ExpressionofInterleukin-4ReceptorAlphainHumanPleural...

Human Cancer Biology

Expression of Interleukin-4 Receptor Alpha in Human PleuralMesothelioma Is Associated with Poor Survival andPromotion of Tumor Inflammation

Bryan M. Burt1, Andrew Bader1, Daniel Winter1, Scott J. Rodig2, Raphael Bueno1, and David J. Sugarbaker1

AbstractPurpose: The origin and pathogenesis of malignant pleural mesothelioma (MPM) are closely aligned

with inflammation.MPM tumors express interleukin-4 receptora (IL-4Ra), the principal subunit of the IL-4receptor. We set out to determine the biologic function and clinical relevance of IL-4Ra in human MPM.

Experimental Design: Expression of IL-4Ra by human MPM tumors was determined by quantitative

real-time PCR (n ¼ 37) and immunohistochemistry (n ¼ 52). Intracellular cytokine analysis of T-cell–

derived IL-4 was carried out on matched tumor and blood samples from eight patients with MPM. Four

human MPM cell lines were used to determine the direct effects of IL-4 on MPM tumor cells.

Results: High tumor mRNA expression of IL-4Ra was an independent predictor of poor survival in

patients with epithelial MPM [HR, 3.13, 95% confidence interval (CI), 1.68–7.15; P ¼ <0.0001]. Ninety-

sevenpercent of epithelialMPM tumors and95%ofnonepithelialMPM tumors expressed IL-4Raprotein by

immunohistochemistry, and strong IL-4Ra staining correlated with worse survival in patients with

epithelial histology (P ¼ 0.04). A greater percentage of tumor-infiltrating T cells produced IL-4 compared

with matched blood T cells (21% � 7% vs. 4% � 2%, P ¼ 0.0002). In response to IL-4, human MPM cells

showed increased STAT-6 phosphorylation and increased production of IL-6, IL-8, and VEGF, without effect

on proliferation or apoptosis.

Conclusions: Tumor expression of IL-4Ra is inversely correlated with survival in patients undergoing

surgical resection for epithelial MPM. Tumor-infiltrating T cells in MPMs are polarized to produce IL-4 and

may provide endogenous activation signals to MPM tumor cells in situ. The IL-4/IL-4 receptor axis is a

potential therapeutic target in human MPM. Clin Cancer Res; 18(6); 1568–77. �2012 AACR.

IntroductionMalignant pleural mesothelioma (MPM) is an aggressive

tumor that arises from the mesothelial lining of the pleura.It is a unique malignancy whose primary mode of spread isinvasion of surrounding tissues and whose proclivity fordistantmetastases is low.MPM is a highly fatal tumor that isresilient to single modality treatment with surgery, chemo-therapy, or radiation.Withmultimodality therapy in select-ed patients, median survival is 19 months (1). Approxi-mately two-thirds of patients with MPM have epithelialhistology. The remaining one third of patients have non-epithelial histology (sarcomatoid or biphasic), which are

more aggressive tumors with significantly worse survival(2).

It recently has become evident that chronic inflammationmay predispose individuals to develop cancer. Inflamma-tion promotes the proliferation and survival of malignantcells, supports angiogenesis and metastasis, and subvertsimmune surveillance and responses to chemotherapy (3).MPM, in particular, is a cancer whose origin and pathogen-esis are closely aligned with inflammation. Occupationalexposure to asbestos is identified as the etiology of MPM in80% of cases (4). Asbestos fibers are thought to initiate acarcinogenic inflammatory reaction marked by an influx ofphagocytes into the tumor and the release of an array ofproinflammatory cytokines, such as TNF-a, that promotemalignant transformation of the mesothelium via NF-kB–dependentmechanisms (5). In humanMPM tumors, ongo-ing inflammation is represented by massive leukocyte infil-trates of T lymphocytes and macrophages whose densitiesare correlated with survival (6, 7).

