Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion Vy M

Published OnlineFirst August 4, 2017; DOI: 10.1158/1541-7786.MCR-17-0352

Signal Transduction Molecular Cancer Research Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion Vy M. Tran1, Anna Wade1, Andrew McKinney1, Katharine Chen1, Olle R. Lindberg1, Jane R. Engler1, Anders I. Persson1,2,3, and Joanna J. Phillips1,2,4

Abstract

Glioblastoma (GBM) is the most common primary malignant heterogeneous HS content and structure across patient-derived brain tumor of adults and confers a poor prognosis due, in part, to tumorsphere lines suggested diverse functions in the GBM tumor diffuse invasion of tumor cells. Heparan sulfate (HS) glycosami- microenvironment. In GBM5 and mPN, elevated expression of noglycans, present on the cell surface and in the extracellular sulfatase 2 (SULF2), an extracellular enzyme that alters ligand matrix, regulate cell signaling pathways and cell–microenviron- binding to HS, was associated with low trisulfated HS disacchar- ment interactions. In GBM, the expression of HS glycosamino- ides, a substrate of SULF2. In contrast, other primary tumorsphere glycans and the enzymes that regulate their function are altered, lines had elevated expression of the HS-modifying enzyme hepar- but the actual HS content and structure are unknown. However, anase (HPSE). Using gene editing strategies to inhibit HPSE, a role inhibition of HS glycosaminoglycan function is emerging as a for HPSE in promoting tumor cell adhesion and invasion was promising therapeutic strategy for some cancers. In this study, we identiﬁed. These studies characterize the heterogeneity in HS use liquid chromatography–mass spectrometry analysis to dem- glycosaminoglycan content and structure across GBM and reveal onstrate differences in HS disaccharide content and structure their role in tumor cell invasion. across four patient-derived tumorsphere lines (GBM1, 5, 6, 43) and between two murine tumorsphere lines derived from murine Implications: HS-interacting factors promote GBM invasion GBM with enrichment of mesenchymal and proneural gene and are potential therapeutic targets. Mol Cancer Res; 1–11. 2017 expression (mMES and mPN, respectively) markers. In GBM, the AACR.

Introduction ment via their binding with diverse partners, including soluble factors, membrane proteins, and the ECM (8). HSPGs consist of a Glioblastoma (GBM) is characterized by tumoral heterogene- core protein and covalently attached heparan sulfate (HS) gly- ity, including differences in tumor cell invasion and receptor cosaminoglycan (GAG) chains comprised of repeating disaccha- tyrosine kinase (RTK) signaling pathway activity. Despite this ride units. Extensive co- and posttranslational enzymatic mod- heterogeneity, transcriptional profiling has identified tumor sub- ifications, particularly involving the HS chains, generate the types (1, 2) with enrichment of specific cell signaling (3), prote- structural diversity important for their many functions, including omic (4, 5), and immune cell characteristics (6, 7). The design of their ability to bind and sequester soluble ligands, act as a improved therapeutics for GBM requires an understanding of coreceptor on the cell surface (9), and influence cell adhesion intertumoral heterogeneity and its potential impact on therapeu- and migration, one mechanism being the regulation of integrin tic response. activity (8, 10). Heparan sulfate proteoglycans (HSPG), present on the cell A major determinant of the specificity and the affinity of HSPG surface and in the extracellular matrix (ECM), are abundant in ligand interactions is the sulfation pattern of the HS GAG side the brain and regulate interactions in the extracellular environ- chains, for which one critical determinant is the 6-O-sulfate (6OS) of glucosamine (8, 11). Regulation of 6OS levels occurs during biosynthesis and postsynthetically by the extracellular endoglu- 1Department of Neurological Surgery, Brain Tumor Center, University of Cali- fornia, San Francisco, San Francisco, California. 2Helen Diller Family Compre- cosamine-6-sulfatases or sulfatases (SULF) whose principle sub- hensive Cancer Center, University of California, San Francisco, San Francisco, strate is trisulfated disaccharides (12). SULF1 or SULF2 desulfa- California. 3Sandler Neurosciences Center, Department of Neurology, University tion of 6OS regulates HS-ligand binding and subsequent cell of California, San Francisco, San Francisco, California. 4Division of Neuropathol- signaling important in normal development and in cancer, ogy, Department of Pathology, University of California, San Francisco, San including Wnt, sonic hedgehog, FGF, VEGF, glial cell line–derived Francisco, California. growth factor, and stromal cell-derived factor 1 (13–17). In GBM, Note: Supplementary data for this article are available at Molecular Cancer the expression of HSPGs and the enzymes that regulate their Research Online (http://mcr.aacrjournals.org/). function, including SULF2, are altered (18). In human GBM cell Corresponding Author: Joanna J. Phillips, University of California, San Fran- lines and murine models for high-grade glioma, SULF2 has been cisco, 1450 Third Street, Room HD492B, Box 0520, San Francisco, CA 94158- directly implicated in promoting tumorigenesis (19–21). Abla- 9001. Phone: 415-514-4929; Fax: 415-514-9792; E-mail: tion of SULF2 in tumor-prone cells resulted in decreased tumor [email protected] growth, prolonged host survival, increased HSPG 6-O-sulfation in doi: 10.1158/1541-7786.MCR-17-0352 tumors in vivo, and decreased activity of PDGFRa and related 2017 American Association for Cancer Research. downstream signaling pathways (21).

