TLR9 Is Critical for Glioma Stem Cell Maintenance and Targeting

2017/02/23

Andreas Herrmann1, Gregory Cherryholmes 1, Anne Schroeder 2, Jillian Phallen 3, Darya Alizadeh 4, Hong Xin 1, Tianyi Wang 1, Heehyoung Lee1, Christoph Lahtz 1 , Piotr Swiderski2 , Brian Armstrong5 , Claudia Kowolik2 , Gary L. Gallia3 , Michael Lim3 Christine Brown6 , Behnam Badie4 , Stephen Forman6 , Marcin Kortylewski1, Richard Jove2 , and Hua Yu 1,7

Abstract

Understanding supports for cancer stem–like cells in malignant glioma may suggest therapeutic strategies for their elimination. Here, we show that the Toll-like receptor TLR9 is elevated in glioma stem–like cells (GSC) in which it contributes to glioma growth. TLR9 overexpression is regulated by STAT3, which is required for GSC maintenance. Stimulation of TLR9 with a CpG ligand (CpG ODN) promoted GSC growth, whereas silencing TLR9 expression abrogated GSC development. CpG-ODN treatment induced Frizzled4-dependent activation of JAK2, thereby activating STAT3. Targeted delivery of siRNA into GSC was achieved via TLR9 using CpG–siRNA conjugates. Through local or systemic treatment, administration of CpG-Stat3 siRNA to silence STAT3 in vivo reducedGSCalongwithgliomagrowth.OurfindingsidentifyTLR9asafunctionalmarkerforGSCandatargetfor the delivery of efficacious therapeutics for glioma treatment. Cancer Res; 74(18); 5218–28. ?2014 AACR.

Introduction

Malignant glioma remains one of the most deadly cancers (1). Despite improved surgical procedures and newer thera- pies, no significant improvement of patients' survival has been observed,underscoringtheurgencyandimportancetoidentify approaches that are not derivatives of current treatment modalities. One of the subpopulations of glioma cells has been recognized as highly tumorigenic and resistant to various therapies (2, 3). This subpopulation of glioma cells exhibits stem cell–like phenotype—capable of sustaining self-renewal with gene expression profiles that resemble those of multi- potentneuralstemcells.Owingtotheirresemblancetonormal stem cells, these aggressive cancer cells are referred to as cancer stem cells (CSC; refs. 4, 5). In addition to glioma, CSCs have been shown in several other types of solid tumors, as well asbloodcancers(6).Althoughtherearestillunansweredissues and questions on the etiology of CSCs, the importance to eliminate glioma stem cells (GSC) for achieving better antitu- mor efficacy is evident.

One of the most crucial factors underlying cancer is signal transducerandactivatoroftranscription3(STAT3).Asasignal transducer, STAT3 is a central node for numerous oncogenic signaling pathways involving cytokines and growth factors (7– 10). STAT3 is also an important transcription regulator, defin- ing a transcriptional program at multiple levels that facilitates tumor cell proliferation, survival, invasion, cancer-promoting inflammation, and suppression of antitumor immune res- ponses (7, 9, 11–14). Furthermore, an essential role of STAT3 in maintaining expression of genes important for stem cell phenotype has been demonstrated (15). For malignant glioma, persistent activation of a STAT3-regulated gene network is critical for tumor progression, mesenchymal transition, and negatively impacts patient survival (16). Highly relevant to the current study, findings from pioneering work have suggested theimportanceoftargetingSTAT3and/oritscriticalupstream activatorsindisruptionofGSCmaintenance(16–18).However, directly targeting STAT3 with small-molecule drugs for the treatment of patients with cancer remains a challenge, due to the lack of enzymatic activity of transcription factors.

Toll-like receptor 9 (TLR9) is expressed in several types of immune cells (19–21). Stimulation by its ligands, single-stran- ded unmethylated DNA containing CpG oligodinucleotides (CpG-ODN),intheimmunesubsetshasbeenshowntoactivate the NF-kB pathway, leading to Th1 immunostimulation and antitumor immune responses, especially in conjunction with other modality of therapies, including radiation (22–24). However, antitumor immune responses induced by CpG-ODN monotherapyarelessthandesirableinseveralpreclinicaltesting models and disappointing in some human trials (22, 25). One of the explanations for the limited antitumor immune responses inducedbyCpG-ODNtreatmentisthattheCpG–TLR9signaling axis also activates STAT3, which serves as a brake to constrain CpG-induced Th1 immune responses (23). The ability of STAT3 to potently suppress Th1 immune responses and antitumor immunity has been well documented (10, 26).

