Mir-504 modulates the stemness and mesenchymal transition of glioma stem cells and their interaction with microglia via delivery by extracellular vesicles

ABSTRACT Glioblastoma (GBM) is a highly aggressive tumor with poor prognosis. A small subpopulation of glioma stem cells (GSCs) has been implicated in radiation resistance and tumor

recurrence. In this study we analyzed the expression of miRNAs associated with the functions of GSCs using miRNA microarray analysis of these cells compared with human neural stem cells.

These analyses identified gene clusters associated with glioma cell invasiveness, axonal guidance, and TGF-β signaling. miR-504 was significantly downregulated in GSCs compared with NSCs,

its expression was lower in GBM compared with normal brain specimens and further decreased in the mesenchymal glioma subtype. Overexpression of miR-504 in GSCs inhibited their self-renewal,

migration and the expression of mesenchymal markers. The inhibitory effect of miR-504 was mediated by targeting Grb10 expression which acts as an oncogene in GSCs and GBM. Overexpression of

exogenous miR-504 resulted also in its delivery to cocultured microglia by GSC-secreted extracellular vesicles (EVs) and in the abrogation of the GSC-induced polarization of microglia to M2

subtype. Finally, miR-504 overexpression prolonged the survival of mice harboring GSC-derived xenografts and decreased tumor growth. In summary, we identified miRNAs and potential target

networks that play a role in the stemness and mesenchymal transition of GSCs and the miR-504/Grb10 pathway as an important regulator of this process. Overexpression of miR-504 exerted

antitumor effects in GSCs as well as bystander effects on the polarization of microglia via delivery by EVs. SIMILAR CONTENT BEING VIEWED BY OTHERS MIR-146A-5P DEFICIENCY IN EXTRACELLULAR

VESICLES OF GLIOMA-ASSOCIATED MACROPHAGES PROMOTES EPITHELIAL-MESENCHYMAL TRANSITION THROUGH THE NF-ΚB SIGNALING PATHWAY Article Open access 30 June 2023 MICRORNA-27A-3P DELIVERED BY

EXTRACELLULAR VESICLES FROM GLIOBLASTOMA CELLS INDUCES M2 MACROPHAGE POLARIZATION VIA THE _EZH1/KDM3A/CTGF_ AXIS Article Open access 14 May 2022 INHIBITION OF HYPOXIC EXOSOMAL MIR-423-3P

DECREASES GLIOMA PROGRESSION BY RESTRICTING AUTOPHAGY IN ASTROCYTES Article Open access 08 April 2025 INTRODUCTION Glioblastomas (GBMs) are the most common and aggressive of the astrocytic

tumors. They are characterized by increased proliferation and angiogenesis, invasion into the surrounding normal tissue and resistance to therapies1. The prognosis of patients with GBM

remains extremely poor, and the median survival of GBM patients has remained around 14–16 months over the past decades2. Gene expression profiling studies identified GBM subtypes that are

classified based on their transcriptional signatures into various molecular groups, including proneural, classical, and mesenchymal3,4. Recently, the profiling of DNA methylation patterns in

glioma has refined these categories5, aligning them with the WHO 2016 diagnostic schema for glioma. These subtypes have distinct differential genetic alterations, molecular signatures,

cellular phenotypes, and patient prognosis5,6,7. GBMs contain a small subpopulation of cancer stem cells (i.e., glioma stem cells [GSCs])8 that are characterized by self-renewal,

multi-lineage differentiation potential, and the ability to generate xenografts that recapitulate the parental tumors9. GSCs have been implicated in tumor infiltration and resistance to

radio- and chemotherapy as well as tumor recurrence10. GSCs share stemness characteristics with neural stem cells (NSCs) but differ in their differentiation and oncogenic potentials9,11,12.

The epithelial-to-mesenchymal transition (EMT) is a process that allows epithelial cells to abandon their polarity and cell-to-cell adhesion properties and acquire mesenchymal cell

phenotypes which are associated with enhanced invasiveness, stemness and metastasis, and treatment resistance13. Recent studies demonstrated a similar process in glioma, and

proneural-to-mesenchymal transition in these tumors is characterized by increased aggressiveness, invasiveness and therapy resistance14,15,16. Mesenchymal transition can occur in recurrent

tumors and in response to radiation17,18 and is associated with poor patient prognosis. Tumor aggressiveness and mesenchymal transition of glioma are induced by cells and secreted factors in

the tumor microenvironment19,20,21. These cells include endogenous central nervous system (CNS) cells such as astrocytes, oligodendrocytes, neurons and microglia, and infiltrating immune

cells21. Microglia are resident immune cells in the brain and together with infiltrating macrophages represent the most prevalent CNS-associated cells in the tumors22. Microglia and

recruited macrophages have been reported to undergo differentiation to cells with M2-type characteristics, in response to factors secreted by glioma cells23. The M2 microglia and macrophages

further support tumor growth via the secretion of growth factors, chemokines and extracellular matrix-modifying enzymes22,24. In addition, recent studies implicated extracellular vesicles

