Distinct Effects of Ligand-Induced PDGFR and PDGFR Signaling in the Human Rhabdomyosarcoma Tumor...

Molecular and Cellular Pathobiology

Distinct Effects of Ligand-Induced PDGFRa and PDGFRbSignaling in the Human Rhabdomyosarcoma Tumor Cell andStroma Cell Compartments

Monika Ehnman1,2, Edoardo Missiaglia3,4, Erika Folestad2, Joanna Selfe3, Carina Strell1, Khin Thway3,Bertha Brodin1, Kristian Pietras5, Janet Shipley3, Arne €Ostman1, and Ulf Eriksson2

AbstractPlatelet-derived growth factor receptors (PDGFR) a and b have been suggested as potential targets for

treatment of rhabdomyosarcoma, themost common soft tissue sarcoma in children. This study identifies biologicactivities linked to PDGF signaling in rhabdomyosarcomamodels and human sample collections. Analysis of geneexpression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in allsubtypes, with PDGF-D as the solely overexpressed PDGFRb ligand. By immunohistochemistry, PDGF-CC, PDGF-DD, and PDGFRa were found in tumor cells, whereas PDGFRb was primarily detected in vascular stroma. Theseresults are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRa signaling associated with genes involved in the reactivation of developmental programs, PDGF-DD/PDGFRb signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations furtheridentified associations between PDGFRb in vascular stroma and the alveolar subtype and with presence ofmetastases. Functional validation of our findings was carried out in molecularly distinct model systems, wheretherapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation,vessel density, andmacrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferationthroughmechanisms involving reduced phosphorylation ofGSK-3a andGSK-3b. Additional tissue culture studiesshowed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness, differentiation,senescence, and apoptosis. In summary, the study shows a clinically relevant distinction in PDGF signalingin human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs onsarcoma progression. Cancer Res; 73(7); 2139–49. �2013 AACR.

IntroductionRhabdomyosarcomas are the most common pediatric soft

tissue sarcoma with 2 main histologic subtypes, embryonal(ERMS) and alveolar (ARMS; ref. 1). ERMS has the mostfavorable prognosis, but the estimated 3-year overall survivalrate is still only 47% in children and adolescents with meta-static disease (2). The ARMS is typically associated with

frequent expression of oncogenic PAX3-FOXO1 or PAX7-FOXO1 gene fusion products and a high propensity to metas-tasize (2). Both subtypes display features of developing skeletalmuscle (3) and their genetic signatures and presence ofPAX3-FOXO1 have been proposed to be useful for patientstratification into low- and high-risk groups (4–7). However,the oncogenic heterogeneity of rhabdomyosarcomas makesthe identification of suitable molecular targets for directedtherapies challenging.

A potential therapeutic candidate for rhabdomyosarcomais the tyrosine kinase platelet-derived growth factor receptor(PDGFR)a. PDGFRa is a direct target of the PAX3-FOXO1fusion protein in p53-deficient cells (8, 9), and its expressionhas been reported to correlate with decreased failure-freesurvival in patients and increased tumorigenicity in mice (9–11). So far, there has been little evidence for other PDGFfamily members contributing to the biology of rhabdomyo-sarcoma. Classically, tumorigenic PDGF signaling follows asa consequence of activating point mutations, amplifications,or translocations, often resulting in autocrine stimulatoryloops (12, 13). PDGF ligand production is another mecha-nism of action to promote both autocrine signaling andparacrine crosstalk with infiltrating stromal cells in varioustumors (14).

Authors' Affiliations: 1Department of Oncology-Pathology, 2VascularBiology Division, Department of Medical Biochemistry and Biophysics,Karolinska Institutet, Stockholm, Sweden; 3Sarcoma Molecular PathologyTeam, Division of Molecular Pathology/Cancer Therapeutics, The Instituteof Cancer Research, Sutton, United Kingdom; 4Bioinformatics Core Facil-ity, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland; and5Department of Laboratory Medicine Malm€o, Center for Molecular Pathol-ogy, Lund University, Lund, Sweden

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

K. Pietras, J. Shipley, and A. €Ostman contributed equally to this work.

Corresponding Author: Monika Ehnman, Department of Oncology-Pathology, Karolinska Institutet, Stockholm 17177, Sweden. Phone:468-51770955; Fax: 468-339031; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-1646

�2013 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst January 21, 2013; DOI: 10.1158/0008-5472.CAN-12-1646

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It has been shown that the 5 homo- or heterodimeric ligands(PDGF-AA, -BB, -AB, -CC, and -DD) have different receptoraffinity in vivo; both PDGF-AA and PDGF-CC signal viaPDGFRa, whereas PDGF-BB and PDGF-DD act preferentiallyvia PDGFRb (12). Moreover, PDGF-CC and PDGF-DD aresecreted as latent growth factors, subsequently proteolyticallyactivated by plasminogen activators before PDGFR binding(15–18). Accordingly, it is becoming increasingly evident thatPDGF signaling is regulated at multiple levels in a context-dependent manner.

