Endostatin gene therapy inhibits intratumoral macrophage M2 polarization

Original

article

Endostatin

gene

therapy

inhibits

intratumoral

macrophage

M2

polarization

Karen

Foguer

a,b,

*

,

Marina

de

Souza

Braga

a,b

,

Jean

Pierre

Schatzmann

Peron

c

,

Karina

Ramalho

Bortoluci

d

,

Maria

Helena

Bellini

a,b

a

NephrologyDivision,FederalUniversityofSaoPaulo,SaoPaulo,Brazil

b

BiotechnologyDepartment,IPEN-CNEN,SaoPaulo,Brazil

c

DepartmentofImmunology,InstituteofBiomedicalSciences,UniversityofSãoPaulo,SãoPaulo,Brazil

d_{DepartamentofBiologyBiologicalSciences,FederalUniversityofSaoPaulo,SaoPaulo,Brazil}

ARTICLE INFO Articlehistory:

Received28October2015

Receivedinrevisedform27January2016 Accepted27January2016

Keywords: Renalcellcarcinoma Endostatin

Tumor-associatedmacrophages

ABSTRACT

Background:Renalcellcarcinoma(RCC)isahighlyvascularizedcancerresistanttochemotherapyand

radiotherapy. RCC is frequently infiltrated with immune cells, with macrophages being the most

abundant cell type. Alternatively activated M2 macrophages are known to contribute to tumor

progression.Endostatin(ES)isafragmentofcollagenXVIIIthatpossessesantiangiogenicactivity.Inthis

study, we investigated the impact of ES gene therapy on the polarization of tumor-associated

macrophages(TAMs)inlungmetastasesfromtumor-bearingmice.

Methods:BALB/cmicedividedintothreegroups:Normal,ControlandES-treated.Tumor-bearingmice

weretreatedwithES-transducedcellsorcontrolcellsovertendays.Attheendofthestudy,plasmawas

collected,andpulmonarymacrophageswereisolatedandusedforFACSorRT-PCR.ELISAtestswereused

toanalyzeplasmaandcellculturesupernatantcytokines.

Results:EStreatmentsignificantlyreducedthelevelsofanti-inflammatoryandpro-angiogeniccytokines,

includingIL4,IL-10,IL-13andVEGF.GeneexpressionofM2markers,suchasIL-10,Arg-1,VEGFandYM-1,

declinedsignificantly.FlowcytometryshowedareductioninthenumberofM2F4/80+CD36+CD206+

CD209+macrophagesandinIL-10secretionbythesecells.ReducedlevelsofIL-10werealsofoundinthe

culturesupernatantsoftheES-treatedgroup.

Conclusions:OurresearchcorroboratespreviousobservationsthatEShasanimportantanti-tumoralrole.

However,asidefrompromotinginterferon-gsecretionandaneffectiveT cellresponse,weshowherethat

thisswitchisextendedtoTAMs,complicatingthemaintenanceofpro-tumorigenicM2macrophagesand

thusfavoringtumorelimination.

ã2016ElsevierMassonSAS.Allrightsreserved.

1.Introduction

Macrophagesareamongthemostimportantcellsoftheinnate immune system due to their capacity to effectively stimulate adaptiveimmuneresponsesand topromotetissue homeostasis andrepair.Infact,macrophagesarewidelydistributedthroughout thewholebodyandareknownastissue-residentcellsthathave tissue-specific nomenclatures: Kupffer cells reside in the liver, microglialcellsinthecentralnervoussystem,Langerhanscellsin the skin, osteoclastsin the bones and many others. It is now acceptedthatmanyofthesecellsarefirstderivedfromtheyolksac,

which maygreatly differ frommonocyte-derived inflammatory macrophagesthatarerecruitedtoinflamedtissues(reviewedin

[1,2]).

In addition to being defined by their tissue specificity, peritoneal [3] or bonemarrow-derived macrophagesmay also becharacterizedbytheirphenotype,ormoreprecisely,bytheir biologicalactivities.Inthissense,macrophagesmaydiffergreatly infunction,anddiscrepantpopulationshavebeendescribed[2,3]. Indeed,itiscurrentlywellacceptedthattissuemacrophageshave a more suppressive phenotype, whereas inflammatory macro-phageshavestimulatoryeffects.Althoughthereisnoestablished consensusregardingthesepopulations(reviewed[4]),themost widelyacceptednomenclatureis M1andM2,or classicallyand alternatively activated macrophages, respectively [5,6]. This nomenclature refers to the activation and functional status of thesecells,anditwasfirstdescribedthatM1areIFN-

g

and/or

LPS-*Correspondingauthorat:BiotechnologyDepartment,IPEN-CNEN,SaoPaulo, Brazil.

