Glioblastoma Multiforme (GBM) is the most aggressive brain tumor that is characterized by the rapid growth, invasiveness, and poor prognosis due to many factors including drug resistance, difficulty to repair damaged brain cells, and the inability of many drugs to cross the blood-brain barrier [1]. Despite multimodal novel treatment options for GBM, the median survival time is still less than 15 months [2]. Currently, many new agents are under investigation to target the cell function and signaling pathways in cancers. Among them are included: Protein kinases, angiogenesis inhibitors, monoclonal antibodies, methyltransferase inhibitors, small interfering RNAs, and mesenchymal stem cells (MSCs) [3].

The therapeutic potential of MSCs in regenerative medicine is relatively well-established but the anti-tumorigenic effect of MSCs has received attention recently. The interest in MSCs for cell therapy is justified by their unique properties [4]. Given their attributes, MSCs are under evaluation in several clinical trials (http://www.clinicaltrials.gov/ct2/results?term = MSC) [5]. Due to the specific homing ability of MSCs to tumor sites in humans, they could be used as delivery vehicles for targeted anticancer drugs or gene-therapy [6–10]. However, this putative approach raised many safety concerns. Firstly, MSCs have intrinsic anti-tumorigenic activities but they also hold pro-tumorigenic properties while suppressing the immune system and modifying the micro-environment for tumor propagation [11]. Secondly, MSCs hold the ability to differentiate which has raised the possibility of their conversion to cancer cells in the tumor microenvironment [12, 13]. Additionally, in the case of allogenic transplantation, MSCs may confront the host immune system which can result in phagocytosis of the transplanted MSCs by immune cells [12, 13]. Moreover, the phenomenon of cannibalism of MSCs by cancer cells, resulting in the dormancy of tumor cells, has been presented as one of the possible reasons for tumor relapse [14]. Furthermore, the delivery of genetic material either by viral or non-viral vectors can increase the chance for induction of insertional mutations in MSCs which have raised the possibility of targeting of the normal cells by MSCs rather than the tumor cells [13, 15]. Therefore, further studies should be done to predispose the MSCs for clinical use in therapy for different types of cancers [16].

The mechanism by which MSCs favor multiple unrelated pathologies remains to be defined. Cell replacement was initially considered their main mechanism of action [17–19]. However, the lack of clear data about the MSCs engraftment at target organs and trans-differentiation [20] led the search for alternative mechanisms. Some experimental studies have reported that the regenerative potential of MSCs is attributed to the paracrine factors and not to their engraftment [21, 22]. This intriguing hypothesis has changed the therapeutic perspectives to develop cell-free strategies based on the use of MSCs secretome as evidenced by various diseases [23, 24]. Therefore, the conditioned media (CM) of MSCs or their purified extracellular vesicles can be used for cancer therapy instead of the injections of MSCs [25]. Very few studies using CM reported the growth factor levels and for most of them, the molecular data are also lacking [26–34]. It has been reported that the same type of cells yielded different growth factor levels relative to cell number, culture medium and condition, and CM processing [27]. That might be the core reason for discrepancies regarding the effect of CM from different sources of MSCs.

Anti or pro-tumorigenic behaviors of MSCs are attributed to types of cells derived from different sources and residing in different tumor environments. Wharton's Jelly/umbilical cord (UC-MSCs/WJ-MSCs) has shown more anti-cancer properties in different glioma cells and solid tumor cell lines [15]. Similarly, adipose tissue-MSCs (AT-MSCs) inhibited the proliferation and induced apoptosis of hepatic cancer cells [35], breast cancer cells [36], prostate cancer cells [37], and melanoma [38]. On the contrary, BM-MSCs have shown both, pro-tumorigenic and anti-proliferative activity on tumor growth [39, 40]. Probably, these conflicting effects of MSCs can be attributed to the experimental settings in different in vitro and in vivo models [41].