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Heparanase (HPSE), a b-endoglucuronidase, cleaves HS chains acetate, 40 mmol/L ammonium acetate, 0.1 mg/mL BSA, pH to reduce HS chain length and release smaller biologically active 7.0) overnight followed by enzyme inactivation at 100C for oligosaccharides. Increased HPSE activity is associated with 5 minutes. Proteins and uncleaved GAG chains were removed increased tumorigenesis, angiogenesis, and invasiveness in using 3 kDa MWCO Amicon columns (12,000 g). HS disac- diverse cancers including multiple myeloma, melanoma, breast, charides were separated by a 5 mm particle size Zorbax SB-C18 and pancreatic cancer (22). In multiple myeloma, a number of HPLC column with a 12 mmol/L TrBA/38 mmol/L ammonium oncogenic functions for HPSE have been identified including acetate, pH 6.5, solvent systems using a gradient curve from 2% to direct effects on tumor cells and indirect effects on tumor- 100% solvent B (65% acetonitrile) at flowrate 10 mL/min with associated endothelial cells (10, 23–25). In the brain, HPSE has extra acetonitrile (0.7 mL/min). The MS was acquired in the been implicated in metastatic tumor cell invasion and medullo- negative ion mode with inline 4000 QTRAP mass spectrometry blastoma cell signaling (26–28), but its role in GBM is less equipped with a TIS (Turbolon Ionspray) source (AB Sciex Instru- established (29, 30), although recent studies suggest its impor- ment. SCIEX) at the following conditions: curtain gas (CUR) at 10 tance in tumor progression (31). psi, ion spray voltage (IS) at 4,500 V, temperature (TEM) at 100C, Given the role for HSPGs in tumorigenesis and the importance ion source gas (GS1) at 10 psi, ion source gas (GS2) at 0 psi, of GAG composition and structure in regulating cell signaling and declustering potential (DP) at –50 V, and entrance Potential (EP) invasion, we investigated HS GAGs in GBM using tumorspheres was set at –10 V. Quantification analysis of heparin/HS disacchar- cultured from four patient-derived GBM lines and two murine ides was performed using calibration curves constructed from GBM. In addition, we examine the biologic function of HS unsaturated HS disaccharide (18.6, 37.2, 74.4, 148.8, 297.6, modification in tumor cell invasion. These data demonstrate 595.2 ng per disaccharide; Supplementary Figs. S2 and S3) as heterogeneity in HS content and structure in GBM and suggest described in Supplementary Materials and Methods. The area of intertumoral differences in proteoglycan expression and function disaccharides and internal standards was quantified using Peak- with potential implications for therapeutic stratification. View 2.2 software (AB Sciex LLC). Total HS amount was calculated as the cumulative total of each disaccharide, and to compare Materials and Methods across tumors, the value was normalized by total protein (ng/mg). Cell culture conditions and reagents RNA and protein analysis Murine GBM tumorspheres established and demonstrated to Tumorspheres were resuspended in an appropriate volume of have tumor-generating and differentiation capacity in Lindberg RLT buffer from an RNeasy mini kit (Qiagen). RNA was isolated as and colleagues (32) were cultured and maintained as described per the manufacturer's instructions and quantified using a Nano- previously (21, 32, 33). GBM43, GBM6, and GBM5 are tumor- drop (NanoDrop Technologies, Inc.) Spectrophotometer. For sphere cultures of patient-derived xenografts (PDX). PDX were quantitative real-time PCR, complementary DNA was synthesized established as previously described (34, 35) and have been using Oligo(dT) primers and Superscript III (18080-051; Thermo genetically and transcriptionally profiled (1, 35, 36). GBM1 Fisher Scientific). Primers were synthesized by IDT (IDT Inc.) and (SF9487) is a newly generated primary patient-derived tumor- are listed in Supplementary Table S2. Real-time PCR was per- sphere culture from UCSF and when injected into the striatum of formed on a 7900 HT Fast Real-Time PCR System (Thermo Fisher nude mice generates highly invasive GBM (Supplementary Fig. S1; Scientific) using FastStart Universal SYBR Green Master (Rox; Supplementary Table S1; and Supplementary Materials and Meth- Roche). C values were normalized to GAPDH C values for each ods). All cell lines were analyzed before using and each subse- t t individual sample and then averaged across experiments. For quent year by short tandem repeat analysis to ensure the identity NanoString analysis of human tumorsphere lines, RNA (200 ng) and validity of cells and checked by PCR for mycoplasma con- integrity was assessed using an Agilent 2100 bioanalyzer (Agilent tamination. Cells were passaged no more than 15 to 20 times and Technologies) and probed using a custom codeset, including four then a new frozen aliquot of cells was used. Unless otherwise genes of interest in GBM (EGFR, EGFRvIII, PDGFRA, and NF1), specified, media and supplements were purchased from Thermo and six normalizing genes (ACTB, B2M, GAPDH, POLR2A, SDHA, Fisher Scientific. Human tumorspheres (2–4 104 cells/1 mL) and TBP). Data were analyzed with the NanoString nCounter were cultured in Neurobasal-A media containing B27-A, N2, and Analysis System at NanoString Technologies according to the glutamine and passaged every 4 to 7 days. bFGF and EGF were manufacturer's protocol (NanoString Technologies), and relative added every third day (20 ng/mL). Tumorspheres were dissoci- expression levels were compared (Supplementary Table S2). Gene ated by adding 2 mL of Accutase (Innovative Cell Technologies; expression data from human GBM were downloaded from http:// hGBM) or 150 mL nonenzymatic dissociation media (NEDM; www.cbioportal.org/index.do. Gene expression analysis of Sigma-Aldrich Corporation; mGBM) followed by mechanical mRNA isolated from FACS-sorted murine tumors was previously trituration. Invasion assays use Corning Matrigel Growth Factor reported (32) and is available in the GEO database (accession Reduced (GFR) Basement Membrane Matrix (Corning Inc.). number GSE87332). To assess enrichment of GBM subtype genes in murine tumors, Gene Set Enrichment Analysis (GSEA) Purification of GAG chains, quantification, and disaccharide was implemented using the Broad Institute GSEA v2.07 soft- compositional profiling of HS chains from tumorspheres ware (http://www.broadinstitute.org/gsea) and subtype signature GAG chains were purified and analyzed from tumorsphere genes (2). For all GSEA analyses, 1,000 gene set permutations cultures following the protocol reported by Yang and colleagues were performed, and a Signal2Noise ranking metric was used to (37) with minor modifications (see Supplementary Materials create the ranked list of genes. An FDR q value of less than and Methods). The HS disaccharides were released from the GAG 0.05 (5%) was considered statistically significant. HPSE protein chains with heparitinase I, II, and III (a kind gift from Dr. Kuberan was detected by Western blotting using anti-HPSE antibody Balagurunathan) in heparitinase buffer (3.3 mmol/L calcium (#APP60766; Aviva Systems Biology). Anti-Hpse antibody

