It has been recently shown that the potent cross talk between cancer cells and tumor-associated stromal cells can be, at least in part, mediated by the release of exosomes [58–61]. These latter are extracellular vesicles, smaller than 150 nm in diameter, that originate in the multivesicular body compartment and are released into the extracellular space and in the body fluids from many cell types [58–62]. Since exosomes can deliver to target cells their content of biologically active molecules (e.g. proteins, mRNAs, miRNAs, lncRNAs), thus modifying their physiological state, they act as a potent intercellular communication system [61, 63–65].

At this regard, Skog’s group showed that glioblastoma tumor cells could release exosomes, containing mRNA, miRNA and angiogenic proteins, able to act on endothelial cells, possibly favoring the development of a tumor-permissive microenvironment [66]. Interestingly, exosomes can be also released into the bloodstream [66] and act on distant sites taking part to the formation of a pre-metastatic niche [59, 67, 68]. Conversely, it has been shown that tumor-associated fibroblasts, through the release of exosomes, could induce, in breast tumor cells, the acquisition of a metastatic phenotype [60].

For this reason, we wondered whether GASC could exert their tumor-supporting action in this way [7]. Exosomes were therefore isolated from GASC culture supernatants and their presence was confirmed by Atomic Force Microscopy and Light Scattering (particles with a diameter ranging from 20 to 110 nm) (Fig. 9.2).

In order to establish whether glioblastoma cells could incorporate GASC-derived exosomes, we labeled these latter with the lipophilic DiD-dye and, as shown in Fig. 9.2, after 4 h of incubation, DiD-labeled exosomes could be identified within the cells [7].

To unequivocally demonstrate that the tumor-supporting fraction of GASC supernatant was indeed due to exosomes and not to other soluble factors, we compared the capacity of: un-fractioned GASC supernatants, exosome-depleted GASC supernatants and GASC-derived exosomes to modify growth kinetic, migration ability and anchorage-independent growth of A172 cells, showing that indeed exosomes were the main responsible for the effects observed. Same effects were observed when we evaluated a clinically more relevant experimental setting, isolating from the same HGG, both GASC and GSC and we verified that GASC-exosomes profoundly modified the growth pattern of GSC as well as their motility and anchorage-independent growth [7]. Exosomes released by non-tumorigenic Wi38 fibroblast did not affect GSC, confirming the specific role of GASC-derived exosomes.

Interestingly, exosomes released both from HGG and LGG were able to increase the in vitro aggressiveness of glioblastoma cells, although those from LGG at a significant lower extent, thus suggesting that the degree of the tumor-supporting ability was proportional to the grade of malignancy of the tumor of origin.

At this regard, unpublished data from our laboratory seem to confirm the existence of differences in the miRNA content of exosomes released by GASC obtained from LGG and HGG, and even from LGG with a different prognosis [7].

Active cell migration and invasion is a peculiar feature of glioma that renders this tumor able to migrate and infiltrate eloquent areas making difficult the achievement of a radical surgery. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. Since we have seen that GASC can modify the motility of glioblastoma cells, we decided to get better insights into the interactions between GSC and GASC isolated from LGG and HGG [57]. By using time-lapse microscopy as well as atomic force microscopy and single cell force spectroscopy, we first demonstrated that, independently from the grade of the glioma of origin, GSC are softer than GASC, in agreement with their neoplastic features [57]. Subsequently we have shown that the adhesion strength of GSC on GASC appears to be significantly lower for cells derived from HGG with respect to those derived from LGG [57]. This is in line with the fact that HGG cells are characterized by a more infiltrative nature with respect to LGG. What was surprising is the fact that when GSC from HGG were cultured, in parallel, on GASC derived from LGG and HGG, they firmly adhere on GASC from LGG but not on those from HGG, suggesting that the grade of GASC plays an important role in modulating cancer cell adhesion, thus possibly affecting glioma cell migration, invasion and thus cancer aggressiveness [57].

The presence of GASC raises issues regarding their origin in humans. In fact, their phenotype share many antigens with tumor cells, making difficult to unequivocally identify the in vivo counterpart of GASC in a tissue. Additionally, lineage-tracing experiments from the cell-of-origin cannot be done in humans [71]. However, some hypotheses on the GASC origin/in vivo counterpart can be done.

Regarding a possible fibroblast origin, in analogy to the tumor-associated fibroblast present in the epithelial tumors, it has to be underlined that there is a limited presence of fibroblasts in the CNS and their proliferation has never been described in the course of pathology [72]. Instead, the cell type that characterizes the CNS’ response to injury is represented by reactive astrocytes [73, 74]. Importantly, in murine models, non-neoplastic astrocytes could be converted, by the glioma microenvironment, into a reactive phenotype [75, 76] that could acquire stem cell features similar to GASC [77]. The activation of astrocytes seems to be mediated by an epithelial-to-mesenchymal like transition process [78], possibly regulated by the presence, in the tumor microenvironment of hypoxia and an increased myeloid cell number [79]. Unpublished data from our laboratory show that GSC-derived exosomes can indeed activate an epithelial-to-mesenchymal like transition process into cultured human astrocytes, which acquire stem cell properties.

Alternatively, GASC may derive from a population of perivascular mesenchymal stem cells endowed with both mesodermal and neuroectodermal differentiation capacities [80]. This opens the question regarding a possible connection between GASC and mesenchymal stem cells.

To date, the concept of “Mesenchymal stem cells” (MSC) is still a developing issue in terms of defining criteria, sites of origin, in vivo counterpart and function in normal and pathological conditions, including cancer. The demonstration of the existence of MSC into the brain opens the questions on the possible role of these cells in glioma and the relationship with GASC.