In this study, autocrine and paracrine PDGF signaling eventsare systematically analyzed with particular attention to theheterogeneity among rhabdomyosarcomas. The data illus-trates that PDGF activity is linked to microenvironmentalchanges with distinct cellular responses observed not alwayspredictable from PDGFR expression levels.

Materials and MethodsGene expression profiles of rhabdomyosarcoma patients

The ITCC/CIT (Innovative Therapies for Children withCancer/Carte d'Identit�e des Tumeurs) gene expression profiledataset has been previously described (6, 7). Additional infor-mation about the gene expression profiling and data handlingcan be found in Supplementary Materials and Methods.

Tissuemicroarrays and clinicopathological correlationsThree independent tissuemicroarrayswere used for analysis

of PDGF ligand and receptor expression in human rhabdo-myosarcoma specimens. First, a commercially available array(Tissue Array Networks), representing rhabdomyosarcomaand leiomyosarcoma samples from 36 patients of various ages,was used to assess tumor compartment–specific protein local-ization. For clinicopathologic correlations, only pediatricmaterial was used. The second array has been previouslydescribed and contained material from 60 alveolar and 171embryonal cases with a mean age of 6.3 years (19). The thirdarray, also previously described, consisted of 25 alveolar and 54embryonal cases with a mean age of 3.8 years (20). Immunos-taining for PDGFRa and b was scored by a consultant pathol-ogist (K. Thway). Each tissue core was assigned a stainingintensity score, where 0, 1, 2, and 3 indicated negative, weak,moderate, and strong staining, respectively. The score for eachpatient was derived from the maximal score in all availablecores for a patient where the percentage of cells stained wasabove 10%. Kaplan–Meier survival analysis of PDGFR expres-sion was conducted separately for tumor cells and tumorstroma, using 1% positively stained cells as cutoff for the latter.Survival characteristics were analyzed on the basis of overallsurvival as clinical outcome using date of diagnosis as timezero.

Cell cultureCells were kept at 37�C in a humidified 5% CO2 atmosphere.

Dulbecco's Modified Eagle Medium (DMEM) was used for allcell lineswith the exception of RMS-YM,whichwasmaintainedin Roswell Park Memorial Institute (RPMI)-1640 medium.Supplements included 10% FBS, 2 mmol/L glutamine, 100

U/mL penicillin, and 100 mg/mL streptomycin unless other-wise stated.

Characterization of cell linesGene expression profiles for cell lines were available (21) and

analyzed to evaluate PDGF-C and -D expression levels.PDGFRa expression was analyzed by immunoblotting of celllysates using a rabbit antibody (Cell Signaling). Equal sampleloading was confirmedwith a calnexin-targeting goat antibody(Santa Cruz Biotechnology).

Modulation of PDGF activity in vitroTyrosine phosphorylation of PDGFa was investigated as

previously described (16). Following PDGF treatment for 7minutes at 37�C, pAkt(Thr308) was detected with a rabbitantibody (Cell Signaling). Phosphorylated GSK-3a(Ser21)and GSK-3b(Ser9) were likewise detected with a rabbit anti-body (Cell Signaling) after a 1 hour pretreatment at 37�C with0.5 mmol/L CP-673,451 or vehicle. CP-673,451 was chosen forits reported ability to inhibit PDGFR phosphorylation withan otherwise limited substrate crossreactivity (22, 23).

Cell proliferation/viability was analyzed using the CyQuantcell proliferation assay (Life Technologies). Prestarved cellswere treated every 24 hourswith vehicle (dimethyl sulfoxide) or0.5 mmol/L CP-673,451 diluted in serum-reduced medium(1.5% FBS) for 96 hours. The amount of nucleic acid presentin lysed cells was normalized to the amount when treatmentwas initiated. Cell proliferation/viability in response to 300ng/mL PDGF-CC (16) was likewise analyzed, but cells werethen kept in serum-free medium and treated twice during a48-hour period.

Apoptosis was studied after 96-hour treatment as above.Cells were then enzymatically detached and labeled using theAnnexin-V-FLUOS Staining Kit (Roche) according to the man-ufacturer's instructions. Samples were analyzed with the BDFACSCalibur flow cytometer and the CellQuest software (BDBiosciences).

A cell-cycle analysis was likewise conducted after 96-hourtreatment. Cell membranes were lysed with a hypotonic buffer(4 mmol/L sodium citrate, 0.1% Triton X-100) containing 0.1mmol/L propidium iodide and 50 mg/mL RNaseA (Life Tech-nologies) for 20 minutes at 4�C. Aggregates were removed byfiltration trough a 40-mm cell strainer. The total nuclei fluo-rescence, FL2-A, was measured under exclusion of debris/aggregates via the FL2-W versus FL2-A plot. Data were ana-lyzed with the ModFIT LT Cell Cycle analysis software (VeritySoftware House).

Rhabdosphere-forming capacity was analyzed in a limiteddilution assay with or without pretreatment with 0.5 mmol/LCP-673,451 or vehicle for 72 hours. The cells were filteredthrough a 40-mm cell strainer to generate a single-cell suspen-sion and then seeded in a descending cell number/well (16, 8, 4,2, 1) in ultra-low attachment 96-well plates (Corning) in 75 mLcomplete stem cell medium (Neurocult, LifeTechnologies)supplemented with 2 mg/mL heparin (STEMCELL Technolo-gies), 2� B27 without vitamin A (Life Technologies), 10 ng/mLEGF, and 10 ng/mL basic fibroblast growth factor (bFGF; bothR&D Systems) containing either 0.5 mmol/L CP-673,451 or

Ehnman et al.

