The water soluble ruthenium(II) organometallic compound [Ru(p-cymene)(bis(3,5 dimethylpyrazol-1-yl)methane)Cl]Cl suppresses triple negative breast cancer growth by inhibiting tumor infiltration of regulatory T cells

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suppresses

triple

negative

breast

cancer

growth

by

inhibiting

tumor

inﬁltration

of

regulatory

T

cells

Maura

Montani

a,1

_,

_Gretta

_V.

_Badillo

_Pazmay

a,b,1

_,

_Albana

_Hysi

c,1

_,

_Giulio

_Lupidi

b,∗∗

_,

Riccardo

Pettinari

b

_,

_Valentina

_Gambini

a

_,

_Martina

_Tilio

a

_,

_Fabio

_Marchetti

d

_,

Claudio

Pettinari

b

_,

_Stefano

_Ferraro

d

_,

_Manuela

_Iezzi

c

_,

_Cristina

_Marchini

a

_,

Augusto

Amici

a,∗

a_School_of_Bioscience_and_Veterinary_Medicine,_University_of_Camerino,_Camerino,_MC,_62032,_Italy b_School_of_Pharmacy,_University_of_Camerino,_Camerino,_MC,_62032,_Italy

c_Aging_Research_Centre,_G._d’Annunzio_University,_Chieti,_66100,_Italy

d_School_of_Science_and_Technology,_University_of_Camerino,_Camerino,_MC,_62032,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21September2015

Receivedinrevisedform11February2016 Accepted1March2016

Availableonline30March2016 Keywords:

Ruthenium(II)

Organometallicarenecomplexes Anticancerdrugs

Triplenegativebreastcancer Invivotests

TumorinﬁltratingregulatoryTcells

a

b

s

t

r

a

c

t

Rutheniumcompoundshavebecomepromisingalternativestoplatinumdrugsbydisplayingspecific activitiesagainstdifferentcancersand favorabletoxicityand clearanceproperties.Here, weshow thattheruthenium(II)complex[Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)methane)Cl]Cl (UNICAM-1)exhibitspotentinvivoantitumoreffects.Whenadministeredasfour-dosecourse,byrepeatingasingle dose(52.4mgkg-1)everythreedays,UNICAM-1significantlyreducesthegrowthofA17triplenegative breastcancercellstransplantedintoFVBsyngeneicmice.Pharmacokineticstudiesindicatethat UNICAM-1israpidlyeliminatedfromkidney,liverandbloodstreamthankstoitshighhydrosolubility,exerting excellenttherapeuticactivitywithminimalsideeffects.Immunohistologicalanalysisrevealedthatthe efficacyofUNICAM-1,mainlyreliesonitscapacitytoreversetumor-associatedimmunesuppressionby significantlyreducingthenumberoftumor-infiltratingregulatoryTcells.Therefore,UNICAM-1appears verypromisingforthetreatmentofTNBC.

1. Introduction

Breastcancerisaheterogeneousdiseaseclassiﬁedinto molec-ularsubtypeswithdistinctivegene expressionsignatures.Ofall themolecularsubtypes,triplenegativebreastcancer(TNBC)has the worst negative outcome and prognosis [1,2]. TNBCs occur mostfrequentlyinyoungwomenandtendtoexhibitaggressive metastatic behavior [3]. TNBCs are estrogen receptor (ER) and

∗ Correspondingauthorat:SchoolofBioscienceandVeterinaryMedicine,via GentileIIIDaVarano,UniversityofCamerino,Camerino,62032,Italy.

∗∗ Correspondingauthor.

E-mailaddresses:giulio.lupidi@unicam.it(G.Lupidi),augusto.amici@unicam.it

(A.Amici).

1 _These_authors_contributed_equally_to_this_work.

progesteronereceptor (PR)-negative and alsolack high expres-sion/ampliﬁcationofHER2,limitingtargetedtherapeuticoptions [4].Newtherapiesagainstthisbreastcancersubtypearetherefore anurgentunmetmedical need.Cisplatin(cis-[PtIICl2(NH3)2])is wellestablishedasaneffectivedrugforthetreatmentoftesticular cancerand,incombinationwithotherchemotherapeuticagents, forovarian,cervical,brain,bladder,lung,andbreastcancers[5,6]. Recently, preclinical and clinical data have revealed encourag-ing anticancer activity of cisplatin as single-agent in patients with TNBC [7,8]. Despite the success of platinum-based drugs, their continued use is greatly limited by severe dose limiting side effects and intrinsic or acquired drug resistance [9–11]. In the search for anticancer agents containing nobel metals other than platinum, ruthenium compounds have turned out to be a cutting-edge class of anticancer compounds [12–18]. Accordingly, two ruthenium(III)-based compounds, namely http://dx.doi.org/10.1016/j.phrs.2016.03.032

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M.Montanietal./PharmacologicalResearch107(2016)282–290 283

Fig.1.Chemicalstructuresofcisplatin,NAMI-AandUNICAM-1.