(EVs) as important mediators of intercellular communication and in the cross talk of tumor cells and their microenvironments25. EVs contain proteins, lipids, and various RNA species and play

important roles in the interaction of glioma cells and microglia23,25. The expression of specific microRNAs (miRNAs) has been shown to be associated with several aspects of glioma

pathogenesis including cell cycle control, invasion, migration, resistance to therapies, and cell apoptosis26. Specific miRNAs have been also implicated in the self-renewal, stemness, and

tumorigenic features of GSCs27. Here, we analyzed the miRNA expression of GSCs in comparison with human NSCs (hNSCs) and identified unique miRNA expression profiles that distinguish these

two cell populations. Focusing on miR-504, which was highly expressed in hNSCs compared to GSCs, we demonstrated that it was upregulated in the G-CIMP glioma subtype compared to other GBM

subtypes. Moreover, we found that miR-504 exerted an antitumor effect in vitro and in vivo and in addition, inhibited the stemness and mesenchymal transit of GSCs. In addition,

overexpression of miR-504 in GSCs exerted a bystander effect on cocultured microglia cells by promoting the differentiation of these cells toward M1 phenotype via EV delivery. MATERIALS AND

METHODS GSC CULTURES All human materials were used in accordance with the policies of the Henry Ford Hospital Institutional Review Board. Generation of GSCs from fresh GBM specimens and

their characterization have been recently described28,29,30,31. Briefly, the GSCs were maintained in neurosphere medium (DMEM-F12 1:1, glutamine 10 mM, HEPES buffer 10 mM, and sodium

bicarbonate 0.025%) supplemented with basic fibroblast growth factor (20 ng/ml) and epidermal growth factor (20 ng/ml). The GSCs expressed markers such as CD44, CD133, Musashi-1, Sox2, and

nestin, exhibited self-renewal, and expressed astrocytic and neuronal markers upon differentiation. The GSCs also exhibited tumorigenic potential and generated glioma xenografts in nude

mice28,29,30,31,32,33. The full information of the GSCs employed in this study is described in Table S1. MICROGLIA AND NSC CULTURES Human microglial cells were obtained from Applied

Biological Material (Richmond, BC, Canada). All cells employed in this study were tested for mycoplasma contamination (Mycoplasma PCR Detection Kit) and found negative. hNSCs (H9, hESC

derived) were obtained from Invitrogen. TRANSDUCTION OF GSCS AND MICROGLIAL CELLS Lentivirus vectors (System Biosciences, Mountain View, CA) expressing the miR-504 reporter, pre-miR-504,

miR-504 antagomiR, Grb10, or control and Grb10 shRNAs were packaged and used to transduce the cells according to the manufacturer’s protocol and as previously described28,29,30. NEUROSPHERE

FORMATION ASSAY To determine the ability of GSCs to form secondary neurospheres, cells were plated in 24-well plates at a density of 10 and 100 cells/well through limiting dilution and the

number of neurospheres/well was determined following 10 days for ten different wells. Spheres that contained more than 20 cells were scored and the results are presented as percentages of

maximal neurospheres formed compared to control cells28,29. IN VITRO LIMITING DILUTION ASSAY For the in vitro limiting dilution assay, GSCs were plated in 96-well plates in decreasing

numbers of cells (50, 20, 10, 5, 2, and 1) per well. Following 10 days, the number of spheres was determined for each well. Extreme limiting dilution was analyzed as recently reported32.

REAL-TIME POLYMERASE CHAIN REACTION (RT-PCR) Total RNA was extracted using RNeasy midi kit according to the manufacturer’s instructions (Qiagen, Frederick, MD). Reverse transcription

reaction was carried out using 2-μg total RNA as previously described28,31. Briefly, reactions were run on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA).