Interleukin-4 receptor a (IL-4Ra) is the major subunit ofthe IL-4 receptor. In addition to being present on immunecells such as B cells, T cells, and macrophages, IL-4Ra isexpressed on a variety of cancer cell lines (8), and in situ on anumber of solid human tumors including non–small cell

Authors' Affiliations: 1Division of Thoracic Surgery, and 2Department ofPathology, The Brigham & Women's Hospital, Boston, Massachusetts

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Bryan M. Burt, Division of Thoracic Surgery,Harvard Medical School, Brigham & Women's Hospital, 75 FrancisStreet, Boston, MA 02115. Phone: 617-840-7411; Fax: 617-566-6434;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-1808

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(6) March 15, 20121568

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lung cancer, pancreatic cancer, ovarian cancer, breast can-cer, prostate cancer, and head and neck cancer (9–12).Furthermore, IL-4 production is augmented in the immunecells of patients with cancer, and serum and tissue levels ofIL-4 are correlated with tumor progression in man (13). InMPM, Beseth and colleagues have shown IL-4Ra expressionon mesothelioma tumors from 13 patients using immuno-histochemistry (14). We set out to determine the clinicalsignificance and biologic function of IL-4Ra in humanMPM.

Materials and MethodsPatients, tissues, and cell linesPatients. Human tumor, blood, and effusion were

acquired from patients undergoing extrapleural pneumo-nectomy (EPP) in accordance with Institutional ReviewBoard policy and following informed consent. Requiredcriteria for EPP include disease confined to the ipsilateralhemithorax and sufficient predicted postoperative pulmo-nary reserve. At our institution, EPP is more commonlycarried out over pleurectomy/decortication for MPM andlikely provides more complete resection, especially forpatients with bulky disease. Patient cohorts for PCR andimmunohistochemical experiments were based on previ-ously published data sets constructed to represent institu-tional experience (15) and their clinicopathologic charac-teristics are shown in Supplementary Tables S1 and S2. Forflow cytometry experiments, tissues were obtained fromthe operating room in patients undergoing cytoreductivesurgery for biopsy-proven MPMs.Tissues. The pathologic diagnosis of MPM was con-

firmed in all cases. For PCR experiments, tumor samples

were snap frozen on the day of resection. For flow cytometryexperiments, tumor and matched blood samples wereobtained fresh and used on the day of surgery. Peripheralblood mononuclear cells were isolated by density centrifu-gation over Ficoll-Paque Plus (GE Healthcare). To isolatetumor-infiltrating mononuclear cells, tumor tissues wereminced into fine fragments in cold Hank’s balanced saltsolution (HBSS) and digested in RPMI [American TypeCulture Collection (ATCC)] containing 10% fetal calfserum (FCS; Gibco), 1 mg/mL Collagenase D (Roche Diag-nostic), and 100 mg/mL DNase (Qiagen) at 37�C for 3hours. The tissue slurry was passed through 70 and 40 mmfilters and washed with cold HBSS. The cells were thenlayered over a Ficoll-Paque density gradient and centrifugedat 1,000� g for 20minutes at which time the interface layercontaining tumor mononuclear cells was harvested.

MPM cell lines. Four humanMPM cell lines were main-tained in RPMImedia containing 10% FCS (Gibco). H2052cells (epithelial origin) were obtained from the ATCC.MS924 (epithelial) and MS428 cells (sarcomatoid) werekindly provided by Dr. Jonathan A. Fletcher, MD, Depart-ment of Pathology, Brigham &Women’s Hospital (Boston,MA). JMN cells (biphasic) were a gift from Dr. JamesRheinwald (16).

Quantitative PCRQuantitative PCR was carried out on MPM tumor tissues

as previously described (17). Briefly, tumors were selectedfor portions with highest tumor density (>50% tumor cellsper high-power field) and stored snap frozen. Total RNA (2mg) was isolated using TRIzol reagent (Invitrogen Life Tech-nologies) and reverse transcribed using Taq-Man ReverseTranscription reagents (Applied Biosystems). Quantitativereal-timePCR (RT-PCR)was carried out using a SYBR-Greenfluorometric-based detection system (Applied Biosystems)with a Stratagene MX 3000P device. The primer sequencesfor IL-4Ra (synthesized by Invitrogen Life Technologies)used for RT-PCR were as follows: 50-TCATGGAT-GACGTGGTCAGT-30 (forward) and 50-GTGTCGGAGA-CATTGGTGTG-30 (reverse). PCR amplification of cDNAsamples was carried out in triplicate using appropriatenegative controls and the melting curves were in agreementwith the expected amplified fragments. Ribosome 18S RNAwas used as an internal control for normalization of data[50-ATGGCCGTTCTTAGTTGGTG-30 (forward) and 30-CGCTGAGCCAGTCAGTGTAG-50 (reverse)].

