Fig. 2.

Bevacizumab is internalised by GBM cells and is detectable at the surface of GBM cells-derived EVs following treatment. a Immunofluorescence detection of bevacizumab and EEA1 in LN18 and U87 GBM cells. GBM cells were allowed to grow on cover slips and then treated with 0.25 mg/mL bevacizumab for 2 h and 24 h. Cells were fixed with 4% PFA and then incubated with antibodies against α-tubulin, EEA1 and human IgG1. Pictures were taken at × 120 magnification. Representative pictures are shown. b Western blotting detection of bevacizumab in LN18 and U87 GBM cells. GBM cells were treated for different times (30 min, 2 h, 6 h and 24 h) with 0.25 mg/mL bevacizumab. Cells were then washed two times with sterile PBS, collected and lysed with RIPA buffer for proteins extraction. β-actin and bevacizumab (IgG1) expression was analyzed by western blotting. Representative pictures are shown. c TEM detection of bevacizumab in LN18 and U87 GBM cells-derived EVs. U87 GBM cells were treated for 24 h with 0.25 mg/mL bevacizumab. Then cells were washed two times with sterile PBS and incubated additional 24 h in serum free conditions without treatment. CM was then collected and EVs were isolated. Immuno-gold labeling was then performed against human IgG in the EVs fractions. Pictures were taken at × 20,000 magnification. Representative pictures are shown. d Western blotting detection of bevacizumab in LN18 and U87 GBM cells-derived EVs. GBM cells were treated for 2 h and 24 h with 0.25 mg/mL bevacizumab. CM was collected after treatment. Cells were washed twice with sterile PBS and incubate for additional 24 h in serum free condition before CM was collected again and EVs isolated. CD9, HSP70 and bevacizumab (IgG1) expression was analysed by western blotting. A representative picture is shown. e Western blotting detection of fibronectin (positive control previously described to be present at the surface of cancer cells EVs) and IgG1 antibody in LN18 GBM cells-derived EVs. Western blotting detection of bevacizumab in LN18 GBM cells-derived EVs. GBM cells were treated for 24 h with 0.25 mg/mL bevacizumab. Then cells were washed two times with sterile PBS and incubated additional 24 h in serum free conditions without treatment. CM was then collected and EVs were isolated. EVs suspension was then diluted in a 2.5 mg/mL trypsin solution or 1% triton X100 or a trypsin/triton combination. Fibronectin and bevacizumab (IgG1) expression was analysed by western blotting. A representative picture is shown. f Western blotting detection of IgG1/bevacizumab antibody, fibronectin and VEGF-A in U87 GBM cells-derived EVs. U87 GBM cells were treated for 24 h with 0.25 mg/mL IgG1 or bevacizumab. Cells were washed twice with sterile PBS and incubate for additional 24 h in serum free condition before CM was collected. Then EVs were isolated from CM. IgG1/bevacizumab antibody was precipitated using an immunoprecipitation matrix. Protein extraction was performed on EVs using RIPA buffer. IgG1, fibronectin and VEGF-A expression was analysed by western blotting. A representative picture is shown. IgG1 HC = IgG1 Heavy chains