We have recently developed an in vivo siRNA delivery technology platform by covalently linking siRNA to the CpG moiety recognized by TLR9. We have demonstrated that CpG- STAT3siRNA treatment in vivo silences STAT3 in dendritic cells, macrophages, and B cells, leading to potent antitumor immunity (27). Our recent data further illustrate that silencing STAT3 in myeloid cells by CpG-STAT3siRNA resulted in anti- tumor effects at both primary tumor and future metastatic sites (14, 27, 28). Although a role of TLR9 in stimulating immune responses has been recognized (21, 29), expression ofTLR9intumors,includingmalignantglioma,correlateswith poor patient survival (30, 31). TLR2 and TLR7 have also been shown to promote tumor growth in a STAT3-dependent manner(32).Inthecurrentstudy,weinvestigatethepossibility thatTLR9hasacriticalroleinpromotingGSCs,which,inturn, also allows inhibition of essential but difficult targets critical for CSC development and maintenance.

Materials and Methods

Animals and treatments

C57BL/6 and immune-compromised IL-2rg(ko)/NOD-SCID micewerepurchasedfromTheJacksonLaboratory. Micewere housed in a pathogen-free animal facility at the Beckman ResearchInstituteattheCityofHopeNationalMedicalCenter. RequiredanimalprocedureswereapprovedbytheInstitution- al Animal Care and Use Committee of the Beckman Research Institute at the City of Hope National Medical Center in compliancewiththeNIHguidelines.Tumor-bearingmicewere treated with 782.5 pmol of CpG-STAT3siRNA, CpG-Luciferase- siRNA, murine CpG1668, or human CpG-D19.

Cell lines and tissue

Mouse glioma cell lines GL261, GL261-luciferase, DBT, as well as human glioma cell lines U251 and U87 (ATCC) were maintained in DMEM (Gibco) supplemented with 10% FBS (Sigma), antibiotics, and antimicotics (Gibco). Human prima- ry, low-passage, stem-like glioblastoma cell lines GSC003–4, GSC008,GSC024,GSC030,GSC036,GSC017–4(kindlyprovided by Dr. Christine Brown, Department of Hematology and Hematopoietic Cell Transplantation, City of Hope Medical Center, Duarte, CA) were cultured in DMEM-F50–50 supple- mented with HEPES (Gibco), B-27 (Invitrogen), Heparin-sodi- um (Sagent), antibiotics and antimicotics (Gibco), 20 ng/mL FGFb, 20 ng/mL EGF (PeproTech), and GlutaMAX (Gibco). Human GSC-like lines 081024, 081110, 090127, JHH 136 were kindly provided by Dr. G.L. Gallia (Johns Hopkins University, Baltimore, MD). Cells used in this study were authenticated by RT-PCR, flow cytometry, and Western blotting assessing var- ious markers specific for neuroectodermal/brain tumor tissue(June 2014). Human brain tumor tissue sections embedded in paraffin were obtained from BioMax (HuCAT018, HuCAT019).

Immunoprecipitation and Western blotting

Whole-cell lysates were prepared using RIPA lysis buffer containing 50 mmol/L Tris–HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA pH 8.0, 0.5% NP-40, 1 mmol/L NaF, 15% glycerol, and 20 mmol/L b-glycerophosphate. Protease inhib- itorcocktailwasaddedfreshtothelysisbuffer(cOmpleteMini; Roche). For immunoprecipitation, antibodies were coupled to rProtein G Agarose beads (Invitrogen) before adding them to the cell lysates. Precipitation was performed by overnight shaking at 4 ? C, then complexes were carefully washed, and proteins were electrophoretically separated by SDS-PAGE followed by Western blot detection using antibodies raised againstTLR9,Sox2,MSI-1(Abcam),pStat3,Stat3,Nestin(Santa Cruz Biotechnology), JAK2 (Cell Signaling Technology), tubu- lin, and b-actin (Sigma); anti-Frizzled 4 used for pulldown was obtained from Santa Cruz Biotechnology.

