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. 2022 Oct 27;10(11):2718. doi: 10.3390/biomedicines10112718

Figure 2.

Figure 2

GBM extracellular vesicles derived from both plasma and tumor cell lines produced cytotoxicity in neurons and brain tissues. (A). Fluorescent microscopy images showing that GBM plasma EVs produced cell death in neuroblastoma cells in a time-dependent manner. SH-SY5Y cells (30–40% confluency) were incubated with GBM plasma and control EVs (50 µg/mL), 5% Normal Human Serum as a complement source, and propidium iodide. GBM pooled plasma EVs (GMB ppEV) produced large numbers of dead cells (red fluorescence) in a time-dependent manner (a–c) compared with control plasma EV (HC ppEV) treated cells (d–f). Scale bar = 100 nm. (B,C). Neuronal death caused by GBM EVs is dose-dependent. (B). A significantly higher number of dead cells were observed in GBM tumor cell line medium EVs (mEV) treated cells compared with controls in a dose-dependent manner (* p = 0.026). (C). Significantly lower number of live cells was detected in GBM mEV-treated cells compared with controls in a dose-dependent manner (** p = 0.0044). (D). GBM cell line EVs produced apoptosis in mouse brain tissues. P1 mouse brain slices (2 mm diameter) were incubated with GBM mEV and apoptosis was measured by luminescence. GBM mEVs produced significantly higher levels of apoptosis compared to controls (* p = 0.01). (E,F). GBM plasma EVs induced higher levels of cytotoxicity in SH-SY5Y cells. Plasma EVs were purified by ExoQuick and incubated with neuroblastoma cells. Cell death was measured by propidium iodide. (E). GBM plasma EVs produced significantly high levels of cytotoxicity compared to MMA (p = 0.002) and HC EVs (p = 0.008). n = 14 for GBM and MMA, n = 8 for HC. (F). Time-dependent cytotoxicity by GBM EVs. (G). GBM medium EVs did not induce significant cytotoxicity to normal human astrocytes (NHA) as compared to Control medium EVs. GBM and Control mEVs, used to treat the neurons and astrocytes, were purified by ultracentrifugation. Cell death was measured by propidium iodide. The results showed no significant cytotoxicity in astrocytes treated with GBM mEV versus Control mEV treated cells (n = 9 for GBM mEV, n = 7 for Control mEV, ns, not significant, **** p > 0.1).