Anti-Oncogenic Activities Exhibited By Secretomes Of Mesenchymal Stem Cells Are Mediated By Modulation Of KITLG and DKK1 Genes In Glioma Stem Cells.

Background. Cancer stem cells (CSCs) use their stemness properties such as self renewal, toxicity, plasticity, and communication with the tumor microenvironment (TME) to perpetuate their lineage and survive chemotherapy. Learning how to interrupt the self renewal ability or modulate the interaction of CSCs with the TME signaling will dramatically improve therapeutic impact on patient’s remission. Antitumor properties of mesenchymal stem cells (MSCs) are currently under investigations and different approaches have been applied to gain benecial effects However, different types of MSCs yielded different conicting results. In order to investigate if different types of MSCs preconditioned in the same culture conditions can exert alike anti oncogenic effect on glioma stem cells, we planned this study. Methods. GSCs were isolated from U87 cell line by FACS cell sorter, characterized and established as gliospheres. Condition media from MSCs of Wharton Jelly (WJ-MSCs) and bone marrow (BM-MSCs) were harvested and used as treatments on glioshperes (3D) to investigate the effect on proliferation, invasion and self renewal properties of GSCs. Microarray analysis was used to determine the effect at molecular level. Specic human CSC gene arrays were applied to validate the ndings of the microarray explicitly the pluripotency of the GSCs. Results. Our results from functional and molecular assays showed that condition media (CM) from both types of MSCs inhibited the metabolism by interrupting oxidative phosphorylation, arrested the cell cycle, induced cell differentiation, targeted the pluripotency and up-regulated the immune response in GSCs. Moreover , media both the same genes (KITLG and DKK1) causing a similar effect while using slightly different routes and signaling pathways signifying their individual effects.

hypoxia, in ammatory stimulus, and other factors/conditions prior to their use in therapy is a new strategy currently being investigated (38).
Based on this knowledge, we planned to investigate the anticancer effect of the secretome from MSCs preconditioned with GSC growth factors to explore the biological mechanisms and signaling pathways that are associated with anti or pro-tumorogenic effects. In doing so, we evaluated the effects of factors secreted (condition media, CM), under same culture conditions, from two different populations of MSCs (bone marrow-derived (BM-MSC) and Wharton's jelly-derived MSC (WJ-MSC) on GSCs survival, invasion and self-renewal capability. Further, the genetic changes at signaling pathway levels were explored by microarray analysis followed by further validation of the effect on the pluripotency of GSCs with human CSC arrays. heat-inactivated Fetal Bovine Serum(GIBCO/Invitrogen Corporation,USA)}. The cells were seeded at 10 4 cells/cm 2 in T75 cultures asks and maintained at 37°C and 5% CO 2 . The culture medium was exchanged every 2-3 days.

FACS Sorting and enrichment of glioma stem cells
Cell line expanded at different passages (P3-P9) was investigated for the percentage of CD133 positive population which was sorted by using FACS JAZZ cell sorter (BD, Biosciences, USA) according to manufacturer's instruction. Brie y, upon reaching the 80-90% con uence, U87 cancer cells were harvested using 0.25% trypsin EDTA (Invitrogen, USA), washed twice with cold PBS (Inivtrogen, USA) and centrifuged at 300xg for 5 minutes. Cell pellet was resuspended in 100 ul of BD FACS staining buffer (BD Biosciences, USA) and 10ul of CD133 antibody (eBioscience, USA) were added. Mixed well and put at 4 o C for about 30 minutes in dark. Next 500 ul of cold PBS were added to wash the cells and centrifuged at 300 xg for 5 minutes and pellet was resuspended in 500 ul PBS for sorting. Sorted populations were divided into positive and negative fractions and were evaluated for the enrichment of CD133 positive cells using FACS Canto ii and analysed by FACS DIVA software Version 7 (BD, Bioscience, USA).
2.2 Characterization of sorted populations.

Glio-subsphere generation.
CD133 + and CD133selected cell populations, derived from rst gliospheres, were dissociated using StemProAccutase Cell Dissociation Reagent (GIBCO, USA) according to manufacturer's instructions. Cells were subsequently re-suspended in GSM and seeded in 24-well ultralow plates (Corning, USA) at 10 3 cells/well. Formation of free oating sub-spheres was observed by phase-contrast microscopy (Zeiss, Germany) and the experiment was conducted till passage 12 (gliosphere, G12). Cells in each passage were kept in culture between 7-10 days.

Monolayer generation
Similarly small fractions of sorted cells from positive and negative populations were cultured in standard culture media (DM-F12+10%FBS, GIBCO, USA) in order to investigate their expansion ability adherently after sorting. The morphological changes were observed under phase contrast microscope (Zeiss, Germany). The experiment was conducted for 6 passages and one passage time was kept between 5-7days.