Cancer Res; 73(7) April 1, 2013 Cancer Research2140




vehicle. Fresh medium, 20 mL/well of 0.5 mmol/L CP-673,451 orvehicle, was added every day. RD rhabdospheres larger than 100mm were counted after 10 days, RUCH2 rhabdospheres largerthan 200 mmwere counted after 14 days. Results were analyzedusing theonlineExtremLimitedDilutionAnalysis software (24).

Xenograft studiesExperimental procedures were approved by the local com-

mittee for animal experiments. TwomillionRUCH2cells in PBSwere implanted subcutaneously into the flank of 8-week-oldfemales and those with palpable tumors were then stratifiedinto 2 groups based on tumor size (width2� length� 0.52). Thefirst cohort of severe combined immunodeficient (SCID) miceincluded 13 tumor-bearing animals and the second, 6 animals.Freshly prepared CP-673,451, or PEG-400 as vehicle, was givenonce daily by oral gavage for 18 days, and the animals weresacrificed 4 hours after the last treatment. After perfusion withPBS followedby 2%paraformaldehyde (PFA), tumorswere keptin 30% sucrose at 4�C overnight and then embedded forcryopreservation, or alternatively, postfixed in 2% PFA at 4�Covernight, dehydrated, and paraffin-embedded. For xenograftsderived from the RMS cell line, 5 million cells in PBS/Matrigel(BD Biosciences) were implanted into 20 SCID mice and CP-673,451 or vehiclewas given for 9 days. The sorafenib treatmenthas been described elsewhere (25).

Immunostaining and immunoquantificationsParaffin-embedded sections were deparaffinized, and

endogenous peroxidase activity was quenched by 3% H2O2 for10 minutes at room temperature. Immunostaining was thenconducted as previously described for PDGFRa and b (26) aswell as PDGF-DD (18). For detection of PDGF-CC, the latterstaining protocol was used with the exception of including anaffinity-purified polyclonal rabbit anti-human PDGF-CC anti-body. Staining specificities were confirmed in pelleted andparaffin-embedded COS-1 cells transiently transfected withhuman full-length PDGF-C or -D as previously described(refs. 16, 18; Supplementary Fig. S1). The PDGFR antibodieswere chosen based on the results from a recent antibodyscreen, where PDGFR isoform-specific reactivities were inves-tigated (27). Both unphosphorylated and phosphorylatedreceptors were recognized.For immunofluorescence-based detection, cryosections

were fixed in 4% PFA (Ki67) or ice-cold acetone (F4/80,CD31/PECAM, pPDGFRs). Paraffin-embedded sections weredeparaffinized and heat-induced antigen retrieval conductedin citrate (Vector Laboratories) or DAKO target retrievalsolution, high pH buffer (DAKO) prior staining (podocalyxinand PDGFRb, respectively). Slides were incubated with block-ing solution (DAKO) and primary antibodies in TNB buffer(PerkinElmer Life Sciences) overnight. Ki67 was detected witha rabbit antibody according to the manufacturer's instructions(Novocastra Laboratories Ltd.). For recognition of macro-phages, a rat F4/80 antibody (Serotec) was used. CD31/PECAMwas detected with a rat antibody (BD Pharmingen) andpPDGFRs with a cross-reacting mouse antibody againstPDGFRb (PY751) (Cell Signaling). Podocalyxin was detectedwith a goat antibody (R&D Systems) and PDGFRb with the

same antibody as above. Appropriate secondary antibodies(Life Technologies) were applied before mounting with Vecta-shield mounting medium with DAPI (Vector Laboratories).The AxioVision Rel. 4.6 Software (Carl Zeiss) was used forautomated quantifications of immunostaining from typically 6RUCH2 tumors per group. Vessel density in RMS tumors wasanalyzed in 3 tumors/group.

ResultsPDGF ligands and receptors are expressed in humanrhabdomyosarcoma

PDGF ligand and receptor expression was systematicallyassessed by microarray analysis of patient material from 101rhabdomyosarcomas and 36 skeletal muscle samples. Forcomparison, other small round blue cell tumors (SRBCT) andmesenchymal stem cells were also included. PDGF-C and -Dwere the only PDGF ligands consistently overexpressed com-pared with skeletal muscle, whereas PDGF-B was consistentlyunderexpressed (Fig. 1A). Only PDGFRb, among the receptors,showed significant overexpression in both ERMS and ARMSP3F-positive samples (Fig. 1B). Altogether, these data areindicative of a ligand-mediated PDGFR activation in rhabdo-myosarcoma, which is also supported by the lack of identifiedPDGFR-activating mutations reported so far (28, 29).