Sphere formation of cell lines and treatments

Sphere formation was induced by subculturing cells in DMEM-F50–50 supplemented with HEPES (Gibco), B-27 (Invi- trogen), Heparin-sodium (Sagent), antibiotics and antimico- tics (Gibco), 20 ng/mL FGFb, 20 ng/mL EGF (PeproTech), and GlutaMAX (Gibco). Cell culture was performed in ultra-low adhesionplates(Corning).PrimaryhumanGSC-likelineswere subculturedusingAccutase(InnovativeCellTechnologies,Inc) tobreakupsphereclusters.Sphericalcellclustersweretreated for 24 hours with 500 pmol/mL CpG-ODN (class A) 5'-GGG GAC GAC:GTC GTG GGG GGG-3', CpG-ODN (class B) 5'-TCG TCG TTT TGT CGT TTT GTC GTT-3', CpG-ODN (class C) 5'- TCG TCG TCG TTC:GAA CGA CGT TGA T-3'purchased from InvivoGen or 500 pmol/mL CpG-ODN-D19 (class A) 5'- GGT GCA TCG ATG CAG GGG GG-3'kindly provided by Dr. Piotr Swiderski, Department of Molecular Medicine, Beckman Re- search Institute at City of Hope Medical Center, Duarte, CA. Interferon-g was purchased from PeproTech; GSCs were trea- tedwith20ng/mLfor30minutesat37 ? C.RNAiagainsthuman Frizzled4 (FZD4)wasintroducedusingRNAiMAX according to the manufacturer's instructions (Invitrogen). Frizzled-4siRNA was obtained from Santa Cruz Biotechnology (sc-39983).

Flow cytometry

Antibodies for flow cytometry were purchased from Santa Cruz Biotechnology (MSI-1, SOX2, Nestin), Cell Signaling Technology (pJAK2), Abcam (GFAP, Tuj1), R&D Systems (oli- godendrocytemarkerO4),orBDPharmingen(pSTAT3,TLR9). Secondary antibodies used were coupled to either Alexa Flu- or647orAlexaFluor488(Invitrogen).Intracellularstainingwas performed after fixation of single-cell suspensions with 2% paraformaldehyde and permeabilization with ice-cold 100% methanol, blocked with PBS/1% BSA for 1 hour at 4 ? C, and stained with an antibody diluted at 1:50 in PBS/1% BSA for 30 to 45 minutes at room temperature. ALDH1 detection was performed according to the manufacturer's instructions (Aldefluor; STEMCELL Technologies). Stained single-cell sus- pensions were analyzed using an Accuri C6 cytometer (BD Biosciences), followed by FlowJo 7.6.1 analysis.

Imaging

Indirect immunofluorescence on frozen tissue sections or paraffin-embedded tissue specimen was carried out as previ- ously described (28). Briefly, after deparaffinization and anti- gen retrieval at low pH citrate buffer (Vector), tissue micro- sections were fixed with 2% paraformaldehyde, followed by permeabilization with ice-cold 100% methanol, treated with signal enhancer(Image-iT; Invitrogen) for30 minutes,blocked with PBS containing 10% goat serum and 2.5% mouse serum (Sigma)for1houratroomtemperature,andstainedwitha1:50 dilution of the primary antibody (TLR9, MSI-1, Sox2, Nestin, GFAP, Tuj1; antibodies were obtained from Abcam; pStat3, Frizzled 4 were purchased from Santa Cruz Biotechnology; pJak2, cleaved caspase-3 antibodies were obtained from Cell Signaling Technology; anti-mouse CD31 was purchased from BD Pharmingen; anti-oligodendrocyte marker O4 was obt- ained from R&D Systems) in PBS/sera overnight at 4 ? C. After washingwithPBS,secondaryantibodieswerediluted(1:100)in PBS/sera containing 100 ng/mL Hoechst 33342 (Invitrogen). After final washing with PBS, specimens were mounted with Mowiol (Calbiochem).