NAMI-A (imidazolium trans-[tetrachloro(dimethylsulfoxide)(1H-imidazole)ruthenate(III)]) [19] and NKP1339 sodium trans-[tetrachloridobis(1Hindazole)ruthenate(III)] [20], are cur-rently ongoing in phase II clinical trials. The different toxicity profiles between platinum- and ruthenium-based compounds couldprobablyduetodifferenttargets.Itiswidelyacceptedthat theantineoplasticpropertiesofplatinumcompoundsrelyontheir interaction withDNA, which, in turn,activates cell death[21]. Instead,themechanismsbywhichruthenium-baseddrugsexert theiranticancereffects remaintobefullyelucidatedbutrecent evidencesuggeststhatrutheniumcompoundsaremostlikelytobe multitargeted[22,23].Thus,theycouldrepresentavalid therapeu-ticalternativetoplatinum-baseddrugswhichareoftenassociated with an unfavorable toxicity profile. Among the ruthenium(II) organometallic complexes, the half sandwich arene–ruthenium subgroup,inparticular,offersagreatpromiseinthefieldofcancer therapy. Two prototypical compounds reported by Aird et al. [24] andRAPTA-Cdeveloped byScolaro etal. [25]have shown relevant therapeutic potential. We have recently reported an extensivestudyonthecoordinationchemistryofrutheniumarene fragmentswithbis(pyrazol-1-yl)methanligands[26].Inthiswork weextendourinvestigationtotheinvivoantitumoractivityofthe prototypecompound, [Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)methane)Cl]Cl,termedUNICAM-1(Fig.1).Theresultspresented hererevealthatUNICAM-1hassignificantanticanceractivityina murinemodelofTNBCandresultstobewelltolerated,showing considerablyreducedside-effectswhencomparedtocisplatinand NAMI-A.Theanalysisoftumorimmuneinfiltratesuggeststhatthe responsetothisnewchemotherapeuticagentreliesmainlyonits capacitytoelicitananticancerimmunosurveillance.

2. Materialandmethods

2.1. Compounds

NAMI-Awaspreparedaccordingtoapatentedprocedure[27]. Cisplatin was obtained by Sigma Chemical Co. (St. Louis, MO). UNICAM-1 was prepared as previously described [26] and its analyticalandspectroscopicdatahavebeenreportedinthe Sup-plementarymaterials.

2.2. Cellcultures,cellproliferationassayandlysatespreparation A17cellswereestablishedfromspontaneouslobular carcino-masthataroseina FVB/neuTmicetransgenicfor theactivated isoformofratHER-2/neuoncogene(FVB/neuT233),aspreviously described [28,29].A17 cells werecultured in Dulbecco’s Modi-ﬁedEagleMedium(DMEM,Invitrogen,Carlsbad,CA)supplemented

with20%fetalbovineserum(FBS,Invitrogen,Carlsbad,CA)and1% penicillin–streptomycin(Invitrogen,Carlsbad,CA).Humanbreast cancer MDA-MB 231 cells were obtainedfrom American Type CultureCollection(Rockville,MD)andculturedinDMEM supple-mentedwith10%FBSand1%penicillin-streptomycin.Cellswere growninahumidiﬁedatmospherewith5%CO2at37◦C.UNICAM-1 effectoncellviability,respecttocisplatinandNAMI-A,was evalu-atedbyseeding2.5×103_cells/well_(A17_cells)_or₇_×₁₀3_cells/well (MDA-MB-231cells)inoctuplicatein96wellplatesincomplete medium.Thedayafter,freshmediumcontainingappropriate con-centrationsofUNICAM-1,NAMI-Aandcisplatin(alldissolvedin isotonicsolution,0.9%NaCl(aq))wereadded.After72hcell viabil-itywasdeterminedusinganMTT(SigmaAldrich,St.Louis,MO) assay,aspreviouslydescribed[30].Thecytotoxicityofthe com-poundswasreportedaspercentageofviablecellsrelativetocontrol cells.Alltheexperimentswererepeatedthreetimes.Forcelllysates preparation, 4×105 _cells/well _(A17 _cells) _or ₈_×₁₀5 _cells/well (MDA-MB-231cells) wereseededin6-wellplates,treated with UNICAM-1, NAMI-Aandcisplatindrugsfor 48h, harvested,and lysedinRIPAbuffer(1%NP40,0.5%Na-deoxycolicacidand0.1%SDS inPBS)withfreshproteaseinhibitors(ProteaseInhibitorCocktail, SigmaAldrich,St.Louis,MO).