E-mailaddress:mhmarumo@terra.com.br(K.Foguer).

http://dx.doi.org/10.1016/j.biopha.2016.01.035

0753-3322/ã2016ElsevierMassonSAS.Allrightsreserved.

Available

online

at

ScienceDirect

stimulatedmacrophages,whereasM2macrophagesacquiretheir phenotypeuponstimulationwithIL-4,IL-10orTGF-

b

[5,6].Dueto the opposing nature and function of the aforementioned cytokines, it has also been observed that M1 and M2macrophageshave opposingtranscriptional and phenotypic profiles[2,3].

M2macrophagesresembletissue-residentmacrophagesthat areinvolvedintissue repair,phagocytosisofcellulardebrisand immunesuppressionandhavehighexpressionofarginase-1[7], IL-10 [8] and SOCS-3 [6], to note a few examples. [9]

M1 macrophages serve as potent stimulators of the adaptive immune response and express important pro-in_flammatory moleculesandtranscriptionfactors,suchasiNOS,IL-12[9],

NF-k

B [10] and STAT-1 [11]. Nonetheless, the importance of this cellularpopulationinseveralphysiologicalaswellaspathological processes,suchasinflammatoryandautoimmunediseases[4,12], pathogenic infections [11] and cancer, has been called into question(reviewedin[13,14]).

Itisverywellestablishedthatthenumberoftumorinfiltrating lymphocytes (TILs) directly correlates with tumor growth, metastasis and patient prognosis [15,16]. Several reports have demonstratedthattheinfiltrationofCD4orCD8IFN-

g

-secreting lymphocytes results in increased tumor cell death, as well as reducedtumorgrowthandmetastasis,resultinginanimproved prognosisandsurvivalrate[15].Thisislikelyaresultofthefact thatseveralimportanttumorkillingmechanismsaretriggeredby IFN-

g

, such as the secretion of NO [17], TNF-

a

, and increased expressionofantigenpresentingmolecules[4,6].

Unfortunately, tumor cells may develop many different mechanisms to circumvent the immune response, mainly by triggering suppressive mechanisms. For example, some solid tumorsmayrecruitTregulatorycellstothetumors,as wellas induce their expansion in draining lymph nodes [18,19]. Interestingly, thisis amplified bytolerogenic dendritic cellsin the draining lymph nodes, whose increased expression of indoleamine-2,3-dioxigenase(IDO)promotesthedifferentiation of Tregs [20], rendering the tumors much more refractory to therapeuticintervention[21,22].Thisevidencedemonstratesthe importanceofthequalityoftumorinfiltrationbyimmunecells, i.e.,Tregsvs.Teffectorcells,andalsooftheinterplaybetween TILs and innate immunity in tumor elimination and patient survival.

TAMshavealsogainedrecentattention,asithasbeenreported that these cells greatly correlate with tumor progression, metastasisand overall outcome, similartoTcells (reviewed in

[14,16]).Indeed,similarlytoTregs,tumor-infiltratingmacrophages mayactuallydampentheimmune responseinseveral different ways.Forinstance,whenTcellsweretransferredtoRAG-deficient animals,thepresenceofTh2lymphocytessecretingIL-4and IL-10greatlyreducedtheimmuneeffectorresponse,convertingTAMs toanM2phenotypeandrenderingtheanimalsmoresusceptibleto lungmetastasis[23].Moreover,TAMsareabletosecretesignificant amounts of the M2 marker CCL2, which in turn may recruit CD4+_CD25+_Foxp3+_{Tregstosolidovariantumors,promotingtheir}

growthandvascularization[24].