Indirect immunofluorescence on cultured cells was carried out as described previously (27). Confocal analysis was per- formed using a LSM 510 Meta (Zeiss). Intravital multiphoton imaging was performed as previously described (27). Blood vasculature of the brain was stained by systemic injection of 100 mg dextran–rhodamine (Invitrogen); cell apoptosis was visualized by systemically injecting 10 mg Annexin V-FITC (BioVision) diluted in sterile HBSS (Gibco). We used anUltima 2-photon Microscope for IVMPM analysis (Prairie Technolo- gies). Acquired Z-stacks were reconstructed to full 3D view using the Amira software (Visualization Sciences Group). Noninvasive imaging of luciferase-expressing glioma cells using D -luciferin was performed according to the manufac- turer's instructions (Caliper Life Sciences; PerkinElmer). Bio- luminescence given by luciferase activity was acquired using IVIS100 imaging systems and analyzed with Living Image 2.50 (Caliper Life Sciences; PerkinElmer).

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation was performed using a ChIP assay kit (Upstate Biotechnology) according to the man- ufacturer's instructions. We performed chromatin immuno- precipitation in cells based on the protocol provided by Upstate Biotechnology. The chromatin was prepared from 5 * 10 6 cells and subjected to the ChIP assay as previously described (13). Of note, 4 mg of anti-STAT3 rabbit polyclonal antibody or control rabbit IgG was used for immunoprecipi- tation. The primers 5'-TGAGCTGCCTGAAAAAGGCT-3', and 5'-GAGCAGAGATGACTCAAAGAC-3'wereusedtoamplifythe mouse Tlr9 promoter fragment (?950 to ?740 bp, from the transcription start site) that contained the putative STATx- binding sites as suggested by the TRANSFAC software.

Limiting dilution assay

Cells were cultured for at least 10 days in sphere forming medium.Beforesphereassays,cellswereplacedintosingle-cell suspension. Cultures free of clumping and being of >=90%viability were used. Cells were serially diluted from 500 to 2 cells per well into 96-well plates as previously described (33). Cells were cultured in DMEM-50–50 supplemented with HEPES (Gibco), B-27 supplement (Life Technologies), Pen- Strep Antibiotics (Life Technologies). Every 3 days, fresh EGF and bFGF (PeproTech) were added. Subsequently, cultures were analyzed for spheres in each well. Data were analyzed using theExtremeLimitingDilutionAlgorithm(ELDA;ref.34).

Statistical analyses

Kaplan–Meier analyses were performed using GraphPad Prism 5. Unless otherwise noted, all statistical comparisons were made by the unpaired two-tailed Student t test and were considered significant if P < 0.05.

Results

TLR9 is overexpressed in glioma stem cells

Elevated TLR9 expression has been shown to correlate with poor survival of patients with malignant glioma (30, 31). We, therefore,testedTLR9expressionlevelsinGSCsubpopulations ofglioblastomatumors.OverexpressionofTLR9aswellashigh expression levels of phospho-STAT3 (pSTAT3) could be found in well-characterized primary, low-passage human GSC-like cells derived from high-grade gliomas (Fig. 1A, and data not shown; refs. 35, 36). Increased TLR9 expression was associat- ed with considerably enhanced expression levels of critical factors favoring GSC maintenance and progeny such as SOX2, Musashi-1 (MSI1), and Nestin. Conversely, low expression of TLR9 was concomitant with reduced expression of SOX2, MSI1, and Nestin in primary human GSCs (Fig. 1B). Using sphereformationassaysinvitroforthedetermination ofstem- like cells and to phenocopy patient-derived GSCs (37), TLR9 expression was dramatically increased upon induction of tumor cell spheres (Supplementary Fig. S1). In addition, TLR9 expression was associated with enhanced expression of the CSC markers Sox2, Msi1, and Nestin in human as well as in murine models (Supplementary Fig. S1). Furthermore, TLR9 ligandation with CpG-ODN propagated tumor sphere forma- tion. However, TLR9 silencing diminished the formation of spheres as well as self-renewal capacity as shown by a limited dilution assay (LDA) assessing the potential of tumorigenic repopulation(SupplementaryFig.S2;refs.33,34).Interestingly, Stat3 was identified to bind to the Tlr9 promoter upon induc- tion of tumor spheres in vitro. These findings from in vitro models indicate a vital role of TLR9 in GSC maintenance. Moreover, SOX2 t and MSI1 t glioma subpopulations of glio- blastoma multiforme (GBM) tumor tissue sections exhibited considerably increased TLR9 expression accompanied by high pSTAT3 expression as assessed by immunofluorescence stain- ing(Fig.1C).WefurthershowedbyWesternblottingthatTLR9 expression was elevated in various human GSC lines (Fig. 1D).

