glioblastoma angiogenesis and tumor cell invasiveness are differentially regulated by ... ·...

Microenvironment and Immunology

Glioblastoma Angiogenesis and Tumor Cell InvasivenessAre Differentially Regulated by b8 Integrin

Jeremy H. Tchaicha1, Steve B. Reyes1, Jaekyung Shin2, Mohammad G. Hossain1,Frederick F. Lang3, and Joseph H. McCarty1

AbstractGlioblastoma multiforme (GBM) is a highly invasive brain tumor that develops florid microvascular

proliferation and hemorrhage. However, mechanisms that favor invasion versus angiogenesis in this settingremain largely uncharacterized. Here, we show that integrin b8 is an essential regulator of both GBM-inducedangiogenesis and tumor cell invasiveness. Highly angiogenic and poorly invasive tumors expressed low levelsof b8 integrin, whereas highly invasive tumors with limited neovascularization expressed high levels of b8integrin. Manipulating b8 integrin protein levels altered the angiogenic and invasive growth properties ofGBMs, in part, reflected by a diminished activation of latent TGFbs, which are extracellular matrix proteinligands for b8 integrin. Taken together, these results establish a role for b8 integrin in differential control ofangiogenesis versus tumor cell invasion in GBM. Our findings suggest that inhibiting b8 integrin or TGFbsignaling may diminish tumor cell invasiveness during malignant progression and following antivasculartherapies. Cancer Res; 71(20); 6371–81. �2011 AACR.

Introduction

Astrocytomas, which arise from presumptive astrocytesand/or neural progenitor cells of origin (1), afflict approxi-mately 20,000 people within the United States each year (2).They represent the most common type of primary braintumor, and in their malignant stages they are one of thedeadliest forms of cancer. Multiple chromosomal abnormal-ities and gene expression defects correlate with astrocytomainitiation and progression (3). For example, low-grade astro-cytomas commonly display loss of p53, and as these tumorsprogress tomoremalignant stages, they often show deletion ofthe Ink4/Arf tumor suppressors (4). Grade IV astrocytomas, orglioblastoma multiformes (GBM), commonly display amplifi-cation of platelet-derived growth factor subunit B (PDGF-B)and/or epidermal growth factor (EGF) receptor expression,leading to hyperactivation of downstream signaling cascadesoften involving Ras (5).

GBMs develop unique angiogenesis pathologies includingmicrovascular hyperproliferation as well as edema and hemor-rhage owing to breakdown of the intratumoral blood–brainbarrier (6). GBM cells are also highly infiltrative, often disper-sing to distal regions of the brain via white matter tracts andvascular basement membranes (7, 8). Antivascular therapiescommonly enhance perivascular GBM cell invasiveness, lead-ing to the development of secondary lesions that are resistantto second-line therapies (9, 10).

GBM-induced angiogenesis and tumor cell invasion areinfluenced by a milieu of growth factors and extracellularmatrix (ECM) cues within the brain microenvironment(6). Most mammalian cells communicate with proteincomponents in the ECM via a family of cell surface re-ceptors known as integrins (11). The 5 members of the avintegrin subfamily—avb1, avb3, avb5, avb6, and avb8—are expressed in neural and vascular cells of the brainand bind to arginine-glycine-aspartic acid (RGD) peptidemotifs present in many shared ECM ligands (12). In parti-cular, genetic studies in mice have revealed that avb8integrin is a central regulator of angiogenesis in thedeveloping brain (13, 14). Targeted ablation of av or b8integrin genes in embryonic neuroepithelial cells causesbrain-specific vascular phenotypes, including endothelialcell hyperproliferation, the formation of vessels with glo-meruloid-like tufts, and intracerebral hemorrhage (15, 16).These pathologies are due to defective activation of latentTGFbs, which are ECM-bound protein ligands for avb8integrin (17, 18). In neurogenic regions of the adult mousebrain, avb8 integrin is expressed in neural stem andprogenitor cells where it governs neurogenesis (19) andneuroblast migration (20).

Authors' Affiliations: Departments of 1Cancer Biology, 2Biochemistryand Molecular Biology, and 3Neurosurgery, University of Texas MDAnderson Cancer Center, Houston, Texas

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for J.H. Tchaicha: Dana Farber Cancer Institute, 44 BinneySt., Boston, MA 02115.

Corresponding Author: Joseph H. McCarty, Department of CancerBiology, Unit 173, MD Anderson Cancer Center, 1515 Holcombe Boule-vard, Houston, TX 77030. Phone: 713-792-0429; Fax: 713-792-0429;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-0991

�2011 American Association for Cancer Research.

CancerResearch

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avb8 integrin expression and functions are dysregulatedin various brain pathologies. For example, diminished expres-sion ofavb8 integrin and reduced TGFb signaling are detectedin brain arteriovenous malformations (21). We have dis-covered that avb8 integrin–activated TGFbs suppress patho-logic angiogenesis in mosaic mouse models of astrocytoma(22). Here, we have analyzed roles for avb8 integrin inangiogenesis and tumor cell invasiveness in human GBM.Using xenograft models as well as cell culture systems, weshow that b8 integrin differentially regulates these GBMpathologies, in part, via autocrine activation of TGFb signalingpathways.