Cancer vaccines comprised of cancer cell lysates are currently under investigation for the generation of antitumor immune response for personalized medicine [15]. Similarly, it has been shown that neurotrophic factors in CM could access affected neurons in the central nervous system (CNS) by either directly crossing the blood-brain barrier or through the retrograde transport mechanism in CNS. CM from MSCs has already been applied for the treatment of many neurodegenerative diseases [42, 43], therefore CM might have a therapeutic effect for gliomas too as a combinatorial therapy. It has been reported that cell lysates and conditioned media from WJ-MSCs can induce inhibition of proliferation in a few cancer cell lines [3, 44]. In another report, conflicting results reported the initial inhibition with simultaneous proliferation with WJ-MSCs’ conditioned media in gliomas [45]. However, cell lysates and conditioned media of MSCs generated under the same culture conditions have not been tested on glioblastoma cell lines which is one of the aggressive cancers with poor prognosis.

To investigate if the conflicting (anti-tumoral or pro-tumoral) effects from MSCs are due to; the type of MSCs, or the time duration for the effect of trophic factors generated under the same culture conditions, we planned this study. We evaluated the anti-tumoral or pro-tumoral effect of the paracrine factors of WJ-MSCs and BM-MSCs in the form of cell extracts such as cell lysate and conditioned media mainly for the proliferation of glioma cells and subsequently for the morphology, and migration potential of the glioma cell line (U87MG). Conditioned media (FBSCM, SFCM) and cell lysates (CL), hereby named as treatments, were generated under the same culture conditions and were applied for the same time points (maximum 120 HR). The fibroblast cell line was added to investigate the effect of treatments on the proliferation (only) of normal cells.

All procedures performed in this study were under the Declaration of Helsinki.

Mesenchymal stem cells (MSCs) could be the next candidate being considered for the treatment of cancer due to the properties of tropism for solid tumors, immune-privileged nature, ease of availability and maintenance, etc [50]. However, the application of MSCs for the possible anti-cancer treatment has divided the scientific society into two groups. One group suggested that MSCs play a crucial role in tumorigenesis by promoting angiogenesis while the other group demonstrated the anti-tumoral effect of MSCs in a few specific cancers [11]. Though, the exact mechanisms of the anti-tumoral property of MSCs are yet to be understood. It has been suggested that the source of MSCs seems to contribute to antitumor properties against particular malignancy. The anti-tumoral effect of umbilical cord blood-derived MSCs was evident against a brain tumor but the same effect was missing with MSCs derived from other tissues [51]. Few studies have reported the particular homing ability of BM-MSCs to tumors that enhanced the metastasis by promoting the migration and angiogenesis and inhibiting apoptosis [52–55]. Many studies also reported that the actual outcome of MSCs' beneficial effects is attributed to the paracrine activities of these cells in the form of cell extracts [21, 22]. To investigate further if the anti-tumoral or pro-tumoral effect of MSCs is subjective to the type of MSCs and whether the cell extracts generated under the same culture conditions may exhibit the same anti-tumoral or pro-tumoral effect, we planned this study.

Regarding morphology, proliferation, and migration potential, our results from cell lysates are following the studies where cells went into autophagy or senescence [3, 44] but we also found the same inhibitory effect of cell lysates on fibroblasts. This is in contradiction to these reports [57, 58]. Perhaps this difference of effect can be attributed to different fibroblast cell lines or the concentrations of cell lysates used in these studies. Recent advancement in the therapeutic potential of cancer vaccines to induce antitumor immune effect caused by cell lysates of cancer cells has opened a novel research horizon [56]. In doing so, this is the first report of the use of cell lysates from MSCs to target glioma cells since glioma patients have a very poor prognosis. However, this area requires further in-depth investigations to rule out specific cytokines, chemokines, or growth factors suitable to cause antitumor immune effect without affecting the normal cells.

About phenotypic results from two types of CM, the treatment did not induce any vacuolation or fragmentation in the glioma cells which conflicts with the previous studies (57.58). Rather we found expanded shapes of cells and a decline in neurite growths. This shows that CM may have induced differentiation or senescence by modulating the microenvironment of the glioma cells and it is supported by our results of the cell cycle analysis where CM treated cells arrested the growth at the G1 phase while control cells were passed into S and G2 phases (Figure 7A).