Immunohistochemistry and tissue microarrayImmunostaining was carried out on a tissue microarray

(TMA) of 58 resected MPM tumors as previously described(7). Each tumor was represented in duplicate or quadru-plicate cores. Six patients were excluded because of damageto the tissue cores, resulting in a sample size of 52. In brief,the TMA slides were soaked in xylene, passed throughgraded alcohols, and then placed in distilled water. Theslides were next treated with citrate buffer in a steampressure cooker (Decloaking Chamber; BioCare Medical)and then washed in distilled water. All further steps were

Translational RelevanceMalignant pleural mesothelioma (MPM) is an aggres-

sive tumor of the pleura that arises in a background ofchronic inflammation. The overall survival of patientswith MPM is poor despite multimodality treatmentstrategies that include surgery, chemotherapy, and radi-ation. In this study, we have identified interleukin-4receptor a (IL-4Ra), the major subunit of the IL-4receptor, as a biomarker with prognostic correlation inMPM. In patients undergoing resection for MPM, hightumor IL-4Ra expression was an independent predictorof decreased survival. Furthermore, freshly isolated Tcells infiltrating human MPM tumors were polarized toproduce IL-4, and IL-4 had direct biologic effects onMPM tumor cells that included promotion of STAT-6phosphorylation and inflammatory cytokine produc-tion. Therefore, we believe that IL-4 endogenously pro-duced in the tumor microenvironment may promotecancer inflammation and progression, and that inter-ruption of the IL-4/IL-4 receptor axismay be a promisingtherapeutic target in human MPMs.

IL-4Ra in Human Pleural MPM

www.aacrjournals.org Clin Cancer Res; 18(6) March 15, 2012 1569




carried out at room temperature in a hydrated chamber. Toquench endogenous peroxidase activity, the slides werepretreated with peroxidase block (DAKO) for 5 minutes.A mouse monoclonal anti-human IL-4Ra antibody (H-4,SC-28361; Santa Cruz Biotechnology) was applied at 1:100in DAKO diluent for 1 hour. Afterward, the slides werewashed in 50 mmol/L Tris-Cl, pH 7.4, and detected withMouse Envision kit (DAKO) as per manufacturer’s instruc-tions. After anotherwashing step, immunoperoxidase stain-ing was developed using a DAB chromogen (DAKO) andcounterstained with hematoxylin. For image analysis, TMAslides stained with IL-4Ra were scanned at 200� magnifi-cation using an Aperio ScanScope XT workstation (AperioTechnology, Inc.). The images were visualized and anno-tated using ImageScope software (version 10.0.35.1800;Aperio Technology). For each core, 3 regions of interest(ROI) were selected by a pathologist (S.J. Rodig) based onareas containing predominantly tumor cells and therebyexcluding the stromal compartment which harbors most ofthe immune infiltrate (Supplementary Fig. S1). Aperiosoftware (positive pixel count v9 algorithm) was used todeconvolute the brown (positive staining for the antigen)andblue (hematoxylin stain) colorations. The percentage ofthe annotated areawas scoredby a software algorithmbasedupon a scale of pixel intensity ranging from 0 to 256, with 0representing black and 256 representing white. Stainingintensity thresholds were set as follows: strong (0–100),not strong (101–256). A representation of strong staining isshown in Supplementary Fig. S1A and a representation ofnot strong staining is shown in Supplementary Fig. S1B.Data were collected for each ROI and the results wereaveraged. Data from duplicate cores were similarlyaveraged.

Flow cytometry and intracellular cytokine detectionFlow cytometry was carried out on mononuclear cell

suspensions and cancer cell lines with a LSR II cytometer(BD Biosciences) using the following monoclonal antibo-dies: CD4-FITC, CD45-PE-Cy5, CD8-PE-Cy7, CD3-PacificBlue (all from BD Biosciences); and IL-4Ra-APC (R&DSystems). Matched immunoglobulin isotype antibodiesand unstained cells were used as controls to set gates.Detection of intracellular IL-4 was carried out on bloodand tumor mononuclear cell suspensions 4 hours afteractivation with 5 ng/mL phorbol 12-myristate 13-acetate(PMA; Sigma-Aldrich) and1mmol/L ionomycin (Sigma), inthe presence of 10 mg/mL Brefeldin A (BD Biosciences).Surface stainingwas completed and then the cells were fixedand permeabilized. Intracellular IL-4 was assayed using anallophycocyanin (APC)-conjugated anti-IL-4 antibody(Intracellular Cytokine Detection kit; BD Biosciences).Flow cytometry data were analyzed with FlowJo software(TreeStar).