2.3. Animals

FemaleFVB/NCrlmicewereobtainedfromCharlesRiverS.r.l. (Lecco,Italy),andhousedundercontrolledconditions.Micewere treatedaccordingtotheEuropeanCommunityguidelines.The Ani-malResearchCommitteeoftheUniversityofCamerinoauthorized theexperimentalprotocol.

2.4. Treatmentsandtumorgrowth

105 _A17 _cells _were _inoculated _in ₂₀₀_␮l _of _PBS _into mam-mary fat pad of 8-week-old FVB females. 10days after tumor challenge,micewererandomlydividedin4groups(10miceper group)andtreatedwithUNICAM-1,NAMI-A,cisplatinorisotonic solution(vehicle) byintraperitoneal injection(via i.p)in accor-dancewithtwodifferentdosageandtreatmentschedules(protocol q3×4orq1×6)aspreviouslyreported[31].Accordingtoprotocol q1×6,35mg/kg/dayofrutheniumcomplexes(210mg/kg/dayfinal amount)and2mg/kg/dayofcisplatin(12mg/kg/dayfinalamount) wereadministeredforsixconsecutivedays.Accordingto proto-colq3×4,52.5mg/kg/dayofrutheniumcomplexes(210mg/kg/day final amount), and 3mg/kg/day ofcisplatin (12mg/kg/day final amount)wereadministeredfourtimes,onceeverythreedays.Body weightof micereceivingUNICAM-1, NAMI-Aand cisplatinwas checkedonceaweekandcomparedwithuntreatedcontrols.Tumor

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Fig.2.UNICAM-1suppressedTNBCgrowthinvivo.FVBfemalemicewereinjectedwithsyngeneicA17cells(105_)._Mice_were_treated_with_UNICAM-1,_cisplatin,_NAMI-A

orisotonicsolution.Treatments(q3×4schedule)startedtendaysaftercellinjections.(A)Tumorgrowthcurvesinmicereceivingq3×4treatmentschedule.Valuesare mean±SEM,n=10.(B)Effectoftreatmentsonmeanbodyweightofmice.Valuesaremean±SEM.(C)Imagesofrepresentativetumorsexplantedfromcontrolandtreated mice36daysaftertumorchallenge.(D)Averagetumordiameterintreatedvscontrolmiceatday36.Valuesaremean±SEM,n=10.*p<0.05;**p<0.01;***p<0.001; ****p<0.0001wascalculatedwithanunpairedtwo-tailedttest.Eachgroupwascomparedtocontrol.

growthwascheckedonceaweekusinganelectroniccaliperuntil theendoftheexperiment.Tumorsweresurgicallyremovedonday 39,intheq1×6protocol,orday36,intheq3×4protocol.

2.5. Histologyandimmunohistochemistry

Tumors,kidneysand liverswereharvestedattheendofthe experimentalperiod,fixedinformalinandembeddedinparaffinor fixedinPFA4%andfrozenincryo-embeddingmedium(OCT Biop-tica,Milan,Italy).Todetectpossibleorgantoxicity,kidneyandliver slideswerestainedwithhematoxylinandeosin.Histopathological evidencesof acutetissue damageweresemi-quantified accord-ingtothemethodologydescribedelsewhere[32].Foreachorgan, twoslideswereanalyzedinablindfashionevaluatingthe follow-ingparameters:centralveincongestion,inflammatoryaggregate, focal hepatic necrosis, dilated sinusoids, breakdown hepato-cytes,perivascularinflammatoryaggregate,degeneratedhepatic cord,apoptoticcells,vacuolationsforliversectionsandatrophic glomerulus, dilated proximal convoluted tubule, degenerated tubules,inflammatorycellsinfiltrate,desquamationofepithelial cellsandcastformationforkidneysections.Theseverityand inci-denceofeachparameterwasscoredasfollows:grade0=absent; grade1=mild;grade2=moderate;grade3=severe.For immuno-histochemistrytumorslides wereincubated withthe following primaryantibodies:rabbitmAbanti-cleavedcaspase-3(MAB835, R&DSystems,Milan,Italy),ratmAbanti-CD4(550278),anti-CD8␣ (550281),anti-CD11b(550282),anti-CD45R/B220(550286), anti-CD31(550274),anti-CD105(550546),anti-Gr-1(550291),(allfrom BDPharmingen,Milan,Italy);anti-CD68(ab53444Abcam, Cam-bridge,UK)and anti-Foxp3 (14-5773,eBioscience,Milan, Italy);

afterwashing, slides wereoverlaid withappropriate secondary antibodies.ImmunostainingwasdevelopedwithDABChromogen System(Dako,Milan,Italy)orVulcanFastRed(Biocare,Milan,Italy) alkalinephosphatasemethod.Thenumberof cleavedcaspase-3 positivecellsandCD31/105positivevesselswasevaluatedon digi-talimagesofcontrols,cisplatin,NAMI-AandUNICAM-1tumors(10 pergroup,5×400microscopicﬁeldspertumor).