2.2.4.Immuno uorescent assay of CD133 positive population
A panel of GSC markers, neuronl lineage markers and late differentiated neuronal markers, was selected (Table.1) to characterize the sorted CD133 + population by immunocytochemistry. Brie y, subspheres at G13 were harvested by treatment with StemPro Accutase Cell Dissociation Reagent (GIBCO, USA), washed with PBS (pH = 7.4 GIBCO,USA) followed by centrifugation. After resuspension, cells were seeded on polyL-.Lysine coated cover slips ( sigma-Aldrich, USA) at the seeding density of 20 4 cell/ cover slip and were immersed in GSM with 2% FBS (GIBCO, USA). After two days the cells were xed with 4% formaldehyde for 20 minutes followed by washing. Cover slips were then permeabilized with 0.3% Triton-X-100 for 15 minutes at room temperature. Blocking was performed with 10% normal goat serum (GIBCO,USA) for 1 hour at room temperature. Cells were stained with the primary antibodies in 1% blocking buffer overnight at 4 0 C with shaking followed by wahing with 0.1% Triton-X-100 in PBS three times for 5 minutes each. Next, cells were stained with secondary antibodies (Table.1) in 1% blocking buffer for 1hour at room temperature in the dark. Cells were washed with 0.1% Triton-X-100 in PBS three times for 5 minutes each. Nuclei were stained with DAPI (Thermo Fisher Scienti c, USA) for 5 minutes, coverslips mounted onto slides. Cell imaging was performed on inverted phase contrast uorescence microscope ( Zeiss Axio Observer Z1. Germany). The description of the primary and secondary antibodies used for the assay is given in the Table 1 Mesenchymal stem cell lines of bone marrow (BM-MSCs) and Wharton's Jelly (WJ-MSCs) were acquired from Cell Therapy Center, University of Jordan which had been expanded and characterized as described previously (39). Three biological samples of WJ-MSCs (WJ1, WJ2, WJ3) and three from BM-MSCs (BM1, BM2, BM3) at passage 3 were used to generate condition media. All six cell lines were plated into T75 asks (TPP, USA) at a density of 6,000 cell /cm 2 in alpha-MEM (GIBCO,USA) + 5% platelet lysate (40). A day before cells reached 80% con uency, monolayers were washed twice with PBS (pH= 7.4) and once with serum free media -SFM (DM-F12). Next GSM was added in the asks and after 48 hours this conditioned media (CM) was collected, centrifuged at 300xg for 10 minutes at 4 o C, ltered through 0.2um lters(BD, Biosciences, USA) and aliquoted. The CM was stored at -80 o C and fresh aliquots were used prior to each experimental assay.

Preliminary data
In order to investigate the effect of condition media (CM) on GSCs, initially one sample of each of BM-MSC and WJ-MSC was used to generate CM and different concentrations of CM were tested with and without addition of fresh GSM. The results are shown in gure (S1.a). Serum free media (DM-F12 only) was kept as another control. Based on the preliminary tests, 100% CM was selected to perform the further experiments in a ratio of 1:9 with fresh gliosphere media (10%GSM: 90%CM). Brie y 10 3 /cm 2 cells from dissociated gliospheres at passage 13(G13) were plated in ultralow attachment six well plates (Corning, USA) containing CM. The experiment was conducted for 7 days and cells growing in normal GSM were kept as control. CM was added every 48 hours and changes in sphere forming ability of GSCs were captured by phase contrast microscopy (Zeiss, Germany).

3D cell proliferation and viability assessment
The proliferation rate of GSCs (at G13) was evaluated by CellTiter-Glo ® 3D Cell Viability Assay (Promega, USA). In brief, the cells were cultured at a seeding density of 5x10 3 cells/well in 100 ul of CM in 96 well ultralow attachment plates (Corning,USA). Every 48 hours fresh CM was added along with control and experiment was conducted for 7 days. The proliferation rate was determined at days 3 and 7 by CellTiter-Glo ® 3D Cell Viability reagent according to the manufacturer's instruction (Promega, USA). Luminescence was measured using a microplate reader (GloMax ® -multi, Promega, USA). The cell survival percentage was measured by dividing the mean value of treated well by the mean value of control wells multiply by 100.
% viability = mean value of treated well / mean value of control wells x100 The decline in viability was measured by deducting the percent viability of treatment from the control viability at that time point.
Decline in viability = 100% viability of control-% viability of treated well.

Invasion assay.
In order to determine the effect of CM on the invasive ability of GSCs, invasion assay was performed using Cultrex® BME Cell Invasion Assay (Trevigen, USA) with a slight modi cation . Brie y, 5x10 4 cells (in triplicate) were treated with CM from both types of MSCs in 6 well ultralow attachment plates (Corning, USA) for 7 days. The media was refreshed every 48 hours. At day 7 the spheres were collected and harvested by accutase (GIBCO, USA), centrifuged at 200xg for 4 minutes and 50 4 cells ( in triplicate) were seeded in the upper chamber of BME 96 well plate. The lower chamber was lled with serum media (DM-F12 +10%FBS, GIBCO,USA). After two days the plates were processed according to manufacturer's instruction and the luminescence was measured using microplate reader (GloMax ® -multi, Promega, USA).

Clonogenic assay
In order to investigate if the effect exerted by CM from both types of MSCs is reversible or irreversible, clonogenic assay was performed for both adherent and spheroid system.
Colony Forming E ciency (CFE) Brie y, 5x10 3 cells/well were treated in 24 well ultralow (Corning, USA) with CM from both types of MSCs for 7 days along with control. After harvesting, single cell suspensions were obtained. 50 cells from each sample were seeded in 6 well plates (TPP, USA) in standard culture media (DM-F12+10%FBS) for two weeks. The medium was exchanged every three days. Colonies were stained using 0.5% crystal violet dye (Sigma-Aldrich, USA) according to the manufacturer's instructions and counted by a light microscope (Zeiss, Germany).