Renalcellcarcinoma(RCC)isthemostcommontypeofkidney cancerand accountsforapproximately3%ofhuman malignan-cies.[25]Inadults,themostcommonhistologicsubtypeisclear cellRCC(ccRCC).MostpatientswithccRCChaveamutationinthe tumorsuppressorgenevonHippel–Lindau(VHL).TheVHLgene encodestheVHLprotein,whichiscapableofdownregulatinga number of intracellular proteins, including hypoxia inducible factor(HIF).HIFisheterodimerictranscriptionfactor(HIF

a

/

b

) that regulates the expression of genes and facilitates the adaptation of tissue during hypoxia. In normoxic conditions, however,VHLproteinisinvolvedinthedegradationoftheHIF

a

subunit. Lossof function of theVHL gene, even under normal oxygenpressure,resultsinanincreaseinHIFandaconsequent increaseinthetranscriptionofseveralangiogenicfactorssuchas vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and basic fibroblast growth factor(FbF)

[26,27]. The presence of these angiogenic factors justifies the intense vascularizationof CCR and contributessubstantially to thedevelopmentandprogressionofthisdisease,explainingthe highprevalenceofmetastases(30–40%)intheinitialdiagnosisof RCC.

Renalcellcarcinoma(RCC)isconsideredtobeanimmunogenic tumor and is frequently in_filtrated by immune cells. However, clinicopathological studies have indicated that patients with higher densities of infiltrating TAMs have significantly poorer relapse-free survival and overall survival rates. Therefore, TAM infiltration appears to be a significant unfavorable prognostic factorincancerpatientsandmaybeapotentiallyusefulprognostic markerofclinicaloutcomes.

In this work,we evaluatedthe functional characteristics of TAMsinmetastaticlungsofmicethatunderwentantiangiogenic genetherapywithES.

2.Materialandmethods 2.1.CellLines

NIH/3T3-LendSN-clone3wasusedforESexpression,andNIH/ 3T3-LXSNwasusedasacontrol,asdescribedinpreviouswork[10]. Bothcelllinesweremaintainedinhigh-glucose(4.5g/Lat25mM) DMEMmedium(LifeTechnologiesCorporation1,GrandIsland,NY, USA)supplementedwith100U/mLpenicillin,100

m

g/mL strepto-mycin(Gibco1LifeTechnologies,NY,EUA),and10%fetalbovine serum (FBS) (Life Technologies Corporation1 Grand Island, NY, USA).

Themurinekidneycarcinomacellline(Renca),wasmaintained in RPMI 1640 medium (Life Technologies Corporation1 Grand Island,NY,USA)supplementedwith10%fetalbovineserum(Life Technologies Corporation1 Grand Island, NY, USA), 2mM L-glutamine (Gibco1 Life Technologies, NY, USA), 1mM sodium pyruvate(Gibco1LifeTechnologies,NY,USA),1%minimalEagle’s mediumnonessentialaminoacids(Gibco1LifeTechnologies,NY, USA),100U/mL penicillinand 100

m

g/mLstreptomycin (Gibco1 LifeTechnologies,NY,USA).

Both cell lines were maintained at 37
C in a humidified atmospherecontaining5%CO2.

2.2.Animals

MaleBALB/cmice,aged8–10weeks,wereobtainedfromthe Animal Facility of IPEN/CNEN-SP, Sao Paulo, Brazil. In these experiments, 24 mice were used (normal group=8, control group=8, ES-treated group=8). All animals were cared for in accordancewiththestandardsoftheinstituteunderaprotocol approved by the Animal Experimentation Ethics Committee (NumberofProcess:0260/12).

2.3.OrthotopicmetastaticRCCtumormodel

Mice were anesthetized by intraperitoneal injection with ketamineand xylazine(100mg/kgbody weight,Ketalar; Parke-Davis,MorrisPlains,NJ,USAand10mg/kgbodyweight,Phoenix Scientific,St.Joseph,MO,USA,respectively).Theleftflankregion was trichotomizedand theleftkidneywasexposedand2105

Rencacellssuspendedin10

m

LofPhosphateBufferedSaline(PBS) were injected under the renal capsule with the aid of a glass

syringe(Hamilton1,Reno,NV,USA).Thekidneywasreturnedto theposteriorregionoftheabdomenandwassuturedattheregion oftheincision.