Materials and Methods

GBM cell lines and human tumor samplesApproval for the use of human specimens was obtained

from the Institutional Review Board (IRB) at The University ofTexas MD Anderson Cancer Center. The IRB waived therequirement for informed consent for previously collectedresidual tissues from surgical procedures stripped of uniquepatient identifiers according to Declaration of Helsinki guide-lines. LNZ208 and LN428 cells were provided by Dr. OliverBogler (MD Anderson Cancer Center) and were authenticatedby short tandem repeat analysis in 2009. U87, LN18, LN229,SNB19, and U373 GBM cell lines were purchased from Amer-ican Type Culture Collection (ATCC). Short tandem repeatanalyses by ATCC have revealed that SNB19 and U373 arefrom the same patient. All cells were grown in DMEM/Ham'sF12 (MediaTech) supplemented with 10% FBS (Atlanta Bio-logicals) and antibiotics. Normal human astrocytes (NHA)were purchased from Lonza, Inc. Transformed human astro-cytes (THA) have been described elsewhere (23). b8 integrinprotein was overexpressed in U87 cells using a pcDNA4.0plasmid harboring a full-length human b8 integrin cDNAfused at the C-terminus with a V5 epitope tag. After selectionfor 10 days in 300 mg/mL zeocin, stable transfectants werepooled and analyzed.

AntibodiesThe following antibodies were purchased from commercial

sources: rat anti-mouse CD34 monoclonal antibody(GeneTex), rat anti-mouse CD31 (Pharmingen), rabbit anti-a-actin (Sigma), and mouse anti-human av integrin mAb (BDBiosciences). Fluorescein isothiocyanate (FITC)-conjugatedphalloidin was purchased from Sigma. The anti-av integrinand anti-b8 integrin rabbit polyclonal antibodies used forimmunoblotting have been described elsewhere (24, 25).Secondary antibodies were goat anti-rabbit, goat anti-chicken,goat anti-rat, and goat anti-mouse, all conjugated to Alexa488or Alexa594 (Molecular Probes). Horseradish peroxidase–conjugated secondary antibodies used were sheep anti-mouse,goat anti-rabbit, and donkey anti-chicken IgY (JacksonImmunoResearch Labs). NIH ImageJ software was used forquantitation of intratumoral blood vessel densities based onanti-CD34 fluorescence intensity.

To generate the anti-b8 integrin antibody used for GBMimmunohistochemistry, a 43-kDa fragment of the b8 integrin

extracellular domain (b8ex) was expressed in bacteria as aglutathione S-transferase (GST) fusion protein using thepGEX-6P expression plasmid (GE Healthcare). The DNA pri-mer sequences used to amplify the b8ex region are as follows:50-TCAGTTGATTCAATAGAATACC-30 and 50-CTGTGTATAT-GAATTTTAGCG-30. GST-b8ex protein (�67 kDa) was foundexclusively in detergent-insoluble inclusion bodies. GST-b8exwas solubilized in buffer containing 6 mol/L urea and sequen-tially dialyzed into urea-free buffer. Soluble recombinantprotein was fractionated with glutathione agarose. The GSTportion was cleaved with PreciScission Protease (GE Health-care) to liberate the 43-kDa b8ex protein, which was used toimmunize rabbits (Covance).

Immunohistochemistry and immunofluorescenceFormalin-fixed paraffin-embedded (FFPE) sections were

deparaffinized in 2 changes of xylene and then dehydratedin 2 changes of 100% ethanol, 95% and 80% ethanol, anddistilled water. For anti-av integrin immunohistochemistry,slides were incubated at 95�C to 100�C with pH 9.0 antigenretrieval buffer (DAKO) for 40 minutes and then transferred toroom temperature before immunostaining. Alternatively,deparaffinized sections were incubated in blocking solutionand then primary anti-b8ex antibody was added. Sectionswere rinsed 3 times with PBS, blocked with peroxidase solu-tion, incubated with secondary antibodies conjugated tohorseradish peroxidase, and then vasoactive intestinal peptideor 3,30-diaminobenzidine chromogens were added to visualizeimmunoreactivity. For quantitation of blood vessel densitiesin tumors generated from U87 cells and THAs, digital imageswere selected from 3 intratumoral regions labeled with anti-CD34 antibodies. Three serial sections were analyzed from 3different mice injected with the various cell types. Meanfluorescence intensity was calculated by NIH ImageJ software.Two investigators, who were blinded to the identities of eachsample, analyzed sections.

Generation of lentivirus-expressing short hairpin RNAsFor lentiviral-mediated gene silencing, the following

synthetic oligonucleotides were used: 50-TGTATCCTCATC-ATGATGTGTTCAAGAGACACATCATGATGAGGATACTTTT-TTC-30 and 50-TCGAGAAAAAAGTATCCTCATCATGATGTG-TCTCTTGAACACATCATGATGAGGATACA-30.

The oligonucleotides were ligated into the pLB lentiviralgene transfer vector (26), using HpaI and XhoI restriction sites.This pLB vector contains a U6 promoter that drives expressionof short hairpin RNAs (shRNA), and a CMV promoter drivinggreen fluorescent protein (GFP) expression. Packaged lenti-viruses were generated by transfecting HEK 293-FT cells(ATCC) with pLB transfer vectors in combination with plas-mids encoding gag/pol and VSV-G envelope proteins.