Cell-based assaysFive thousand cells were plated on 96-well cell culture

plates in RPMI containing 10% FCS. Twenty-four hourslater, recombinant human IL-4 (Invitrogen) was added at a

concentration of 20 ng/mL. Cellular proliferation wasmea-sured at indicated time points using an MTT cell prolifer-ation assay (ATCC), and apoptosis was assessed via mea-surement of caspase-3/7 activity with a luminescence-basedassay (Promega). ELISA was used to assay the supernatantfor cytokines IL-6, IL-8, and VEGF, 24 hours after theaddition of IL-4. In-Cell ELISA assays were used to quantifychanges in the concentrationof STAT-6 andphosphorylatedSTAT-6, after 24 hours of culture in 20 ng/mL of IL-4. Finalvalues were normalized to cell number using Janus Greenreagent. To detect IL-4 in tumor tissues, snap-frozen MPMtumors were homogenized in buffer containing Iscove’sMedium (Gibco), 10% FCS, 0.5 mmol/L amino-n-caproicacid (Sigma), and 0.05 mmol/L EDTA (Sigma). An ultra-sensitive ELISA detection kit was used to detect IL-4 in thetissue homogenate. All ELISA kits were purchased fromInvitrogen.

Statistical analysisStatistical analysis was conducted using JMP statistical

software (version 8.0; SAS Institute). Distributions wereplotted to examine compliance with distributional assump-tions. Nonparametric tests were used to compare dichoto-mous variables in univariate analyses. The Wilcoxon testwas used to compare 2 groups of continuous variablesand one-way ANOVA was used to compare 3 groups ofcontinuous variables. Kaplan–Meier plots were used toillustrate the cumulative proportion surviving as a func-tion of months since surgery, and differences in survivalwere tested for significance by the log-rank test. Cox pro-portional hazards regression was used to test the predictivecapacity of IL-4Ra expression in univariate and multi-variate models and to estimate 95% confidence intervals(CI) influencing survival. P values less than 0.05 wereconsidered significant.

ResultsIncreased expression of the IL-4Ra message ispredictive of poor survival in patients undergoingresection for epithelial MPM

Tumor expression of IL-4Ra was determined by RT-PCR of MPM tumors from 37 patients undergoing EPP.Greater than median expression levels of IL-4Ra wereassociated with significantly worse survival for patientswith epithelial (n ¼ 22), but not nonepithelial MPM (n ¼15; Fig. 1A). Univariate Cox proportional hazards model-ing identified increased IL-4Ra expression as a predictorof worse survival in patients with epithelial MPM; whenadjusted for age, gender, and stage, IL-4Ra expressionwas retained as an independent predictor of survival(Table 1). Interestingly, a slightly higher level of IL-4Raexpression was found in nonepithelial tumors than inepithelial tumors (Fig. 1B). The expression level of IL-4Radid not correlate with pathologic stage in the epithelialgroup but could not be assessed in the nonepithelialgroup owing to the high distribution of stage III tumorsin these patients (Fig. 1C).

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Increasedexpressionof the IL-4Raprotein ispredictiveof poor survival in patients undergoing resection forepithelial MPMTo confirm the expression of IL-4Ra on tumor cells from

human MPM tumor specimens, we carried out flow cyto-metry on the CD45-negative cell fraction of tumor mono-nuclear cell suspensions, where high frequencies of IL-4Ra–positive cells were found (Fig. 2A). To determine the degreeof expression of the IL-4Ra protein on epithelial and non-epithelial MPM, and if expression of the IL-4Ra protein wasassociated with survival in MPM, we stained our TMA oftumors from 52 patients with an anti-IL-4Ra antibody (Fig.2B). IL-4Ra staining was present on the tumors of 31 of 32patients with epithelial tumors (97%) and 19 of 20 patientswith nonepithelial tumors (95%). Although a small, scat-tered fraction of cells in the stromal compartment of thetumor stained positive for IL-4Ra, the great majority of IL-4Ra staining was found on the tumor cells themselves.Similar to the RT-PCR data, strong IL-4Ra staining wasassociated with worse overall survival following EPP inpatients with epithelial MPM (Fig. 2C). A greater fractionof epithelial tumors showed strong staining for the IL-4Ra

protein than nonepithelial tumors (27% � 4% vs. 10% �2%, Fig. 2D), and strong IL-4Ra staining was not associatedwith pathologic stage (Fig. 2E). Because IL-4Ra is expressedon tumor-infiltrating macrophages in humanMPM (7), wecompared IL-4Ra and CD68 staining on epithelial andnonepithelial mesothelioma tumors (Supplementary Fig.S1). While IL-4Ra strongly stained the tumor cell compart-ment, there was less marked staining in the tumor stroma.Conversely, CD68 staining was predominantly found onstromal cells.

