Shedding of bevacizumab in tumour cells-derived extracellular vesicles as a new therapeutic escape mechanism in glioblastoma

Abstract

Glioblastoma (GBM) is the most aggressive type of primary brain tumours. Anti-angiogenic therapies (AAT), such as bevacizumab, have been developed to target the tumour blood supply. However, GBM presents mechanisms of escape from AAT activity, including a speculated direct effect of AAT on GBM cells. Furthermore, bevacizumab can alter the intercellular communication of GBM cells with their direct microenvironment. Extracellular vesicles (EVs) have been recently described as main acts in the GBM microenvironment, allowing tumour and stromal cells to exchange genetic and proteomic material. Herein, we examined and described the alterations in the EVs produced by GBM cells following bevacizumab treatment. Interestingly, bevacizumab that is able to neutralise GBM cells-derived VEGF-A, was found to be directly captured by GBM cells and eventually sorted at the surface of the respective EVs. We also identified early endosomes as potential pathways involved in the bevacizumab internalisation by GBM cells. Via MS analysis, we observed that treatment with bevacizumab induces changes in the EVs proteomic content, which are associated with tumour progression and therapeutic resistance. Accordingly, inhibition of EVs production by GBM cells improved the anti-tumour effect of bevacizumab. Together, this data suggests of a potential new mechanism of GBM escape from bevacizumab activity.

Keywords: Bevacizumab; Extracellular vesicles; Glioblastoma; Resistance.

Fig. 1

IgG1/Bevacizumab antibody can affect LN18 and U87 GBM cells-derived EVs concentration and their proteomic content. NTA of a LN18 or b U87 GBM cells-derived EVs following treatment with bevacizumab (0.25 mg/mL). LN18 or U87 GBM cells were treated for 24 h with 0.25 mg/mL IgG1 or 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, EVs were isolated and re-suspended in 100 μL filtered sterile PBS. EVs suspension was 1/5 diluted and infused to a Nanosight© NS300 instrument. 5 captures of 60s each were recorded. Particles concentration (particles/mL) and size (nm) were measured. Particles concentration was normalised to the number of cells after treatment (particles/mL/cell). The mean ± SEM of 3 independent experiments is shown. c Western blotting validation of the human IgG, Annexin A2 and CD44 in EVs derived from LN18 and U87 GBM cells. d Gene expression distribution of Annexin A2 and CD44 among the different GBM subtypes has been obtained from TCGA. The mean ± SEM is shown (*p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001; ANOVA, compare to ‘normal’)

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

Fig. 3

Inhibition of EVs production increases effects of bevacizumab on U87 GBM cells' viability. a NTA of LN18 GBM cells-derived EVs following treatment with GW4869 (20 μM). LN18 GBM cells were treated for 24 h with 20 μM GW4869 or DMSO. CM was then collected, EVs were isolated and re-suspended in 100 μL filtered sterile PBS. EVs suspension was 1/5 diluted and infused to a Nanosight© NS300 instrument. 5 captures of 60s each were recorded. Particles concentration (particles/mL) and size (nm) were measured. Particles concentration was normalised to the number of cells after treatment (particles/mL/cell). b LN18 and U87 GBM cells' viability assay in response to bevacizumab combined with GW4869. Cells were seeded in a 96well plate and allowed to grow for 24 h. Cells were then treated with different bevacizumab concentrations (0.25 mg/mL or 1.5 mg/mL) with or without GW4869 (10 μM or 20 μM) for 24 h and 48 h. CellTiter-Glo® luminescent cell viability assay was then performed. Results are expressed as normalised Relative Light Unit (RLU). The mean ± SEM of 4 independent experiments is shown. (*p < 0.05, ***p < 0.01; ANOVA). c LN18 and U87 GBM cells' invasiveness assay using a hyaluronic acid (HA) hydrogel. Cells were incubated within a HA hydrogel for 7 days. Cells were treated with bevacizumab (0.25 mg/mL) with or without GW4869 (20 μM). 5 pictures (capture) per gel were taken following the treatments. Colony counting followed by CellTiter-Glo® Luminescent Cell Viability Assay were then performed. Results are expressed as normalised number of colonies / capture and normalised RLU, respectively. The mean ± SEM of 3 independent experiments is shown (*p < 0.05; ANOVA)