7daysafterRencacellinoculation,thekidneywasremovedby unilateral nephrectomy. Then, mice were divided into two experimentalgroups:controlgroupandES-treatedgroup.These groupsreceivedasubcutaneousinjectionof3.6106_cellsNIH/

3T3-LXSNorNIH/3T3-LendSN-clone3,respectively. 2.4.Obtainingplasmafromwholeblood

Attheendoftheexperiment,whichtotaled17days,micewere euthanizedinaccordancewithcurrentstandardsoftheAmerican VeterinarianMedical Association. Bloodsampleswerecollected intotesttubescontainingheparin.Theplasmacomponentswere separatedfromwholebloodbycentrifugationandthenstoredina freezerat20
_C.

2.5.DeterminationofplasmaESandcytokinelevels

Plasma ES levels were measured using a Mouse Endostatin ELISAKit(Novateinbio1,AcceleratesResearchandDevelopment, USA) according to the manufacturer’s instructions. The ES concentrations were determined at least in duplicate, and the assayreproducibilitywasconfirmed.ELISAplateswerereadusing theMultiskan EX Microplate Reader (Labsystems, Milford,MA, USA).

The cytokine concentrations were determined using the Milliplex1 (Mouse Cytokine Magnetic Bead Panel, Millipore), accordingtothemanufacturer'sinstructions.Theplatewasreadon Luminex200TMdeviceanddatawereprocessedusingtheXponent software.Weanalyzeddatafromthemeanfluorescenceintensity (MFI).

2.6.IsolationofTAMs

Thelungsofthemicewereperfusedwithcoldsalinesolution, andafterremovaltheyweremaintainedinpureDMEMmedium. Thelungs were mincedwith surgicalscissors, treatedwithan enzymesolutionofcollagenaseD(2.5mg/mL)(Roche,Mannheim, Germany) diluted in DMEM and incubated at 37
C for 45min withcontinuousagitation.DMEMwith10%FBSwasthenadded, andthesampleswerecentrifugedat450gfor5minat4
C.The tissue suspension was passed through a 70-mm cell strainer, subjected to discontinuous Percoll gradient separation (GE Healthcare1,Uppsala,Sweden)using37and 70%dilutions,and centrifugedat950gfor20minat4
Cwiththebrakesoff.The cells were collected from the interface between the different gradientsofPercollandresuspendedintheappropriatemedium supplementedwith10%FBS.Thecellswerecentrifugedat450g at 4
_{C for 5min and resuspended in 1mL of the appropriate}

culture medium without serum and counted in a Neubauer chamber.

Theisolatedcellswereseededin100-mmcellcultureplatesin RPMI-1640mediumfor40min.Non-adherentcellswereremoved by PBS washes, and adhered cells (TAMs) were used for the followingexperiments.

2.7.Reversetranscription-polymerasechainreaction(RT-PCR) RNAwas isolated fromthe adherentcells using thereagent Qiazol(Qiagen, Germantown, MD, USA).cDNAwas synthesized from 2

m

g total RNA with the first-strand cDNA Synthesis kit (Promega, Heidelberg, Germany), following the manufacturer's instructions. All RT-PCR reactions were performed using the ABsoluteMix SYBR Green qPCR kit (Thermo Scientific1 _Grand

Island,NY,USA).The20

m

Lreactionscontained10

m

Lof1SYBR Green qPCR Mix, containingthe fluorophoreSYBRGreen I, Taq polymerase, dNTPs, MgCl2 and buffer the enzyme, and 10

m

L containing primers and H2O. The amplification reaction was

performedbytheRealTimeStepOnePlusPCRSystem(Applied Biosystems1)asfollows:50
Cfor2minutes,95
Cfor10min,and 40cyclesof95
Cfor15sand60
Cfor1min.Theexpressionlevels oftargetgenescalculatedusingthefollowingequation:relative quantification=2DDCt_{. A Ct value was calculated for both the}

targetgeneandthereferencegene(HPRT)foreachsample.This valuerepresentsthepointatwhichtheamplificationsignalwas detected. From there, the

D

Ct was calculated (CttargetgeneCt

normalizing[HPRT]). Subsequently, the

DD

Ct was determined

(

D

Ctsample

D

CtHPRT) using normal (healthy lung) tissue to

comparetheleveloftargetgeneexpression.The

DD

Ctwasused toquantifytherelativeexpressionlevel,wherethenumber2isthe sumoftheprimerefficienciesofthetargetreferencegenes,where both have100% efficiency.Allprimers sequencesare shownin

Table1.