Tumor-infiltrating T cells in humanMPM are polarizedto produce IL-4

Because T lymphocytes are a chief source of IL-4 intumors, we carried out intracellular cytokine analyses todetermine whether tumor-infiltrating T cells in MPM werea potential source of endogenous IL-4. Mononuclear cellswere freshly isolated from matched tumor and blood sam-ples from 8 patients undergoing EPP for MPM (epithelial,n¼ 4; nonepithelial, n¼ 4).We found that high frequenciesof tumor-infiltrating CD3 T cells produced IL-4 after stim-ulation (21% � 7%) when compared with matched blood

Table 1. Association of IL-4Ra message with survival in MPM

Nonadjusted Adjusteda

Variable HR (95% CI) P HR (95% CI) P

All (n ¼ 37) 1.78 (1.32–2.47) <0.0001 1.81 (1.28–2.68) 0.0003Epithelial (n ¼ 22) 2.97 (1.70–6.02) <0.0001 3.13 (1.68–7.15) <0.0001Nonepithelial (n ¼ 15) 0.91 (0.50–1.66) 0.8 1.16 (0.57–2.51) 0.7

aAdjusted for age, gender, and stage.

Figure 1. Expression of the IL-4Ramessage predicts survival in humanMPM. A, IL-4Ra mRNA expressionwas determined by RT-PCR in MPMtumor specimens obtained after EPPin patients with all (n ¼ 37), epithelial(EPI; n ¼ 22), and nonepithelialhistology (NE; n ¼ 15). Patients weredivided into groups of either greaterthan or less than median tumor IL-4Ra expression. Expression of tumorIL-4Ra was compared betweenepithelial and nonepithelial tumors(B) and among tumors of varyingpathologic stage (C). TNM, tumor-node-metastasis.






CD3 T cells from the same patients (4% � 2%; Fig. 3A andB), suggesting that T cells withinMPM tumors are skewed toproduce IL-4. Although both CD4 and CD8 tumor-infil-trating T cells produced IL-4 (Fig. 3C), CD8 T cells were themajor T-cell subset responsible for IL-4 production within

the tumor, representing 63%� 12%of tumor CD3þIL-4þ Tcells, and CD4 T cells were the major T-cell subset respon-sible for IL-4 production in the blood, representing 56% �14% of blood CD3þIL-4þ T cells (Fig. 3D). This differencewas duemainly to a greater number of CD8 thanCD4T cells

Figure 2. Expression of the IL-4Ra protein is associated with survival in human MPM. A, IL-4Ra expression on tumor cells from human MPM tumors wasconfirmed by flow cytometry on CD45-negative tumor mononuclear cells (n ¼ 4). IL-4Ra protein expression was determined by immunohistochemistry onMPM tumor specimens obtained after EPP for patients with all (n¼ 52), epithelial (EPI; n¼ 32), and nonepithelial histology (NE; n¼ 20). B, representative coresfrom epithelial and nonepithelial MPM stainedwith IL-4Ra. C, Kaplan–Meier survival curves are shown for patients with either high or low tumor expression ofIL-4Ra. D, the percentage of patients with high tumor expression of IL-Rawas compared between epithelial and nonepithelial histology. E, the percentage ofpatients with high tumor expression of IL-4Ra was stratified by pathologic stage. TNM, tumor-node-metastasis.

Figure 3. Tumor-infiltrating T cells in human MPM are polarized to produce IL-4. Mononuclear cells were freshly isolated from matched blood and tumorspecimens from patients undergoing surgical resection of MPM. Intracellular IL-4 production was determined in CD3 T cells by intracellular cytokine analysisafter 4 hours of nonspecific stimulation with PMA and ionomycin. A, flow cytometric dot plots from one representative patient. B, summary data of matchedtumor and bloodCD3þIL-4þ cells from 8patients. C, representative contour plot of intratumoral CD4 andCD8 T-cell subsets and intracellular IL-4 production.D, CD4 and CD8 subset composition of CD3þIL-4þ T cells (n ¼ 5). E, the percentage of CD4 and CD8 T cells that stain positive for IL-4 (n ¼ 5). IL-4concentrations (F) and IL-13 concentrations (G) in tumor (n¼ 8), effusion (n ¼ 8), and serum (n ¼ 10) of patients with MPM was determined by ultrasensitiveELISA. Values represent pg/mg of tissue and pg/mL of effusion and serum.