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binding to both the 50 and 61 kDa bands could be blocked with Results an HPSE blocking peptide (Supplementary Fig. S4A). Human GBMs are heterogeneous and can be divided into subtypes based upon transcriptional profiling (1, 2). Our CRISPR/Cas9 gene editing of HPSE previous studies in GBM (21) and our analysis of transcrip- Human HPSE targeting CRISPR guide RNA 6 (CCTTC- tional data from The Cancer Genome Atlas (18) suggested TTGGGATCGAAAATT) in the pLenti-Guide-puro backbone and differences in HS proteoglycan expression and function across lentiCas9-Blast (GenScript) were used to make lentivirus using tumor transcriptional subtypes. HS disaccharides consist of fi standard procedures (21). Target sites and speci city were repeating units of a glucosamine (N-acetylglucosamine or N- validated using the UCSC Genome Browser. Transduced sulfate glucosamine) and uronic acid (glucuronic acid or iduro- GBM43 cells were clonally plated, sequenced, and comparisons nic acid) that undergo extensive posttranslational modifica- were made between GBM43-Cas9 and GBM43-gRNA6. tions including sulfation. These modifications greatly alter their fi Sequencing the targeted HPSE loci con rmed introduction by functional properties (8). To determine the diversity of HS the Cas9 nuclease of small, mostly frameshift indels/WT, content and structure in human GBM, we profiled HS disac- including gRNA6-1 (NM_001098540, c.395-414del) and charides isolated from patient-derived GBM lines maintained gRNA6-2 (NM_001098540, c.389-415del). The human HPSE as tumorspheres using liquid chromatography–mass spectrom- cDNA construct, pIRES2-EGFP/HPSE1 (38), was a kind gift of etry (LC/MS). Tumor cells were cultured as tumorspheres, using Dr. Ralph Sanderson (University of Alabama at Birmingham, neural stem cell conditions including nonadherent growth and Birmingham, AL). minimal essential media, to promote retention of tumor-generating cells (40, 41). The four human lines were selected based Invasion and adhesion assays on their diversity of molecular and transcriptional properties, Three-dimensional invasion assays were performed as including EGFR amplification and EGFRvIII expression in described previously (ref. 39; see Supplementary Materials and GBM6, PDGFRA amplification in GBM5, and CDKN2C/p18 Methods). Briefly, 500 to 1,000 cells per well were plated in a (INK4c) homozygous deletion in GBM43 (35, 36, 42). Nano- 96-well round-bottom ultra-low attachment plate (Corning string analysis of tumorsphere mRNA confirmed differences Life Sciences) in standard culture media (100 mL/well) for between the lines, including EGFR and EGFRvIII expression in 3 days. For drug treatment, the cells were treated with heparin GBM6, low expression of NF1 in GBM43, and robust expres- (24 hours), necuparanib (formerly M402, a kind gift from sion of PDGFRA in all four lines (Supplementary Table S3). Momenta Pharmaceuticals, Cambridge, MA; 6 h), or an equiv- Individual disaccharides were identified and quantified based alent volume of PBS prior to matrigel invasion assay using on standard curves generated from known quantities of indi- growth factor–reduced matrigel. Images were acquired at 0, 16, vidual disaccharides (Supplementary Figs. S2 and S3). Each 24, and 40 hours after plating. Relative invasion was deter- of the four independent human GBM lines had a unique mined from representative images of each sphere using ImageJ HS disaccharide ion chromatography profile (Fig. 1A–D) (http://imagej.nih.gov/ij/) by subtracting the solid sphere area and different total HS abundance (ng disaccharide/mg from the maximal invaded sphere area and normalizing by the protein; Fig. 1E). GBM5 had 2-fold greater absolute abundance initial sphere area at 0 hour (Supplementary Fig. S5). Adhesion of HS disaccharides compared with the other three GBM lines assays were performed in 96-well plates precoated with laminin (P < 0.005). HS structure also differed between GBM as dem- (2 mg/cm2) and blocked with 1% BSA in PBS-D for 1 to 2 hours onstrated by differences in relative disaccharide abundance at 37C. Cells were treated with necuparanib (Necu) or PBS (Fig. 1F). Although many disaccharide structures are regulated (control) for 24 hours, dissociated to single cells, counted x3, during biosynthesis, trisulfate disaccharides can be reduced dilutedto2.5 104 cells per 100 mLoffullgrowthmedia,and postsynthetically in the extracellular environment by the extra- transferred to the laminin-coated wells. Cells were allowed to cellular endosulfatases, or SULFs, which remove 6-O sulfate adhere for 2 hours at 37C, after which media and nonadherent from trisulfate disaccharides (12, 43). GBM5 had the lowest cells were gently removed, wells were gently washed with ice- trisulfate abundance relative to the other GBM (Fig. 1F, inset; cold PBS, and adherent cells were fixed with 4% paraformal- P < 0.05). GBM5 also had the highest expression of SULF2 dehyde, incubated on ice for 10 minutes, and stained with mRNA across the four lines (Fig. 1G; P < 0.0001). In contrast, crystal violet as previously described (32, 39). Transwell inva- SULF1 was expressed at similar levels (Fig. 1H). sion assays were performed using BD BioCoat Growth Factor HPSE, an HS-degrading enzyme, can directly alter cellular HS Reduced Matrigel Invasion chambers (BD Biosciences) as content (44) and indirectly change cell surface proteoglycan described previously (ref. 39; see Supplementary Materials and core protein composition via HS-dependent regulation of pro- Methods). HPSE or control-conditioned media were generated teases (45). In several cancers including GBM, HPSE expression from 293T cells incubated in minimal essential media as is upregulated and has been associated with increased tumor described in Supplementary Materials and Methods. Images cell invasion and increased tumor growth in vivo (22, 46). In for 3D invasion, adhesion, and transwell invasion were human GBM, HPSE was most highly expressed in the mesen- acquired using a DMI IL LED Leica microscope (Leica). chymal GBM subtype (Fig. 2A; P < 0.0001), a subtype proposed to be the most therapy resistant (47). To examine potential functions for HPSE in GBM43, CRISPR-Cas9 gene editing was Statistical analysis used to introduce deletions in exon 3 of HPSE. Clones with All statistics were performed using Graph Pad Prism 6.0 soft- heterozygous deletions in HPSE and reduced HPSE expression ware (Graph Pad Software). All data are presented as mean SEM. relative to control clones were isolated (Fig. 2B, P < 0.001). One-way ANOVA, the Student t test analysis, and Mann–Whitney HPSE-reduced clones did not exhibit a growth defect relative to test were performed at confidence interval levels of 95%.

