Small extracellular vesicles promote invadopodia activity in glioblastoma cells in a therapy-dependent manner

Abstract

Purpose The therapeutic efficacy of radiotherapy/temozolomide treatment for glioblastoma (GBM) is limited by the augmented invasiveness mediated by invadopodia activity of surviving GBM cells. As yet, however the underlying mechanisms remain poorly understood. Due to their ability to transport oncogenic material between cells, small extracellular vesicles (sEVs) have emerged as key mediators of tumour progression. We hypothesize that the sustained growth and invasion of cancer cells depends on bidirectional sEV-mediated cell–cell communication. Methods Invadopodia assays and zymography gels were used to examine the invadopodia activity capacity of GBM cells. Differential ultracentrifugation was utilized to isolate sEVs from conditioned medium and proteomic analyses were conducted on both GBM cell lines and their sEVs to determine the cargo present within the sEVs. In addition, the impact of radiotherapy and temozolomide treatment of GBM cells was studied. Results We found that GBM cells form active invadopodia and secrete sEVs containing the matrix metalloproteinase MMP-2. Subsequent proteomic studies revealed the presence of an invadopodia-related protein sEV cargo and that sEVs from highly invadopodia active GBM cells (LN229) increase invadopodia activity in sEV recipient GBM cells. We also found that GBM cells displayed increases in invadopodia activity and sEV secretion post radiation/temozolomide treatment. Together, these data reveal a relationship between invadopodia and sEV composition/secretion/uptake in promoting the invasiveness of GBM cells. Conclusions Our data indicate that sEVs secreted by GBM cells can facilitate tumour invasion by promoting invadopodia activity in recipient cells, which may be enhanced by treatment with radio-chemotherapy. The transfer of pro-invasive cargos may yield important insights into the functional capacity of sEVs in invadopodia.

Proteome of human glioblastoma and meningioma tissue small extracellular vesicles

“…When these were compared against the MNG EV proteins we identified, a total of 11 common proteins were identified (PGK1, ALDOA, ANAXA1, HSPB1, PFN1, PKM, S100A11, SPTBN1, SPTAN1, PLEC and TF). Our study not only confirms the presence of proteins previously identified in MNG EVs (4,(7)(8)(9)(10)(11)(12)25), but also identified novel proteins that have not been previously reported in association with MNG EVs.…”

“…Differential expression analysis was utilized to reveal the relative abundance of proteins that were enriched in the EVs compared to the tumour tissue. Our study not only confirms the presence of proteins previously identified in GBM EVs from various sources (including CUSA washings, cell lines, and plasma) (4,(7)(8)(9)(10)(11)(12)25), but also crucially identified novel proteins that have not been reported before in association with GBM EVs before including proteins associated with solute carrier transporters and fatty acid transport. Recently it has been shown that solute carrier family members can promote the progression of GBM by activating the PI3-AKT signalling pathway (44).…”

“…Importantly, analysis of our exclusive and enriched proteins in GBM EVs in the Glioma Bio Discovery Portal (38) demonstrated that a higher expression of these proteins had a significant impact in reducing GBM patient survival. We have previously demonstrated that GBM cells can form invadopodia to facilitate GBM cell invasion (54) and that extracellular vesicles can promote invadopodia activity in recipient GBM cells in vitro, in response to their increased secretion post-radiation and temozolomide treatment (11).…”

“…Despite progress in understanding the significance of EVs in brain function and the genesis and progression of brain tumours, there is a gap in our knowledge. The majority of prior investigations have primarily been on EVs obtained from patient fluids or cell cultures (4,(7)(8)(9)(10)(11)(12), rather than directly from the tumour tissue itself, which is important as these EVs may not adequately represent the complexity and specificity of those in tumour tissue. Relative to the routine isolation of EVs from fluids, including conditioned cell culture media, blood, urine or CSF, many technical issues must be addressed when isolating EVs directly from solid tissue.…”