Cycle threshold (Ct) values were obtained from the ABI 7000 software. S12 levels were used as controls. The primer sequences are described in Table S2. WESTERN BLOT ANALYSIS Cell pellet

preparation and Western Blot analyses were performed as previously described28,29,30. TRANSWELL MIGRATION ASSAY Transwell chambers (BD Biosciences, San Jose, CA) were used for analyzing cell

migration as recently reported29,34 In brief, cells (25,000/well) were incubated for 3 h in culture medium with 10% fetal bovine serum in the bottom chambers. The total number of the

migrated cells was determined in fixed and stained cells (0.05% crystal violet for 5 min). CELL VIABILITY ASSAY Cells were washed with phosphate-buffered saline (PBS), centrifugated in 3000 

rpm for 5 min and the cell pellet was incubated in PBS containing 0.4% trypan to stain the dead cells. The number of Trypan-blue stained cells was determined using a Countess II FL (Thermo

Fisher, MA, USA). ISOLATION OF GSC-DERIVED EVS EVs were prepared as previously described31,35 using sequential centrifugation at 300 × _g_ for 10 min, 2500 × _g_ for 20 min, 10,000 × _g_ for

30 min and 110,000 × _g_ for 90 min. The pellet was then resuspended in PBS and washed twice followed by filtration using a 0.22-μm filter. The protein content of the enriched EV fractions

was determined using the Micro BCA assay kit (ThermoFischer Scientific, Oregon City, OR). The expression of the exosome markers CD63, CD81, and CD9 was analyzed by Western blot and the

quantification of the isolated EVs was performed using the ExoELISA-Ultra CD63 kit according to the manufacturer’s instructions. For the exosome treatment, 0.5 × 108 EVs were added to the

cultured cells. IMAGESTREAMX ANALYSIS Microglial cells were treated with GSC-derived EVs labeled with CellTracker Red (ThermoFisher, Waltham, MA) for 24 h. Cells were excited using 561-nm

laser, and cell fluorescence of approximately 104 cells per sample was captured and photographed using an ImageStreamX high-resolution imaging flow cytometer (Amnis Co., Seattle, WA) as

previously described35. The samples were gated to obtain a population of captured single-cell images of living cells, then gated for the cells in focus using the gradient root mean square

feature. Cells incubated with or without labeled EVs were compared for the intensity of the red channel fluorescence. Images were analyzed using IDEAS 6.0 software (Amnis Co., Seattle, WA).

MIR-504 REPORTER For analyzing miR-504 delivery, a miR-504 luciferase reporter plasmid was employed as previously described for miR-12436. A unique miR-504 binding site, which is a fully

complementary sequence of mature miR-504, was cloned downstream of luciferase reporter gene of the pMiR-Luc reporter vector from Signosis, Inc. (Santa Clara, CA). For the mCherry reporter,

the luciferase gene of pMiR-Luc reporter vector was replaced with mCherry-N1 obtained from Clontech (Mountain View, CA). PHAGOCYTOSIS ANALYSIS Human microglial cells were plated alone or in

coculture with GSCs. Phagocytosis was determined using the pHrodo™ Green zymosan bioparticle assay (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly,

microglia plated alone and in the presence of GSCs were incubated with a solution of pHrodo Green zymosan bioparticles in Live Cell Imaging Solution (0.5 mg/ml). Phagocytosis was determined

after 120 min using fluorescence plate reader at Ex/Em 509/533. MIRNA ARRAY PROCESSING AND ANALYSIS All experiments were performed using Affymetrix HU GENE1.0st oligonucleotide arrays and

GeneChip miRNA 4.0 Array (ThermoFisher). Sample processing was performed according to the protocol provided by the company. The rest of the analysis was performed using Partek® Genomics

SuiteTM software, version 6.6 (©2012 Partek, Inc.). miRNA data were summarized using RMA and standardized by sketch-quantile normalization. Differential expression was performed via ANOVA.

Significant miRNAs were selected to have at least 1.5-fold change and a _P_ value < 0.05. Results were visualized by volcano plot. Functional analysis was conducted by Ingenuity software

using the core analysis on differential miRNA lists. The panel of measured miRNAs (a list of all measured miRNAs) was used as the background set for enrichment tests. Networks included up to

35 miRNAs and mRNAs. TCGA DATA ANALYSIS Expression data were downloaded for TCGA cases from the Broad Firehose portal (http://gdac.broadinstitute.org/). GBM cases were assayed by microarray

for miRNA expression6. The level 3, batch-adjusted, expression data file captured mature miRNA quantification (file date: 12/10/2014). Low-grade glioma (LGG) cases were assayed by

miRNA-sequencing37. The level 3 data file contained expression data per mature miRNA as reads per million miRNAs mapped (file date: 12/10/2014). GBM and LGG cases were assayed by

mRNA-sequencing5. The level 3 data file contained RSEM normalized data38, quantified per-gene as the normalized count (file date: 12/10/2014). Expression data are used continuously,

discretized by quantile, or dichotomized at the median as high/low as appropriate for the research question. Clinical data and molecular classifications were taken from the recent

publication of the TCGA Glioma Analysis Working Group6. Comparison of mean expression between groups was performed by one-way ANOVA followed by Tukey’s corrected two-sample tests, which

adjust for multiple comparisons to maintain the family-wise error rate. XENOGRAFT STUDIES Following the guidelines of Henry Ford Hospital’s Institutional Animal Care and Use Committee,