Sphere Forming E ciency (SFE)
Similarly, 2x10 3 cells/ well were cultured in 24 well ultralow plates (Corning, USA) in GSM for about 7days. GSM was added every 48 hours and sphere formation was monitored under light microscope (Zeiss, Germany). On day 8 the spheres were counted and photographed using Zeiss microscope. Spheres were dissociated as described before and were plated again for successive passage. Fresh GSM was added every 48 hours and passage time was kept for 7 days. Brie y, 10 4 cells/cm 2 (G13) were seeded in 25 ml ultralow attachment asks (Corning, USA) containing CM from two types of MSCs and control (gliospheres). Sorted population that did not make spheres was taken as a negative control. On day 7 the spheres were harvested using the accutase (Gibco,USA). Total RNA was extracted using trizol ® reagent (Invitrogen, CA, USA) and cleaned up with RNeasy Mini kit (Qiagen, USA) following the instructions of the manufacturer. Extracted RNA was quanti ed using NanoDrop 2000c spectrophotometer system (ThermoFisher Scienti c, USA). RNA quality analysis was performed using Agilent 2100 Bioanalyzer instrument with Agilent RNA 6000 Nano Kit according to the manufacturer's instructions.

Global gene expression pro ling (Transcriptome analysis)
Whole Transcriptome analysis was performed in triplicate for gliospheres , control group (-Ve and cell line) ,gliospheres treated with; CM from BM-MSCs (BMT) and CM from WJ-MSCs (WJT). GeneChip ® Human Transcriptome Array (HTA) 2.0 (Affymetrix. Inc., Santa Clara, CA, USA) was used for gene expression pro le analysis. The procedure was followed as described by manufacturer. The microarray data can be accessed via Gene Expression Omnibus-GEO (GSE149216) (www.ncbi.nlm.nih.gov/geo). For simplicity, we will use terms BMT and WJT for treated gliospheres from here onward.

RT 2 Pro ler TM PCR Array
In order to validate further the speci c genes and pathways involved in certain mechanisms exhibited by min, (ii) 40 cycles of 95 °C for 15 seconds, (iii) 60 °C for 1 minute. The data analysis was performed using the 2-ΔΔCt method available by the SABiosciences company (Qiagen, USA) web portal, www.SABiosciences.com/ pcrarraydataanalysis.php. The data were normalized, across all plates, to the average of arithmetic mean of the following housekeeping genes: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Beta-2-microglobulin (B2M) and actin beta (ACTB). The threshold cycle values of the control wells were all within the ranges recommended by the PCR array user manual.

2.6.Statistical analysis
All experiments were performed in triplicates(n=3). Data were analyzed using Microsoft Excel and GraphPrism software. Quantitative data were expressed as mean ± standard deviation. Data were evaluated by two way ANOVA and Dunnet's post-test was used to analyze multiple comparisons (P<0.05).

CM concentration optimization assays
Figure (S1.a) shows the results of preliminary tests for different concentrations of CM. It was noted that cells treated with different concentrations of CM changed their morphology at 48 and 72 hrs and started to become linear rather than making spheres . It was also noted that GSCs kept on growing even in serum-free conditions (DM-F12 only) without the addition of growth factors and supplements under ultralow attachment culture conditions (3D). This showed the plasticity of GSCs, modifying them according to the change in the environment (41). However, to determine the effect of CM from MSCs speci cally for GSCs in 3D culture system under normal growth conditions, we no longer used the SFM as a control for further assays.

Enrichment and characterization of CD133 positive population.
Figure (S2.a) depicts the ow cytometric evaluation of the CD133 + population present in U87 cell line (P3-P7) before sorting and enrichment of a positive population after sorting. Before cell sorting, we found that the average percentage of CD133 + population was 5.1% in cell line. After sorting CD133 positive population was enriched to 48% while the negative population still showed 2.1% of CD133 + cells (Table. 2). In order to determine if successive cultures of sorted cells have affected the enrichment of the CD133 + population in cancer stem cell media (GSM), we also determined the enrichment of sorted positive population at a random passage (G5), which was above 90% with almost uniform sphere morphology ( Fig. S2.b). The enriched glioma stem cell line with 90 % CD133 positive population was used for all the experiments.

Morphological characteristics of sorted populations
Sorted populations were divided into positive and negative fractions and were cultured in adherent (2D) and sphere system (3D). In adherent cultures, CD133 positive population made a mesh network and started forming connections from passage1, and at passage 3, cells showed a conspicuous stellar morphology, which is a typical morphological feature of U87 cells (Fig1.a P3,arrow). While CD133 negative population showed more large cells indicating the presence of a differentiated progeny (Fig1.a P3, arrow). Moreover, the negative population showed a slight stellar morphology at later passages (P5-P6), which might be due to the presence of residual CD133 positive cells in negative fraction or some CD133 negative cells may have acquired the state of CD133 positive cells (42).
Similarly, in spheroid culture, positive fraction started making spheres from G1 and continues till passage G12. However, the negative fraction did not make spheres after G3, and at latter passages cells tend to be adherent rather than spheroids (same as in Fig S1.b). This negative population, failed to form spheres, was kept as a negative control for molecular analysis.