2.8.Immunofluorescence

The isolated cells were plated in 24-well plates and after adhesionwerefixedin4%paraformaldehydeatroomtemperature for10min.Then,thecellswerewashedwithPBSandincubated with 1% BSA at 37
C for 1h toblock nonspecific interactions. Primary antibody to F4/80 (A3-1, MCA497G, AbD Serotec, Kidlington, UK) was incubated at 4
C overnight. After washes with PBS, the cells were incubated with anti-IgG secondary antibodyandconjugatedwithAlexafluor-645(Invitrogen, Carls-bad,USA)atroomtemperaturefor1.5h. Subsequently,thecells werewashedwithPBSandkeptinmountingmedia(ProlongGold Antifade Reagent, Invitrogen, Carlsbad, USA) containing 40

,6-diamidino-2-phenylindole (DAPI) in the darkroom at 4
C until acquisitionofimages.

2.9.Flowcytometryanalysis

Cellswereseededatadensityof1106perwellina96-well plate, and incubated with lipopolysaccharide (LPS) (1

m

g/mL) (Sigma–Aldrich, St. Louis, MO). Cells were incubated with brefeldin-A(e-Biosciences,SanDiego,CA,USA)for10htoavoid cytokine secretion, for subsequent intracellular staining. Cells surfaceswerethenstainedwithanti-F4/80antibodyconjugatedto FITC(BM8,cat.number11-4801,e-Bioscience,SanDiego,CA,USA), anti-CD11cantibodyconjugatedtoPE(N418,cat.number12-0114, e-Bioscience,SanDiego,CA,USA),anti-CD206antibodyconjugated

Table1

SequenceofPrimersusedforRT-PCR.

HPRT Forward(F)50_{-TGGACAGGACTGAAAGACTT-3}0

Reverse(R)50_{-AATGTAATCCAGCAGGTCAG3}0

IL-10 Forward(F)50_{-CCTCGTGGAGCCTCAGTTTTC-3}0

Reverse(R)50-GAGCACGTCAGGTACATTTCAATT-30 IL-12 Forward(F)50-GCGTGGGAGTGGGATGTG-30

Reverse(R)50_{-GCAAAACGATGGCAAACCA-3}0

Arg-1 Forward(F)50_{-AACGGGAGGGTAACCATAAGC-3}0

Reverse(R)50_{-GCGCATTCACAGTCACTTAGGT-3}0

iNOS Forward(F)50_{-GGGCAGCCTGTGAGACCTT-3}0

Reverse(R)50_{-TGAAGCGTTTCGGGATCTG-3}0

VEGF Forward(F)50-CCAGACCTCTCACCGGAAAG-30 Reverse(R)50_{-CTGTCAACGGTGACGATGATG-3}0

to AlexaFluor 647 (MR5D3, cat. number MCA2235A647, AbD Serotec,Kidlington,UK),anti-CD80antibodyconjugatedtoPE(cat. number 12-0801, e-Bioscience, San Diego, CA, USA), anti-CD36 antibody conjugated to PE (cat. number 12-0361, e-Bioscience,SanDiego,CA,USA),oranti-CD209antibody conjugat-edtoPE(cat.number122091,e-Bioscience,SanDiego,CA,USA) and fixed with Cytofix/Cytoperm (BD Biosciences, New Jersey, USA).Cellswerethenintracellularproteinswerestainedwith anti-IL-10 antibody conjugated to PE (cat number 554467, BD Biosciences,NewJersey,USA),anti-IL-12antibodyconjugatedto PE(catnumber554479,BDBiosciences,NewJersey,USA), anti-IL-12 antibody conjugated to PE-Cy7 (cat number 25-7123, e-Bioscience, San Diego, CA, USA) and anti-Arginase I antibody conjugatedtoPE(catnumberIC5868P,BDBiosciences,NewJersey, USA).

FlowcytometrywasperformedonaFACSAccuriC6(BD,San Diego,CA)withCsamplersoftwarefordataanalysis.

2.10.Cultureofmacrophages

Following tissue digestion and isolation of macrophages by densitygradientcentrifugation,5105macrophageswere sepa-ratedandplatedin96-wellplatesinRPMI.After2htoallowcellsto adhere, a wash was performed withPBS, and thesecells were stimulated with1

m

g/mL of LPS in RPMI for 48h. Cell culture supernatantwascollectedandstoredat20
_C.

