EVs from hypoxic NB cells stimulate migration and invasion of target cells via miR-210-3p

Tumor hypoxia stimulates release of extracellular vesicles (EVs) that facilitate short- and long-range intercellular communication and metastatization. Albeit hypoxia and EVs release are known features of Neuroblastoma (NB), a metastasis-prone childhood malignancy of the sympathetic nervous system, whether hypoxic EVs can facilitate NB dissemination is unclear. Here we show that EVs from hypoxic NB cells promote pro-metastasis features in vitro and in an in vivo model. EVs from NB cells cultured at different oxygen tensions did not differ for type and abundance of surface markers and for biophysical properties. However, EVs derived from hypoxic NB cells were more potent than their normoxic counterpart in inducing NB cells migration and colony formation. We characterized and compared the microRNA (miRNA) cargo of EVs from NB cells cultured at different oxygen tensions and found that miR-210-3p was the most upregulated. Mechanistically, we identified that the pro-metastatic features of EVs from hypoxic NB cells derived from a miR-210-3p enrichment in their cargo. Indeed, overexpression of miR-210-3p in normoxic EVs conferred them pro-metastatic features, whereas miR-210-3p silencing suppressed the metastatic ability of hypoxic EVs both in vitro and in in vivo zebrafish model. Our data identify a role for hypoxic EVs in the cellular and microenvironmental changes favoring NB dissemination.

肿瘤缺氧可刺激细胞外囊泡（extracellular vesicles, EVs）的释放，此类囊泡可介导短程与长程细胞间通讯，并促进肿瘤转移。尽管缺氧与细胞外囊泡释放已是神经母细胞瘤（Neuroblastoma, NB）的已知特征——神经母细胞瘤是一种好发于儿童的、易发生转移的交感神经系统恶性肿瘤——但缺氧来源的细胞外囊泡是否能促进神经母细胞瘤的播散仍尚不明确。本研究证实，源自缺氧环境下神经母细胞瘤细胞的细胞外囊泡，可在体外及体内模型中促发转移相关表型。在不同氧分压条件下培养的神经母细胞瘤细胞所释放的细胞外囊泡，其表面标志物的类型与丰度、以及生物物理特性均无显著差异。但相较常氧环境下的对应囊泡，缺氧来源的神经母细胞瘤细胞外囊泡在诱导神经母细胞瘤细胞迁移与集落形成方面活性更强。我们对不同氧分压条件下培养的神经母细胞瘤细胞来源的细胞外囊泡所携带的微小RNA（microRNA, miRNA）载荷进行了表征与比较，发现miR-210-3p的上调幅度最为显著。从机制层面而言，我们证实缺氧来源的神经母细胞瘤细胞外囊泡所具备的促转移表型，源于其载荷中富集的miR-210-3p。确实，在常氧来源的细胞外囊泡中过表达miR-210-3p，可赋予其促转移表型；而敲低miR-210-3p则可在体外及活体斑马鱼模型中，抑制缺氧来源细胞外囊泡的转移能力。本研究数据证实，缺氧来源的细胞外囊泡可通过改变细胞与肿瘤微环境，促进神经母细胞瘤的播散。