Stereotactic injectionsAll animal procedures were conducted under Institutional

Animal Care and Use Committee–approved protocols.NCR-nu/nu male mice (Jackson Laboratories) were anesthe-tized, and a single incision was made from the anterior pole ofthe skull to the posterior ridge. We targeted the striatum for

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cell implantation, using the following stereotactic coordinates:1.5 mm rostral, 1.5 mm anterior, and 4 mm below the pialsurface. An automated micropump (Stoelting Instruments)was used to dispense cells in 3 mL PBS over a 5-minute period.To generate intracranial tumors from THAs, we injected2.5 � 105 cells for the analysis of tumor-induced angiogenesis,tumor cell invasiveness, and tumor volumes. Kaplan–Meiersurvival analysis was conducted after injecting 5 � 104 THAsper mouse. Human intracranial xenograft tumors were gen-erated by injecting 5 � 105 U87 or SNB19 cells into thestriatum of NCR-nu/nu mice. Animals were monitored fortumor-induced neurologic deficits, and moribund animalswere euthanized with gaseous CO2. Mice were perfused with4% paraformaldehyde/PBS, brains were coronally sliced at1-mm intervals, and paraffin-embedded tissues were seriallysectioned at 7-mm intervals.

PAI1-luciferase assaysTHAs were transiently transfected with a PAI1-luciferase

plasmid (27). A total of 4 � 105 cells were seeded onto 6-wellplates coated with poly-D-lysine (Sigma), and 4 hours later, themedium was removed and serum-free medium was added for24 hours. Cell lysates were prepared and firefly luciferaseactivities were quantified (Enhanced Luciferase Assay Kit;BD Biosciences).

Invasion and migration assaysA total of 5 � 104 THAs were added in the serum-free

medium to the top chambers of Matrigel invasion Transwellsystems (BD Biosciences; 8-mm pore sizes). Normal growthmedium containing 10% FBS was added to the bottom wellsand invasion was quantified after 18 hours by staining theTranswell filters with crystal violet. Alternatively, THAswere plated onto laminin-coated coverslips in 24-well dishes(1 � 105 cells per well). After reaching confluence, cells werewounded with a 10-mL pipette tip. Cell migration wasanalyzed at various time points by fixing cells and labelingwith phalloidin-FITC or anti-paxillin antibodies. Alterna-tively, migration was imaged by time-lapse microscopy withan Olympus IX81 inverted microscope mounted on anautomated stage and equipped with a humidified chamberand a DP25 digital camera. SlideBook software (IntelligentImaging Innovations) was used to quantify migration.Migration was analyzed in separate frames, and woundclosure was defined when 5 migrating cells from oppositesides of the scratch (within a 200� field) made contactswithin the wound region.Alternatively, siRNA pools (Dharmacon) targeting human

b8 integrin were transfected into LN229 GBM cells usingmanufacturer-supplied transfection reagents and protocols.After 48 hours, transfection cells were either lysed to confirmintegrin silencing by immunoblotting or analyzed in theMatrigel invasion system using 5 � 104 cells per Transwellas detailed earlier.

Statistical analysesThe Student t test was conducted to determine sta-

tistically significant differences between groups. The

Wilcoxan rank-sum test was used for analysis of Kaplan–Meier survival results. Microsoft excel was used to calculatestatistics.

Results

Analysis of b8 integrin protein expression patterns inhuman GBM cell lines in vitro and GBMs in situ

We used anti-b8 integrin and anti-av integrin antibodiesto screen NHAs, THAs, and 7 different human GBM celllines for levels of integrin protein expression. As shown inFigure 1A, all cell types expressed similar levels of avintegrin protein whereas b8 integrin protein was expressedat varying levels in the different cell types. NHAs expressedrobust levels of b8 integrin, and oncogene-mediated trans-formation of these cells resulted in a significant reductionin integrin protein expression. The human GBM cell linesU373, LNZ208, LN428, SNB19, and LN229 all expressedrobust levels of b8 integrin protein. In contrast, U87 andLN18 cells expressed low or undetectable levels of b8 integ-rin protein (Fig. 1A). Plasma membrane expression of avb8integrin protein was confirmed using a membrane-impermeable reactive biotin derivative to label cells. Deter-gent-soluble lysates were then immunoprecipitated withanti-av and anti-b8 integrin antibodies to reveal cell surfaceexpression (Supplementary Fig. S1).

We also compared integrin expression levels by immuno-blotting lysates prepared from 7 different freshly resectedGBM samples. All samples expressed similar levels of avintegrin protein, and 6 of 7 samples expressed robust levelsof b8 integrin protein, with 1 sample expressing lower levels ofb8 integrin (Fig. 1B). Robust levels of avb8 integrin proteinwere also detected in lysates prepared from 3 freshly resectedgrade III anaplastic astrocytomas, 2 grade IV oligodendroglio-mas, and 1 gliosarcoma (data not shown).