2.6. Metaltraceexamination

Rutheniumandplatinumcontentinexplantedliversand kid-neys from mice treated with UNICAM-1, NAMI-A, cisplatin or isotonicsolutionwereevaluatedbyInductivelyCoupled Plasma-MassSpectrometry(ICP-MS).Metalcontentinseraofmicewere measuredat7,14,30and60daysfromthebeginningofthe treat-ments.Allsolutionswerepreparedusingultrapurewaterobtained froma MilliporeMilli-Qsystem(resistivity 18.2M cm). Opti-mizeddigestionofsamples(SupplementaryTableI)wascarried outin amicrowavedigestor(Berghof Speedwavefour,Berghof, Eningen,Germany)with5mlofHNO3 (65%).Fiftymicrolitersof iridiumsolution(20mg)wasaddedasrecoverystandard.Digested solutionsweredilutedwithultrapurewatertoobtainasolution withthecorrectacidconcentrationinordertoperformanalysis. Theconcentrationsofrutheniumintheprocessedsampleswere measuredbyICP-MS (7500cxseries)withthe operating condi-tionsdescribedelsewhere[33].Calibrationcurveswereobtained usingaqueousstandard solutions (1.5%nitricacid) with appro-priatestockstandardsdilutions(FlukaAnalytical,Aldrich,Milan, Italy).

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Table1

Half Maximal Inhibitory concentration(IC50)values were calculatedfor A17

andMDA-MB231cellstreatedfor72hwithincreasingconcentrationsoftested compounds.

IC50(␮M)±SD

Cisplatin NAMI-A UNICAM-1 A17 6.93±0.14 485.58±0.02 230.66±0.02 MDA-MB231 38.70±0.03 840.21±0.03 409.89±0.04

2.7. Immunoblottinganalysis

Tumor lysates were obtained as previously described for celllysatespreparation. Lysates(40␮g/lane)wereseparatedby 4–20%gradientprecastSDS-PAGE(Bio-Rad)andtransferredonto polyvinylidenediﬂuoride(PVDF)membranes(ImmobilonP, Mil-lipore).RabbitmonoclonalantibodiesagainstCOX2andcleaved caspase-3andrabbitpolyclonalantibodyagainstcaspase-3were fromCellSignaling(Beverly,MA,USA).Secondaryantibody con-jugatedwithperoxidasewasfromSigma–Aldrich(St.Louis,MO, USA).TheimmunoreactivebandsweredetectedbyusingLiteAblot PLUS(Euroclone)detectingreagentsandpictureswereacquired withChemiDocImagingSystem(Bio-Rad).

2.8. Statisticalanalysis

Quantitativedataarepresentedasmeans±SEMfromatleast threeindependentexperiments.Thesigniﬁcanceofdifferenceswas evaluatedwithtwo-tailedStudentst-test,oronewayANOVA fol-lowedbyBonferroniposttest.Statisticalanalysiswascarriedout withGraphPadPrism5Software(SanDiego,CA,USA).p≤0.05was usedasthecriticallevelofsigniﬁcance.

3. Resultsanddiscussion

3.1. UNICAM-1inhibitedTNBCgrowthinvitro

TheeffectofUNICAM-1,compared withthat ofNAMI-Aand cisplatin, on cancer cell viability was estimated by MTT assay at72htreatmentusingthe humanMDA-MB-231cells andthe murineA17cellsasmodelsofTNBC.A17cellswereestablished fromanFVB/neuTtransgenicmammarytumorand were previ-ouslydescribed as strongly related toTNBC [34]. Althoughthe

ruthenium(II)complexesshowedalowereffectivenessrespectto cisplatin,theyareabletoinhibitviabilityofboth celllinesina dose-dependentmanner,whereUNICAM-1provedmoreefﬁcient thanNAMI-AasreportedinTable1andSupplementaryFig.1.We nextstudiedwhethertheseinhibitoryeffectsofruthenium(II) com-plexesoncellsviabilityweretheresultofapoptosis.Interestingly, onlyUNICAM-1resultedintheinductionofcelldeathbythe acti-vationoftheapoptotic“executioner”caspase-3(Supplementary Fig.2)whencomparedtoNAMI-Aorcisplatintreatments.Taken together,theseresultssuggestthatUNICAM-1leadstoapoptosis ofTNBCcells,showingpromisinginvitroanticanceractivity. 3.2. UNICAM-1inhibitedTNBCgrowthinvivo

