Multiple populations of glioblastoma (GBM) cells coexist within a single tumour and communicate by a variety of extracellular signals increasing the complexity of the disease, thus suggesting a potential significance in understanding how signals produced by a population of glioma cells affect the surrounding tumour cells response.
In the context of GBM, EVs have been shown to be able to affect phenotypes of stromal counterpart cells. EVs derived from GBM cells have been implicated in endothelial cell (EC) proliferation, migration and tubulogenesis via delivery of angiogenic proteins and RNA to microvascular ECs . Furthermore, the crosstalk between GBM and astrocytes via EVs is critical in the evasion of tumour cell apoptosis, contributing to GBM aggressiveness and proliferation .
In the present study, we demonstrate that glioma cells expressing AQP4 can export their metastatic or apoptotic phenotypes toward tumour surrounding cells, and this phenomenon is, at least in part, mediated by intercellular transfer of EVs.
The interest in acquaporins (AQPs) trafficking is justified by the role demonstrated for AQPs in brain tumour pathogenesis. In particular, aquaporin-1 (AQP1) is important in tumour growth and spread  and AQP4 protein has a crucial role in vasogenic oedema that increases the mortality related to brain tumours .
Moreover, in the last five years, in five different studies, AQP4 has been found in association with EVs in CNS tissues in Alzheimer’s Disease (AD), stress-induced exhaustion disorder (SED), traumatic brain injury (reviewed here ), suggesting the growing interest in these fields.
These studies, based on data from CSF and blood samples, demonstrated the increase of AQP4-containing EVs release in pathological conditions in diseased patients and animal models (www.microvesicle.org). However, none of these studies investigated in detail the AQP4 aggregation state in EVs.
Besides the role in brain edema, we have previously demonstrated that the aggregation state of AQP4, ranging from tetramers to different sized OAPs, can influence glioma cell fate as follows: AQP4-OAP expression leads to cell shrinkage with alteration in the actin cytoskeleton and apoptotic outcome being therefore "deleterious" for glioma cell survival, while AQP4-tetramer expression increases glioma invasive capability being therefore "beneficial" for glioma cells . This finds its basis in the functional role amply reported for M1-AQP4 in favoring cell migration both in healthy astrocytes and in glioma cells [31, 32].
Furthermore, considering the reduced amount of OAPs found in the human GBM sample, we have previously speculated that this could be considered a survival strategy adopted by the glioma cells that exert the decrease in OAPs, through disaggregation of OAPs in tetramers, to escape apoptosis and to increase the grade of malignancy .
The different phenotypic features activated by AQP4 aggregation/disaggregation state in glioma cells prompted us to hypothesize that different signal cascades could be transferred, via EVs, to surrounding tumour cells from metastatic or apoptotic glioma cells expressing AQP4-tetramers or AQP4-OAPs, respectively.
The study has been conducted using the most widespread experimental model of glioma: the U87 cell line as recipient cells and U87 cells transfected with AQP4–tetramers or AQP4-OAPs as donor cells, in serum withdrawal conditions. It is well known that tumour cells undergoing serum starvation in vitro try to adapt to the modified environment supporting the tumour growth . Moreover, it is also likely that cells having acquired constitutive tolerance for nutrient and oxygen deficiency show an increase in malignancy . First, we obtained direct evidence that AQP4 aggregation states trigger different morphological changes, also under starving conditions. If AQP4-OAP expression induced the AVD like shape , the expression of AQP4 tetramers led to a significant cell elongation.
Given the well-known ability of glioma cancer cells to generate EVs [24, 23], we hypothesize that morphological adaptation of U87 expressing AQP4–tetramers is predictor of the EV secretion that is facilitated by the greater cell plasma membrane surface and by more endosomal machinery available in the larger cells.
The advanced apoptotic phenotype of U87 cells expressing AQP4-OAPs is a predictor of EV release, given that apoptotic cells release more EVs than viable cells. Moreover, we found that OAP expression in U87 cells induces the formation of “beads-on-a-string” vesicles released in the extracellular space through fragmentation of beaded apoptopodia. These membrane protrusions, peculiar of apoptotic cells, have recently been reported for other cell lines such as apoptotic monocytes . Since tumour cells acquire tolerance for nutrients and oxygen deficiency and increasing malignancy, it is not surprising that U87 cells also exhibit features, namely the enhanced ability to grow under serum-starved conditions and altered cell shapes .
The U87 cell line is well known to be prone to produce a large and heterogeneous population of EVs also without any treatment and, at the same time, to uptake autologous EVs [37, 23].
To date, the studies available on EVs derived from untransfected U87 cells reveal that EVs are capable of conferring on normal counterpart cells the transformed characteristics of cancer cells; promoting radiation resistance; and increasing proliferation and invasion [38, 38] (reviewed in ).
The ability of glioma cells to generate EVs is also sustained by the presence of actin rings at the membrane level that facilitate membrane blebbing either in exocytosis or endocytosis processes . The physical dynamics that fold sub-regions of the plasma membrane into vesicles involves modulation of the actin cytoskeleton playing a key role in the formation and the release of EVs .
The actin rings form the neck of growing EVs when they are still in contact with the plasma membrane, next contributing to reducing the diameter of the neck of budding vesicles before being shed from the plasma membrane. Therefore, the presence of actin rings is predictive of continuity between the plasma membrane and forming EVs such as in endocytosis [21, 20].
From the analysis of actin cytoskeleton we demonstrate that glioma cells expressing AQP4-OAPs show a higher density of actin rings and many F-actin-rich regions resembling vesicular structures completely coated in filamentous actin. This is in line with previous experiments that showed that glioma cells expressing OAPs display a higher content of F-actin, in turn compatible with their very low migration potential being directed to apoptosis rather than invasiveness .