dissociated GSCs (3 × 105 cells) transduced with a lentivirus vector expressing a control pre-miR or pre-miR-504 were inoculated intracranially into nude mice (Nu/Nu) as previously

described32. Briefly, animals were anesthetized and injected with the GSCs through a 3-mm hole to the right of the bregma at a depth of 2.5 mm and a rate of 5 μL/30 s. All animals were

monitored daily and sacrificed at the first signs of neurological deficit. STATISTICAL ANALYSIS The results are presented as the mean values ± SD. The data of patient specimens are presented

graphically with median and interquartile range noted. Data were analyzed using ANOVA or a Student’s _t_ test with correction for data sets with unequal variances. Data were analyzed on a

log 2 scale as appropriate. Kaplan–Meier estimates of the survival time from diagnosis until death or last follow-up were used for outcome analysis. Differences in survival curves between

groups were assessed by the log-rank test. Cox regression was used to construct multivariable models of survival including miRNA expression, age at diagnosis, IDH mutation status and grade.

RESULTS FUNCTIONAL CLUSTERING AND NETWORKS ASSOCIATED WITH MIRNAS THAT DISTINGUISH GSCS FROM HNSCS To define the patterns of miRNA expressions that are unique to GSCs and associated with

their tumorigenicity and mesenchymal characteristics, we used a miRNA array chip for 12 GSCs and three different cultures of hNSCs. We first compared the miRNA expression of GSCs and hNSCs.

miRNAs were identified using cutoffs for ≥1.5-fold differential expression and a significant _P_ value (_P_ ≤ 0.05), as listed in Table S3 and as shown in a volcano plot (Fig. 1A). Thirty

miRNAs were significantly upregulated, and 55 miRNAs were downregulated in GSCs relative to hNSCs. These miRNAs were further analyzed by functional enrichment and network analysis using

Ingenuity Pathway Analysis (IPA; Ingenuity Systems, http://www.ingenuity.com). IPA analysis identified clusters of miRNAs that are associated with well-known oncogenic pathways including

cell cycle, cellular development, cellular growth and proliferation, cell-to-cell signaling and interaction and cell death and survival (Fig. 1B). IPA was also used to generate three

networks of altered miRNA interactions consisting of at least 15 miRNAs from the miRNA lists (Figs. 1C, S1A, B). These networks are associated with miRNA biogenesis including regulation of

Dicer1 and AGO2 (Figs. 1C, S1A) and of oncogenes such as TERT, MYC, CASP2, CASP10, BCL2, and TP73 (Fig. S1a). An important oncogenic pathway that was also identified is associated with

increased regulation of classical EMT mediators such as Smad2/3, Smad6/7, TGFβ1, and Dicer (Figs. 1C, S1B). We then identified miRNAs that were expressed in GSCs but not in hNSCs and found

that 10 miRNAs were significantly upregulated in GSCs while 37 were downregulated (Fig. S1D). The IPA of these miRNA clusters generated two networks, each containing more than 10 miRNAs

(Figs. 1D, S1C). These networks consist of proteins related to cell survival (p53 and TERT) and miRNA biogenesis (Dicer and AGO2) similar to the pathways that were obtained in the initial

comparison of the GSCs and hNSCs (Fig. 1D). In addition, the two key mesenchymal markers ZEB2 and RUNX1 were also identified in these networks (Fig. S1C). MIR-504 IS DOWNREGULATED IN GBMS

AND GSCS Using RT-PCR analysis we first validated some of the miR array results (Figs. 2A, S2). Since miR-504 was one of the most downregulated miRNAs in GSCs compared with hNSCs (Fig. 1A),

we focused on this miRNA as a potential inhibitor of the tumorigenicity of GSCs. The expression of miR-504 in GBM specimens was also significantly increased in normal brain compared with

astrocytic tumor specimens (Fig. 2B). We then analyzed the relative expression of miR-504 in the different subtypes of GBM using TCGA6. There are 339 GBM cases in our study that have

IDH/Methylation subtyping. This subtyping splits the IDHmut-noncodel class into two groups according to methylation pattern (Fig. 2C). The G-CMIP-low class has a lower level of methylation

globally and has been found to have worse prognosis. The IDHwt GBM tumors are split into three groups. Two align with expression class, as mesenchymal-like and classic-like, and the third

has a distinct methylation pattern, denoted as LGm6–GBM (Fig. 2C, ANOVA, _P_ < 0.0001). Gray lines indicate significant difference between groups (post hoc _t_ test, _P_ < 0.05).