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AB NAc 1.8 1.8 GBM 1 GBM 43 NS

NAc6S/NAc2S

7 1.2 1.2 NS6S/NS2S IS Trisulfate 0.6 0.6

Intensity × 10

0 0 0 15 30 45 0 15 30 45 Time (min) Time (min) C D 1.8 Figure 1. GBM 6 1.8 GBM 5 Analysis of HS disaccharide structure

7 and amount from human GBM 1.2 1.2 tumorspheres. A–D, Extracted ion chromatography proﬁle of HS disaccharides isolated from human 0.6 0.6 GBM tumorspheres demonstrating intensity proﬁles (counts per second)

Intensity × 10 Intensity × 10 over time (minutes) across tumors. 0 0 Based on the analysis of disaccharide 0 15 30 45 0 15 30 45 standards (Supplementary Figs. S2 and Time (min) Time (min) S3), each peak could be assigned a disaccharide with different sulfate E F patterns. E, Absolute HS abundance *** GBM1 *** * normalized to total protein extracted *** GBM43 ** (ng/mg) across tumorspheres. F, 400 50 GBM6 *** Relative HS disaccharide abundance as GBM5 6 a percentage (%) of HS disaccharide 40 composition. Inset highlights the 300 4 comparison of the relative percentage 30 of trisulfate content. G and H, Relative 2 SULF1 and SULF2 gene expression 200 20 Trisulfate abundance (%) 0 levels in tumorspheres, normalized to GBM43 (n ¼ 3). Representative data (A–D) from biological replicates. NAc, 100 10 N-acetylglucosamine; NS, N-sulfate