Immunocytochemistry of Gliospheres
Figure (1.c) shows three panels of markers selected for the characterization of the glioma stem cells in actively growing spheres (gliospheres). The markers were; glioma stem cell markers (CD133 and Msh1), neuronal lineage markers (nestin, sox1, sox2 and pax6) and differentiated markers (GFAP, Tuj1 and Olig123). Since the glioma spheroids contain heterogeneous populations (43) comprising of stem and differentiated cells, so we found the substantial presence of all the selected markers in various combinations.

CM inhibited sphere formation, proliferation, invasion and clonogenicity of GSCs.
Gliospheres were treated with CM from two types of MSCs for 7 days and the results are shown in Figure  2. Morphologically, spheroids tend to change their form as early as on day 3 (D3) where cells started to become linear rather than spheroids. At D7, cells started to branch out just like adherent culture, despite growing in ultralow stem cell environment ( Fig. 2.a.) In relation to this, 3D Glow viability assay showed that cells' proliferative ability was signi cantly decreased at D7 (p<0.0005) as compared to control (

c)
As a fraction of total, the reduction in invasion was 27% (WJ) and 26% (BM) as compared to 45% from control ( Fig. S1.c).
In order to investigate if the effect of CM from both types of MSCs is irreversible, a clonogenic assay was performed. It was noted that the colony-forming e ciency of GSCs was signi cantly reduced both adherently and in spheroids compared to control(p<0.005, Fig.2 d,e). It was found that the average sphere/colony formation from control was 39/19, while the reduction in sphere/colony formation was 5/11 from BM-MSCs and 7/6 from WJ-MSCs( Fig.S1.d).
3.6.1. CM inhibited the metabolism and cell cycle while activated the immune response at the Molecular level Principal component analysis (PCA) was performed to demonstrate global gene-expression changes among different treatments (BMT vs. gliospheres, WJT vs. gliospheres, gliospheres, and -VeS) and to identify any outliers.PCA (Fig 3.a) shows that each type of group clustered together with a clear separation between them, and no outlier was observed. Both BMT and WJT gliospheres are closer to the gliospheres (+VeS) while -Ve control has its unique ancestor.
To generate a list of differentially expressed genes (DEGs) between different types of treatments in comparison to controls, ebayes ANOVA method was used, and a ltering criterion of; Fold Change (FC) ≤ -2 or ≥ 2 and p-value ≤ 0.05 were applied. The detailed description of DEGs has been provided in supplementary le and shown in Venn diagram (excel 1, Fig.3b ).
Gliospheres. For 1483 DEGs between gliospheres and negative control (Fig.3c), 81 signi cant canonical pathways were identi ed by Ingenuity Pathways Analysis software (IPA, Qiagen, USA). Based on the zscore evaluation, supremely activated pathways included cholesterol biosynthesis, mevalonate pathway 1, systemic lupus erythematosus in T cells signaling pathway, Toll-Like receptor signaling while PD-1/PD-L1 cancer immunotherapy pathway was inhibited.
WJT gliospheres. For 579 DEGs between WJT and gliospheres applied to IPA (Fig 4.a), 57 canonical pathways were identi ed as signi cant. These include TREM1 signaling, GP6 signaling, and NF-κB activation by virus as activated, while Oxidative phosphorylation was inhibited.
It was noted that both treatments commonly inhibited oxidative phosphorylation at complexes IV-V(green ,down-regulated Gliospheres. For up-regulated genes in gliospheres, Ribosome biogenesis in eukaryotes, Steroid biosynthesis, Antigen processing and presentation, Asthma, and Terpenoid backbone biosynthesis were identi ed as signi cantly up-regulated Kegg pathways. While the down-regulated genes were involved in HIF-1 signaling pathway, Protein export, Protein processing in the endoplasmic reticulum, Ferroptosis, Glycosphingolipid biosynthesis -ganglio series, and Autophagy (Fig. S3.).
Similarly, up-regulated genes were represented by top biological processes of lipid, steroid and cholesterol metabolic process, antigen processing and presentation, fatty acid biosynthesis and metabolic process, and immune response while the down-regulated genes were represented by top biological processes of negative regulation of the apoptotic process, cell death, angiogenesis, cell adhesion, rhythmic process, and synaptic vesicle exocytosis (Fig. S3).
BMT and WJT gliospheres. From both types of treatment (CM of BM-MSCs and WJ-MSCs), we found almost the same signi cantly up-regulated kegg pathways such as: ECM-receptor interaction, AGE-RAGE signaling pathway in diabetic complications, Focal adhesion, Protein digestion and absorption, PI3K-Akt signaling pathway and proteoglycans in cancers. While Ribosome biogenesis in eukaryotes, Systemic lupus erythematosus, Asthma, and antigen processing and presentation were signi cantly downregulated or inhibited ( Fig. S4 and S5) In the same manner, GO terms for up-regulated biological processes were enriched in cell adhesion, angiogenesis, receptor-mediate endocytosis, heart and skeletal system development, cellular defense response, cell differentiation and blood coagulation while the down-regulated genes were represented by top biological processes of DNA replication, regulation of cell cycle, RNA splicing, DNA repair, cell cycle, antigen processing and presentation, RNA splicing, and protein transport ( Fig. S4 and S5).
Based on pathway analysis and biological processes ,it was noted that CM from both types of MSCs inhibited the metabolism, arrested cell cycle and activated immune response in GSCs. In order to validate the ndings of microarray analysis, speci c human CSC array was used to investigate if the cell cycle was arrested and multipotency or pluripotency of glioma stem cells was affected and which speci c genes and pathways are involved in causing this effect. The results of CSC array for gliospheres are summarized in (Table.3a). After normalization, out of 84 genes of the arrays, 29 genes were identi ed as DEGs (24-upregulated, 5-down-regulated) in gliospheres. A Scatter plot for gliospheres (gliospheres vs. Control, Fig.5a) shows the distribution of gene expression changes along the central diagonal line. Signi cant up-regulated genes (p <0.05) were DNMT1, GSK3B, IKBKB, ITGA6, LATS1, LIN28B, WWC1, ZEB2, and YAP1. While the genes observed with higher fold regulation were LIN28B, JAG1, EPCAM, TWIST2, ATM, NANOG and CD38 (Table 3a). However, one gene was found signi cantly up-regulated (p <0.05) and with high fold regulation LIN28B (7.72).
Scatter plots for WJT and BMT gliospheres have shown the same pattern as depicted in Figure (5.b and  c, Table.3b). Based on fold regulation, it was noted that both treatments down-regulated most of the genes that were up-regulated in gliospheres. We found almost the same up-regulated genes by both treatments such as CXCL8 and FOXP1, while PLAUR was up-regulated by WJT, and NANOG remained unchanged by BMT. However, we found some variations in down-regulated genes by both treatments.
Uniquely down-regulated genes based on fold regulation by WJT were ABCG2, EPCAM and LIN28B, while uniquely down-regulated genes by BMT were JAG1 and POU5F1 (Table 3.b).