Analysis of miR-504 expression in LLGs demonstrated that the expression of miR-504 was higher on average in grade II glioma compared with grade III (Fig. 2D). There are 509 LGG cases in our

study that have IDH/methylation subtyping. This subtyping splits the IDHmut class into three groups according to methylation pattern (Fig. 2E). The G-CMIP-high class has a highest miR-504

expression. The IDHwt LGG tumors are also split into three groups. The classic-like and PA-like are two groups with the lowest and highest expression, respectively. While the

mesenchymal-like group has intermediate expression (Fig. 2E). Gray lines indicate significant difference between groups (post hoc _t_ test, _P_ < 0.05). While survival differences were

observed by IDH-mutation status (data not shown), there was no evidence that miR-504 expression has independent prognostic value beyond the two new WHO 2016 diagnostic groups, GBM with IDHwt

and GBM with IDH mutant. A Kaplan–Meier plot demonstrates that the quarter of patients with lowest expression of miR-504 has the worst survival outcome among grade II and III glioma (Fig.

2F; log-rank _P_ = 0.00136 overall; log-rank _P_ = 0.00402, 0.00123, 0.0111 Q1 vs. Q2–4, respectively). Collectively, these results indicate that miR-504 expression is inversely correlated

with tumor aggressiveness and poor prognosis. MIR-504 INHIBITS THE STEMNESS AND MESENCHYMAL TRANSIT OF GSCS To examine the effect of miR-504 we overexpressed it in GSCs (Fig. 3SA) and found

that pre-miR-504 markedly decreased the expression of the stemness markers Oct4 and Nanog and increased the expression of the astrocytic marker GFAP in both GSC-1 and GSC-2 (Fig. 3A). The

role of miR-504 in the stemness of GSCs was further examined on the self-renewal ability and neurosphere formation of these cells. Overexpression of miR-504 in GSCs decreased their ability

to form neurospheres as indicated by analyzing secondary neurosphere formation (Fig. 3B), the extreme limiting dilution assay (Fig. 3C) and spheroid size (Fig. 3D), suggesting that miR-504

inhibited the stemness potential of GSCs and increased their differentiation. miR-504 overexpression did not induce cell death in the GSCs as determined by trypan blue staining (data not

shown). In addition, overexpression of miR-504 also decreased GSC migration (Fig. 3E) and the expression of the mesenchymal markers CTGF, fibronectin 1 (FN), and YKL-40 (Fig. 3F). These

results demonstrate that miR-504 acts as a negative regulator of the stemness, mesenchymal phenotypes, and migration of GSCs. We further examined the effects of miR-504 overexpression on the

tumorigenic capacity of GSCs in vivo. For these experiments we employed GSC-1 transduced with lentivirus vectors expressing pre-miR-504. Intracranial implantation of these cells into

immunocompromised mice resulted in significantly smaller xenografts (4.35 ± 1.33 mm2; _n_ = 5) compared with those expressing a control pre-miR (11.96 ± 4.20 mm2; _N_ = 5, _P_ < 0.005)

(Fig. 3G). In addition, overexpression of miR-504 increased the mean survival of tumor-bearing mice compared with the control miR group (66 days vs. 42 days, _P_ < 0.0001, _N_ = 11/group)

(Fig. 3H). These data demonstrate that overexpression of miR-504 in GSCs decreased their tumorigenicity which is in line with the favorable prognosis of patients with tumors that express

high levels of this miR. GRB10 IS A TARGET OF MIR-504 AND MEDIATES THE INHIBITORY EFFECTS OF THIS MIR ON GSCS Bioinformatics analysis identified Grb10 as a potential target of miR-504 which

was also recently reported as a validated one39. Using the Grb10 3′-UTR- tagged to luciferase, we demonstrated a direct targeting of Grb10 by miR-504 (Fig. 4A). We then analyzed the

expression of Grb10 in hNSCs and GSCs (_N_ = 10) and found that this gene was highly expressed in GSCs compared to hNSCs (Fig. 4B). Using TCGA we analyzed the expression of Grb10 in various

subtypes of glioma and found that the expression of Grb10 was significantly higher in GBM compared with low-grade tumors as determined by histology criteria (Fig. 4C) and the WHO grade (Fig.

4D). In addition, analysis of methylation glioma subtypes demonstrated that Grb10 was highly expressed in the mesenchymal subtype and exhibited lowest expression in the G-CIMP high tumors

(Fig. 4E). Kaplan–Meier analysis (Fig. 4F) shows that the quarter of patients with highest expression of Grb10 have the worst survival outcomes (log-rank _P_ = 6.39e−12 overall; log-rank _P_

 = 5.13e−11, 6.15e−07, 2e−05, Q4 vs. Q1–3, respectively). Dichotomizing miR-504 at the 25th and Grb10 at the 75th percentiles, demonstrates that the low/high pattern of miR-504 and Grb10,

respectively, more clearly identifies a subset of patients with poor overall survival compared to each expression alone (Fig. 4G, log-rank _P_ = 2.67e−8 overall; log-rank _P_ = 0.0012,