The anti-b8 integrin antibody used for immunoblotting(Fig. 1A and B) was not compatible for immunohistochemicallabeling of FFPE tumor sections. Therefore, we generatedpolyclonal antisera directed against the extracellular portionof human b8 integrin. The antiserum is highly specific forhuman b8 integrin protein and shows minimal cross-reactiv-ity with mouse b8 integrin (Supplementary Figs. S2 and S3).FFPE samples corresponding to 3 of 7 GBM samples analyzedin Figure 1B were analyzed by anti-av and anti-b8 integrinimmunohistochemistry. As shown in Figures 1C, a commer-cially available anti-av mAb revealed integrin protein expres-sion in intratumoral blood vessels as well as in GBM cells. Theblood vessel immunoreactivity was likely due to avb3 integrin,which is expressed in vascular endothelial cells of GBM andother malignant neural tumors (28, 29). We detected b8integrin protein expression in most GBM cells (Fig. 1C);however, little if any b8 integrin protein was detected inthe intratumoral vasculature. These data are consistent witha prior report showing b8 integrin mRNA and protein expres-sion in human GBM cells but not blood vessels (30). Theseimmunohistochemical results confirm our analyses of av andb8 integrin protein expression in lysates prepared from NHAs,GBM cell lines, and GBMs (Fig. 1A and B).

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Exogenous expression of b8 integrin in U87 GBM cellsleads to diminished intratumoral vascular pathologies

To analyze b8 integrin functions in GBMs in vivo, weforcibly expressed V5-tagged human b8 integrin protein inU87 GBM cells, which express low levels of endogenous b8integrin (Fig. 1A). In comparison to control U87 cells trans-fected with empty vector, increased b8 integrin proteinexpression was detected in pools of cells stably overexpres-sing b8-V5 integrin (Fig. 2A). Next, stably transfected controlcells or U87 cells overexpressing b8-V5 protein were injectedinto the brains of NCR-nu/nu mice (n ¼ 5 mice per geno-type). After 4 to 6 weeks, mice were sacrificed, cardiac-perfused with fixative, and brains were sliced coronally toanalyze integrin-dependent differences in tumorigenicity.As shown in Figure 2B, U87 cells stably transfected withempty plasmid and U87 cells stably expressing b8-V5 integ-rin both generated large intracranial tumors. Tumors gen-erated from control U87 cells lacking b8 integrin expression,however, showed grossly obvious hemorrhage and edema.In contrast, U87 tumors expressing b8-V5 protein were

vascularized but did not contain severe hemorrhage andedema. Intratumoral blood vessel densities were quantifiedby immunolabeling U87 tumor sections with anti-CD34antibodies (n ¼ 3 different tumors per cell type, n ¼ 3sections per tumor). As shown in Figure 2C, we detecteddistended, sinusoid-like vessels throughout GBMs generatedfrom U87 cells stably transfected with empty plasmids.U87 cells stably overexpressing b8-V5 protein also yieldedwell-vascularized tumors; however, the abnormal bloodvessel morphologies were not evident, with most vesselsdisplaying small, capillary-like morphologies. In addition,intratumoral vascular densities, based on quantitation ofCD34 fluorescence, were diminished significantly in U87tumors overexpressing b8 integrin protein (Fig. 2D).

RNA interference–mediated silencing of b8 integringene expression impacts angiogenesis and tumor cellinvasiveness in human glioblastoma multiformes

We selected LN229 and SNB19 GBM cell lines thatexpressed high levels of endogenous b8 integrin protein

Figure 1. Analysis of b8 integrin protein expression in human GBM cell lines and freshly resected tumor samples. A, detergent-soluble lysates from NHAs,THAs, and 7 different human GBM cell lines were immunoblotted with anti-av or anti-b8 integrin antibodies. Note that NHAs express robust levels of av andb8 integrin proteins, with oncogene-induced transformation leading to reduced integrin expression in THAs. In addition, av and b8 integrin proteins areexpressed at varying levels in the different human GBM cell lines. B, detergent-soluble lysates from 7 different primary GBM samples were immunoblottedwith anti-av or anti-b8 integrin antibodies, revealing integrin protein expression in the different samples. C, FFPE sections from 3 different grade IVastrocytomas (GBM) stained with H&E (top), anti-av integrin antibodies (middle), or anti-b8 integrin antibodies (bottom). Note that av integrin protein isexpressed in GBM cells as well as in intratumoral blood vessels. b8 integrin protein is also expressed in GBM cells but is largely absent in intratumoralblood vessels. Images are shown at 400� magnification.

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(Fig. 1A) and attempted to stably silence integrin expression,using lentiviral-delivered shRNAs. These efforts resulted inonly approximately 60% reduction in b8 integrin proteinexpression in pools of stably transfected cells (data notshown). Analysis of in vitro and in vivo growth and invasiveproperties in LN229 and SNB19 cells expressing diminishedb8 integrin protein, however, did not reveal obvious differ-ences in comparison with control cells (data not shown).Our prior studies of cultured astrocytes and neural progeni-tors from b8þ/� mice, which express 50% less b8 integrinprotein in comparison with wild-type controls, revealed nodifferences in cell behaviors (data not shown), suggesting

that nearly complete silencing of integrin expression islikely required to reveal functional roles in cells. Therefore,we stably silenced b8 integrin expression in THAs, whichexpress lower levels of b8 integrin protein (Fig. 1A) andrepresent a more genetically tractable model system forin vitro and in vivo studies. THAs were generated by in-fecting NHAs with retroviruses engineered to expresshuman papilloma virus E6 and E7 proteins, oncogenicH-Ras (G12VH-Ras), and human telomerase reverse tran-scriptase (hTERT; ref. 23). E6/E7 oncoproteins inhibit theexpression and function of the p53 and Rb tumor sup-pressors, respectively. p53 is commonly mutated or deleted