Pathway analysis and GO terms of CSC arrays
Gliospheres. Figure 6 shows the activated and inhibited pathways and biological processes of signi cantly DEGs in gliospheres. It was noted that most signi cant activated pathways in gliospheres were Hippo signaling, P13/Akt signaling, Pathways in cancer and MicroRNAs in cancer (Fig. 6 a). In relation to this,highly signi cant biological processes (based on p values) in gliospheres were protein phosphorylation, negative regulation of apoptotic processes, glycogen and carbohydrate metabolic processes,, cell proliferation, circadian rhythem, chtomatin organisation and cell matrix adhesion (Fig. 6 b).
Based on high fold regulation, the top activated pathways were, Proteoglycans in cancer, Notch signaling pathway, P53 signaling pathway, Endocrine resistance, Th1, and Th2 cell differentiation, Haematopoietic cell lineage, homologous combinations, platinum drug resistance etc (Fig. 6 c). In addition to this, top activated biological processes were related to cell communication, hemopoises, negative regulation of apoptotic processes regulation of cell cycle, DNA replication, angiogenesis, cell proliferation, and DNA repair (Fig.6 d).
WJT and BMT gliospheres. Figure 7 gathers the information about activated and inhibited pathways and biological processes of DEGs of treated gliospheres with two types of CM of MSCs. We found similar activated KEGG pathways (non signi cant) from both treatments (Fig S6 a,b) except complement and coagulant cascade pathway in WJT gliospheres. It was found that both treatments signi cantly downregulated almost similar pathways with a slight variation (Fig.7). Among down-regulated pathways from WJT, the signi cant ones were Rap1 signaling, Hippo signaling, ABC transporters, Signaling pathways regulating pluripotency of stem cells and TGF-beta signaling pathways etc. In relation to this, the top most down-regulated biological processes were negative regulation of apoptotic processes, regulation of cell cycle, cell proliferation and angiogenesis.
Similarly, Figure 7 b shows the up and down-regulated pathways and biological processes of DEGs of BMT gliospheres. It was noted that there was a slight difference in up-regulated biological processes with respect to genes (FOXP1,CXCL8,NANOG). Nanog also up-regulated cell differentiation, cell proliferation and transcription by RNA polymerase II while FOXP1 and CXCL8 reamined the same as in WJT gliospheres. However, none of these up-regulated genes from both treatments were statistically signi cant (p<0.05).
On the other hand, the signi cant down-regulated pathways observed from both treatments were signaling pathways regulating pluripotency of stem cells, Rap 1 signaling, pathways in cancer, Notch signaling and TGF-beta signaling etc. Similarly, signi cantly down-regulated biological processes noted were the same, with the highlighted ones being the negative regulation of apoptotic processes, cell communication, angiogenesis and regulation of cell cycle etc.
Among up-regulated pathways, AGE-RAGE signaling and Nf-kB signaling were the same as shown by microarray analysis as well. This shows that the results of the CSC array are consistent with the ndings from the microarray and are being validated. Oveer all , CSCs array analysis depicted the inhibition of cell proliferatipon, pluripotency, induced differentiation and activated immune response in GSCs.