Relative abundance (%) glucosamine; 6S, 6-O-sulfated

HS absolute abundance (ng/mg) glucosamine; 2S, 2-O-sulfated iduronic 0 0 acid; and trisulfate, NS2S6S. Mean SEM. , P < 0.05; , P < 0.01; NS NAc NS6S , P < 0.001; and , P < 0.0001. NAc6S NAc2S NS2S Trisulfate G **** H **** 7 **** 7 6 SULF1 5 SULF2 5 3 4 3 1 2 0.1 1 Relative expression 0.0 Relative expression 0 GBM: 1 43 56 GBM: 1 436 5

control clones in vitro (Supplementary Fig. S4B). To assess cell three-dimensional matrix requires coordinated regulation of invasion in the context of a three-dimensional matrix, tumor- cell attachment, migration, and ECM breakdown. Given the spheres were embedded in matrigel, and tumor cell invasion reported roles for HPSE and HS in cell invasion and adhesion, away from the sphere was imaged and quantiﬁed over time. we asked whether decreased invasion in HPSE-reduced cells Reduced HPSE conferred a marked decrease in tumor cell was associated with reduced cell adhesion. HPSE-reduced invasion (Fig. 2C and D; P < 0.0001). Cell invasion in a GBM43 had markedly reduced cell adhesion to laminin relative

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A BC Human GBM GBM43 0 h 24 h 1.0 1.2 *** ****

0.5

0.8 Control

Figure 2. HPSE expression is increased in the 0 0.4 mesenchymal GBM subtype and can promote tumor cell invasion. A, Increased expression of HPSE in Relative HPSE expression Relative HPSE human GBM of the mesenchymal HPSE expression (z-score) -0.5 0

l HPSE del. (MES) transcriptional subtype. Bars Ctr denote the mean SEM Z-score for MES tumors in the speciﬁed subtype (1) non-MES HPSE del. with mesenchymal (black; n ¼ 56) and nonmesenchymal (white; n ¼ 139); The D E F 1.2 Cancer Genome Atlas (TCGA) Data 2 h ** Portal (63). B, Focal deletion in HPSE HPSE confers decreased mRNA **** expression in human GBM43 (HPSE 1.2 del., n ¼ 7; Ctrl, n ¼ 3). C and D, 0.8 Control Decreased 3D invasion of GBM43 with 0.9 HPSE deletion as compared with Ctrl. Representative images and quantiﬁcation (biologic triplicate) at 0.6 0.4

24 hours after plating. E and F, Relative adhesion Decreased laminin adhesion of GBM43 0.3

with HPSE deletion relative to Ctrl. G Relative invasion HPSE del. and H, Increased invasion of HPSE 0.0 0 deletion GBM43 cells treated with . conditioned media from HPSE- Ctrl Ctrl overexpressing cells as compared with control vector (Ctrl) overexpressing HPSE del HPSE del. cells (HPSE, n ¼ 5; Ctrl, n ¼ 5). Mean SEM. Representative images and data G 16 h H are from biological triplicate HPSE del. experiments; , P < 0.05; , P < 0.01; ** , P < 0.001; and , P < 0.0001. C, F, and H, Scale bars denote 100 mm; E, 2.0 m Scale bar denotes 20 m. HPSE del. 1.5

1.0

0.5 Relative invasion HPSE del. (+) HPSE 0.0 (+) HPSE – +

to control cells (Fig. 2E and F; P < 0.01). Addition of exogenous 3C; P < 0.01). Thus, an HS mimetic could phenocopy HPSE HPSE promoted invasion of HPSE-reduced GBM43 cells rela- knockdown, supporting a role for HPSE in GBM tumor cell tive to control-treated cells (Fig. 2G and H; P < 0.05). invasion and cell adhesion to laminin. Heparin, a highly sulfated HS, can bind to and inhibit HS- Human GBM can be subtyped based on transcriptional signa- interacting factors including enzymes such as HPSE (48, 49). Due tures (1, 2); however, multiple sampling of tumors and single-cell to the anticoagulant properties of heparin, however, HS mimetics analysis suggest tumors retain signiﬁcant intratumoral heteroge- have been developed with reduced anti-coagulant properties. neity even within a given subtype (50). To investigate associations These mimetics have been used for their ability to inhibit HPSE between HS GAG content, structure, and role in invasion in a and the function of other HS-interacting factors in therapeutic more homogeneous system, we used a murine model for GBM trials for cancer, including necuparanib (formerly M402), PI-88, driven by deﬁned alterations in neural progenitor cells (refs. 21, PG-545, and SST0001 (https://clinicaltrials.gov; Supplementary 32; Fig. 4A). First, tumor-prone cells, containing alterations com- Table S4). Treating GBM43 with necuparanib (Necu), an HS mon to human GBM (deletion of Ink4a/Arf and expression of mimetic re-engineered from heparin (49), reduced three- EGFRvIII), were implanted orthotopically in mice and allowed to dimensional tumor cell invasion (Fig. 3A and B; P < 0.001) form invasive tumors. Second, tumor cells were isolated and and reduced cell adhesion to 53% of control-treated cells (Fig. cultured from tumors as tumorspheres under nonadherent

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B C A Figure 3. 0 h 40 h 1.5 1.2 *** Inhibition of GBM43 invasion and ** adhesion by a heparan sulfate mimetic. A and B, Decreased 3D invasion of GBM43 following treatment with the 1.0 0.8 heparan sulfate mimetic necuparanib Control (Necu; 240 mg/mL). Representative images and quantiﬁcation at 40 hours after plating (Necu, n ¼ 13; 0.5 0.4 Ctrl, n ¼ 4). C, Decreased adhesion of Necu-treated GBM43 as compared Relative invasion Relative adhesion with Ctrl (biological triplicate). Mean