2.89e−12, 0.0003, low/high vs. high/high, high/low and low/low, respectively). We then examined the role of Grb10 in GSC functions and demonstrated that its overexpression in GSCs increased

(Fig. 4H), while its silencing decreased (Fig. 4H) the stemness and mesenchymal phenotypes of GSCs, similar to the effects of miR-504. Overexpression of a Grb10 plasmid lacking 3′-UTR

induced a modest upregulation of self-renewal of the GSCs and partially abrogated the inhibitory effect of miR-504 on the mesenchymal phenotype (Fig. 4I) and the self-renewal (Fig. 4J) of

the treated GSCs. These results demonstrate that targeting Grb10 by miR-504 mediates at least in part the inhibitory effects of miR-504 on the stemness and mesenchymal phenotypes of GSCs.

OVEREXPRESSION OF MIR-504 IN GSCS PROMOTES M1 (PRO-INFLAMMATORY) PHENOTYPES OF COCULTURED MICROGLIAL CELLS Glioma cells and GSCs have been demonstrated to induce polarization of microglia

toward the M2 phenotype (anti-inflammatory/pro-tumorigenic) by the secretion of cytokines and EV-derived miRNAs23,40,41. In addition, we recently reported that EVs can deliver exogenous

miRNAs both in vitro and in vivo31,36. We therefore hypothesized that the overexpressed miR-504 in GSCs can be transferred to neighboring cells and therefore affects not only the oncogenic

functions of the GSCs but also their interactions with neighboring cells such as microglia. For these studies we employed co-cultures of microglial cells and GSCs overexpressing pre-miR-504

or a control pre-miR in transwell plates with 1-μm filters that allow only the transfer of soluble factors and EVs as described in Fig. 5A. Coculturing of microglia with control GSCs or

cells transduced with lentivirus expressing a control pre-miR (Fig. 5A, D) resulted in a relative increased expression of the M2-associated markers, CD209 and TGF-β and in the decreased

expression of CD86 and TNF-α (Fig. 5B, E). GSC coculturing also increased the phagocytosis of microglia cells (Fig. 5C). In contrast, transduction of the GSCs with pre-miR-504 (Fig. 5D)

decreased the expression of the M2-associated markers, increased the expression of CD86 and TNF-α (Fig. 5E) and decreased phagocytosis (Fig. 5F). To verify that miR-504 was delivered by the

GSCs to the cocultured microglial cells, we employed a miR-504 reporter tagged to luciferase that allows the quantification of the transferred miRNA as was previously reported33. Microglial

cells were transduced with a lentivirus vector expressing the miR-504-luciferase reporter and the GSCs were transduced with lentivirus vector expressing either control pre-miR or pre-miR-504

(Fig. 5D). As presented in Fig. 5G, microglia that were co-cultured with GSCs overexpressing pre-miR-504 exhibited decreased luciferase activity indicating that miR-504 was transferred by

the cocultured GSCs. These results indicate that miR-504 was transferred by GSCs to cocultured microglial cells. We further analyzed the expression of miR-504 in the cocultured microglial

cells and found that they expressed high levels of this miR compared with microglia cocultured with GSCs expressing a control miR (Fig. 5H). Finally, we demonstrated that overexpression of

miR-504 in microglial cells upregulated the relative expression of the M1 markers CD86 and TNF-α (Fig. 5I). EV-ASSOCIATED MIR-504 DERIVED FROM GSCS INDUCES MICROGLIA M1 PHENOTYPES

EV-associated miRNAs are implicated in the cross-talk of GSCs and microglia33,35. To determine the role of EVs in the delivery of miR-504 to microglial cells, we first analyzed the

expression of miR-504 in EVs derived from GSC-1 that were transduced with lentivirus vectors expressing control pre-miR or pre-miR-504. EVs were isolated from GSC-1 cultures using

differential ultracentrifugation as previously described35 and were analyzed for the expression of CD63, CD81, and CD9 (Fig. S3B). The amount of the secreted EVs was determined using ELISA

of CD63 antibodies and was found to be comparable in GSC-1 overexpressing con-miR or miR-504 (data not shown). We found that EVs isolated from GSC-1 overexpressing miR-504 expressed

significantly higher levels of miR-504 compared with EVs isolated from GSC-1 expressing a control pre-miR (Figs. 6A, S3C). We next demonstrated the transfer of EVs from GSC-1 to microglial

cells using ImageStreamX analysis. In these studies, GSC-1 derived EVs labeled with CellTracker Red were added to microglial cells and their fluorescence was determined 12 h later. The EVs

were efficiently internalized and accumulated in the microglial cells (Fig. 6B). Incubation of microglial cells that express the miR-504 reporter with EVs that were isolated from GSC-1

expressing either the con-miR or miR-504 (Fig. 6C) demonstrated the functional delivery of the miR-504 to the microglial cells (Fig. 6D), which resulted in decreased expression of CD209 and