2.11.Determinationofcytokinelevelsinthecellculturesupernatant

IL-12andIL-10levelswereanalyzedbycaptureELISAusingkit MouseIL-12ELISASetandmouseIL-10ELISASet(BDBiosciences, NewJersey,USA),accordingtomanufacturer'sinstructions.

2.12.Statisticalanalysis

SimplecomparisonsofmeanswereperformedbyStudent’sT tests.Themultiplemeancomparisonswereperformedusing one-wayortwo-wayANOVAfollowedbyBonferroni’stestusingthe GraphPad Prism program Version 5.0 Windows (GraphPad1 Software,SanDiego,CA,USA),http://www.graphpad.com. Differ-ences with a p value of p<0.05 were considered statistically significant.

3.Results

ESplasmalevelsofthenormalandexperimentalgroupswere measured. The normal group,control group and ES group had plasma levels of 127.19.43

m

g/mL, 234.026.46

m

g/mL and 333.9

m

g/mL9.21,respectively.Thepresenceoftumorcellsled toa1.84-foldincreaseinendogenousESlevelsinthecontrolgroup comparedwiththenormalgroup(normalvs.controlp<0.001). TheESgrouphada2.62-foldincreaseinESplasmalevelscompared withthenormalgroup(normalvs.ESp<0.001)anda1.42-fold increase compared with the control group (control vs. ES p<0.001). Circulating ESlevelscorroborated our previousdata

[29–36].

3.1.EStreatmentdecreasesanti-inflammatoryandpro-angiogenic cytokines

Webeganourresearchbymeasuringthecirculatinglevelsof cytokines in all three experimental groups using multiplex cytokinepanels.Severalcytokinesweredetectedabovethelower limitsofdetectionoftheassayinthenormalgroup.Interestingly, wedidnotdetectdifferencesinTh1-relatedcytokines,suchas IL-12 and TNF-

a

in ES-treated animals compared with control animals (Fig. 1A and F). However, ES treatment significantly reduced anti-inflammatory and pro-angiogenic cytokines,

Fig.1.DeterminationofplasmalevelsofIL-12,IL-10,VEGF,IL-4,IL-13andTNF-a.Circulatinglevelsofprooranti-inflammatorycytokinesweredeterminedintheplasmaof controlandES-treatedanimalsaccordingtothematerialsandmethods.N=4.One-wayANOVAfollowedbyBonferronibyBonferroni'stest.*

p<0.05;**

p<0.01;***

including IL4, IL-10, IL-13 and VEGF (Fig. 1B–E), which was expectedduetoitsanti-angiogeniccapacity.

3.2.ExpressionofM2-associatedgenesweredecreasedbyES treatment

We next evaluated the gene expression profile of TAMs. Accordingly,TAMswereisolatedfrommetastaticlungsandplated toenrichthemacrophagesbyadherence.Thelungweightofthe samples ranged from 0.1g to 0.5g and the number of TAMs collectedrangedfrom1.0106to2.0106cells.F4/80labeling confirmed the enrichment of the isolated macrophages of the lungs(Fig.2A).RNAwasisolatedfromthemacrophagestocarry outgeneexpressionanalysis.

ThegeneexpressionoftheclassicM1markersIL-12,iNOSdid notchangesignificantlyinresponsetoEStreatment(Fig.2BandC). Interestingly,EStreatmentsignificantlyreducedtheexpressionof several M2 markers, such as IL-10, Arg-1, VEGF and a novel phenotypemarker,YM-1(Fig.2D–G).

3.3.MacrophagerecruitmentinmetastaticlungswasnotalteredbyES treatment

To corroborate previous findings, we next evaluated the frequencyofM1andM2macrophagesamongtheTAMsbyflow cytometry.Initially,analysisofthesidescatterprofilebeam(SSC) and forward scatterprofile (FSC)was performedfor gating. As showninFig.3A,TAMsaccountedforapproximately51.2%ofthe totalpopulation(Fig.3A).Thispopulationwasthenanalyzedfor theexpressionofF4/80(Fig.3B).Therewasasignificantincreasein thepopulationofF4/80+_{TAMsinthecontrolgroupcomparedwith}

thenormalgroup(17.265.98vs.12.010.46F4/80+%,p<0.05) (Fig.3C).Nochangeswereobservedamongtheothergroups.