From the results of the proliferation of the glioma cells, we found significant inhibition from two types of CM (FBSCM and SFCM) at 96HR and 120HR (Figures 5, 6). For WJ-FBSCM, we also found an inhibitory effect at 24HR from some samples. This initial decrease can be explained by the presence of heterogeneous cells at different phases of the cell cycle and the cell's response to arrest or die or proliferate in response to the treatment [57]. Both concentrations of BM-FBSCM and WJ-FBSCM (100% and 50%) showed significant inhibition in comparison to FBS control at 96HR and 120HR. This shows that paracrine factors collected in the presence of FBS can exert inhibitory effect without being modulated by the serum proteins. Though FBS provides a cheap and easily accessible source for essential growth factors and other proteins for the propagation of different types of cells [58]. From a clinical application point of view for cell-free therapy, the collection of paracrine factors in the presence of xenogenic compounds might not be the right choice since immunological reactions have been reported in patients due to the internalization of xenogenic proteins [59]. In this regard, SFCM can be a safe choice for further in vivo research since MSC-CM has already been implicated in the treatment of neurodegenerative diseases [42, 43].

On the other hand, the effect of both CM from BM-MSCs on the fibroblasts was stimulatory while FBSCM from WJ-MSCs induced a slight inhibitory effect. It was evident that CM from two types of MSCs in serum-free conditions holds a specific inhibitory effect for glioma cells only. Fibroblasts are an integral part of the tumor microenvironment where they secrete an extracellular matrix (ECM) providing a natural barrier against tumor progression [16, 60]. Contrarily, the same ECM can promote tumor progression and metastasis by modifications caused by secreted proteins of fibroblasts [61]. In this context, the role of cell lysates and CM can be fundamental and need further evaluation in terms of creating an inhibitory effect for the glioma cells only.

In general, we found significant inhibition of migration potential and cells’ metabolic activity (MTT) from all three treatments (CL, SFCM, FBSCM) of two types of MSCs on U87MG glioma cells. Our results are following the previous studies for the anti-tumoral effect from WJ-MSCs [62–67] except for apoptosis. However, our results from BM-MSCs are in contradiction to some earlier studies [13, 52, 67]. Perhaps the reason for this contradiction of biological difference lies with the heterogeneity of MSCs from donors which can be attributed to a diverse repertoire of sub-populations both intra-population or within the same aspirate [68]. Many anti-cancer therapies are targeting cell cycle checkpoints arrest using different modalities. Cell cycle arrest was first identified by lower concentrations of HDAC inhibitors that induced G1 arrest by blocking deacetylase activity in tumors [69]. Many enzymes related to cyclin-dependent kinases (Cdks) are responsible for the correct ordering of cell cycle and progression through the cell cycle is controlled by reversible Cdk phosphorylation [69]. However, Cdk1 alone appears to be the major contributor to cell cycle progression as it can bind to all cyclins [70, 71]. We propose that cell extracts of both MSCs might have arrested the cell cycle by interrupting the pathways related to reversible phosphorylation of Cdk proteins. Cell cycle arrest leads to alter many mechanisms like proliferation, migration, and induction of senescence and differentiation. That might be the reason for the change in morphology, inhibition of proliferation, and inhibition of the migration ability of the glioma cells in our results. However, we did not find any apoptosis as a result of cell cycle arrest which can be explained by the fact that glioma cells might have exhibited cell-cycle quiescence due to the inherent ability of stemness [72].

Unregulated proliferation without stimulation from exogenous mitogens has been the hallmark of cancer which is mediated by the presence of cancer stem cells [73]. It has been reported that glioma CSC/CS-LCs can form spheres in serum-free media without mitogens such as FBS [34, 74]. The short period of serum deprivation can lead to the enrichment of CSCs as a side population (spheres) in cell lines [75, 76]. In our study, sphere formation was quite evident in SFM control as shown by morphology (Figure 4). However, SFCM seems to inhibit the sphere formation in comparison to SFM control (Figure 4). Besides, FBSCM and CL also inhibited the sphere formation in comparison to FBS control (Figures 3A, 4) This shows that cell lysates and conditioned media could have modified the sphere-forming ability of glioma CSCs. However, the functional assay for CSCs along with molecular evaluation (in vivo) is required to further strengthen this concept.

We conclude that cell extracts from two types of MSCs (BM and WJ) successfully induced the inhibition of growth and migration potential of U87MG glioma cells by arresting the cell cycle at the G1 phase. We also present serum-free conditioned media as a preferred source for the collection of paracrine factors since its effect was persistent on the glioma cells from both types of MSCs. Moreover, serum-free media with only a small fraction of exogenous mitogens (1–2%FBS) may provide an important experimental model to investigate the regulation and maintenance of stemness properties of glioma cells in the 2D system (monolayer).