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Figure 2. b8 integrin suppresses microvascular pathologies and hemorrhage in GBM. A, U87 cells were stably transfected with empty plasmid or aplasmid containing a cDNA encoding for a b8-V5 fusion protein. Detergent-soluble lysates were then immunoblotted with antibodies directed against anti-b8integrin or anti-actin antibodies. Note the increased expression of b8 integrin protein. B, U87 cells stably transfected with empty plasmid (top) or plasmidharboring a b8-V5 cDNA (bottom) were stereotactically injected into the striatum of immunocompromised mice (n ¼ 5 mice per cell type). Shown aregross images of slices from representative brains harboring tumors generated from each cell type. Note that the intratumoral hemorrhage evident in the U87tumors is not evident in tumors overexpressing b8-V5 protein. C, U87 tumors stably transfected with empty plasmid or plasmid expressing b8-V5 wereimmunostained with an anti-CD34 antibody to visualize vascular endothelial cells (n ¼ 3 different brains per cell type). Note the increased numbers ofblood vessels and abnormal morphologies in U87 tumors transfected with empty plasmid versus the capillary-likemorphologies in U87 tumors overexpressingb8 integrin. D, immunofluorescence intensities were quantified on the basis of anti-CD34 antibody staining (shown at 200�magnification). Note the significantreduction of fluorescent intensity in U87 tumors forcibly expressing b8-V5 integrin protein (*, P < 0.0001). Error bars, SDs.






in human GBMs, and both p53 and Rb negatively regulatethe functions of the Ink4a/Arf tumor suppressors, which arealso commonly deleted in malignant astrocytomas (31, 32).Ras is often hyperactivated in GBM, owing to gene mutationsor elevated expression of EGF receptors (33), and hTERToverexpression is necessary for maintaining telomeres inhuman cancer cells (34).

THAs were stably transduced with either scrambledshRNAs (controls) or 2 different shRNAs targeting b8 integrin,and cells were fractionated on the basis of GFP expression(Supplementary Fig. S4A and B). Integrin gene silencing wasconfirmed by immunoblotting detergent-soluble lysates(Fig. 3A) or immunoprecipitating integrins from biotinylatedcell lysates with anti-av and anti-b8 antibodies (Fig. 3B). BothshRNAs significantly reduced b8 integrin expression, althoughone was used to analyze THA behaviors in vitro and in vivo.In comparison with THAs expressing scrambled shRNAs, cells

expressing b8 shRNAs produced significantly more coloniesin 3-dimensional soft agar assays (Supplementary Fig. S4C).We next analyzed b8 integrin–dependent effects on tumorgrowth, angiogenesis, and tumor cell invasiveness in vivo.THAs expressing scrambled shRNAs or b8 shRNAs werestereotactically injected in the striatum of immunocompro-mised mice (n ¼ 5 mice per cell type, 2.5 � 104 cells permouse). Within 5 to 6 weeks after injection, all mice developedobvious signs of tumor burden including weight loss andneurologic deficits such as ataxia. Nearly all mice (n ¼ 4 of5 animals) injected with THAs expressing b8 shRNAs alsodeveloped severe hydrocephaly (data not shown). Gross ana-lysis of intracranial tumors derived from THAs expressingcontrol shRNAs reveal localized lesions adjacent to the lateralventricles; in contrast, astrocytomas formed from THAsexpressing diminished b8 integrin protein were significantlylarger and showed severe edema and hemorrhage (Fig. 3C).

Figure 3. Silencing b8 integrin expression in THAs leads to enhanced tumor growth. A, THAs were transduced with pLB lentivirus expressing nontargetingscrambled shRNAs (control) or shRNAs targeting human b8 integrin (b8 shRNA). Detergent-soluble lysates were immunoblotted with anti-integrin antibodies,revealing a significant reduction in b8 integrin protein expression. B, THAs were incubated with an amine-reactive biotin to label cell surface proteins.Detergent-soluble lysates were then immunoprecipitated with anti-av or anti-b8 integrin antibodies. THAs expressing control and b8 shRNAs containrobust levels of av-containing integrins on their cell surfaces (left). In contrast, note the low levels of cell surface–expressed avb8 integrin protein inTHAs expressing b8 shRNAs (right). IP, immunoprecipitation. C, NCR-nu/nu mice (n ¼ 8 per cell type) were stereotactically injected with THAs expressing b8shRNAs (right) or scrambled shRNAs (left). Shown are images of 2 representative brains containing astrocytomas. Tumors generated from THAs expressing b8shRNAs are larger and display severe hemorrhage (arrows). D, significantly larger astrocytoma volumes, as determined by measuring cross-sectionalareas of H&E-stained tumors expressing b8 shRNAs versus scrambled shRNAs (left). Kaplan–Meier analysis reveals that mice harboring tumors generatedfrom THAs expressing b8 shRNAs die earlier than animals harboring tumors generated from THAs expressing control shRNAs (right); *, P < 0.05. The meansurvival times for mice bearing tumors derived from control THAs is 84 days, whereas the mean survival for mice harboring tumors derived from THAsexpressing b8 shRNAs is 62 days.