We next investigated the in vivo antineoplastic effects of UNICAM-1 against A17 cells, able to give rise to aggressive mesenchymal tumors when injected into syngeneic mice. A17 cellssharemolecularsignaturewithTNBC,includingexpression of vimentin, cytokeratin 14, and N-cadherin as we previously described[28,34].AkeyfeatureofA17-signatureisalsothe over-expressionofCOX2,amesenchymalhallmarkintumors,whose relevance in growth,vasculogenesis and invasiveness has been widelydocumentedinvarioustypesofcarcinoma,bothin clin-icalandexperimentalstudies[35,36].Herewehaveshownthat UNICAM-1isabletosignificantlyreduceA17transplantabletumors growthinFVBsyngeneicmice(Fig.2).Theefficacyof UNICAM-1asantitumoralagentwascompared withthatofcisplatinand NAMI-A.Inanattempttooptimizeatreatmentschedule,resulting in a powerful anticancer action associated with minimal side-effects, the same final concentration of UNICAM-1(210mg/kg) orcisplatin(12mg/kg)orNAMI-A(210mg/kg)wasadministered accordingtotwo differentprotocols:a)treatmentcalledq1×6 (intraperitoneal (i.p.) injection of UNICAM-1 (35mg/kg/day) or cisplatin(2mg/kg/day) or NAMI-A(35mg/kg/day), repeatedfor 6consecutivedays);b)treatmentcalledq3×4(i.p.injectionof UNICAM-1(52.5mg/kg/day)orcisplatin(3mg/kg/day)orNAMI-A (52.5mg/kg/day),repeated4timesatintervalsof3days).Although bothprotocolswereeffective(SupplementaryFig.3),tumorgrowth inhibition became more significantin q3×4 treatment, where UNICAM-1 displayed the same anticancer activity of cisplatin (Fig.2),suggestingthattheprotocolofadministration,notonlythe dosages,arecriticalfeatures.Focusingonq3×4drugregimen,itis

Table2

Semiquantitativeanalysisofmorphologicalinjuryparametersinliverandkidneyofcontrol,UNICAM-1,cisplatinandNAMI-Agroups.

CONTROLS UNICAM-1(52.5mg/Kg) CISPLATIN(3mg/Kg) NAMI-A(52.5mg/Kg) Liver

Centralveincongestion(CVC) 0.8333±0.4082 0.3333±0.2582*,*** _1.167_±_0.7528 _0.5833_±_0.5845

Inﬂammatoryaggregate(IA) 0.8333±0.4082 0.5833±0.5845# _1.417_±_0.7360 _1.667_±_0.9309

Focalhepaticnecrosis(FHN) 0.4167±0.3764 0.6667±0.5164 0.7500±0.6892 0.6667±0.4082 Dilatedsinusoids(DS) 0.6667±0.4082 0.7500±0.5244 1.000±0.4472 0.9167±0.3764 Moderatebreakdownhepatocytes(MBH) 0.7500±0.5244 0.4167±0.4916 1.000±0.6325 1.000±0.7071

Perivascularinﬂammatoryaggregate(PIA) 0.9167±0.5845 0.5000±0.3162***,# _1.417_±_0.8612 _1.500_±_0.7071

Degeneratedhepaticcord(DHC) 0.6667±0.2582 1.250±0.2739** _1.250_±_0.6892 _1.500_±_0.6325*

Apoptoticcells(AC) 0.0±0.0 0.5000±0.4472* _1.167_±_0.6831** _0.3333_±_0.2582*,***

Vacuolations(V) 0.08333±0.2041 1.000±1.581 0.8333±1.211 0.4167±0.8010 Kidney

Atrophicglomerulus(G) 0.5833±0.3764 1.083±0.2041* _0.7500_±_0.6892 _1.167_±_0.6055

Dilatedproximalconvolutedtubule(PCT) 0.8333±0.2582 0.9167±0.3764 0.7500±0.6892 0.9167±0.5845

Degeneratedtubules(T) 0.3333±0.2582 0.8333±0.4082* _0.6667_±_0.4082 _1.250_±_0.6124**

Inﬂammatorycellsinﬁltrate(I) 0.5833±1.201 0.3333±0.4082 0.9167±1.281 0.08333±0.2041 Desquamationofepithelialcells(D) 0.2500±0.4183 0.8333±0.4082* _0.7500_±_0.4183 _0.7500_±_0.8216

Castformation(C) 0.08333±0.2041 0.1667±0.2582 0.1667±0.2582 0.1667±0.2582

Valuesaregivenasmean±SD.Thegroupwiththehigherscoreforeachanalyzedinjuryparameterisevidencedbyboldcharacter.

*_p_<_0.05_vs._Control_group. **_p_<_0.01_vs._Control_group. ***_p_<_0.05_vs._Cisplatin_group.