PDGF ligands are confined to the rhabdomyosarcomatumor cell compartment, while PDGFRs are expressed byboth tumor cells and infiltrating stromal cells

Tumor compartment–specific expression of PDGF familymembers was analyzed by immunostaining of 3 different tissuemicroarrays. On the basis of the results from the RNA expres-sion profiling data, PDGF-CC was selected as the only consis-tently overexpressed PDGFRa ligand andPDGF-DD as the onlyoverexpressed PDGFRb ligand. Production of PDGF-CC andPDGF-DDwas shown to be almost exclusively located to tumorcells, whereas PDGFRa was frequently expressed in both thetumor and stromal cell compartment (Fig. 2A and C). PDGFRbwas occasionally detected in tumor cells but mainly expressedin vascular stroma (Fig. 2A–C).

Stromal PDGFRb expression associates with the alveolarsubtype

Protein levels of PDGFRs were assessed in 2 different tissuemicroarrays for clinicopathologic correlations. PDGFRb wasagain rarely detected in tumor cells, whereas its stromalstaining was associated with the alveolar subtype (Table 1).PDGFRa expression was observed in tumor cells and stromaand was in both cases associated with the embryonal subtype(P < 0.0033 and P < 0.0329).

The material in the third and largest array (19) was sepa-rately analyzed for associations with proliferation and meta-static status. No significant association was seen betweeneither PDGFR staining and the proliferation marker Ki67(evaluated by the reference monoclonal antibody MIB-1, datanot shown). Stromal expression of PDGFRa and PDGFRb was,respectively, negatively and positively associated with thepresence of metastases at diagnosis (Table 1).

PDGFR Signaling in Rhabdomyosarcoma

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PDGF-C expression is associated with genes involved indevelopmental programs, while PDGF-D is associatedwith those involved in wounding and immune systemprocesses

To further investigate ligand-dependent PDGFR activa-tion, the gene expression profile of the primary tumors wasanalyzed to identify sets of genes, which correlated to eitherPDGF-C or PDGF-D. On the basis of Gene Ontology (GO)enrichment analysis, several genes positively correlatingwith PDGF-C expression were associated with various devel-opmental processes (Table 2). This suggests that PDGF-C

expression is linked with the reactivation of embryonicsignaling pathways, which could facilitate tumor progres-sion. PDGF-D expression correlated with genes associatedwith wounding, leukocyte differentiation, and cell adhesion.When individual genes were investigated, PDGF-C was foundto correlate with, for example, HOX genes, GLI3 and FZD7(Supplementary Table S1). PDGF-D, on the other hand,correlated with genes such as CXCL12, VWF, and CD302.Our findings are consistent with some of the previouslyreported activities mediated by PDGFRa and PDGFRb sig-naling (30).

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Figure 1. PDGF ligands and receptors are expressed in human rhabdomyosarcomas. Box andwhisker plots representing the expression ofPDGF-A,PDGF-B,PDGF-C, PDGF-D (A) and their cognate receptors, PDGFRa and PDGFRb (B), in 36 skeletal muscle control samples (Sk. muscle), 32 mesenchymal stem cellsamples (MSC), 50 neuroblastomas (NB), 18 Ewing sarcomas (ES), 20 Wilms tumors (WT), 39 medulloblastomas (MBL), 36 ERMS (ERMS), 34 ARMSPAX3-FOXO1 and 1 PAX3-NCOA1 fusion-positive (ARMS_P3F), 10 ARMS PAX7-FOXO1 fusion-positive (ARMS_P7F), and 20 ARMS fusion-negative(ARMS_NEG) samples based on gene expression profiling using the Affymetrix chip HG-133plus2 (�,P < 0.05; ��, P < 0.01; ��, P < 0.001; Wilcoxon rank-sumtest between Sk. muscle and each rhabdomyosarcoma subtype).

Ehnman et al.





To further analyze PDGF-DD/PDGFRb signaling, patientswith tumors with high (top quartile) and low (bottom quartile)PDGF-D expression were compared using predefined gene sets(31). High expression of PDGF-D was associated with expres-sion of genes involved in cell migration (Supplementary Fig.S2A), leukocyte transendothelial migration, and vascularsmooth muscle contraction (Supplementary Fig. S2B). Theseassociations were not seen in the corresponding PDGF-Cexpression analysis (data not shown).

Therapeutic targeting of PDGFR signaling results indecreased cell proliferation, cell-cycle arrest, andapoptosis in a cell-type–specific mannerTo explore ligand-dependent PDGFR activity, cell lines

were screened for PDGF-C and -D expression (Supplemen-

tary Table S2), and a subset (Supplementary Table S3) ofhigh- and low-expressing cell lines was analyzed for PDGFRaprotein expression. RD and RUCH2 cells displayed highPDGFRa expression and were responsive to PDGF stimuli(Fig. 3A and B, Supplementary Fig. S3A and S3B). By qRT-PCR analysis (Supplementary Table S4), these 2 cell linesdisplayed the highest expression levels of PDGF-C and -Dcompared with PDGF-A and -B (Supplementary Fig. S3C).They also decreased their proliferation rate in response tothe PDGFR inhibitor CP-673,451, whereas the PDGFR-neg-ative cell line RMS did not (Fig. 3C). Furthermore, Akt andthe downstream key regulators GSK-3a and GSK-3b wereidentified as targets after ligand-induced PDGFRa stimula-tion (Fig. 3D and E). This phosphorylation response wascompletely abolished when cells were pretreated with CP-

