Animals and regents
C57BL/6J mice were purchased from Shanghai Laboratory Animal Center (Chinese Academy of Sciences) and were housed and maintained in the Comparative Medicine Facility of the Tongji University School of Medicine (Shanghai, China). All procedures were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee at Tongji University School of Medicine.
Mouse NPCs (mNPCs) culture and treatment
Mouse cortical NPCs were isolated from gestational day E14 brain tissue as previously described . Brain tissues were dissected and mechanically dissociated using forceps to remove the membranes and large blood vessels. Brain tissues were digested by Trypsin-EDTA (Life Technologies) and then plated on cell culture flasks in mouse NeuroCult NSC Proliferation Medium (StemCell Technologies, Vancouver, BC, Canada), supplemented with epidermal growth factor (EGF, 10 ng/ml, Novus Biologicals), basic fibroblast growth factor (bFGF, 20 ng/ml, Novus Biologicals) for selective neurosphere cultures and penicillin/streptomycin (1% v/v, Gibco). Neurospheres were passaged when they reached 100-150 μm in diameter.
U87 and A172 culture
The human glioblastoma cell lines U87 and A172 (American Type Culture Collection, Manassas, VA, USA) were authenticated by American Type Culture Collection using the short tandem repeat genotyping method. U87 and A172 cells were cultured in DMEM GlutaMax (Gibco) containing 10% FBS (Sigma-Aldrich) and penicillin/streptomycin (1% v/v) in humidified chamber (37 °C, 5% CO2 incubator). U87 cells were passaged at 3-4 day intervals.
Isolation of EVs
The method for the isolation of extracellular vesicles has been described previously . Briefly, 1 × 106 U87 or A172 cells were plated on 10 cm dish and grown to 70-80% confluence. Then, U87s or A172 were rinsed with PBS three times and incubated with serum-free DMEM GluMax for 24 hours. Media were harvested and first centrifuged at 300 × g for 10 minutes to remove free cells, at 3,000 × g for 20 minutes to remove cellular debris, and then at 10,000 × g for 30 minutes to remove intracellular organelles. Lastly, EVs were collected by ultracentrifugation at 100,000 × g for 2 hours. All centrifugation steps were performed at 4 °C.
Transmission electron microscopy (TEM)
EVs were fixed with paraformaldehyde, negatively stained, and then spread on the copper grids. The droplets of EVs were removed with filter paper and air-dried at room temperature. Images were obtained using transmission electron microscopy (JEM-1230, JEOL Ltd.).
Dynamic light scattering (DLS)
EVs were re-suspended in 100 μl PBS and then diluted as 1:10 in PBS. 500 μl sample were added into a microcuvette (ZEN0118, Malvern Instruments, UK) to measure size using Nano ZS90 (Malvern Instruments, UK) at 25 °C.
Nanoparticle tracking analysis (NTA)
The size and concentration of extracellular vesicles were measured with NanoSight NS300 system (Malvern Instruments, UK). Briefly, U87 and A172 cells were cultured in 10 cm culture dishes for 48 hours. Then, the medium was changed to serum-free medium for 24 hours. The supernatants were differentially centrifugated and resuspended with 100 μl PBS and diluted at 1:10 in PBS, and then 1 ml solution was used for NanoSight analysis.
EV uptake assay
For EVs tracking, EVs or PBS were fluorescently labeled with PKH26 Red Fluorescent Cell Linker Mini Kit (SigmaAldrich) according to manufacturer's protocol. The labeled EVs or PBS were added into mNPCs for 8 hours. Then, the samples were stained with F-Actin (Phalloidin) and DAPI and images were captured by Zeiss AX10 fluorescence microscope.
EV pellets or cells were lysed in M-PER mammalian protein extraction reagent (Thermo Scientific) containing protease inhibitor (Thermo Scientific). Protein concentration was determined using the BCA (bicinchoninic acid) Protein Assay Kit (Pierce). An analytical 10% SDS polyacrylamide gel electrophoresis (SDS PAGE) was prepared and then transferred to polyvinyldifluoridene (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking in 5% fat-free milk for 1 hour, the membrane was incubated with purified primary antibodies for phospho-PI3K (p-PI3K, 1:1,000; Cell Signaling Technologies), PI3K (1:1,000; Cell Signaling Technologies), phospho-Akt (Ser473) (p-Akt, 1:1,000; Cell Signaling Technologies), Akt (1:1,000; Cell Signaling Technologies), phospho-mTOR (p-mTOR, 1:1,000; Cell Signaling Technologies), mTOR (1:1,000; Cell Signaling Technologies), phospho-C-Raf (p-C-Raf, 1:1000; Cell Signaling Technologies), phospho-MEK1/2 (p-MEK1/2, 1:1,000; Cell Signaling Technologies), MEK1/2 (1:1,000; Cell Signaling Technologies), phospho-ERK1/2 (p-ERK1/2, 1:1,000; Cell Signaling Technologies), ERK1/2 (1:1,000; Cell Signaling Technologies), β-actin (Actin, 1:5,000; Proteintech), flotillin-1 (1:1,000; BDbiosciences), flotillin-2 (1:5,000; BDbiosciences), TSG101 (1:5,000; Abacm), Alix (1:2,000; Bioworld), Calreticulin (1:1,000; Abcam) overnight at 4 °C followed by a horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibody (1:5,000; Icllab). Antigen-antibody complexes were visualized by Pierce ECL Western Blotting Substrate (Thermo Scientific).