3.4.EStherapydecreasesM2macrophagesinthetumor microenvironment

Next, TAMs were quantified according to M1 and M2 phenotypes. F4/80+_/CD11c+ _{and F4/80}+_/CD80+ _populations Fig.2.TAMgeneexpressionprofileofM1andM2markers.TAMswereobtained,enrichedandanalyzedaccordingtomaterialsandmethodsandanalyzedfortheexpression ofIL-12,iNOS,IL-10,arginaseI,YM-1andVEGF.(A)EnrichmentofTAMswasevaluatedbyF4/80(anti-F4/80Pe)andDAPI(blue)staining.TAMswerevisualizedat40 magnification.(B)ThegeneexpressionprofilewasevaluatedbyqPCR.N=4.Student’st-test**_p<0.01;***_{p<0.001.(Forinterpretationofthereferencestocolorinthisfigure}

increasedsignificantlyin themetastaticlungsof controlgroup mice(Fig.4A–D).However,EStreatmentcausednochangeinthe frequencyof thesepopulations.Similarly, M2markers(CD206, CD209,CD36andarginase1)weresignificantlyincreasedinthe lungsofcontrolgroupmice.However,EStreatmentsignificantly decreasedthenumberofM2-polarizedmacrophages.(Fig.4E–L). Theproductionofpro-andanti-inflammatorycytokineswas measuredinthemacrophages.F4/80/CD11candF4/80/CD80cells wereselected(Fig.4AandC),andIL-12wasidentifiedfromthem. The presence of lung metastases resulted in a significant increase in F4/80+_/CD11c+_/IL-12+ _{and F4/80}+_/CD80+_/IL-12+ _cells.

However,treatmentwithESdidnotcauseasignificantchangein anyofthesepopulations(Fig.5A–D).Thecontrolgroupmicehad significantlyelevatedlevelsofIL-10-producingmacrophages(F4/ 80+_/CD206+_/IL-10+_{, F4/80}+_/CD209+_/IL-10+ _{and F4/80}+_/CD36+

/IL-10+_{)comparedwiththenormalmice.TreatmentwithESpromoted}

asignificantreductioninthesepopulations(Fig.5E–I).

3.5.Determinationofcytokinelevelsinthecellculturesupernatant ThedeterminationofIL-12andIL-10inthesupernatantsofthe macrophagecultureswasperformedafterstimulationwithLPS. IL-12levelswerenotsignificantlydifferentbetweengroups(normal vs. control, p=0.45;normal vs. ES, p=0.07; and control vs. ES, p=0.45). However, the amountof IL-10 present in the culture supernatants was significant increased in the control group compared with the normal (p<0.05) and ES (p<0.01) groups (Fig.6).

4.Discussion

Inthepresentwork,wesoughttoevaluatewhethertreatment withrecombinantESwouldmodulatethepro_fileofTAMsinour

modeloflungmetastasis.ESisa20-kDafragmentresultingfrom cleavageof thecollagenXVIII COOHterminus. Thisportionhas beendescribedtoinhibitendothelialcellproliferation,migration and invasion, and to dramatically reduce tumor growth and metastasisinseveralmousemodelswithnoserioussideeffects

[28–36]. Of note, using the same model, we have previously published that ES treatment increased the frequency of pro-inflammatoryTILs,mainlyIFN-

g

-secretingCD4,CD8andNKcells, resulting ina reduced number of lung tumornodules, reduced metastasisandahighersurvivalrate[17].Thus,besidesits anti-angiogenicactivity,wehavepreviouslydemonstratedthatESalso displays an important stimulatory function on T lymphocytes. Here,ourapproachwastoevaluatewhetherthiseffectcouldbe extended,eitherdirectlyorindirectly,toTAMs.

Ourresultsclearlydemonstrateacontractionoftheregulatory M2macrophageswithinthetumorallungnodules;however,we observed nochangein pro-inflammatory M1macrophages. We observedadecreaseinseveralimportantM2markersinvivo,such asarginase-1,Ym-1andVEGF.Previousresearchhasshownthat reducedintra-tumoralO2tensioninduceshighexpressionof

HIF-1

a

thatspecificallyup-regulatessuppressiveandpro-angiogenic molecules,suchasPD-1andVEGF[37].TheimportanceofVEGFin tumorgrowthandestablishmentofmetastasesisworthnoting. Accordingly,weobservedthatEStreatmentnotonlyreducedthe expressionofVEGFRinmacrophagesisolatedfromthemetastatic nodulesbutalsothattheES-treatedanimalshadlesssolubleVEGF inplasmathanthecontrolgroup.