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Figure 2. PDGF ligands are confined to the rhabdomyosarcoma tumor cell compartment, whereas PDGFRs are expressed by both tumor cells and infiltratingstromal cells. A, immunostaining of PDGF-CC, PDGF-DD, PDGFRa, and PDGFRb in consecutive sections of human ERMS tissue cores. B, PDGFRbexpression in vascular stroma (top) and in tumor cells (bottom). C, stromal expression of PDGFRa and PDGFRb in ERMS and ARMS, respectively, used forscoring and clinicopathologic correlations. PDGFRa was notably also found in tumor cells.

Table 1. Associations of PDGFR expression in tumor stroma with metastatic status and subtype usinga c2 test for trend

PDGFRa in tumor stroma PDGFRb in tumor stroma

Stainingintensity score

MetastasisSubtype

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MetastasisSubtype

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No Yes ERMS ARMS No Yes ERMS ARMS

0 88 30 125 62 0 76 9 121 171 14 3 25 8 1 23 8 36 262 16 0 22 3 2 29 16 44 273 4 0 3 1 3 12 4 12 10

PDGFRa stromal staining isnegatively associated with

metastasis.

PDGFRa stromalstaining is positivelyassociated with theERMS subtype

PDGFRb stromal staining ispositively associated with

metastasis

PDGFRb stromalstaining is positivelyassociated with theARMS subtype

c2 test for trend statistic6.4005; P ¼ 0.0114

c2 test for trendstatistic 4.551;P ¼ 0.0329

c2 test for trend statistic8.6819; P ¼ 0.0032

c2 test for trendstatistic 19.02;P < 0.0001

NOTE: Metastatic status implies that the patient presented at stage IV with metastasis.






673,451. A striking heterogeneity was observed between the 2PDGFR-positive cell lines; the RUCH2 cell line displayed anapoptotic response accompanied by a G2–M cell cycle arrest,whereas RD cells only responded with a modest S-phasearrest (Fig. 3F and G).

Impaired rhabdosphere-forming capacity anddifferentiation in cells devoid of PDGF activity

So far, the data indicated that PDGF-CC/PDGFRa signalingis involved in the reactivation of developmental pathways.Stemness potential in the presence of CP-673,451 or vehiclewas therefore analyzed in an anchorage-independent growthassay. CP-673,451 impaired rhabdosphere-forming capacity inboth RD and RUCH2 cultures (Fig. 4A) and this was likewiseobserved in a rhabdosphere-forming assay where adherentRUCH2 cells were pretreated for 72 hours with CP-673,451before seedingwithout treatments (Fig. 4B). In light of previousstudies, RD cells were also used as a model system for rhab-domyosarcoma differentiation (32). In the presence of CP-673,451 under conditions facilitating cellular differentiation,hallmarks of senescence, that is, enlarged cell size andpresenceof perinuclear senescence-associated bgalactosidase (SA-bgal)activity, were observed (Supplementary Fig. S4A). The absenceof elongating cells initiating myogenesis suggested dysfunc-tional differentiation (Supplementary Fig. S4B) further sup-ported by an expression analysis of the myogenic markersMYL1 and MYOGENIN and the myogenic repressor HEY-1(ref. 33; Supplementary Fig. S4C).

PDGFR inhibition reduces tumor growth and stromalcell infiltration

On the basis of the in vitro results, RUCH2 cells expressedPDGF-C and -D and showed PDGFR dependency. Xeno-

graft-bearing mice were treated with CP-673,451 or vehicleand growth inhibition due to the therapeutic regimen wasdetected after 10 days of treatment (Fig. 5A). Tumorvolume after dissection was analyzed in both cohorts asa separate endpoint. Control tumors had then reached anaverage size of 66 mm3 and tumors from mice treated withthe active compound had reached a size of 32 mm3 (Stu-dent t test, P ¼ 0.017, n � 9 mice/group). No metastaseswere detected.

Immunostaining revealed a decreased number of cells in theproliferative phase in CP-673,451–treated animals (Fig. 5B).The effects on PDGFR phosphorylation were specifically visu-alized by immunostaining for phosphorylated PDGFRs. Areduced staining intensity was observed in sections fromanimals treated with the active compound (Fig. 5C).

Before evaluating the therapeutic effect on host-derivedstroma, it was confirmed that human PDGF-DD (34) couldactivate mouse PDGFRb (Supplementary Fig. S5). Thereaf-ter, immunostaining of RUCH2 xenografts revealed that CP-673451 reduced the number of F4/80-positive macrophages(Fig. 5D) and CD31-positive vessels (Fig. 5E). Fibroticstreaks composed of collectively migrating fibroblasts werenot found and consequently not quantified (data notshown).