For immunofluorescence staining, mNPCs were plated on the coverslips and fixed using 4% paraformaldehyde (PFA) for 20 minutes, then permeabilized with 0.4% Triton-X in PBS for 15 minutes. Subsequently, the coverslips were blocked with1% BSA for 1 hour. mNPCs were incubated with primary antibodies overnight including Nestin (1:500; Novus Biologicals) and Ki67 (1:1,000; Cell Signaling Technologies). Coverslips were washed with PBS three times and incubated for 1 hour at room temperature with secondary antibodies including anti-rabbit, mouse or chicken IgG (coupled with Alexa Fluor 488 or 568, Life Technologies). Nuclei were counter-stained with DAPI. Coverglasses were fixed on glass slides with Mounting Medium (Sigma-Aldrich). The images were captured by Zeiss AX10 fluorescence microscope. For quantification, the numbers of stained cells were quantified by Image-Pro Plus 6.0.
8 mm pore size transwell system (Costar) were coated with diluted matrigel (1:80, matrigel:NPC basal medium) in humidified chamber (37 °C, 5% CO2 incubator) for 30 minutes. Briefly, NPCs were dissociated into single cells and 2 × 105 cells/ml were resuspended in Mouse NeuroCult Proliferation Medium. The top chamber of the transwell was loaded with 100 μl of cell suspension containing either EVs or PBS. In the lower chamber, 600 μl of NPC proliferation medium was added. After 12 hours, the transwell inserts member was fixed with 4% PFA, and cells were removed by a cotton swab from the upper chamber. Migrated cells on the bottom of the membrane were stained with DAPI (Sigma Aldrich). For each insert, cells migrated through the pores were captured by Zeiss AX10 fluorescence microscope. Cell numbers were counted using Image-Pro Plus 6.0. The cell number of each insert-treated group was normalized to the cell number of the control group to analyze migration index.
Wound healing assay
mNPCs were seeded at 80% confluence in a 24-well plate coated with diluted matrigel. Each well was scratched using a 200 µl pipette tip. Each well was washed with PBS for three times and added NPC proliferation medium containing EVs or PBS. Images were captured at 0 hour and24 hours after the initial scratch. Images were captured by Olympus light microscopy.
EdU incorporation assay
Click-iT® EdU Imaging Kits (Thermo Scientific, #C10338) was used to analyze DNA synthesis according to the manufacturer’s instructions. 2.5 × 105 mNPCs cell were planted on 35 mm Coverglass-Bottom Dish (Cellvis, #D35-14-1-N). After 24 hours, medium was changed with fresh medium containing 50 μg/ml EVs or PBS for 24 hours. EdU was added to medium before 2 hours of fixation. Then, cells were fixed using 4% PFA for 20 minutes, and permeabilized with 0.5% triton-X100 in PBS for 15 minutes. Click-iT® reaction cocktails (Thermo Scientific) was added to dish for reacting for 30 minutes at room temperature and protected from light. Subsequently, cell nuclei were stained with DAPI. For each dish, 6 fields were randomly taken using Zeiss AX10 fluorescence microscope. The numbers of EdU-labled and DAPI-stained cells were counted by Image-Pro Plus 6.0.
Cell proliferation assay
Briefly, 15 μg/ml glioblastoma-derived EVs were co-cultured with 5000 cells/well mNPCs on 96-well plates for 24 hours, then changed with NPC proliferation medium without EVs and cultured for 48 hours. Cell viability was measured by CCK-8 (Yeasen, #40203ES80) assays at different time points. Experiments were handled according to the manufacturer's instructions. Absorbance was measured at 570 nm and 450 nm and analyzed using SpectraMax M5 microplate readers (Molecular Devices).
Protein identification and bioinformatics analysis
The resulting MS/MS data were processed using Maxquant search engine (v.220.127.116.11). Tandem mass spectra were searched against uniprot database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages. FDR was adjusted to < 1% and minimum score for modified peptides was set > 40. Proteins were classified by GO annotation into three categories: biological process, cellular compartment and molecular function. For each category, a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. The GO with a corrected p-value < 0.05 is considered significant. Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially expressed protein against all identified proteins. The pathway with a corrected p-value < 0.05 was considered significant.
All results are the means of at least three independent experiments ± SD. Data from two groups were evaluated statistically by two-tailed, paired or unpaired student t test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001, in comparison to control.