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Fig.3. Histopathologicalanalysisofliverusinghematoxylinandeosin(H&E)staining.Representativesectionsofliverfromdifferenttreatmentgroupsareshown.CV, centralvein;CVC,centralveincongestion;IA,inﬂammatoryaggregate;FHN,focalhepaticnecrosis;DS,dilatedsinusoids;DHC,degeneratedhepaticcord;PIA,perivascular inﬂammatoryaggregate.

possibletoappreciatehowA17tumorgrowrate(Fig.2A)andthe finaltumordimensions(Fig.2CandD)weresignificantlyreduced intheUNICAM-1-andcisplatin-treatedgroupscomparedtothe controlgroup,whereasNAMI-Awaslesseffective.Whileall con-trolmicedevelopedpalpabletumorsaftertwo weeksfromA17 cellchallenge,twoofthetotaltenmicetreatedwithUNICAM-1 didnotdeveloppalpabletumorsuntiltheendoftheexperiment. Ofnote,asshowninthetumorgrowthgraph,tumorsdeveloped incontrolmicedisplayedarapidgrowthrate,reachingan aver-agediameterof7mm attheend ofthestudy(on day36 after challenge),whereastumorsdevelopedintheUNICAM-1treated miceremainedverysmall,withanaveragediameternever exceed-ing3mm.Interestingly,body weightdidnot significantlydiffer betweenUNICAM-1-treatedandcontrolmice(Fig.2B), suggest-ingtheabsenceofdrugtoxicityattheselecteddoselevel.Onthe contrary,bothNAMI-A-andcisplatin-treatmentswereassociated withweighloss(Fig.2B).

3.3. UNICAM-1exhibitedmarkedlyreducedlivertoxicity comparedtocisplatin

Anextensivehistopathologicalanalysiswiththeevaluationof severaltissue injury parameters was carried out on explanted liverandkidney,commontargetsofchemotherapydrugs toxic-ity.Table2showsmoderatekidneytoxicitylevelssimilarforthe threedrugs,exceptforthepresenceofinﬂammatorycellsinﬁltrate onlyincisplatintreatedkidneys.Importantly,livertoxicitywas muchlessevidentinUNICAM-1thanincisplatinorNAMI-Atreated mice(Table2andFig.3).Pharmacokineticstudiesindicatedthat UNICAM-1wasrapidlylostfromtheorgansandthebloodstream

thankstoitshighhydrosolubility,inagreementwithlackofserious sideeffects.Inparticular,analysisoftracesofrutheniumand plat-inumintheliversandkidneysoftreatedmiceclearlydemonstrated ahigheliminationofUNICAM-1.Incontrast,repeated administra-tionsofNAMI-Aandcisplatinleadtoanaccumulationofruthenium andplatinumrespectively,preferentiallyinthekidneys(Fig.4A). Moreover,bloodanalysisdemonstratedthatrutheniumand plat-inumwerestillcirculatingsevendaysafterthelastadministration ofNAMI-Aandcisplatin,whereasrutheniumtraceswerealmost undetectableoneweekafterthelastUNICAM-1treatment(Fig.4B). 3.4. UNICAM-1impairedtumorangiogenesisandinduced

apoptosisinTNBC

Histologicalandimmunohistochemicalanalysescarriedouton explantedtumorsshowedseveraldifferencesamongcontroland treatedmice.Notably,whileallthreetreatmentsprovokea reduc-tioninthenumberofvessels,asigniﬁcantlyincreasednumberof apoptoticcells(cleavedcaspase-3positivecells)wasvisibleonly inUNICAM-1treatedtumors(Fig.5),suggestingthattheinhibitory activityofUNICAM-1ontumorgrowthwasdue,atleastinpart,to celldeathinductionthroughapoptosis.

3.5. UNICAM-1suppressedTNBCgrowthbyinhibitingtumor inﬁltrationofregulatoryTcells

Chemotherapyseemstobeefﬁcientifitsucceedsineliciting anticancerimmunosurveillance,thatimpliestheactivationofan immuneresponsespeciﬁcfor malignantcells[37]. Antineoplas-ticagentsmaystimulateimmunosurveillancebyactingoncancer

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Fig.4.Rutheniumandplatinumcontentinliverandkidney(A)andserum(B)frommiceinjectedwithUNICAM-1,NAMI-AorcisplatinevaluatedbyInductivelyCoupled Plasma-MassSpectrometry(ICP-MS).Thedataarepresentedas%injecteddosepergramoftissue(%IDperg).Valuesaremean±SEM,n=10.*p<0.05;**p<0.01;***p<0.001; ****p<0.0001wascalculatedwithanunpairedtwo-tailedt-test.Eachgroupwascomparedtocontrol.