Vessel density in tumors derived from the RMS cell line isreduced by the multitargeting tyrosine kinase inhibitorsorafenib but not by CP-673,451

In primary tumors, PDGF-D expression correlated to setsof genes involved in blood vessel function. Vessel densitywas therefore separately analyzed in tumors derived fromthe RMS cell line after treatment with CP-673,451 or sor-afenib. PDGFRs were detected in infiltrating stroma

Table 2. PDGF-C expression is associated with genes involved in developmental programs, whereasPDGF-D is associated with those involved in wounding and immune system processes

GOBPID P OR Exp count Count Size Term

PDGF-CGO:0009954 0.000 32.961 0 6 18 Proximal/distal pattern formationGO:0009952 0.000 7.919 1 10 95 Anterior/posterior pattern formationGO:0045110 0.000 193.875 0 3 4 Intermediate filament bundle assemblyGO:0048856 0.000 2.228 25 46 1695 Anatomical structure developmentGO:0007275 0.000 2.145 25 44 1705 Multicellular organismal developmentGO:0007389 0.000 4.427 3 11 178 Pattern specification process

PDGF-DGO:0007155 0.000 6.203 3 12 477 Cell adhesionGO:0030318 0.000 75.597 0 3 11 Melanocyte differentiationGO:0009611 0.000 6.940 2 9 302 Response to woundingGO:0050878 0.000 15.424 0 5 73 Regulation of body fluid levelsGO:0042246 0.000 60.464 0 3 13 Tissue regenerationGO:0002763 0.000 54.961 0 3 14 Positive regulation of myeloid leukocyte differentiation

NOTE: A gene to GO BP conditional test for overrepresentation, where Exp Count is the expected number of genes to be foundassociated with each specific process (term). This is obtained considering the number of genes (size) associatedwith that process andhow many of those being selected by the analysis (count). The first 6 categories associated with each gene are shown.

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(Supplementary Fig. S6A) but not in tumor cells or vessels(data not shown). CP-673,451 neither altered tumor growth(Supplementary Fig. S6B) nor vessel density (Fig. 5F), where-as sorafenib treatment almost completely eliminated allblood vessels (Fig. 5G).

DiscussionTherapeutic inhibition of PDGF activity has proven benefi-

cial in several types of sarcomas (35–37). PDGFRa was alsorecently associated with acquired resistance to IGF-1R anti-body therapy in rhabdomyosarcoma (38). However, little is

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Figure 3. Therapeutic targeting of PDGFR signaling results in decreased cell proliferation, cell-cycle arrest, and apoptosis in a cell-type–specific manner. A,immunoblotting of PDGFRa protein in rhabdomyosarcoma cell lysates. Calnexin was used as loading control. B, immunoblotting of pPDGFRa and totalPDGFRa after ligand stimulation as indicated. C, cell proliferation response following PDGFR inhibition in 2 PDGFR-positive cell lines and 1 negative.D, immunoblotting of pAkt(Thr308) after PDGF ligand stimulation. PDGFRa was used as loading control. E, immunoblotting of pPDGFRa, total PDGFRa,pGSK-3a(Ser21), and pGSK-3b(Ser9) following treatments as indicated. Calnexin was used as loading control. F, apoptotic response analyzed by flowcytometry in PDGFR-positive cell lines following treatment with CP-673,451 or vehicle. G, cell-cycle analysis by flow cytometry of PDGFR-positive cell linesfollowing treatment with CP-673,451 or vehicle. Data are presented � SD (�, P < 0.05; ��, P < 0.001; Student t test).






known about the pathobiology associated with PDGF signalingin rhabdomyosarcoma. We have therefore systematically ana-lyzed PDGF ligand and receptor expression in human rhab-domyosarcoma samples and used animal and cell culturemodels for functional studies. The analyses suggest the exis-tence of tumor compartment–specific effects of ligand-depen-dent PDGFRa and PDGFRb signaling. An important aspect ofthe study is the indication of clinically relevant variations in thestromal compartment of rhabdomyosarcoma. Stromal PDGFRsignaling has previously been extensively described in tumorsof epithelial origin (27, 39, 40).

Supported by an analysis of the gene expression profile of101 pediatric primary tumors and 36 normal skeletal musclesamples, PDGF-C and PDGF-Dwere identified as the only PDGFligands with consistently elevated expression relative to skel-etal muscle. Accordingly, we confirmed their presence in thehyperchromatic tumor cell compartment. PDGFRa seemed tobe the predominating receptor isoform expressed by tumorcells, whereas PDGFRb was mainly found in vascular stroma.Consistent with our findings, autocrine PDGFRa signaling hasrecently been associated with both rhabdomyosarcoma tumorcell survival and regulation of differentiation in pediatricmalignancies (9, 41), whereas PDGFRb is classically associatedwith blood vessel morphogenesis (42–44). Accumulating datahereby suggest that PDGF signaling in rhabdomyosarcomaincludes both autocrine and paracrine signaling in differentcell types.