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within the tumor, as a similar number of CD8 and CD4 Tcells in each compartment stainedpositive for IL-4 (Fig. 3E).Similar numbers of IL-4þ tumor-infiltrating T cells werefound in epithelial and nonepithelial tumors (not shown).To further characterize the phenotype of tumor-infiltratingT cells in humanMPMs, we assayed for T-cell production ofIFN-g and IL-10. Interestingly, we found that tumor-infil-trating T cells in human MPMwere significant producers ofIFN-g in response to PMA/ionomycin when compared withmatched circulating T cells (Supplementary Fig. S2A, 47%�4% tumor, 20%� 12%blood, P¼ 0.02). A small frequencyof both tumor-infiltrating T cells and matched circulating Tcells produced IL-10 in response to PMA/ionomycin stim-ulation (Supplemental Fig. S2B, 5%� 1% tumor, 3%� 1%blood,P¼0.2). Todeterminewhether the ligands for the IL-4 receptor are present in MPM tumors, we used ELISA todetect IL-4 and IL-13 concentrations within the tumortissue. Low levels of IL-4 were measured in tumor tissues(n ¼ 8), whereas IL-4 was undetectable in the serum (n ¼10) and effusion (n¼ 8) of patients withMPM (Fig. 3F). IL-13 was present in all compartments, with higher levelsfound in tumors and effusions than in serum (Fig. 3G).

STAT-6 signaling is activated in human MPM cancercells in response to IL-4Because human MPM tumor cells express high in situ

levels of IL-4Ra and tumor-infiltrating T cells in MPM areskewed toward an IL-4–producing phenotype, we askedwhether IL-4 had a biologic effect on MPM tumor cells. Toinvestigate the function of IL-4Ra on human MPM cancercells, we studied 4 human MPM cell lines. We first con-firmed that each of these cell lines showed cell surfaceexpression of IL-4Ra using flow cytometry (Fig. 4A). Wethen cultured MPM tumor cells in the presence of IL-4 todetermine whether IL-4 caused activation of STAT-6 signal-ing. Culture with IL-4 increased the levels of phosphory-lated STAT-6 in each of the 4 MPM cells lines by 29% to46%, suggesting that the IL-4Ra receptor complex onMPMtumor cells is functional (Fig. 4B).

Human MPM cancer cells respond to IL-4 viaproduction of inflammatory and angiogenic cytokinesIL-4 is a pleiotropic cytokine that has been shown to have

varied and sometimes opposing effects on tumors thatinclude both increasing and decreasing the proliferationand survival of malignant cells (18). To determine whetherIL-4 could provide a direct survival benefit to human MPMcells, we cultured MPM tumor cells in the presence of IL-4and found no change in proliferation or apoptosis (Fig. 5Aand B). To determine whether IL-4 had proinflammatory oranti-inflammatory effects onMPM tumors cells, we assayedcell culture supernatants for production of various cyto-kines including IL-6, IL-8, and VEGF. Addition of IL-4resulted in statistically significant increases in productionof the inflammatory cytokine IL-6 on each of the 4MPMcelllines (Fig. 5C). MS924 and JMN cells similarly increasedproduction of the inflammatory cytokine IL-8, whereas nochange in IL-8 production was seen in H2052 or MS428

MPM cells. Culture in IL-4 resulted in increased productionof the angiogenic cytokine, VEGF, in H2052, MS924, andJMN cells. Although all MPM cell lines made significantquantities of TGF-b, no increase in productionwas found inresponse to IL-4, and no MPM cell lines made significantquantities of TNF-a,macrophage colony-stimulating factor,or monocyte chemotactic protein 1 (MCP-1) while restingor in response to IL-4 stimulation (not shown).

DiscussionIL-4Ra is the major subunit of the IL-4 receptor and a

primary IL-4–binding protein. After binding of IL-4 andreceptor complex formation, intracellular signaling ensuesthrough phosphorylation of STAT-6 (19, 20). IL-4Ra isexpressed on a number of cancer cell lines derived fromnonhematologic human malignancies that include pancre-atic adenocarcinoma, renal cell carcinoma, melanoma,ovarian carcinoma, Kaposi sarcoma, glioblastoma, coloncarcinoma, gastric carcinoma, breast carcinoma, lung car-cinoma, and head and neck cancers (8, 21). Moreover,elevated expression of IL-4Ra has been found on severalhuman tumors in situ. For example, IL-4Ra has beendetected in 66% to 79% of non–small cell lung cancertumors (9), 60% of ovarian carcinomas (10), 77% of headand neck cancers (11), and is also present on pancreatic,breast, bladder, and prostate carcinomas (12).