Fig.5.AnalysisofapoptoticcellsandvesselnumberinA17tumors.Representativeimagesofcleavedcaspase-3andCD31/105positiveimmunohistochemicalsectionsof control,cisplatin,NAMI-AandUNICAM-1treatedtumorsonday36(upperpanel).Quantiﬁcationofcleavedcaspase-3positivecellsandnumberofvesselsincontroland treatedtumorswasdeterminedasdescribedintheexperimentalsection(lowerpanel).Resultsarerepresentedasmeans±SEMfrom5×400microscopicﬁeldspertumor (n=10).*p<0.05;**p<0.01;***p<0.001;****p<0.0001wascalculatedwithanunpairedtwo-tailedttest.Eachgroupwascomparedtocontrol.

cellsin severalways, forexample,bydepletionof immunosup-pressivecellsfromthetumorbed[38],orthroughdirecteffects onmalignant cells that then elevate theirimmunogenicityand theirsusceptibility to immune control by cytotoxicT and nat-uralkiller(NK) cells.Certainchemotherapeuticsenhancetumor immunogenicitybecausetheyinduceaformofapoptosisin can-cer cells, known as “immunogenic cell death”, that favors the transfer of tumor antigens to dendritic cells (DC), which ulti-mately“cross-prime” and activate anti-tumorigenic CD4+/CD8+ T-cell immunity. Accordingly, numerous studies have reported

a linkbetween response toconventional therapies, and tumor immuneinfiltrateinseveraldifferentsolidtumortypes, includ-ingbreastcancer[39].Immunohistochemicalanalysisoftumors explantedfrommicetreatedwithcisplatin,NAMI-Aand UNICAM-1revealedasignificantlyhigherleveloftumorimmuneinfiltrates incomparisonwithcontrolmice(Fig.6).Ourresultsare concor-dantwitharecentstudybyDenkertetal.[40],showingthattumor infiltratinglymphocytelevelscorrelatepositivelywithpatient sur-vivalandpredictpathologiccompleteresponsetochemotherapyin humanTNBC.Infact,thepresenceoftumor-infiltratingT

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lympho-Fig.6. SemiquantitativeanalysisofinflammatoryinfiltrationinA17tumorsfromcontrol,cisplatin,NAMI-AandUNICAM-1treatedmice(protocolq3×4).Datawereanalyzed asdescribedintheexperimentalsection.Resultsarerepresentedasmeans±SEMfrom5×400microscopicfieldspertumor(n=5).CD4+cells:T-helperlymphocytes;CD8+ cells:cytotoxic-Tlymphocytes;CD11b+cells:dendriticcells;CD68+cells:macrophages;B220+cells:Blymphocytes;Foxp3cells:regulatoryTlymphocytes.*p<0.05; **p<0.01;***p<0.001;****p<0.0001wascalculatedwithanunpairedtwo-tailedttest.Eachgroupwascomparedtocontrol.

cytesindicatesthehostimmuneresponsetotumorantigensandis consideredasapositivemarkerofresponsetochemotherapy. How-ever,humantumorsalsopromoteaccumulationofCD4+CD25+ immunosuppressiveregulatoryTcells(Treg,Foxp3positivecells) inthetumorbedorintheblood.Thesecellsareknownasthekey contributortomaintenanceofimmunetolerance[41].Emerging evidencesuggeststhatTregcellshaveanimportant immunopatho-logic role in humantumor growthby suppressing endogenous tumor-associatedantigen-specificT-cellimmunity[42,43].Tregs cansuppressproliferationofactivatedeffector Tcellsby direct contact,inhibitingtheirclonalexpansion[44],orcaninducedirect killingofeffectorcellsthroughreleaseofgranzymeandperforin [45,46].Transforminggrowthfactor-b(TGF-b)andIL-10secreted byTregshavealsobeeninvolvedininhibitionoftumor-specificCTL cytotoxicityinvivo[47].Interestingly,followingUNICAM-1 treat-mentthelevelsofCD4+andcytotoxicCD8+Tlymphocyteswere increased,concomitantlywithastrongreductionofFoxp3 regu-latoryT-cellinfiltration(Fig.6,SupplementaryFigs.4,5).Recently, animmunologicsignatureconsistingoftheabsenceofFoxp3cells andthepresenceofahighnumberofCD8Tcellsonfinalsurgical biopsyofbreasttumortreatedbyneoadjuvantchemotherapyhas beenassociatedwithpathologiccompleteresponses[38,48].Ahigh CD8/Tregratiohasbeenalsoassociatedwithfavorableprognosis inepithelialovariancancer[49].However,althoughinductionand expansionofTregsinthetumormicroenvironmentareconsidered criticalstepsinevasionoftheimmune responseand tumorcell survival,themolecularmechanismsunderlyingTregcells recruit-mentintheprimarytumorremainunclear.Clinicalstudiesofbreast cancerpatientshaveshownacorrelationbetweenincreasedCOX2 expressionwithhighTregrecruitment[50]andrecently Karavi-tisetal.provideddirectevidencethatCOX2,andsubsequentPGE2

