Glioblastoma Cells Expressing Oncogenic EGFR Release Multiple Extracellular Vesicle Subpopulations Positive or Negative for EGFR

Extracellular vesicle (EV) heterogeneity is well documented but poorly defined. This is especially important in cancer where EVs serve as carriers of unique oncogenic macromolecules that can be transferred to recipient cells or targeted for liquid biopsy diagnostics. Here we employed a series of human glioma cell lines to test the content and distribution of oncogenic epidermal growth factor receptor (EGFR) including its mutant (EGFRvIII) among different subsets of tumour-derived EVs. Our results suggest that the global levels of EGFR packaged into EVs parallels its expression in parental cancer cells. However, while in glioma cells expressing high levels of EGFR (GSC83) this receptor was uniformly distributed on cellular surfaces, only a small fraction of their derived EVs contained EGFR, as documented by nano-flow cytometry, ExoView and super-resolution microscopy. Using only three protein markers (CD63, CD81 and EGFR) these single EV platforms revealed the existence of seven different EV subsets, of which four contained EGFR. Purified EGFR-positive and negative EVs contained both shared and distinctive protein markers. EGFR packaging into EVs of GSC83 cells was independent of syntenin 1 expression, but was suppressed upon treatment with pharmacological inhibitor of neutral sphingomyelinase (GW4869). Exposure of human microglial cells (HMC3) to EVs released from GW4869-treated and control glioma cells triggered distinctive changes in cellular proteome including transfer of EGFR. Overall, our results suggest that multiple pathways of EV biogenesis may operate in glioma cells resulting in formation of complex EV landscapes consisting of EGFR-positive and EGFR-negative EV subsets. This heterogeneity may have important implications for EV functions and EV-based diagnostics.