Signi cant genes down-regulated by both treatments in CSC array
It was noted that two genes KITLG and DKK1, were signi cantly down-regulated by CM of two types of MSCs. The details of their relevant pathways and biological processes are shown in Figure 8 (a & b).
Signi cantly down-regulated pathways found with KITLG were, hematopoietic cell lineage, melanogenesis, Rap1 signaling, MAPK signaling, P13/AKT signaling, pathways in cancer , PLD signaling and Ras signaling . On the other hand, DKK1 was involved in the down-regulation of Wnt signaling pathways. With regard to this, the main down-regulated biological processes were negative regulation of the apoptotic process, cell proliferation, cell adhesion, and endoderm development.
Overall, CM from both MSCs inhibited the cell proliferation, invasion, and pluripotency of GSC, but illicit the immune response that can activate angiogenesis by modulating the tumor microenvironment (Table   4).

Discussion
In Glioblastoma, GSCs were rst identi ed by Singh et al., as a population of cells capable of initiating tumor growth in vivo (44). The crucial role played by GSCs in tumor initiation, progression, recurrence, and resistance to therapy indicates that new therapeutic strategies require the eradication of this population (45,46,47). It is also important to note that tumor cells are heterogeneous; therefore, it may be more advantageous to target multiple elements of various cellular pathways, to eradicate GBM (48). A possible solution to speci cally target GSCs might be to force them to acquire a non-self-renewing state. In this non-stem cell-like state, the cells should lose their tumorigenic nature and become vulnerable to therapies. Many therapies fail to have the expected bene cial effects due to the blood-brain barrier and the presence of active e ux pumps that prevent drug entry into the brain. New treatment modalities, including novel agents and small molecule inhibitors, are currently under investigation. Remotely, mesenchymal stem cells (MSCs) and their soluble factors are reported to exhibit bene cial anticancer effects (49,50,51). In order to investigate further if the soluble factors of MSCs may affect different pathways related to proliferation and stemness of GSC to transform them into non-stem-like cells prone to therapies, we planned this study.
In the rst step CD133 positive cells were sorted and characterized according to morphological and immunocytochemical assays. These cells exhibited a high expression of all the known markers established for GSC pro le. Actively growing GSCs in spheroids were treated with paracrine factors of manipulated MSCs and showed morphological changes, reduced proliferation, viability and invasion.
Clonogenic assay revealed a remarkable decrease in self renewal ability of GSCs signifying the e cacy of paracrine factors of MSCs. Gene ontology results of DEGs from microarray analysis were further evaluated by speci c human CSC array and we found consistent results of arrested cell cycle, inhibited metabolism, inhibited pluripotency of GSC and activated immune response. IPA analysis of treated spheres indicated inhibited oxidative phosphorylation from both treatments. However, slightly variable canonical pathways were up-regulated for immune response, from two types of treatments.
Metabolic reprogramming has been the hallmark of cancer stem cells. Growing evidence has demonstrated that slow-cycling GSCs possess a preference for mitochondrial oxidative metabolism.
Few studies have demonstrated that CSCs can rely on fatty acid oxidation for their maintenance and function (55) and lipid catabolism seems critical for CSCs self-renewal (56). Similarly, the mevalonate pathway is an essential metabolic pathway in providing cells with bioactive molecules, crucial for different cellular processes, including cell proliferation, differentiation, survival (57) and CSCs enrichment (58). Since CSCs have been shown to be enriched in mitochondrial mass and relying heavily on OXPHOS, disrupting this pathway has become an attractive therapeutic strategy. Oxidative phosphorylation (OxPhos) plays a central role in cellular energy. The OxPhos electron transport chain (ETC) constitutes four complexes (CI-CIV) that transfer electrons from donors generated by the TCA cycle and fatty acid oxidation to oxygen. Complex V (ATP synthase) uses the stored energy in the proton gradient to generate ATP (59,60,61). As shown by our results of IPA, the main activated metabolic pathways involved in gliospheres were those with fatty acid and mevalonate pathways that were inhibited due to disruption at C4 and C5 complexes of ETC in OxPhos (Fig. 4.c). This may have, in turn, inhibited the proliferation, viability, invasion and sphere-forming ability of GSC consistent with the study done by (62).
IPA analysis of WJT gliospheres showed activation of TREM1 signaling, GP6 signaling and Nf-kB signaling. It has been shown that TREM1 had been up-regulated only in infectious in ammatory responses (63). In tumors, TREM1 seems to be induced on tumor-associated macrophages, which has been correlated with cancer recurrence and poor survival (64). Immunohistochemical analysis of breast tumor tissues con rmed co-localization of TREM1 protein expression with the pan-macrophage marker, CD68. These ndings established the role of tumor in ltrating macrophages in promoting in ammation by immune evasion (65). It has also been investigated that TREM1 expression is regulated by Nf-kB at the transcriptional level [66], emphasizing the contribution of Nf-kB pathway activation in bridging in ammation and tumor promotion and progression (67). Hypoxia regulated genes mediate blood vessel formation by stimulating encoding of chemotactic molecules such as CCL2, IL8 and VEGF that recruit macrophages and exert tumor-promoting effects such as angiogenesis (68,69,70). Our results are in agreement with the above-mentioned studies in view of macrophage activation and angiogenesis, as con rmed by the biological processes of treated spheres (Fig. 7). We propose that CM from WJ-MSCs might have caused a hypoxic effect, which may contribute to angiogenesis through the involvement of macrophages in the tumor microenvironment.
GPVI (Glycoprotein VI) is exclusively expressed on platelets and megakaryocytes and together with integrin α 2 β 1 mediates collagen-induced aggregation and adhesion (71,72,73). The role of platelets in the pathophysiology of GBM appears to be two-edged. On the one hand, activated platelets and their secretome can modulate immune responses, thereby prolonging overall survival in a GBM model in mice (74). On the other hand, platelet activation needs to be avoided since GBM patients have an increased risk for systemic cardiovascular events, and the intratumoral occlusion of numerous vessels leads to a hypoxia-induced tumor progression (75). We found that WJT spheres have up-regulated a coagulation cascade to recruit platelets as a survival strategy. Therefore suitable antithrombotic and antiplatelet concepts may be a valuable addition to future individualized, targeted therapies (76). However, the details of the molecular mechanism in platelet activation require further studies.
