Fig. 2: Protracted expression of activated EGFR in endothelial cells subjected to extracellular vesicle-mediated transfer of EGFR/EGFRvIII transcript from mesenchymal glioma stem cells.

a Quantification of the human phospho-RTK expression array; analysis performed with EVs from either PN or MES EVs incubated with primary endothelial cells (HUVEC) for 7 days (n = 4 wells/2 independent experiments). Data are presented as mean values ± SD; b Endothelial cells incubated with EVs from PN or MES EVs followed by protein extraction and western blot to analyse the expression of EGFR. The transfer resulted in lasting ectopic activation of EGFR in HUVEC up to 6 days. β-Actin was used as loading control. (n = 3 independent experiments); c Glioma stem cell-derived EGFR+ EVs were incubated with mouse endothelial cells. The transfer of human-specific EGFRvIII was detected for up to 6 days. Mouse β-Tubulin was used as loading control (n = 3 independent experiments); d Glioma stem cell-derived EVs were incubated with mouse endothelial cells enabling transfer of human EGFR. The transfer of human-specific EGFR mRNA was detected only after treatment with MES GSC EVs (n = 3 independent experiments); e Western blot and RT-PCR analysis showed that GW4869 treatment selectively inhibited the shedding of EVs carrying EGFR protein, while EGFR mRNA EVs were not affected (n = 3 independent experiments); f Schematic diagram illustrating the immunoprecipitation approach using magnetic beads crosslinked with anti-EGFR antibody. Western blot and RT-PCR analysis showed a population of EVs enriched for EGFR protein and mostly depleted for EGFR mRNA, while unbound EVs with no EGFR protein were enriched for EGFR mRNA (n = 3 independent experiments); g EGFR status in endothelial cells treated with EVs carrying only EGFR mRNA. Western blot analysis reveals that transfer of EGFR mRNA is sufficient to express EGFR protein in primary endothelial cells for up to 6 days (n = 3 independent experiments); Source data are provided as a Source Data file.