TGF-β, and increased expression of CD86 and TNF-α (Fig. 6E), similar to our observations with cocultured GSCs (Fig. 5D). To demonstrate that the delivered miR-504 mediated the effects of the

GSC-1 derived EVs, we examined the effects of EVs isolated from GSC-1 overexpressing miR-504 on the differentiation of microglial cells transfected with a miR-504 antagomir (Fig. 6F) and

found that these effects were markedly abrogated, whereas, no significant inhibition was observed in microglia transfected with a control antagomir (Fig. 6G). These results indicate that the

transfer of miR-504 by GSC-derived EVs mediated, at least partly, the increased M1 phenotypes of the microglial cells induced by the cocultured GSCs. DISCUSSION GBM is one of the most

aggressive, infiltrative and incurable tumors with an average patient survival of around 14–16 months1,2. GBM therapy resistance and recurrence are primarily attributed to the existence of

GSCs8,9,10. Therefore, targeting these cells is an essential component of any successful therapeutic approach. Recent studies demonstrated that the mesenchymal differentiation of GBM is

associated with acquisition of stemness characteristics, tumor aggressiveness, therapy resistance, and poor clinical outcome16,17,18,42. Thus, deciphering the mechanisms underlying the

mesenchymal differentiation of GSCs is essential for identifying novel therapeutic targets and improving patient survival. miRNAs have emerged as attractive therapeutic targets due to their

critical roles in the regulation of major cell processes such as cell proliferation, stemness, and apoptosis that are key components in cancer initiation and progression29,30,31,43. In

addition, specific miRNAs have been implicated in controlling the mesenchymal differentiation of tumor cells26,44,45. Using miRNA microarray analysis of GSCs in comparison to hNSCs, we

identified 85 miRNAs that were significantly altered in the GSCs compared with hNSCs. These miRNAs were found to be associated with well-known tumorigenic pathways including cell cycle,

cellular development, cellular growth and proliferation, cell-to-cell signaling and interaction, and cell death and survival. These findings indicate that alterations in miRNA expression are

associated with deregulation in pathways which contribute to the tumorigenic phenotypes of GSCs. Additional differences in miRNA expression between GSCs and hNSCs were also associated with

the EMT process and included pathways regulating Smad2/3, Smad6/7 TGFβ146, and Dicer47, suggesting that the expression of specific miRNAs in GSCs regulate their own expression in parallel

with the tumorigenic characteristics of these cells. Finally, other IPA networks identified ZEB2 and RUNX1, two major regulators of the EMT pathway7, as mainly enriched in GSCs. One of the

most downregulated miRNAs in GSCs compared to hNSCs was miR-504. We further found that miR-504 expression was significantly lower in GBM as compared to normal brains and exhibited a

grade-dependent expression. In addition, using the TCGA portal, we found that miR-504 expression was significantly increased in the G-CIMP high glioma and more generally in IDH-mutant GBM,

whereas, it was considerably lower in the IDH-wt glioma classes including the mesenchymal-like subtypes. In agreement with the lower expression of miR-504 in more high grade tumors and in

the mesenchymal subtype, we found that overexpression of miR-504 inhibited the self-renewal and mesenchymal phenotypes of GSCs, Collectively, the current results highlight miR-504 as a

potential tumor suppressor miRNA and as a negative regulator of the tumorigenicity of GBM and GSCs. Our results of a role of miR-504 as a tumor suppressor in glioma and as an inhibitor of

mesenchymal transformation are in agreement with recent publications48,49,50,51,52. The current studies present new data regarding the expression of miR-504 in patient-derived GSCs compared

with NSCs and in specific subtypes of gliomas including patient survival data. In addition, the current studies focus on the functions of miR-504 in GSCs including their tumorgenicity using

intracranial xenografts. The role of miR-504 in oncogenic processes appears to be tumor dependent. Thus, in gastric cancer miR-504 expression was decreased by the tumor suppressor gene

Trefoil factor 1 (TFF1) that leads to the activation of p5353, whereas miR-504 had a dual function in oral squamous cell carcinoma54. Moreover, serum expression of miR-504 were demonstrated

to differentiate between primary and metastatic brain tumors55, suggesting a role of this miR as a diagnostic marker and a mediator of the interaction of glioma and non-CNS cells. The

inhibitory effects of miR-504 on GSCs were at least partly mediated by Grb10, which was recently reported as a validated miR-504 target in vascular endothelial cells39. Grb10 is an imprinted

gene that is differentially expressed from two promoters and in the brain it is paternally expressed56. The role of Grb10 in tumorigenesis is just beginning to be understood57,58. Using