In addition to its expression on tumor cells, IL-4Ra isdetected on circulating and tumor-infiltrating myeloidimmune cells with immunosuppressive properties in can-cer-bearing hosts. IL-4Ra is a marker of the recently

Figure 4. Human MPM cells respond to IL-4 by activation of STAT-6signaling. A, the presence of IL-4Ra on 4 human MPM cell lines wasdetermined by flow cytometry. Shaded histograms represent IL-4Rastaining and empty histograms represent isotype controls. B, eachhuman MPM cell line was cultured for 24 hours in the presence of20ng/mLof IL-4, and the level of STAT-6 andphosphorylated (p-) STAT-6was determined by intracellular ELISA. These experiments were carriedout a minimum of 3 times.






described myeloid-derived suppressor cells in tumor-bear-ing mice and also identifies a fraction of circulating mono-nuclear and polymorphonuclear cells with T-cell suppres-sive function in patients with melanoma and colon cancer(22, 23). In human MPM, we have found that IL-4Ra ishighly expressed on a population of tumor-infiltratingmacrophages that displays an immunoregulatory pheno-type (7). Given the high expression of IL-4Ra on multiplehuman cancers and the tumor-promoting effects of IL-4 onhuman cancer cells (18), it is likely that IL-4Ra plays a rolein tumor progression in humans. For example, IL-4Racompetent murine tumors exhibit increased tumor growthwhen compared with tumors from IL-4Ra–deficient coun-terparts (24). Herein, we show that patients with epithelialMPM whose tumors show high IL-4Ra expression have asignificant survival disadvantage after surgical resection.The reason for this difference between epithelial and none-pithelial MPM is not exactly clear; however, nonepithelialMPM is a particularly aggressive tumor and the immuneresponse to epithelial and nonepithelial MPM is distinct(7). It should be noted that our PCR data represent IL-4Ra

expression of the entire tumor mass (tumor cells andstromal cells that include macrophages), whereas ourimmunohistochemical experiments were designed to focuson tumor cell expression of IL-4Ra. To our knowledge, thisis the first study to equate tumor expression of IL-4Ra withsurvival indices in patients with cancer.

The distinction of Th1 and Th2 immune responses hasimportant implications for patients with cancer. Antitumorimmune responses are sustained principally by tumor-infil-trating lymphocytes consisting primarily of T cells. WhileTh1 responses typically signify the production of IL-2 andIFN-g with activation of cytotoxic T-cell responses, Th2responses generally represent a cytokine profile includingIL-4, IL-5, IL-6, and IL-13 (25), of which IL-4 is the proty-pical Th2 cytokine. IL-4 has differential effects on immunitythat could be advantageous to tumor growth. These includeinhibition of Th1 cell differentiation and cellular immunity,abrogation of CD8 T-cell cytotoxicity, accumulation of Th2cytokines, and facilitation of Th2 CD4 T-cell development(26, 27). Most clinical studies support the finding of anabnormal Th1 to Th2 ratio in patients with various cancers

Figure 5. Human MPM cellsproduce proinflammatory andangiogenic cytokines in responseto IL-4. Four human MPM cell lineswere cultured in the presence of 20ng/mL of IL-4. On days 1, 3, and 5thereafter, cellular proliferationwasassayed byMTT (A), and apoptosiswas assayed by determination ofcaspase-3/7 activity measured byrelative light units (B). Twenty-fourhours after the addition of IL-4,supernatants were assayed for IL-6, IL-8, and VEGF via ELISA (C).These experiments were carriedout a minimum of 3 times. RLU,relative light units.

