fig6

Figure 6. Effects of GBM-derived EVs on maturation/activation of BMDCs and BV-2 microglial cells. (A) Downstream effects analysis of EV protein datasets revealed the predicted activation of the “Immune Response of Cells” biofunction. Orange and blue shapes represent predicted activation or inhibition, respectively. Instead, red and green shapes represent increased or decreased measurements of EV-quantified proteins, respectively, whose expression values are reported in the figure (ratio: EVs +TMZ/ EVs -TMZ). Continuous lines represent direct relationships, dotted lines represent indirect relationships, and grey and yellow lines indicate non-predicted and inconsistent relationships. (B) Expression of CD80 and CD206 in BV-2 cells treated with GBM-derived EVs detected by Western blot analysis. Data are normalized to β-actin and expressed as fold change compared to the untreated (CTRL) group. Data are presented as mean ± SD of three independent biological replicates (n = 3). Original uncropped blots are reported in Supplementary Figure 6. Relative mRNA expression levels of IL-1β (C) and IL-10 (D) in BV-2 cells treated with EVs derived from untreated and TMZ-treated U87MG and U251MG cells. Data are expressed as fold change compared to control (CTRL, untreated) and normalized to the housekeeping gene Tbp. Statistically significant differences between groups were determined using one-way ANOVA followed by Tukey’s post hoc test ^***P < 0.001. Data are presented as mean ± SD of three independent biological replicates (n = 3). (E) Microscopy fluorescence images showing the uptake of PKH26-labelled EVs derived from untreated (EVs -TMZ) and TMZ-treated (EVs +TMZ) U87MG and U251MG cells in recipient BMDCs after 30 min of incubation. Internalized EVs are indicated by white arrows (scale bar: 20 μm); CD86 (F), CD80 (G), and MHCII (H) expression on BMDC surfaces was assessed by flow cytometry. LPS-treated BMDCs were used as positive controls. Statistically significant differences between groups were determined using one-way ANOVA followed by Tukey’s post hoc test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Data are presented as the mean ± SD of three independent biological replicates (n = 3). ANOVA: Analysis of variance; BMDCs: bone marrow-derived dendritic cells; CD80: cluster of differentiation 80; CD86: cluster of differentiation 86; CD206: cluster of differentiation 206; CTRL: control; EVs: extracellular vesicles; GBM: glioblastoma; IL-1β: interleukin-1 beta; IL-10: interleukin-10; LPS: lipopolysaccharide; MHCII: major histocompatibility complex class II; PKH26: red fluorescent cell membrane dye; SD: standard deviation; TMZ: temozolomide; Tbp: TATA-box binding protein.