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Quantitation of tumor volumes in hematoxylin and eosin(H&E)-stained histologic sections revealed that astrocytomasexpressing b8 shRNAs were significantly larger (Fig. 3D, left).Kaplan–Meier survival comparisons revealed that mice har-boring astrocytomas expressing b8 shRNAs died significantlyearlier (Fig. 3D, right), likely due to larger tumor sizes andassociated neurologic deficits.Coronal sections were H&E stained to reveal that THAs

expressing control shRNAs were well vascularized, with intra-tumoral endothelial cells expressing laminin (Fig. 4A, left).Astrocytomas expressing b8 shRNAs contained obviousabnormalities in blood vessel morphologies as revealed byH&E and anti-laminin staining (Fig. 4A, right), with manyvessel displaying distended, sinusoid-like morphologies.Quantitation of intratumoral blood vessels by anti-CD34immunofluorescence and quantitation using ImageJ softwarerevealed significantly higher numbers of vessels in astrocyto-mas expressing b8 shRNAs (Fig. 4B). Astrocytomas expressing

scrambled shRNAs grew in periventricular regions of thebrain and were infiltrative, with invading astrocytoma cellsdetected several micrometers away from the main tumormass (Fig. 4C, left). In contrast, THAs expressing b8 shRNAsgenerated larger, noninfiltrative astrocytomas. Dissemina-tion of GFP-expressing tumors cells into the surroundingbrain parenchyma was not detected in the absence of b8integrin expression (Fig. 4C, right). Invasion assays through3-dimensional ECM were also used to quantify integrin-dependent THA invasion. As shown in Figure 4D, in com-parison with THAs expressing scrambled shRNAs, cellsexpressing b8 shRNAs showed a significant decrease ininvasiveness. This integrin-dependent defect in cell invasionwas not specific for THAs, as LN229 human GBM cells, whichexpress robust levels of b8 integrin (Fig. 1A), showed asignificant reduction in invasiveness following transientsiRNA-mediated silencing of b8 integrin expression (Supple-mentary Fig. S5).

Figure 4. Microscopic analysis of integrin-dependent tumor-induced angiogenesis and tumor cell invasiveness. A, coronal sections from control (left, top)and b8 shRNA (right, top) astrocytomas were stained with H&E. Sections were also immunolabeled with an antibody directed against laminin (LM; bottom).Note the enlarged blood vessel sizes and abnormal morphologies in tumors derived from THAs expressing b8 shRNAs (arrows, right bottom). Images areshown at 200� magnification. B, coronal sections from control or b8 shRNA astrocytomas (n ¼ 3 tumors per cell type, n ¼ 5 sections per tumor) wereimmunostained with anti-CD34 to visualize vascular endothelial cells, and fluorescence intensities were quantified; *, P < 0.001. Error bars, SDs. C,H&E-stained coronal sections from astrocytomas generated from THAs expressing control shRNAs (left, top) or shRNAs targeting b8 integrin (right, top).Coronal sections from control (left, bottom) and b8 shRNA tumors (right, bottom) were immunofluorescently labeled with an antibody directed against GFP,revealing robust expression in tumor cells. Note the well-defined and noninvasive tumor peripheries in b8 shRNA samples (arrows, right bottom) as comparedwith the infiltrative tumor cells in control astrocytomas (arrows, left bottom). Images are shown at 200� magnification. D, THAs expressing b8 shRNAsshowed a significant reduction in invasiveness through 3-dimensional extracellular matrices as compared with THAs expressing scrambled shRNAs.Triplicate samples were analyzed for each cell type; *, P < 0.001. Error bars, SDs.






Hypoxia within the tumor microenvironment has beenreported to influence GBM cell growth and invasiveness;for example, mouse astrocytoma cells genetically null forhypoxia-inducible factor-1 a (HIF-1a) or the HIF-1a–inducible gene, VEGF-A, show highly invasive intracranialgrowth patterns (35). To address potential functional linksbetween hypoxia and avb8 integrin–mediated astrocytomacell invasiveness, HIF-1a protein levels were interrogated inastrocytomas derived from control THAs or THAs manipu-lated to express diminished levels of b8 integrin (n¼ 3 tumorsper cell type, n ¼ 3 sections per tumor). In comparison withtumors expressing control shRNAs (Supplementary Fig. S6A),we detected elevated levels of HIF-1a protein in tumorsexpressing b8 shRNAs (Supplementary Fig. S6B). Integrin-dependent increases in HIF-1a protein expression were notdetected in vitro (Supplementary Fig. S6C). We detected anapproximately 50% reduction in VEGF-A protein in THAsexpressing b8 shRNAs (Supplementary Fig. S6D), althoughintegrin-dependent differences in VEGF-A protein expression

in astrocytoma sections were not evident (data not shown).Taken together, these data suggest that b8 integrin is inter-connected with the HIF-1a/VEGF-A signaling cascade andthese events drive astrocytoma cell invasiveness.

b8 integrin–mediated TGFb activation and signalingare essential for astrocytoma cell polarity anddirectional migration