Associatedwiththesefindings,wealsodemonstratedthrough flowcytometryandintracellularstainingthatthereisnotonlya reduction of M2 F4/80+_CD36+_CD206+_CD209+ _{macrophages but}

also a reduction in IL-10 secretion by these populations. The quantificationofthecytokinesinculturesupernatantalsorevealed reducedlevelsofsecretedIL-10intheES-treatedgroup.IL-10is

Fig.3.AssessmentofthefrequencyofTAMsinthelungsofmicebyflowcytometry.(A)Representativegateshowingmacrophagegatingstrategy.(B)Representativescatter chartsshowtherespectivepercentageofF4/80+

Fig.4.AssessmentofM1andM2macrophagesubsetsamongTAMsbyflowcytometry.(A,C,E,G,I,K)RepresentativescatterchartsshowthepercentageofF4/80+_CD11+_,F4/

80+_CD80+_,F4/80+_CD206+_,F4/80+_CD209+_,F4/80+_CD36+_,andF4/80+_Arg1+_{cells.(B,D,F,H,J,L)Statisticalanalysisforthedatarepresentedineachpanelwasperformedusinga}

one-wayANOVAfollowedbyaBonferroni'stest.N=4.*

p<0.05,**

p<0.01,***

Fig.5.AssessmentofIL-10-andIL-12-producingmacrophagesinthelungsofnormal,controlandES-treatedmicebyflowcytometry.(AandC)Representativescattercharts showthepercentagesofF4/80+_/CD11c+_/IL-12+_andF4/80+_/CD80+_/IL-12+_{cells.(B)(E,G,I)RepresentativescatterchartsshowthepercentagesofF4/80}+_/CD206+_/IL-10+_,F4/80+_/

CD209+

/IL-10+

andF4/80+

/CD36+

/IL-10+

cells.(B,D,F,H,J)Statisticalanalysisforthedatarepresentedineachpanelwasperformedusingone-wayANOVAfollowedby Bonferroni’stest.N=4.*

p<0.05,**

p<0.01,***

one of the most important suppressive cytokines that directly correlateswithimmunosuppressionandtumoraggressiveness,as showninbreasttumors[38]andHPV-relatedcancer[39,40].These findingsmayindicatethatthechangesobservedforTILsdescribed inourpreviousresearch(i.e.,theincreaseinIFN-

g

-secretingTand NK cells) may be responsible for these changes observed. Furthermore, we may not exclude the possibility that ES acts directlyonmacrophagesoreveninamoresystemicway,asithas recentlybeendescribedasa androgenreceptorantagonist[41]. ThereductioninIL-10iscoincidentwithreductionofIL-4,whichis alsoimportantinskewingmacrophagestowardtheM2phenotype. Itisworthmentioning,however,thatalthoughthesesuppressive cytokineswerereduced,nochangeswereobservedforIL-12and IL-1

b

secretion, leading us to speculate that ES may be more effectiveinabrogatingordampeningM2expansion,ratherthan promotingM1differentiation.

5.Conclusion

Insummary,ourresearchcorroboratespreviousobservations thatEShasimportantanti-tumoralactivity.However,asidefrom promotinganeffectiveTcellresponseandinterferon-

g

secretion, weshowherethatthisswitchisextendedtoTAMs,complicating themaintenance ofpro-tumorigenicM2macrophagesand thus favoringtumorelimination.Althoughwerefertotumorinfiltrating immunecellsasan“inflammatoryinfiltrate”,manyofthesecellsin factdisplaymoreofasuppressivephenotyperatherthanan anti-tumoralprofile.Ourfindingshighlighttheconundrumthatisthe anti-tumorimmuneresponseandthepleiotropiceffectofESin thismodel.

Con_flictofinterest

The authors declare that they have no conflicts of interest concerningthisarticle.

Acknowledgements

WewouldliketothankMs.EvelinCarolinedaSilvafortheir valuable technical support on this project. This study was supported by FAPESP (Process number: 2011/18703-2) CNPq (Processnumber:473102/2012-9)andCAPES/PNPD.

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