IPA analysis of BMT spheres showed up-regulation of PD1/PDL1 pathway and inhibition of calciuminduced T lymphocyte apoptosis. PD-L1 is not constitutively expressed in tumor cells but rather is inducibly expressed (i.e., adaptive immune resistance) in response to in ammatory signals (77,78).
Immune checkpoint inhibitors, PD-1 and PD-L1, have shown clinical e cacies against many different solid and hematologic malignancies (79). Binding of PD-L1 to its receptor suppresses T cell migration, proliferation, and secretion of cytotoxic mediators, and restricts tumor cell killing. Inhibitors of PD-1 and PD-L1 disrupt PD-1 axis, thereby reverses T cell suppression and enhances endogenous antitumor immunity to unleash long-term antitumor responses in a wide range of cancers (80). Our results show that CM from BM-MSCs has exerted an adaptive immune response, which induced PDl/PDL1 expression on cancer cells. Ca 2 + signaling plays an essential role throughout vertebrate development, from fertilization to organogenesis. It has been shown that the main checkpoints controlling the fate of a cell are mainly controlled by Ca 2 + signaling pathways (81). Few studies have shown that some tumors develop an immune evasion strategy based on FasL-mediated destruction of invading lymphocytes (82,83,84). Invading T lymphocytes that express Fas is stimulated to apoptosis by tumor cells that express FasL.
The expression of FasL has recently been demonstrated in GBMs (85. It has also been reported that T lymphocytes were present in GBMs and would account for the aggressive growth of tumors (86). Our results show that the calcium-induced T lymphocyte apoptosis pathway has been inhibited, which indicates that CM from BM-MSCs might have affected the Fas-FasL combination and inversed the reaction of immune evasion by GSCs which in turn has up-regulated apoptosis in GSC as shown by biological process analysis. (Fig. 7) From gene ontology results of speci c CSC array, we found two common genes signi cantly downregulated from CM of both MSCs such as; KITLG and DKK1. DKK1 has downregulated the Wnt pathway (Fig. 9a). A large number of studies have suggested that WNT signaling is aberrantly activated in GBM, and it promotes GBM growth and invasion via the maintenance of stem cell properties (87,88,89,90). Dickkopf (DKK) acts as an antagonist of WNT signaling via binding to its co-receptor LRP (91,92). Interestingly, the MSC-induced pro-tumorigenic effect seems to be regulated by the Wnt/β-catenin signaling in breast cancer (93.94), whereas the inhibition of tumor proliferation occurs by MSC induced secretion of DKK-1, an inhibitor of the same pathway (95,96). Furthermore, the MSC-derived CM exerts its effect by targeting the Wnt/β-catenin signaling pathway (97,98). Our in silico results are in accordance with these studies as we found inhibition of growth and stemness properties of GSC through downregulation of the Wnt pathway (Fig. 8a).
KITLG gene encodes the ligand of receptor tyrosine kinases (RTKs) by the KIT locus.RTKs are a family of cell surface receptors, which upon activation, signal through two major downstream pathways Ras/MAPK/ERK and Ras/PI3K/AKT. These pathways are involved in the regulation of cell proliferation, survival, differentiation, and angiogenesis (99). We found that CM from MSCs inhibited these pathways through the downregulation of the KITLG gene. In addition, Phospholipase D (PLD) activity has been suggested to function as a sensor of metabolites, including lipid pools (100) and a critical regulator of autophagy (101). Keeping this in mind, we predict that the metabolism of the GSCs was deregulated by inhibition of PLD pathway, which might have up-regulated apoptosis and differentiation also shown by biological processes (Fig. 8.b) mediated by KITLG gene.
Among uniquely up-regulated genes of CSC array of gliospheres LIN28B was found to be the most signi cant. Lin28, along with Oct4, Sox2, and Nanog, has corroborated its role in pluripotent stem cells (102). In addition to tumor initiation, LIN28B is necessary for the maintenance of cancers too (103). Recent advances have shown that Lin28 regulates let-7 microRNA biogenesis and mRNA translation, to coordinate both cellular metabolism and proliferative growth pathways for stem cell self-renewal (104).
Our results of WJT spheres show the down-regulation of LIN28B that has contributed to the inhibition of metabolism and the self-renewal capacity of GSC. In addition, we also found the downregulation of ABCG2 related to chemoresistance and SP formation (105) and EPCAM regulating the proliferation and invasion (106).
From BMT spheres, uniquely down-regulated genes were JAG1 and POU5F1. JAG1 is the ligands of the Notch signaling and has been shown to promote glioma-initiating cells (GICs) in glioblastoma. Notch signaling mediates direct cell-cell interactions and plays a crucial part in cell fate maintenance and selfrenewal of GICs (107). Moreover, studies have shown that down-regulation of Jagged1 induces apoptosis and inhibits proliferation in glioma cell lines (108). Similarly, different variants of OCT4 (POU5F1) have been related to colony formation and regulation of cell survival in GSC (109). We found inhibition of these two vital stem cell markers from CM of BM-MSCs.
One common pathway that has been identi ed by both arrays was the AGE-RAGE signaling pathway. RAGE was rst identi ed as a receptor for AGEs in relation to diabetes, renal diseases, and aging (110,111). In glioblastomas, RAGE is expressed on tumor cells, endothelial cells, stromal cells, and tumorassociated macrophages comprising microglia and myeloid-derived macrophages (112). RAGE binding activates downstream signaling pathways that stimulate cell proliferation, survival, and migration via increased angiogenesis, in ammation, and reduced apoptosis, While blocking RAGE signaling suppresses tumor growth and metastasis (113)(114)(115)(116). Despite the inhibition of oncogenic mechanisms at the cellular level, we found the macrophage activation and immune response as activated biological processes in treated gliospheres. We presume that AGE-RAGE signaling might be the contributing factor for this response, which can modulate the tumor microenvironment for angiogenesis. Combining inhibition of RAGE signaling with CM might be a novel strategy to inhibit tumor growth.
There are several challenges involved in treating glioma, including the immunosuppressive nature of GBM itself with high inhibitory checkpoint expression, the immunoselection blood brain barrier impairing the ability for peripheral lymphocytes to tra c to the tumor microenvironment and the high prevalence of corticosteroid use which suppress lymphocyte activation. However, by simultaneously targeting multiple costimulatory and inhibitory pathways, it may be possible to achieve an effective antitumoral immune response (117). This is where a combination of manipulated MSCs-secreted factors has the most signi cant potential.