The observed compartmentalization of PDGFRa andPDGFRb expression urged us to elucidate clinical parametersassociated with their expression in tumor stroma. PDGFRawas then found to associate with the embryonal subtype,whereas PDGFRb associated with the alveolar subtype. Thisdifference was not detected in our gene expression analysis oftumor lysates but is likely explained by the relatively smallproportion of infiltrating cells compared with the tumor cellmass. In light of previous findings about PDGF signaling inblood vessel morphogenesis (43) and metastatic spread ofrhabdomyosarcoma cells (9, 10), associations with the pres-ence of metastases were analyzed in a similar way. StromalPDGFRa was then found to negatively associate with metas-tasis, whereas PDGFRb positively associated with metastasis.These findings could reflect the difference in PDGFR expres-

sion observed between ERMS and ARMS and the higherpropensity of the latter to metastasize. Accordingly, multivar-iate analyses of data from even larger cohorts are needed toclarify whether the investigated correlations are independentof histology.

By analyzing gene expression profiling of primary tumorsamples, tumorigenic PDGF activities were characterized; GOenrichments for biologic processes suggested thatPDGF-Cmaybe involved in developmental processes, whereas PDGF-Dassociated with genes active in cell adhesion, wounding, andimmune system processes. These findings support a role ofPDGF-DD/PDGFRb signaling in stromal cell recruitment andare in line with previous studies on PDGFRb as a regulator ofinterstitial fluid pressure, a well-established mechanisminvolving stromal cells and extracellular matrix constituentswith direct implications for the delivery of therapeutic agentsto tumor cells (45–47). To explore this further, we comparedhigh PDGF-D–expressing tumors with low PDGF-D–expressingtumors on the basis of metagenes generated using GO andKEGG annotations. In this analysis, we observed enrichmentfor terms such as cell adhesion molecules, leukocyte transen-dothelial migration, and vascular smooth muscle contraction.This was detected for PDGF-D, and not PDGF-C, once againhighlighting in vivo differences between PDGFRa and PDGFRbsignaling.

To mechanistically explore autocrine PDGFRa signaling,cell lines were screened for PDGF-C and -D expression. HighPDGFRa protein expression was confirmed in RD and RUCH2cells. These 2 cell lines were responsive to PDGF-CC stimula-tion and they displayed decreased cell proliferation/survivalafter PDGFR targeting by the PDGFR tyrosine kinase inhibitorCP-673,451. However, in line with our gene set enrichmentsbased on human material, our in vitro data revealed a hetero-geneity associated with PDGF signaling in rhabdomyosarco-ma. Following PDGFR inhibition, the RUCH2 cell line mor-phologically displayed clear signs of cell death, mechanisticallyidentified as an increase in apoptosis and a G2–M cell-cyclearrest. RD cells, on the other hand, were largely unaffectedunder proliferative conditions. Under conditions facilitatingmyogenic differentiation; however, morphologic changes,including signs of senescence, were observed in the presenceof CP-673,451. An impaired differentiation capacity was also

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Figure 4. Impaired anchorage-independent rhabdosphere-forming capacity in cells devoid ofPDGF activity. A, rhabdosphereformation in the presenceof CP-673,451 or vehicle. B,rhabdosphere formation of cellspretreated 72 hours with CP-673,451 or vehicle in adherentcultures before seeding.

Ehnman et al.





evident, which is in line with a recent study describing the needfor active PDGFRa signaling in neuroblastoma differentiation(41). This is supportive of PDGF-CC/PDGFRa being involved inthe intricate interplay between proliferation and differentia-tion as our findings from the data mining suggested. At thesame time, in an anchorage-independent stem cell assay, bothRDandRUCH2 cells displayed impaired ability to form rhabdo-spheres in the presence of CP-673,451, indicative of PDGFRasignaling being required for the maintenance of stemnesscharacteristics of embryonal rhabdomyosarcoma cells.Together these results suggest that PDGFRa signaling regu-lates both cancer cell stemness and myogenic differentiation.For further validation of the identified correlations in the

rhabdomyosarcoma patient dataset, the RUCH2 cell line,

with comparably high PDGF-D expression, was selected fordevelopment of a mouse xenograft model. These cellsdisplayed the highest response to PDGF-CC stimulation invitro and in the corresponding in vivo model, therapeutictargeting of PDGFRs decreased tumor burden. Additionaleffects were noted on vessel density and the number ofinfiltrating macrophages. Whether our therapeutic regimenmechanistically targeted PDGFRb-positive pericytesinvolved in the angiogenic process (22, 48), or alternatively,downregulated VEGF expression in tumorigenic cells (49) isnot known. It is however likely that both these previouslycharacterized PDGF/PDGFR-driven processes wouldexplain how PDGF signaling contributes to angiogenesisin RUCH2 xenografts.

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Figure 5. PDGFR inhibition reduces tumor growthand stromal cell infiltration. A, growth ofRUCH2xenografts in response toCP-673,451 or vehicle (n�6mice/group). B, quantification of Ki67 immunopositive cells in RUCH2 tumor sections. C, quantification of pPDGFR immunopositive cells inRUCH2 tumor sections.D, quantification of F4/80 immunopositive macrophages in RUCH2 tumor sections. E, quantification of vessel density in RUCH2 tumor sectionsimmunostained forCD31/PECAM.FandG, quantification of vessel density in tumors derived from theRMScell line and treatedwithCP-673,451, sorafenib, orvehicle. Sections were immunostained for the vessel marker podocalyxin. A compensatory threshold was set to disregard the autofluorescence generatedfrom massive cell death in tumors treated with sorafenib. Data are presented � SD (�, P < 0.05; ��, P < 0.01; ��, P < 0.001, Student t test).