Triggering TLR9 with CpG-ODNs induces Frizzled 4-dependent JAK2 activation

OurdatashowthatCpG-ODNsactivateSTAT3(23).Because JAK2 is a tyrosine kinase contributing to STAT3 activation, we examined whether CpG-ODN could activate JAK2. We treated human primary GSCs with various classes of CpG-ODNs, followedbyflowcytometricanalysisforJAK2phosphorylation. Data obtained from these experiments showed that treatment with CpG-ODNs for 24 hours activates JAK2 effectively, which wascomparablewiththeimmediateearlyresponseinducedby IFNg (Fig. 2A). We next explored the molecular mechanism(s) by which CpG-ODN/TLR9 signaling activates JAK2/STAT3. Because Wnt/Frizzled class receptor 4 (Frizzled 4) signaling is critical for CSC maintenance and has been shown to be important for GSC stemness (38, 39), we hypothesized that CpG/TLR9 signaling may, directly or indirectly, stimulate the Wnt/Frizzled pathway. We showed that silencing Frizzled 4 (Fig. 2B) dramatically inhibited pJAK2 and pSTAT3 levels (Fig. 2C). Furthermore, silencing Frizzled 4 reduced CpG- ODN–induced sphere formation (Fig. 2D). To provide addi- tional evidence that CpG-ODNs induce interaction between pJAK2andFrizzled4,weperformedmicroscopicanalysis.Data from these experiments showed that pJAK2 colocalized with Frizzled 4 upon CpG-ODN treatment, and silencing Frizzled 4 (FZD4) abrogated their colocalization as well as JAK2 activity (Fig.2E,top).Interestingly,Frizzled4andpJAK2werelocalized in the same subcellular domains upon CpG-ODN stimulation (Fig. 2E, bottom). Furthermore, immunoprecipitation with an anti-Frizzled4antibodyfollowedbyWesternblottingtodetect JAK2 showed that JAK2 and Frizzled 4 were in the same complex upon CpG-ODN stimulation (Fig. 2F).

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CpG-STAT3siRNA inhibits human GSC-like cells and human glioblastoma growth

We next investigated whether a human version of CpG- STAT3siRNA (40) diminishes the GSC phenotype in GBM. To assess whether undifferentiated populations can be pushed toward differentiation, human primary GSCs (35, 36) were treated in vitro with CpG-ODN or CpG-STAT3siRNA and ana- lyzed via confocal microscopy for expression of cell differen- tiation markers. Upon treatment with CpG-STAT3siRNA, the cell differentiation markers O4, GFAP, and Tuj1 were substan- tially increased compared with those of CpG-ODN and non- treated controls. Interestingly, primary human GSCs treated with CpG-ODN reduced O4 expression (Fig. 3A and B). More- over, whereas CpG-ODN treatment stimulated tumor sphere growth (Fig. 3A, middle), administration of CpG-STAT3siRNA resultedindisruptionoftumorsphereintegrity(Fig.3A,right).

TLR9 is necessary to maintain murine GSC-like phenotype (Supplementary Fig. S2). To determine whether STAT3 is necessary for maintaining human GSC phenotype, primary human GSC cells were treated with CpG-STAT3siRNA and placed into a LDA to determine the frequency of CSCs within a tumor cell population (33, 34, 41). Treatment of primary human GSCs with CpG-STAT3siRNA resulted in a significant decrease in the ability of GSC populations to form sphere cultures upon extreme dilution of cells plated, suggesting a reduction in GSC frequency upon treatment (Fig. 3C). We further assessed the effects of CpG-STAT3siRNA on expression of an enzymatic CSC marker, aldehyde dehydrogenase 1 (ALDH1). Using a fluorescent substrate of ALDH, bodipy- aminoacetaldehyde, a substantial decrease in ALDH1 + cells was observed upon introduction of CpG-STAT3siRNA as ana- lyzed in two different primary human GSC lines (Fig. 3D).

