From the ‡Research Institute of the McGill University Health Centre, Glen Site, McGill University, Montreal, Quebec, H4A 3J1, Canada.
§Donnelly Centre and Departments of Molecular Genetics and Computer Science, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
Mol Cell Proteomics. 2018 Oct;17(10):1948-1964. doi: 10.1074/mcp.RA118.000644. Epub 2018 Jul 13.
Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.
多形性胶质母细胞瘤（GBM）是一种高度侵袭性和异质性的原发性脑肿瘤，由一系列致癌改变驱动，包括持续激活的表皮生长因子受体（EGFRvIII）。EGFRvIII 影响 GBM 进展的细胞内在和非细胞自主方面，包括细胞侵袭、血管生成和肿瘤微环境的调节。这至少部分归因于细胞外囊泡（EVs）的释放和细胞间运输，EVs 是含有多种生物活性大分子的异质膜结构。在这里，我们使用具有强转化活性的同基因肿瘤细胞系分析了 EGFRvIII 对神经胶质瘤 EV 谱的影响。我们观察到 EGFRvIII 的表达改变了 EV 调节基因（vesiculome）和 EV 特性的表达，包括它们的蛋白质组成。使用质谱、定量蛋白质组学分析和基因本体术语过滤器，我们观察到 EGFRvIII 转化细胞释放的 EV 富含细胞外外泌体和焦点黏附相关蛋白。在这些蛋白中，我们验证了侵袭前蛋白（CD44、BSG、CD151）与表达 EGFRvIII 的神经胶质瘤细胞 EV 的关联，以及相对于 EGFRvIII 阴性细胞的 EV 中 exosomal 标记物（CD81 和 CD82）的下调。纳流细胞术显示，单个神经胶质瘤细胞系的 EV 输出高度异质，以至于只有一部分囊泡含有特定的蛋白质（包括 EGFRvIII）。值得注意的是，表达 EGFRvIII 的细胞释放的 EV 对 CD44/BSG 双阳性，并且这些蛋白也在细胞丝状伪足中聚集。我们还检测到同源性黏附分子的表达和 EGFRvIII 阳性神经胶质瘤细胞对同源性 EV 的摄取增加。这些结果表明，致癌性 EGFRvIII 重新编程了 GBM 相关 EV 的蛋白质组和摄取，这一概念对它们的生物学活性和与 EV 为基础的癌症生物标志物发展相关的特性具有重要意义。