Angiogenesis was also explored in xenografts derived fromthe RMS cell line. These cells were not responsive to PDGFRinhibition in vitro or in vivo. Blood vessel density was also notaltered in these tumors following treatment with CP-673,451.PDGFRb expression was only detected in stroma, and conse-quently, these results indicate that targeting of PDGFR signal-ing in stroma is insufficient to affect rhabdomyosarcomatumor growth. They further support that if PDGF signalingregulates blood vessel characteristics, it is most likely via anintimate crosstalk between PDGFR-positive mural cells andendothelial cells, mechanistically distinct from VEGF-drivenangiogenesis acting directly on the vascular endothelial cells.In a comparative analysis, tumors derived from the RMS cellline were in contrast highly responsive to sorafenib, known forits ability to target VEGFRs.

Whether or not vascular PDGFRs are transiently expressedduring rhabdomyosarcoma tumor formation and progressionis still unclear, but this was at least not evident from atherapeutic perspective in our study. Overall, very little isknown about stromagenesis, including blood vessel morpho-genesis and immunoregulatory processes, in rhabdomyosar-coma biology. Macrophage infiltration has been linked toPDGF-D expression in skeletal muscle (50), and our findingsindicate that PDGF-D has similar immunoregulatory functionsin the corresponding tumor tissue. Immunomodulation wasalso recently investigated in a conditional mouse model ofrhabdomyosarcoma (9). PDGFRa expressionwas in thesemicelinked to disease progression but did not regulate immuneresponses. This is in line with our results that rather suggestPDGFRb to be involved in this regulation not PDGFRa.

Taken together, through analysis of gene expression profil-ing of 101 rhabdomyosarcomapatient samples and subsequentin vitro and in vivo validation, we found that PDGF activity cansupport rhabdomyosarcoma growth and regulate fundamentalcellular behavior. This includes tumor cell proliferation/sur-vival, apoptosis/senescence, cancer cell stemness/differentia-tion, immune system processes and blood vessel character-istics—typical processes known to require extracellular matrixremodeling. We could also link these activities to PDGF-CC/

PDGFRa signaling and PDGF-DD/PDGFRb signaling in atumor compartment–specific manner. Our findings suggestthat stromal PDGFR signaling should be further studied inrhabdomyosarcoma subtypes and other sarcomas.

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

Authors' ContributionsConception and design: M. Ehnman, K. Pietras, A. €Ostman, U. ErikssonDevelopment of methodology: M. Ehnman, C. StrellAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. BrodinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Ehnman, E. Missiaglia, E. Folestad, J. Selfe, C.Strell, K. Thway, K. Pietras, J. Shipley, A. €Ostman, U. ErikssonWriting, review, and/or revision of the manuscript: M. Ehnman, E. Mis-siaglia, C. Strell, K. Pietras, J. Shipley, A. €Ostman, U. ErikssonAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Ehnman, E. Folestad, J. Selfe, J.ShipleyStudy supervision: M. Ehnman, J. Shipley, A. €Ostman, U. Eriksson

AcknowledgmentsThe authors thank Inger Bodin for technical assistance and, Carina Hellberg

and Carl-Henrik Heldin (Ludwig Institute for Cancer Research Ltd.) who kindlyprovided PDGF-BB and antiserum against human PDGFRs. CP-673,451, ownedby Arog Pharmaceuticals LLC, was a valuable gift from Pfizer. The Children'sCancer and Leukaemia Group assisted with tumor collection.

Grant SupportThis study was supported by the Ludwig Institute for Cancer Research Ltd.

(M. Ehnman, E. Folestad, U. Eriksson), The Swedish Cancer Society (U. Eriksson,A. €Ostman, K. Pietras), The Childhood Cancer Foundation (M. Ehnman, K.Pietras), The Swedish Research Council Linnaeus grant to the STARGET con-sortium (U. Eriksson, A. €Ostman, K. Pietras), Karolinska Institutet (U. Eriksson),The Chris Lucas Trust (J. Selfe, E. Missiaglia), The Institute of Cancer Research (J.Shipley), The Royal Marsden Hospital Charity Panel (K. Thway), and Anders OttoSw€ards stiftelse (E. Folestad). Cancer Research UK (J. Shipley C5066/A10399)funded tumor collection and the Carte d'Identite Program of the Ligue NationaleContre le Cancer supported the expression profiling. The authors thank NHSfunding to the NIHR Biomedical Research Centre.

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

Received May 2, 2012; revised November 6, 2012; accepted November 26, 2012;published OnlineFirst January 21, 2013.

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2013;73:2139-2149. Published OnlineFirst January 21, 2013.Cancer Res Monika Ehnman, Edoardo Missiaglia, Erika Folestad, et al. Compartmentsin the Human Rhabdomyosarcoma Tumor Cell and Stroma Cell

Signalingβ and PDGFRαDistinct Effects of Ligand-Induced PDGFR

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