We next determined the effects of in vivo treatment with CpG-STAT3siRNA of primary human glioma cells enriched for theGSCphenotypeexvivobeforeengraftment.Primaryhuman GSCs were implanted in immunodeficient (NSG) mice to form tumors. Treatment with CpG-STAT3siRNA resulted in a sig- nificant delay in tumor growth compared with the nontreated and CpG-ODN–treated controls (Fig. 3E). When analyzed for inhibition of pSTAT3 via confocal microscopy, pSTAT3 levels were considerably decreased in CpG-STAT3siRNA–treated human GSC primary cells when compared with CpG-ODN– treated and nontreated controls (Fig. 3F, top). Furthermore, tumorapoptosiswasincreased,asshownbyenhancedcleaved caspase-3 expression (Fig. 3F, middle), and proliferative activ- ity indicated by Ki67 was significantly decreased in the CpG- STAT3siRNA–treated cells compared with controls (Fig. 3F, bottom).Thesedataindicateasignificanttherapeuticeffecton glioblastoma highly enriched with GSC-like cells when treated with CpG-STAT3siRNA in vivo. Because the siRNA in the conjugate is against human STAT3, and the mice used in the experiments are severely immune-deficient, the antitumor effects observed are likely due to direct effects on the human glioblastoma/GSC-like cells in vivo, which are also supported by results generated in vitro (Fig. 3A–D). Moreover, the anti- tumoral efficacy of CpG-Stat3siRNA could be validated in rodent models (Supplementary Fig. S3), and a significantly reduced expression of factors involved in GSC maintenance, such as Sox2 and SSEA1, could be confirmed in mouse tumors whenmiceweretreated withCpG-Stat3siRNA (Supplementary Fig. S4).

Systemic delivery of CpG-Stat3siRNA targets GSC in orthotopic brain tumor

Local treatments with CpG-Stat3siRNA of subcutaneous gliomaresultedininhibitionoftumorgrowthandStat3activity, inductionoftumorcellapoptosis,aswellasprolongedsurvival (Supplementary Fig. S3). The local treatment also decreased expression of CSC-associated factors (Supplementary Fig. S4). However, the blood–brain barrier represents a major obstacle for therapeutic intervention for brain tumors. After systemic injection,fluorescentlylabeledCpG-Stat3siRNAcouldbefound in an intact, tumor-free brain as fast as 2 hours after admin- istration (Fig. 4A), and appeared to internalize into cells (Fig. 4B). Systemically treating mice bearing luciferase-expressing tumors with CpG-luciferase-siRNA also showed that the CpG- siRNA could pass the blood–brain barrier to reach the tumor (Supplementary Fig. S5). Furthermore, systemic CpG-Stat3- siRNA–treated mice had a significantly prolonged survival compared with the control groups (Fig. 4C). A concomitant decrease in Stat3 activityand TLR9 expression, compared with CpG-scrambled-RNAtreatments,wasobserved(Fig.4D).More- over, systemic treatments of orthotopically engrafted brain tumors with CpG-Stat3siRNA drastically reduced the expres- sion of Nestin, Msi1, as well as pStat3 (Fig. 4E).

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Discussion

OurcurrentstudyidentifiesTLR9asakeyfunctionalmarker for glioma cancer stem–like cells. The role of TLR9 is mostly characterized in immune cells and linked to inflammation (21, 42–44). Although CpG-TLR9 stimulation in immune cells leadstotheactivationofantitumorimmuneresponses,STAT3 alsoactsasabrake/checkpointforthispathwayintheimmune cells, constraining antitumor immunity (23). The ligands of TLR9, CpG-ODNs, have been extensively studied in animal models and in clinical trials as cancer immunotherapeutics (25, 29). However, evidence has been accumulating that TLR9 can be elevated in tumors and associated with cancer pro- gression (30, 31). Our results suggest that TLR9, an important inflammatory signaling molecule, is crucial for CSC develop- ment/maintenance in malignant glioma. Furthermore, CpG/ TLR9andSTAT3formafeed-forwardloopinGSCs:CpG-TLR9 activates STAT3, whereas activated STAT3 upregulates TLR9 expression. These results provide a possible molecular expla- nation about why TLR9 upregulation correlates with poor survival of some patients with cancer, including patients with GBM (30, 31).