In the developing brain, b8 integrin is a receptor for ECM-bound latent TGFbs and mediates their activation and sub-sequent receptor engagement (36). To investigate roles foravb8 integrin–mediated TGFb activation in astrocytoma cells,THAs expressing scrambled shRNAs or b8 shRNAs weretransiently transfected with a reporter plasmid consistingof the TGFb-responsive PAI1 promoter driving expressionof firefly luciferase (27). Levels of firefly luciferase activitywere analyzed after 24 hours. As shown in Figure 5A, approxi-mately 4-fold less luciferase activities were detected in THAsexpressing b8 shRNAs, reflecting diminished endogenous b8

Figure 5. Integrin-activated TGFbs promote astrocytoma cell migration. A, THAs expressing scrambled shRNAs or b8 shRNAs were transiently transfectedwith a PAI1-luciferase plasmid to measure TGFb signaling. Triplicate samples were analyzed for each cell type; *, P < 0.001. Error bars, SDs. B,confluent monolayers of control or b8 shRNA THAs were scratched, and 24 hours later, cells were labeled with phalloidin-FITC. Note that THAs expressingscrambled shRNAs fill the wound area within 24 hours, whereas THAs expressing b8 shRNAs show diminished migration. Exogenous addition of TGFb1significantly enhanced THA directional migration. Double dashed lines indicate wound boundaries and single dashed white lines indicate closedwounds. Images are shown at 100� magnification. C, quantification of integrin-dependent cell migration. Confluent monolayers of THAs expressingscrambled or b8 shRNAs were scratched and migration was imaged over 48 hours by time-lapse microscopy (n ¼ 3 different samples per cell type).THAs expressing scrambled shRNAs (gray bars) filled the wound area within 24 hours, whereas THAs expressing b8 shRNAs (black bars) show diminishedmigration and required nearly 36 hours to fill the wound region. Exogenous addition of TGFb1 significantly enhanced THA directional migration. *, P ¼ 0.006;**, P ¼ 0.03. The migration rates between control cells with or without TGFb1 are not statistically significant. Error bars, SDs.

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integrin–mediated TGFb activation and signaling. Theseresults showing b8 integrin as a mediator of latent TGFb1activation and signaling in astrocytoma cells are consistentwith our prior data revealing that neural stem and progenitorcells also use b8 integrin to activate latent TGFbs to controlcell growth and differentiation in neurogenic regions of themouse brain (19).Astrocytoma cell polarity and directional migration were

next analyzed by in vitro scratch-wound assays (37). Confluentmonolayers of THAs expressing scrambled shRNAs or b8shRNAs (n ¼ 3 different samples per cell type) were woundedwith a pipette tip. After 2 hours, polarity was monitored bylabeling cells with phalloidin or anti-paxillin antibodies tovisualize the actin cytoskeletal network and cell–ECM con-tacts, respectively. THAs expressing control shRNAs displayedpaxillin-positive adhesion sites at the leading edge (Supple-mentary Fig. S7A), whereas THAs expressing b8 shRNAsdisplayed fewer paxillin-containing focal adhesion complexesat their leading edges (Supplementary Fig. S7B). THAs expres-sing scrambled shRNAs polarized in the direction of thewound and formed well-organized actin cytoskeletal networks(Supplementary Fig. S7C); in contrast, THAs expressing b8shRNAs showed diminished polarization at the wound edge,with few cells forming actin-rich projections into the woundregion (Supplementary Fig. S7D). Over a 24-hour period, THAsexpressing scrambled shRNAs largely filled the wound region,whereas migration by THAs expressing b8 shRNAs was sig-nificantly impaired, as evidenced by failure to fill the woundarea at 24 hours (Fig. 5B).TGFbs have been reported to promote cell migration in

development and cancer (38, 39); therefore, we analyzedwhether active TGFbs might enhance THA migration in vitro.As shown in Figure 5B, the addition of 1 ng/mL of TGFb1enhanced directional migration in THAs expressing b8shRNAs. To quantify roles for integrin-mediated TGFb activa-tion in THA migration, we imaged cell motility over 48 hoursby time-lapse bright field microscopy (Supplementary Fig. S7).THAs expressing scrambled shRNAs required approximately

24 hours to fill the wound regions, whereas THAs expressingb8 shRNAs required nearly 36 hours to fill the wound (Fig. 5C).Exogenous addition of TGFb1 showed a statistically signifi-cant rescue of the migration defects in THAs expressing b8shRNAs (Fig. 5C and Supplementary Fig. S7). These data revealthat the b8 integrin–dependent astrocytoma cell polarity andmigration defects are due, in part, to defective integrin-activated TGFb signaling. These results also support ourin vivo data showing diminished invasion by astrocytomacells lacking b8 integrin (Fig. 4) and support a model in whichb8 integrin–mediated TGFb activation is differentiallyinvolved in GBM neovascularization and tumor cell invasive-ness (Fig. 6).