The preliminary analysis of protein released in extracellular space by glioma cells, either expressing AQP4-OAPs or AQP4–tetramers, confirms the presence of AQP4, suggesting that, apart from its role in cell physiology, AQP4 also exists as a secreted protein. Although it has been postulated that brain cancer cells distribute aquaporins (AQPs) between cells via EVs [43, 44], as occurs in the kidney where AQP1 and aquaporin-2 (AQP2) have been found in urinary exosomes, the presence of an AQP, namely AQP4, in EV cargoes derived from glioma cells has been reported here for the first time.
Based on the evidence that EVs are released from all cells in varying sizes and with different contents, we isolated them by exploiting their pelletting properties.
Glioma-secreted EVs mainly appear to be of plasma membrane/ ‘shed-vesicle’ origin and belonging to three distinct subpopulations. Despite the numerous studies, the nomenclature and the boundaries between subpopulations of EVs are still under debate. Here we have focused on two (large and small) subpopulations of EVs, because of their abundance compared to the smallest vesicle fraction and their genesis from plasma membrane where AQP4 protein resides.
Our findings indicate that both AQP4-M1 and AQP4-M23 proteins are readily detected in both subpopulations. In detail, AQP4-M1 and AQP4-M23 contents are comparable in large EVs, while M23 protein has higher levels in small EVs. Interestingly, we found a reduced amount of AQP4 expression levels in the cell lysate of U87 expressing M1 compared to U87 expressing M23 protein. This could be ascribed to the sub-optimal translation initiation signal for the M1 start codon [45, 46] and to perturbations in the translation initiation mechanism that occur in cancer cells in stress conditions such as serum withdrawal [47, 48].
Several studies in the past decades have shown that large and small EVs, by transferring several bioactive molecules, affect the phenotypic features of receiver cells, increasing their migratory capability, proliferation and therapy resistance .
Finally, in line with this, we demonstrated that EVs derived from U87 expressing AQP4-tetramers or from AQP4-OAPs are able to reach receiving cells, exporting to them the pattern of their cells of origin.
In detail, large EVs derived from more invading glioma cells expressing AQP4-tetramers potentiate the migratory response of receiving glioma cells, while EVs shed by apoptotic glioma cells expressing AQP4-OAPs favour the apoptotic path of receiving cells.
Notably, these large vesicles are more than twofold the size of small and micro vesicles, therefore they can virtually include a larger number of tumor-derived molecules, with a distinct impact on the recipient cells.
It is reasonable that U87 aggressive cells may preferentially load some proteins in large vesicles more than in small EVs to improve cell-to-cell messages aimed to cancer progression.
Intriguingly, large vesicles, such as oncosome, have recently been shown to be enriched in a set of several enzymes involved in cancer cell metabolism and cell cycle and are able to transfer both adhesion and invasion properties from aggressive cell line to the less aggressive counterpart. Moreover, large vesicles have been shown to contain miRNA, mRNA and DNA and genetic aberrations belonging to the cell of origin including copy number variations of genes frequently altered in aggressive tumour cells.(reviewed in ).
Since GBM cells exert the redistribution of OAPs in favour of tetramers, the mainly transferred phenotypic trait is invasiveness. Moreover, the apoptotic activation of surrounding cells could be addressed toward less malignant cells or stromal cells.
Therefore, AQP4 EV-mediated transfer could be a tumour-supporting mechanism by which glioma cells can export their tumour-enhancing- phenotype or can promote a phenotypic switch either between the tumour and less malignant tumour cells or among tumour cells and stroma. Also in this latter case, the mirroring of glioma cell traits is useful for tumour propagation.
Therefore, in trying to understand the patho-physiology of glioma, the general increase in AQP4 expression and the redistribution of OAPs in favour of tetramers is useful for tumour propagation as they affect both glioma cells expressing AQP4 and tumour or normal surrounding cells with which they communicate. As it is well known that the impact of EVs may not be often fully caused by any single molecule, one possibility is that EVs contain multiple proteins including AQP4 and other components such as various miRNAs  with overlapping functional roles acting in a concerted mechanism to affect the phenotype of recipient cells.
A comprehensive proteome profiling of glioblastoma-derived extracellular vesicles derived from six GBM, including U87, cells lines revealed that levels of 14 EV proteins significantly correlated with cell invasion. Gene levels corresponding to invasion-related EV proteins showed that five genes (annexin A1, actin-related protein 3, integrin-β1, insulin-like growth factor 2 receptor and programmed cell death 6-interacting protein) were significantly higher in GBM tumours .
Further studies are needed to investigate the change in gene levels and invasion-related proteins in EVs from GBM cells expressing AQP4-tetramers compared to expressing AQP4-OAPs.
In conclusion, this study demonstrates that invasiveness or apoptosis traits of glioma cells expressing AQP4 protein affect the signal transferred to surrounding cells. By EV-mediated crosstalk, the phenotypic features of donor cells are exported to receiving glioma cells, amplifying the role of AQP4 in glioma cells also in surrounding cells. In terms of the biology of glioma, EV-mediated transfer of AQP4 to surrounding cells acts as a tumour-supporting mechanism, emphasizing the role of AQP4 as a determinant of cell fate and confirming that the redistribution of OAPs in favour of tetramers is useful in propagating tumours and in spreading malignancy.
Thus, it is conceivable that the phenotype previously described as being dependent on AQP4 membrane expression (17) could also be generated by the AQP4 circulating fraction.