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and it has been shown that the nature of the immuneresponse (Th1 vs. Th2) can affect outcome in patients withcancer. Specifically, high serum and tumor IL-4 levels, andhigh frequencies of IL-4–secreting T cells are elevated inpatients with cancer and parallel worse prognosis (28–30).Our data show that the natural immune reaction to humanMPM involves tumor-infiltrating T cells skewed toward anIL-4–producing phenotype.The direct effects of IL-4 on tumor cells are varied and

sometimes opposing. In human colon cancer cell lines,treatment with IL-4 results in dose-dependent increases incellular proliferation (31), and IL-4 is an autocrine growthfactor made by pancreatic cancer cells (21). IL-4 protectshuman prostate, breast, and cancer cell lines from chemo-therapy and death receptor–induced apoptosis throughupregulation of antiapoptotic proteins cFLIP/FLAME-1 andBcl-xL (12). Similar results are seen in murine in vivo tumormodels where IL-4 increases tumor growth, reduces apopto-sis, and promotes metastases (24, 32). Conversely, IL-4 isrequired for the development of effective antitumor immu-nity (33). For instance, IL-4 can have a direct inhibitory effecton proliferation of human cancer cells (34, 35), and IL-4 canbeapotent antitumor agent in some animalmodels (36, 37).It has been suggested that the differential positive and neg-ative effects of IL-4 on tumor developmentmay be explainedby the source of IL-4, with exogenously delivered IL-4 exert-ing tumor-suppressing effects and endogenously producedIL-4 exerting tumor-promoting effects (18).Our data show that IL-4 did not have a direct effect on

MPM tumor cell line proliferation or apoptosis. Activationof human MPM cells with IL-4 resulted in upregulation ofSTAT-6 phosporylation and an increase in production ofinflammatory and angiogenic cytokines including IL-6, IL-8, and VEGF. Each of these cytokines has been shown to beproduced by MPM cell lines where they act as potentautocrine growth factors and are each detected at significantlevels in the effusions of patients with MPMs (38–42). IL-6is a multifunctional, proinflammatory cytokine involved inacute phase inflammatory responses and additionally sti-mulates VEGF production by MPM cells (43). It has beensuggested that IL-6 may be responsible for the systemicmanifestations of MPM that include fever, cachexia, andthrombocytosis (44). IL-8 is a member of the C-X-C che-mokine family and has direct growth-potentiating andangiogenic effects on tumors. VEGF is a powerful mitogenfor the vascular endothelium, and in patients with MPM,elevated serum levels of VEGF correlate with advanceddisease stage and worse survival (45). It is reasonable tosurmise that each of these factors has tumor-promotingeffects in the tumor microenvironment.

Taken together, the high expression of IL-4Ra on humantumor cells, its association with protumor function, and itscorrelation with survival reported here make it a promisingtherapeutic target. Puri’s group has targeted tumor IL-4Rawith a chimeric protein consisting of a circularly permu-tated IL-4 molecule that is fused to a truncated from ofPseudomonas exotoxin. This agent has been shown to havepotent cytotoxic activity in vitro against numerous IL-4Raexpressing cell lines and in vivo in xenograft models ofvarious human cancers including MPM (8, 14, 46, 47).Ultimately, this molecule has been brought to clinical trialsand has shown encouraging results in patients with glio-blastoma (48).

In summary, MPM is a malignancy whose origin anddevelopment are deeply rooted in inflammation. We havefound that IL-4Ra is highly expressed in situ by tumor cellsin human MPM tumors, tumor expression of IL-4Ra ispredictive of survival in patients undergoing cytoreductivesurgery for epithelial MPM, and that MPM tumor cellsrespond to IL-4 via upregulation of STAT-6 signaling andinflammatory cytokine production.We hypothesize that IL-4 endogenously producedbyMPMtumor-infiltrating T cellsprovides stimulus to the IL-4 receptor on MPM tumor cellsin vivo and promotes inflammatory and angiogenic signalsthat stimulate tumorigenesis. Taken together, it seems thatmesothelioma tumors with high IL-4Ra expression areclinically more aggressive cancers that have worse outcomeafter surgical resection. These data have important implica-tions for future research and clinical applications. IL-4Ra isa potentially useful marker of prognosis in patients under-going surgical resection and possibly also for patientsundergoing definitive chemotherapy; however, thisrequires further investigation. Moreover, the IL-4/IL-4Raaxis represents a promising target for directed therapy inman, with potential application for IL-4 and IL-4 receptorblocking agents and IL-4Ra–specific cytotoxins.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe authors thank Dr. Christina Wei for technical assistance.

Grant SupportThis study was supported by the International Mesothelioma Program of

the Brigham & Women’s Hospital.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 20, 2011; revised December 22, 2011; accepted December31, 2011; published OnlineFirst January 18, 2012.

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2012;18:1568-1577. Published OnlineFirst January 18, 2012.Clin Cancer Res Bryan M. Burt, Andrew Bader, Daniel Winter, et al. Tumor InflammationMesothelioma Is Associated with Poor Survival and Promotion of Expression of Interleukin-4 Receptor Alpha in Human Pleural

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