Discussion

The data in this report reveal for the first time that avb8integrin, via activation of its latent TGFb protein ligands, playscentral roles in regulating GBM-induced angiogenesis andperivascular tumor cell invasiveness. Although various reportshave shown elevated levels of TGFb receptor signaling (40–42)in GBM cells, the factors that activate latent TGFbs within thetumor microenvironment remain obscure. Our data also showthat avb8 integrin is a positive regulator of the TGFb signalingcascade in invasive GBM cells. We propose that distinctpopulations of GBM cells selectively express high versuslow levels of avb8 integrin, with integrin protein expressionlevels directly determining tumor cell behaviors (Fig. 6).Although the exact mechanisms that regulate b8 integrinexpression in GBM cells remain unknown, a recent reporthas shown that b8 integrin is a direct target for miR-93, withmicroRNA (miR)-mediated inhibition of b8 integrin expres-sion correlating with enhanced angiogenesis and tumorigeni-city (43). Therefore, it is enticing to speculate that duringtumor growth and progression, subpopulations of GBM cellsselectively express high levels of miR-93, thus leading todiminished levels of b8 integrin and the development ofangiogenesis pathologies. We also postulate that invasive

Figure 6. A model for b8 integrin–mediated regulation of GBM angiogenesis versus tumor cell invasiveness. A, in the normal brain, astrocytesand neural progenitor (NP) cells express avb8 integrin and regulate cerebral blood vessel development and physiology via activation of TGFb signalingpathways. B, genetic mutations in NPs induce their transformation and initiate development of astrocytomas that maintain avb8 integrin expression.C and D, as astrocytomas progress to GBM, subpopulations of tumor cells express different levels of avb8 integrin protein. Cells with low avb8 integrinprotein (avb8lo) activate diminished levels of TGFbs and contribute to angiogenesis and vascular permeability pathologies. C, in contrast, cellsthat express elevated levels of avb8 integrin protein (avb8hi) activate more TGFbs and drive invasive GBM growth properties. D, differential levels ofintegrin-mediated TGFb activation and signaling likely cooperate with the HIF-1a/VEGF-A pathway to regulate angiogenesis versus tumor cellinvasiveness.






GBM cells express low levels of miR-93 and high levels of b8integrin, leading to proinvasive behaviors likely via autocrineactivation of TGFb signaling pathways. Interestingly, p-38and Sp-1 have also been reported to regulate b8 integringene expression (44), suggesting that genetic and epigeneticevents may cooperatively control b8 integrin expressionand functions in GBM-induced angiogenesis and tumor cellinvasiveness.

Putative tumor suppressor–like roles for avb8 integrin havebeen reported in malignant cancers of the lung (45) and skin(46). Although adult b8�/� mice do not develop spontaneousbrain tumors (19), we have found that THAs expressingdiminished levels of b8 integrin form more colonies in vitroin soft agar assays and generate significantly larger astrocy-tomas in vivo, suggesting growth-suppressive functions forthis integrin. Tumor suppressor–like roles for integrin avb3have been reported in mouse models of GBM. For example, inmouse astrocytoma cells, avb3 integrin normally suppressescell proliferation and genetic ablation of b3 integrin expres-sion leads to enhanced tumorigenicity (47). However, humanGBMs often display elevated levels of avb3 integrin, suggest-ing roles for this integrin in promoting tumor cell growthand/or invasion (48, 49). Indeed, small-molecule inhibitors ofavb3 cause diminished GBM cell growth and invasivenessin vitro and in vivo (50). avb3 integrin has been reported toactivate focal adhesion kinase in cultured GBM cells (51) andinhibition of focal adhesion kinase activities leads to dimin-ished GBM cell growth in vivo (52). It will be interesting todetermine whether avb8 integrin cooperatively signals withother integrins in GBM cells to regulate the activation ofcommon intracellular effectors such as focal adhesion kinase.

Hypoxia within the tumor microenvironment also influ-ences GBM cell growth and invasiveness. For example, loss ofHIF-1a–inducible VEGF-A expression in mouse astrocytomacells causes enhanced perivascular dispersal (35, 53). Inter-estingly, astrocytoma cells that express b8 shRNAs and are

poorly invasive show elevated levels of HIF-1a protein,although in these cells we detected reduced levels of VEGF-A (Supplementary Fig. S6). Prior reports have shown thatTGFb-activated Smads function in concert with HIF-1a todrive VEGF-A gene expression (54, 55), and THAs expressingb8 shRNAs show diminished TGFb activation (Fig. 5A). There-fore, we propose that these lower levels of VEGF-A expressionare due, in part, to diminished integrin-activated TGFb signal-ing, resulting in impaired functional links between Smads andHIF-1a. Finally, although our data point to critical roles inavb8 integrin–activated TGFbs in GBM-induced angiogenesisand tumor cell invasiveness in mouse models, these resultsrequire further corroboration in human GBM samples. It willbe important to analyze levels of integrin protein expressionand TGFb signaling in invasive tumor cells in situ to determinethe relative importance of this pathway in GBMs and otherinvasive human cancers.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Dr. Russell Pieper (UCSF) for providing the transformedhuman astrocytes and Dr. Nami McCarty (UT-HSC) for helpful advice onlentivirus manipulations.

Grant Support

This research was supported by grants awarded to J.H. McCarty from theEllison Medical Foundation (AG-NS-0324-06), the National Institutes of Neu-rological Disease and Stroke (R01NS059876-03), and the National CancerInstitute (P50CA127001-02).

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

Received March 21, 2011; revised August 1, 2011; accepted August 16,2011; published OnlineFirst August 22, 2011.

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2011;71:6371-6381. Published OnlineFirst August 22, 2011.Cancer Res Jeremy H. Tchaicha, Steve B. Reyes, Jaekyung Shin, et al.

8 IntegrinβAre Differentially Regulated by Glioblastoma Angiogenesis and Tumor Cell Invasiveness

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