Conclusion
Taken together, the results of microarray and CSC arrays in vitro elucidate the possible mechanism of action by which MSCs secretome inhibited the 3D formation of GSCs observed in culture. The inhibition was translated into decreased oncogenic activities, including stemness of GSCs through different pathways mediated by KITLG and DKK1 genes. We conclude that CM from two sources of MSCs holds the antitumor properties, which are mediated by different routes of signaling pathways while causing the same effect. It has been shown that neurotrophic factors in CM could access affected neurons in central nervous system (CNS) by either directly crossing the blood-brain barrier or through the retrograde transport mechanism in CNS. Since CM has already been implicated for many neurodegenerative diseases (118,119 ), therefore, CM from MSCs can be a subject of combinatorial therapy for gliomas. Regarding the preference of choice, we propose that CM from BM-MSCs may contribute more valuable effect as a combinatorial therapy in conjunction with antitumor immune therapy to treat gliomas. This study provided valuable information regarding the potential ability of acclimatized MSCs with GSC environment to interrupt the growth and pluripotency of GSCs with the potential for translation to new The study was approved by the ethics committe at the Cell Therapy Center. No animal or human was included in this study so consent to participate is not applicable.

Consent for publications
Not applicable Availability of data and materials Microarray data has been submitted and can be accessed via Gene Expression Omnibus-GEO (GSE149216) (www.ncbi.nlm.nih.gov/geo). Additional excel le of DEGs-PCA has been provided as supplementary material.
Competing interest.
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.      Effect of CM from MSCs on GSCs' (gliospheres) morphology, proliferation, invasion and clonogenicity. a.
Representative images of the morphological changes occurring in gliospheres with CM at day 3 and 7.
Scale bar (100um). Cells started adherence at day 3 and become branched out at day7 rather than spheroids as compared to control. b. Effect of CM from two types of MSCs on the proliferation of gliospheres at day 3 and 7. Proliferative ability of GSCs' was signi cantly inhibited at day7(P<0.0005) in comparison to control. c. Effect of CM from two types of MSCs on the invasive ability of gliospheres at Day 7.Both treatments signi cantly inhibited the invasive potential of GSCs (p<0.05). Clonogenicity of treated glioma stem cells was signi cantly reduced both in d) sphere culture, SFE e) adherent culture,CFE.
This shows that treatment of condition media from both MSCs caused irreversible effect on the self renewal ability of glioma stem cells. Signi cantly enriched canonical pathways were identi ed with a right-tailed Fisher's Exact Test that calculates the P-values. The P-values were corrected for multiple testing using the Benjamini-Hochberg method for correcting the FDR. The z-score indicates predicted activation state of the canonical pathway.
Blue color or lighter shades of blue indicate a negative z-score and down-regulation of the pathway, and orange color or lighter shades of orange indicate a positive z-score and up-regulation of the pathway.

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