STAT3 has been demonstrated extensively to be a crucial transcription factor for the development and maintenance of stemcellsandCSCs(45–47).Recently,aroleofJAK2asatarget to induce cell-cycle arrest and apoptosis of GSCs has alsobeen indicated (17). Although in immune cells CpG-ODN is known to induce STAT3 (23), the detailed mechanisms underlying CpG-induced STAT3 activation remain to be further explored. A important role of Wnt/Frizzled signaling in stem cells/CSCs is evident, and Frizzled 4 has been shown to be important for GSCs (38, 39, 48). Our current studies suggest that CpG-TLR9 signaling induces JAK2 activation in a Frizzled 4–dependent manner,andthatactivatedJAK2andFrizzled4formacomplex as demonstrated by coimmunoprecipitation/Western blot- ting.Atthistime,howCpG-ODNinducesFrizzled4expression remains to be demonstrated. However, a role of STAT3 in upregulating expression of Wnt is known (49), suggesting that induction of Frizzled 4 by CpG/TLR9 could be through a STAT3/Wnt connection. Although more mechanistic studies are required to fully understand how CpG-TLR9 signaling supportsGSCs,ourcurrentstudiesprovideevidencethatJAK2 and Frizzled 4 interact, and that Frizzled 4 plays an important role in JAK2-STAT3 activation in glioma stem cells induced by CpG/TLR9.

Several excellent molecular targets to inhibit GSCs have been shown (5, 6, 18). At the same time, broadly targeting STAT3 and other GSC targets in patients may cause undesir- ableeffects.IdentifyingTLR9asacriticalfunctionalmarkerfor GSCs allows the use of the CpG-siRNA technology to more specifically target malignant glioma. First of all, CpG-siRNA is uptaken by immune cells of myeloid origin, such as microglia, in the GBM tumor microenvironment. By silencing STAT3, CpG-STAT3siRNA should reduce tumor-induced immunosup- pression and boost antitumor immune responses triggered by CpG-ODN (23, 27, 28). Second, because malignant glioma cells, especially glioma stem cells, display elevated TLR9 expression as demonstrated by our current study, CpG-STAT3siRNA can be efficiently internalized by GSCs. When STAT3 is silenced, tumor growth and the GSC-like population are reduced. The experiments done in immune-deficient mice using human STAT3-siRNA further show that in the absence of immune responses and other effects from the tumor microenviron- ment, CpG-STAT3siRNA reduces human stem–like glioblasto- ma cells. The current study indicates that CpG-siRNA can also pass the blood–brain barrier. Our studies, therefore, provide the proof-of-principle evidence that supports clinical use of CpG-STAT3siRNAfortreatingmalignantglioma.BecauseCpG- STAT3siRNA also modulates the tumor immunologic environ- ment to favor antitumor immune responses, combinatorial therapy with CpG-STAT3siRNA and immunotherapies such as T-cell therapy can be highly desirable.

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Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Disclaimer

The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Authors' Contributions

Conception and design:A. Herrmann, G. Cherryholmes, P. Swiderski, M. Kortylewski, H. Yu

Development of methodology: A. Herrmann, G. Cherryholmes, B. Badie,M. Kortylewski

Development of methodology: A. Herrmann, G. Cherryholmes, B. Badie, M. Kortylewski

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Herrmann, G. Cherryholmes, A. Schroeder, J. Phallen, D. Alizadeh, H. Xin, T. Wang, C. Lahtz, B. Armstrong, C. Kowolik, G.L. Gallia, M. Lim, C. Brown, B. Badie

Analysisandinterpretationofdata(e.g.,statisticalanalysis,biostatistics, computational analysis): A. Herrmann, G. Cherryholmes, A. Schroeder, T. Wang, H. Lee, M. Lim, H. Yu

Writing, review, and/or revision of the manuscript:A. Herrmann, G.L. Gallia, M. Lim, C. Brown, B. Badie, S. Forman, R. Jove, H. Yu

Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): A. Herrmann, J. Phallen, D. Alizadeh Study supervision: A. Herrmann, H. Yu

Acknowledgments

The authors thank the staff members at the flow cytometry core, pathology core, and light microscopy core at Beckman Research Institute, City of Hope Comprehensive Cancer Center, for their technical assistance. The authors also thank Dr. J. Wu for his expertise in mouse tumor models, and the staff members at the animal facilities at City of Hope for their contributions.

Grant Support

This work is funded in part by R01CA146092 and a grant from the Marcus Foundation, R01CA155367 and W81XWH-12–1–0132 as well as Billy and Audrey L. Wilder Endowment. Research reported in this publication was supported by the National Cancer Institute of the NIH under grant number P30CA033572.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 14, 2014; revised June 24, 2014; accepted July 1, 2014; published OnlineFirst July 21, 2014.

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