ABSTRACT: Glioblastoma multiforme (GBM) is an aggressive, heterogeneous and highly vascularized brain tumor. GBM is thought to arise from glioblastoma stem-like cells (GSCs) which are characterized as being either proneural or mesenchymal. The former isolates of GSCs are tight sphere forming and slow growing while mesenchymal GSCs are lose sphere forming, fast growing, highly invasive and when dominant yield poorer patient prognosis. GSCs are known to be plastic in nature and can therefore evolve from a proneural to a mesenchymal state. Here, we observed that factors secreted by endothelial cells (which make up the brain vasculature) alter several properties of GSCs resulting in the acquisition of a more mesenchymal and invasive phenotype coupled with changes at the level of secretory and cellular proteome. Thus, using mass spectrometry, quantitative proteomic analysis and GO term filters, we identified several mesenchymal traits in proneural GSCs exposed to endothelial cell secretome. Specifically, proneural cells treated with the conditioned media derived from human umbilical vein endothelial cells (HUVEC) upregulated the expression of mesenchymal proteins such as CD44 and VIM, while downregulating the expression of the proneural proteins such as NOTCH1, activated NOTCH intracellular domain (NICD), SOX2 and NESTIN, which were validated using flow cytometry (FACS) and western blots (WB). Using DAVID analysis tool, we detected the features of cellular proteome indicative of the activation of NFkB, Wnt and several other pathways in the proneural cells treated with HUVEC conditioned media. Using conditioned media fractionation through several centrifugation steps we identified the extracellular vesicles (EV) sedimented at 100,000 x g using ultracentrifugation, as the source of activity in endothelial conditioned media capable of triggering mesenchymal shift in proneural GSCs. EVs are heterogeneous membrane structures containing multiple bioactive macromolecules, which have the ability to carry multiple bioactive proteins, transfer them to recipient cells and alter their function, signalling and biological programmes. We compared the effects of EVs, soluble fraction and unfractionated conditioned media in terms of their ability to trigger mesenchymal changes in the phenotype of proneural GSCs. Once the cultures were established, the culture medium was removed and replaced with HUVEC-derived material (conditioned media, supernatant or EV fraction) and responses evaluated over 7 days by microscopical analysis of sphere structures, and biochemically by following the aforementioned proneural or mesenchymal markers (WB, FACS). We observed an upregulation of mesenchymal proteins, as well as downregulation of the proneural proteins, mentioned above. These effects recapitulated those of unfractionated conditioned media and were absent from target cells exposed to EV-depleted conditioned media. The data analysis of EV proteome included canonical markers and pathways of cellular vesiculation as well as markers and pathways of interest with regards to the biological effects associated with treatment of GSC recipient cells. In this regard we observed several EV related tetraspanin markers, which were validated using western blot including CD9, CD63, CD81 and a purity control, BIP. Although we identified several potential effectors associated with endothelial cell EVs that could impact proneural cell phenotype, we focused on MMPs for at least three reasons: (i) evidence in the literature (see text) indicated that MMPs may induce differentiation programs in neural stem cells; (ii) MMPs in EV cargo were relatively abundant and have been implicated in various biological processes; (iii) MMPs released from endothelial cells could be functionally involved in disrupting proneural cell sphere structures that we observed in the presence of endothelial cell secretome. We noted the expressions of MMP1, MMP2, MMP11 and MP14 in our HUVEC-EV mass spectrometry dataset, the activity of which was validated using the MMP activity assay kit from abcam (ab112146). Using GO terms we also detected a signal for NFkB pathway activation in the proteome of endothelial (HUVEC) conditioned media-treated proneural GSCs. NFkB activation is regarded as hallmark of mesenchymal phenotype in GSCs and GBM cells. We validated that the upregulation of NFkB, was also true for the proneural cells treated with HUVEC derived EVs. Moreover, upon blocking MMP expression in proneural cells treated with endothelial cell EVs, we inhibited the activation of NFkB activity thereby documenting that the initial effects of MMPs trigger a shift in cellular phenotype toward NfkB activation and mesenchymal reprogramming. Briefly, we compared GO terms of GSC157 cells treated with their own or HUVEC-derived EVs. Validation of the NFkB pathway activation was analysed using WB and immunofluorescence for levels of NFkB and phosphor-NFkB.
Lata Adnani 
