In this study, we examined the effects of NDV-infected MSCs from different sources on glioma cells and GSCs and the mechanisms involved in their effects.

NDV has been reported to induce selective apoptosis of various cancer cell lines including glioma [11, 14, 16, 51]; however, its effects on human astrocytes have not been examined. We demonstrated that infection of U87, U251 and A172 cells with NDV induced cell apoptosis already at 1 MOI and that normal human astrocytes were resistant to the cytotoxic effect of NDV even at 10 MOI, further suggesting that NDV effects are tumor cell selective.

GBMs contain a population of GSCs that contribute to therapy resistance and tumor recurrence despite successful surgical removal of visible tumor [52]. Thus, identifying treatments that can selectively target these cells is of utmost importance in the treatment of GBM. Our results show for the first time that NDV can also target GSCs and that although these cells are less sensitive than differentiated tumors cells to the cytotoxic effect of NDV, they exhibit a decreased self-renewal ability when infected with low virus titers. Importantly, also for these cells, the effects of NDV appear to be tumor selective and human NSCs are resistant to NDV infection as indicated in Fig. 1c.

MSCs have been reported to migrate to tumor sites, such as glioma, and to deliver various anti-cancer treatments including oncolytic viruses [31, 32, 5355]. Our results indicate that MSCs can be also used to deliver oncolytic NDV to glioma cells and GSCs. We found that NDV infected the different MSCs and that these cells underwent apoptosis after 4 days of infection and in response to higher virus titers as compared to glioma cells. These results suggest that NDV-infected MSCs can deliver the virus effectively and can be used safely for the eradication of NDV-sensitive GSCs with no residual presence of MSCs that could theoretically inhibit the local immune response elicited by the infected cells [5, 30].

Interestingly, treatment of glioma cell lines and GSCs with conditioned medium of NDV-infected MSCs induced a larger cytotoxic effect as compared to glioma cells infected with similar NDV titers. The most pronounced effect was obtained with UC-MSC infected cells compared with BM and AD-derived MSCs. We found that the infected UC-MSCs secreted high levels of TRAIL and that treatment of glioma cells with conditioned medium of NDV-infected UC-MSCs in the presence of anti-TRAIL neutralizing antibody abrogated the enhanced cytotoxic effect of the conditioned medium. Moreover, infection of glioma cells and GSCs with NDV sensitized these cells to the apoptotic effect of TRAIL, further supporting our conclusion that this factor mediated at least some of the enhanced cytotoxic effects of the NDV-infected MSCs on glioma cells.

TRAIL has been reported to induce a selective cell apoptosis in tumor cells and has been considered a promising anti-tumor agent [23, 29]. Indeed, multiple studies demonstrated the apoptotic effect of TRAIL on a variety of tumor cells including glioma cells [2528, 56]. Despite the selective effects of TRAIL on tumor cells there are some cells (in particular cancer stem cells) that exhibit resistance to the apoptotic effect of this ligand [57]. Thus, the current results that demonstrate an increased cytotoxic effect of TRAIL and NDV in both glioma cells and GSCs may provide a mechanism to bypass the relative resistance of GSCs to both TRAIL and NDV. Indeed, a recent study demonstrated enhanced anti-tumor effects of NDV engineered to express TRAIL and IL-2 [58, 59]. The delivery of TRAIL by MSCs engineered to overexpress this protein was recently reported to exert cytotoxic effects in glioma xenografts [60, 61]. Our findings demonstrate a novel approach to increase the secretion of endogenous TRAIL by NDV infection of UC-MSCs, which may further enhance the anti-tumor effects of these cells as was recently reported [62, 63].

Our results of sensitizing GSCs to γ-radiation by the conditioned medium of NDV-infected MSCs demonstrated a mechanism to overcome the resistance of GSCs to radiation, which is one of the first-line treatments for GBM. Indeed, a combined treatment of TRAIL and γ-radiation has been reported to exert a synergistic effect in vitro and in vivo [50]. Importantly, recent studies demonstrated that radiation increased the homing of MSCs to glioma xenografts [50], therefore suggesting that not only NDV-infected MSCs are expected to home to the tumor site more efficiently, but they can also enhance the response of resistant tumor cells to γ-radiation.

Considering the preferential migration of MSCs to tumor sites, it seems reasonable to hypothesize that using MSCs as a delivery system is likely to provide an effective method for the selective targeting of NDV to glioma cells and GSCs. Thus, encapsulating NDV in MSCs can overcome the current limitations in virus delivery and lack of extravasion into the tumor and can shield the viruses from sequestration in the liver and neutralization. Since treatment of GBM patients with NDV seems to be effective in a small fraction of cases [64], it seems reasonable to assume that a more efficient transfer of NDV into the tumor cells may result in a more effective anti-cancer impact for a larger number of patients. Thus, a targeted delivery of NDV by MSCs may represent a substantially more effective approach for transfer of NDV into the malignant cells, rather than relying on random delivery of the circulating viruses.

Preliminary clinical studies suggest that treatment with NDV is safe [16]. Similarly, recent preclinical and clinical studies using both autologous and allogeneic MSCs from various sources indicate that treatment of patients with various neurological and inflammatory disorders is safe and has some therapeutic impact [6569]. Based on these studies and our results showing the lack of toxicity of NDV or MSCs loaded with NDV on normal astrocytes and NSCs, we expect that treatment with MSCs loaded with NDV is also likely to be harmless against normal neural cells in vivo. Thus, infection of MSCs with NDC may represent a novel approach for the treatment of minimal residual disease and the eradication of GSCs following or along with conventional modalities.