Necu SEM. Representative data are from 0.0 0 biological triplicate experiments; , P < 0.01; , P < 0.001. Ctrl Ctrl Necu Necu Scale bars denote 100mm.

conditions in minimal essential media. Murine tumorspheres (ng disaccharide/mg protein) relative to mMES (Fig. 5C, P < 0.05). maintained in this manner retain tumor-forming capacity and Although overall HS structure of mMES and mPN was more biological features of the original tumor (32). Previously, we similar than when comparing the human GBM, mPN had identified and transcriptionally profiled tumors from two murine decreased levels of trisulfate disaccharides relative to mMES (Fig. tumorsphere lines with different biological behaviors (32). GSEA 5D, P < 0.01). Similar to GBM5 with reduced levels of trisulfate of differentially expressed genes (GSE87332) demonstrated sig- disaccharides, the mPN cells had increased expression of the nificant enrichment for mesenchymal signature genes in tumors extracellular endosulfatase Sulf2 for which HS trisulfate disac- designated murine mesenchymal-like (mMES) and proneural charides are a substrate (Fig. 5E, P < 0.001). SULF2 protein signature genes in tumors designated murine proneural-like expression was also elevated in mPN tumors in vivo (Fig. 5F), (mPN; Fig. 4B, NES 1.59, FDR q value < 0.001% and Fig. 4C, suggesting that SULF2 may be one determinant of HS trisulfate NES 2.66, FDR q value < 0.0001%, respectively). Using these two abundance in GBM. tumorsphere lines generated from syngeneic neural progenitor To investigate the factors that may influence the reduced HS cells, we investigated differences in HS content and HS structure. content in mMES tumor cells, we compared expression of HPSE HS disaccharides isolated from murine tumorspheres were and HS biosynthetic enzymes and core proteins. HPSE expression analyzed by LC/MS. Similar to the human tumor cells, each was markedly different in the two lines with increased Hpse murine line had a unique HS disaccharide ion chromatography mRNA (Fig. 6A, P < 0.001) and protein expression (Fig. 6B and profile (Fig. 5A and B). mPN had increased total HS abundance C, P < 0.05) in mMES relative to mPN tumorspheres, including the

A Figure 4. Gene expression Gene expression analysis of murine Selected in vivo analysis of tumor cells. A, Murine tumorspheres EGFR* + ZsGreen sorted tumor were derived from brain tumors cells formed from Ink4a/Arf/ neural progenitor cells (NPC) transduced with a constitutively active deletion mutant of EGFR (EGFRvIII; EGFR) Ink4a/Arf-/- NPC Glioma progenitor cells and a ﬂuorescent tag (ZsGreen). GSEA maintained as tumorspheres of differentially expressed genes in tumorsphere-generated tumors [n ¼ 6 B mice per line (32)], isolated by FACS mMES C mPN and revealed differential enrichment of genes associated with human GBM transcriptional subtypes (2). B, Tumors enriched for the human mesenchymal GBM subtype gene signature were denoted mMES (NES 1.59, FDR q value < 0.001%). C, Tumors Proneural enriched for the human proneural GBM Mesenchymal subtype gene signature were denoted mPN (NES 2.66, FDR q value < 0.0001%). NES 1.59 NES 2.66 FDR q-value <0.001% FDR q-value <0.0001%

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AB NAc 1. 0 07 1.8 07 1.8 mMes 1.8 mPN NS

7 NAc6S/NAc2S

1. 07 10 07 NS6S/NS2S 10 10 1.2

1.2 × 1.2

× IS Trisulfate 16001600

6. 06 06 010 1500 6.0

0.6 Intensity, cps 0.6

1400 Intensity

Intensity

00 1 00 0153045 0153045 0 15 0 30 45 0 15 30 45 Figure 5. Time (min) Time (min) 6 43 M5 M BM G GB Time (min)GB HS disaccharide analysis of mMES and Time (min) mPN tumorspheres. GAG chains isolated from mMES and mPN C D tumorspheres were analyzed as 500 60 in Fig. 1. A and B, Extracted ion * mMes chromatography proﬁle of HS mPN disaccharides demonstrating 40 differences in HS structure, intensity proﬁles (counts per second) over time 400 20 (minutes). C, Elevated total HS content in mPN as compared with 6 mMES normalized to total protein extracted (ng/mg). D, Relative 300 ** abundance of HS disaccharide 3 composition in tumorspheres Relative abundance (%) demonstrating decreased trisulfate Absorlute abundance (ng/mg) content in mPN. E, Increased 0 0 Sulf2 expression of mRNA in mPN NS mPN NAc relative to mMES. F, Increased SULF2 NS6S NS2S mMES NAc6S NAc2S protein expression in mPN brain Trisulfate tumors as compared with mMES. A E 6 F and B, F, Representative data from *** four biological replicates. Mean SEM. , P < 0.05, n ¼ 4; , P < 0.01, n ¼ 4; and mMES mPN , P < 0.001 n ¼ 5. 4

