumor-derived exosomes that modulate tumor vasculature. Funding: The research was supported by National Natural Science Foundation of China (No.81671210; No. 30801180; No. 81371380). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The use of human tissues was approved by the Human Research Committee of Huazhong University of Science and Technology (S898). Written informed consent was obtained from each patient. All animal experiments were performed according to the guidelines of care and use of laboratory animals and were approved by the Tongji Medical College Animal Experiments Committee (S2130).