TCGA analysis, we demonstrated that Grb10 was highly expressed in more aggressive glioma tumors and its expression was directly correlated with worse prognosis. In addition, overexpression

of Grb10 in GSCs promoted their aggressiveness, whereas silencing exerted an opposite effect and abrogated the inhibitory effect of miR-504. Thus, our studies demonstrated the miR-504/Grb10

pathway as an important regulator of the stemness-EMT process in GSCs. Additional targets of miR-504 were reported in glioma and a recent study reported that miR-504 inhibited EMT by

targeting the Frizzled-7-mediated the Wnt-β-catenin pathway51. GSCs have been reported to promote the differentiation of microglia toward the M2/anti-inflammatory phenotype, an effect that

is more pronounced in mesenchymal GSCs23,40,41. The M2 microglia phenotype in turn acts to support the migration and aggressiveness of the tumor cells and inhibition of anti-tumor immune

response24,59,60. The cross talk between microglia and GSCs is mediated by secreted cytokines and EV-derived miRNAs23,40,41,61 In view of our recent reports that EVs can also deliver

exogenous miRNA to neighboring cells31,36, we hypothesized that overexpression of miR-504 in GSCs may be transferred to neighboring cells via EVs. Using a miR-504 reporter that can directly

detect changes in miRNA levels, we found that GSCs overexpressing miR-504 delivered this miRNA to cocultured microglia via EVs. We also found that the increased relative expression of M2

markers induced by GSCs was abrogated in microglia co-cultured with GSCs overexpressing a miR-504 mimic. The mechanisms by which the EV-derived miR-504 exerts its effects on the microglial

cells are currently being studied. Since miR-504 decreased the stemness and mesenchymal differentiation of GSCs, it is possible that other factors or miRNAs that are secreted by the

transduced GSCs can also contribute to the induction of this change in the microglial phenotype. The mutual crosstalk between glioma cells and microglia highlights the importance of soluble

factors as potential therapeutic targets. Indeed, EV-associated miRNAs have been reported to play an important role in intercellular interactions in both physiological and pathological

conditions62,63. In addition, overexpressed miRNA mimics have been also reported to be delivered by EVs to neighboring cells36,39. Our data indicate that overexpressing miR-504 in GSCs

affects not only the functions of the tumor cells but also the tumor-promoting activity of microglia and probably macrophages in the tumor microenvironment, thereby amplifying the

therapeutic effect of miR-504 (Fig. 7). CONCLUSIONS The acquisition of mesenchymal phenotypes has been associated with increased stemness, infiltration, and aggressive phenotypes in GSCs64.

Therefore, identifying therapeutic targets that can interfere with this process is of utmost importance. Performing comparative analyses of hNSCs and GSCs, we identified novel miRNAs and

potential target networks that are associated with the stemness and mesenchymal transit of GSCs. miR-504 is downregulated in GSCs and exerts inhibitory effects on the functions of these

cells via the targeting of Grb10 that acts as an oncogene in GBM and GSCs. Importantly, the overexpression of miR-504 in GSCs not only inhibits the tumorigenic potential of GSCs in vitro and

in vivo but can be also transferred to microglial calls and promote their M1 polarization. Thus, the antitumor effects of RNA-based therapy in cancer cells can further exert a bystander

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(2017). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Dr. Hae Kyung Lee and Susan Finniss for their technical support and Susan McPhee for her diligent

editing of the manuscript. This work was supported by the William and Karen Davidson Fund, Hermelin Brain Tumor Center and by ICRF (C.B.). AUTHOR INFORMATION Author notes * These authors

contributed equally: Ariel Bier, Xin Hong, Simona Cazacu AUTHORS AND AFFILIATIONS * The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Ariel Bier, 

Hodaya Goldstein, Daniel Rand, Hiba Waldman Ben-Asher, Aharon Brodie & Chaya Brodie * Davidson Laboratory of Cell Signaling and Tumorigenesis, Hermelin Brain Tumor Center, Department of

Neurosurgery, Henry Ford Hospital, Detroit, MI, USA Xin Hong, Simona Cazacu, Cunli Xiang, Wei Jiang & Chaya Brodie * Department of Neurosurgery, Sheba Medical Center, Henry Ford

Hospital, Detroit, MI, USA Moshe Attia * Department of Public Health Sciences, Henry Ford Hospital, Detroit, MI, USA Ruicong She & Laila M. Poisson Authors * Ariel Bier View author

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Bier, A., Hong, X., Cazacu, S. _et al._ miR-504 modulates the stemness and mesenchymal transition of glioma stem cells and their interaction

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