2 Sulf2 Sulf2 mRNA

mPN mMES

52 kDa active enzyme. Biosynthetic enzymes important in HS Similar to treatment with heparin, HS mimetic treatment inhib- chain initiation (Xylt1) and elongation (Ext1) were also decreased ited invasion of Hpse-high mMES cells (Fig. 7D and E, P < 0.05). in mMES relative to mPN (Fig. 6D, P < 0.0001). In addition, expression of the core protein Syndecan 3 (Sdc3) was signiﬁcantly decreased in mMES cells (P < 0.05; Fig. 6E). These data suggest Discussion that several factors may contribute to the lower abundance of GBMs are highly malignant, invasive tumors that are highly total HS in mMES versus mPN, including decreased expression of resistant to current therapies and associated with a dismal prog- HS biosynthetic enzymes, decreased expression of core proteins nosis. HS proteoglycans on the cell surface and in the ECM such as Sdc3, and increased expression of the HS-degrading regulate interactions in the extracellular environment such as enzyme Hpse. ligand-mediated signaling, a potential mechanism of resistance Based on our data with human tumorspheres, we treated to targeted therapies (51). Analysis of tumorspheres derived from murine tumorspheres with either heparin or the HS mimetic four patient-derived GBM (hGBM) lines and two murine GBM necuparanib to investigate HS function in tumor cell invasion. lines demonstrates heterogeneity in the content and the structure Treatment of Hpse-high mMES cells with heparin inhibited inva- of HS GAGs. Patient-derived GBM5 and mPN demonstrated sion in three-dimensional matrigel invasion assays in a dose- increased HS content, increased SULF2 expression, and reduced dependent manner (Fig. 7A and B, P < 0.05). In contrast, invasion HS trisulfate groups, the major substrate for SULF2. These data of Hpse-low mPN cells was not decreased by heparin (Fig. 7C). support previous studies in human GBM demonstrating that

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ABC 40 25 mMes mPN *** * 30 20

60 kDa 20

15 50 kDa Figure 6. 10 Increased HPSE and decreased HS biosynthetic enzyme and core protein Hpse mRNA Expression Actin Active HPSE expression expression in mMES. Tumorspheres 0 0 from mMES demonstrate increased Hpse mRNA (A) and protein (B) expression as compared with mPN. mPN mPN mMES mMES C, Quantiﬁcation of 52 kDa active enzyme levels. D, Decreased D E expression of HS biosynthetic mMes enzymes Xylt1 and Ext1 in mMES mPN **** relative to mPN. E, Decreased Sdc3 5 30 expression in mMES relative to mPN. **** **** , P < 0.05; , P < 0.01; , P < 0.001; 4 and , P < 0.0001. Mean SEM, at 20 least biological triplicate. 3

2 10 1 0 0 Relative mRNA expression Relative mRNA Relative mRNA expression Relative mRNA

Xylt1 Xylt2 Ext1 Ext2 Extl1 Extl2 Extl3 Gpc6 Sdc1 Sdc2 Sdc3 Sdc4 Gpc1 Gpc4 Hspg2

SULF2, an extracellular endosulfatase that removes 6-O-sulfate lines derived from both human and murine GBM, we identify from trisulfate disaccharides and can promote PDGFRA signaling, elevated HPSE expression in a subset of tumors, and HPSE is most highly expressed in a subset of GBM (21). In contrast, knockdown in human GBM43 or inhibition of HS function in GBM43 and mMES tumorspheres exhibited elevated expression human GBM43 or mMES tumorspheres reduced tumor cell of the HS-modifying enzyme HPSE. Heterozygous gene deletion invasion. Although the regulators of HPSE expression in GBM of HPSE in human GBM tumorspheres decreased tumor cell are not known, the NF-kB pathway, active in a subset of GBM with invasion and adhesion. Chemical inhibition of HS function a radioresistant and mesenchymal phenotype (47), can drive reduced human and murine tumorsphere invasion, and this was HPSE expression following viral infection (44). most pronounced in HPSE-high cells. These data support a role for HSPGs regulate signaling pathways important in cell prolif- HS-interacting factors, including HPSE, in tumor invasion and eration, inflammation, angiogenesis, and invasion and are highlight the heterogeneity of HS content, structure, and function dysregulated in cancer (reviewed in refs. 18 and 55). As extra- in GBM. cellular molecules, HSPGs and the extracellular enzymes that HPSE specifically liberates biologically active GAG chains from modify them, SULF2 and HPSE, are amenable to therapeutic HSPGs, and its activity is associated with both cell-autonomous targeting. HS mimetics, highly sulfated oligosaccharides, inhib- and non–cell-autonomous functions (reviewed in ref. 22). HPSE itHS-bindingenzymessuchastheSULFsandHPSE,aswellas can promote tumor cell invasion in several ways, including sequester HS-binding ligands, making them attractive candi- actions on HSPGs on the cell surface and in the ECM (10, 26, dates for cancer therapy (56, 57). In preclinical studies, HS 27, 52, 53). In multiple myeloma, HPSE promotes tumor cell mimetics have effectively targeted multiple HSPG-dependent adhesion and invasion via SDC1 ectodomain shedding and phenotypes, as indicated by their ability to decrease tumor subsequent alterations in RTK signaling and integrin binding growth, invasion, metastasis, and angiogenesis (49, 58). (10, 23, 54). Few studies have investigated the role for HPSE in Although HS mimetics have not yet been tested in GBM primary brain tumors (28–31, 46), but the data suggest that HPSE in vivo, our data suggest that HS mimetics or more selective can promote tumor growth and progression in vivo. HPSE knock- inhibitors of proteoglycan function may have antitumor effi- down in human GBM43 cells cultured under neural stem cell cacy in subsets of GBM patients. conditions did not alter proliferation. The role for HPSE in Using human and murine GBMs, our study identifies different proliferation in vitro may be complex, and potential modifiers patterns of proteoglycan expression and modification in tumors include cell culture conditions, cell lines used, and method to alter and demonstrates a role for HPSE in tumor invasion. Recent HPSE levels (31, 46). In human GBM, HPSE is most highly advances in glycan analysis from cells and tissue (59–62) open the expressed in the mesenchymal GBM subtype. In tumorsphere way for a detailed analysis of glycan structure and content in

OF8 Mol Cancer Res; 2017 Molecular Cancer Research

Heparan Sulfate Structure and Functional Modiﬁcation in GBM

ABC 0 h 40 h mMES mPN 3 **

1.5 N.S. Ctrl 2 Figure 7. 1.0 Inhibitors of HS function inhibit * invasion in HPSE-high tumorspheres. A and B, Heparin inhibits mMES invasion in a dose-dependent manner 1 Relative invasion 0.5 in 3D spheroid invasion assays. A,

Representative images of Control Relative invasion Heparin (Ctrl) and heparin (300 and m 150 g/mL)-treated mMES 0 0.0 tumorspheres at 40 hours (h) and (B) mMES 16 40 16 quantification at 16 and 40 hours. Time (h) Time (h) C, In contrast, heparin treatment of Ctrl mPN tumorspheres had no effect on Heparin 150 µg/mL invasion even at the highest Heparin 300 µg/mL concentration (300 mg/mL) at 16 hours. D and E, The heparan sulfate D 0 h 40 h E mimetic necuparanib (Necu) also reduced invasion of mMES mMES tumorspheres in 3D invasion assays. * Representative images at 40 hours (E) 2.0 and quantification at 16 and 40 hours (F). Assays performed in biological Ctrl triplicate. Mean SEM. B, , P < 0.05 1.5 and , P < 0.01, Ctrl (n ¼ 6), heparin Ctrl * 150 mg/mL (n ¼ 5), and heparin 300 Necu mg/mL (n ¼ 6). , P < 0.05, Ctrl and Necu (n ¼ 5 each). N.S., not significant 1.0 (n ¼ 6). Scale bars denote 100 mm. Necu Relative invasion 0.5

0.0 mMES 16 40 Time (h) human tumors. The integration of detailed glycomic, genomic, Administrative, technical, or material support (i.e., reporting or organizing and proteomic data will facilitate the identification of novel data, constructing databases): V.M. Tran, A. McKinney, K. Chen tumor biomarkers and help to identify tumors most likely to Study supervision: J.J. Phillips respond to inhibitors of HSPG function. Acknowledgments TheauthorsthankDrs.KuberanBalagurunathan,StevenD.Rosen,and Disclosure of Potential Conflicts of Interest Ralph Sanderson for sharing their expertise and reagents. We also thank Dr. Balagurunathan for providing us with heparitinase I, II, and III. No potential conflicts of interest were disclosed. Necuparanib (formerly M402) was generously provided by Momenta Pharmaceuticals with special thanks to Amanda MacDonald, Birgit Disclaimer Schultes, and Silva Krause. The content is solely the responsibility of the authors and does not neces- sarily represent the official views of the NIH. Grant Support This study was funded by NIH/NINDS R01 NS081117 and NIH/NCI U01 CA168878 (to J.J. Phillips), and resources were provided by NIH Grant PO1- Authors' Contributions HL107152 (to BK). Mass spectrometry was performed at the UCSF Mass Spec Conception and design: V.M. Tran, A. McKinney, J.J. Phillips Facility (NIH NCRR P41RR001614). The UCSF Brain Tumor SPORE Tissue Core Development of methodology: V.M. Tran, A. Wade (P50CA097257) provided deidentified GBM tissue for establishment of GBM1 Acquisition of data (provided animals, acquired and managed patients, tumorspheres. provided facilities, etc.): A. McKinney, K. Chen, O.R. Lindberg, J.R. Engler The costs of publication of this article were defrayed in part by the payment Analysis and interpretation of data (e.g., statistical analysis, biostatistics, of page charges. This article must therefore be hereby marked advertisement computational analysis): V.M. Tran, A. Wade, K. Chen, O.R. Lindberg, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. A.I. Persson, J.J. Phillips Writing, review, and/or revision of the manuscript: V.M. Tran, A. McKinney, Received July 6, 2017; revised July 28, 2017; accepted August 1, 2017; K. Chen, A.I. Persson, J.J. Phillips published OnlineFirst August 4, 2017.

www.aacrjournals.org Mol Cancer Res; 2017 OF9

Tran et al.

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www.aacrjournals.org Mol Cancer Res; 2017 OF11

Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion

Vy M. Tran, Anna Wade, Andrew McKinney, et al.

Mol Cancer Res Published OnlineFirst August 4, 2017.

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