overexpression,resultsinatumorenvironmentthatpromotesTreg recruitmentandattenuationofthenormalimmuneresponse[51]. COX2isassociatedwithabasal-liketranscriptionpatterninhuman breasttumors[34]andit wasfoundsignificantlyoverexpressed inA17tumors,whereitcorrelateswiththeirmesenchymal signa-ture[29,34,35,52,53].Besideitsimmunosuppressiveeffect,COX2 expressionhasbeenalsolinkedtocancerprogressionduetoits ability to promote cell proliferation and angiogenesis [36]. We thereforeevaluated ifthereis a correlationbetweenCOX2 lev-elsandTregrecruitmentinA17tumorsexplantedfromcontrol andtreatedanimals.Ofnote,westernblotanalysesrevealeda sig-nificantreductionofCOX2expressionuponUNICAM-1treatment (Fig.7),supportingtheassociationbetweenCOX2levelsandTreg infiltrationandunravelingthemolecularmechanismwhichmight mediatetheantitumoral and immunomodulatoryaction of this rutheniumcompound.Inaddition,UNICAM-1inducesan enhance-mentofrecruitmentandinfiltrationintotumorsofcellsexpressing thedendritic markerCD11band themacrophagemarkerCD68 (Fig.6).Althoughtheroleoftumorassociatedmacrophagesisstill controversial,tumor-infiltratingmacrophagesoftenparticipatein thehostresponsetowardthetumor,killingtumorcells [54,55]. Inagreementwiththisfinding,highmacrophageinfiltrationhas been correlated to improvedsurvival in colorectal cancer[56]. Overall,wereportthatUNICAM-1hasarelevanttherapeutic effi-cacyagainstTNBCanditsuniqueimmunomodulatoryactionmight explaintheobservedanticancereffects.

4. Conclusion

Cancer remains a major cause of mortality worldwide. Chemotherapyisoneofthemostpotentstrategiestotreattumors,

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Fig.7. UNICAM-1targetedCOX2expressioninA17transplantedtumors.Western blotanalysisfortheexpressionlevelsofCOX2and␤-actin(loadingcontrol)inA17 tumorsfromFVBmicereceivingisotonicsolution(control),cisplatin,UNICAM-1,or NAMI-A(upperpanel).DensitometricquantiﬁcationofCOX2expressionnormalized on␤-actinwasperformedwithImageJSoftware(lowerpanel).Statisticalanalysis wasexecutedwithGraphPadPrismSoftware(SanDiego,CA,USA),usingp<0.05as thecriticallevelofsigniﬁcance(****p<0.0001;***p<0.001).

howeverthetoxicityofcurrentlyavailableanticancerdrugs repre-sentsoneofthemostcriticalissues.Newtherapies,characterized byhighefficiencyandlowtoxicity,arethereforeanurgentunmet medicalneed.Conventionalcancertherapies,suchas chemother-apies, have been developed based on the concept that cancer constitutesacell-autonomousgeneticorepigeneticdisease.Asa consequence,theircytostaticand/orcytotoxiceffectshavebeen testedinvitro,onculturedhumantumorcells,andonhuman can-cerxenograftsgrowinginimmunodeficientmice.Littleattention hasbeengiventothehostimmune systemintermsof progno-sisandpotentialresponsetotherapy[57].Ontheotherhand,the roleoftheimmunesystemincancerhaslongbeenknownand accumulatingevidenceindicatesthatthetherapeuticefficacyof severalantineoplasticagentsreliesontheircapacitytoinfluence thetumor-hostinteractionandtofavorablymodifytheimmune microenvironment.

Our studies were focused on the development and valida-tionofanorganometallicruthenium(II)-arenecomplex,wecalled UNICAM-1,asnovelanticancerdrug.Overall,UNICAM-1exhibitsa markedanti-tumoralactivityinvivoagainstanexperimentalTNBC model,associatedwithlowtoxicityandfavorableclearance proper-ties.TherapeuticefficacyofUNICAM-1seemstorelyonitscapacity toinfluencethetumor-hostinteraction,leadingtoactivationof animmuneresponsespecificformalignantcells,supportingthe hypothesisthatchemotherapyisefficientifitsucceedsineliciting anticancerimmunosurveillance[37].

Acknowledgments

ThisstudywassupportedbyA.Amici’sF.A.R.2015ofthe Uni-versityofCamerino.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.phrs.2016.03. 032.

References

[1]T.O.Nielsen,F.D.Hsu,K.Jensen,M.Cheang,G.Karaca,Z.Hu,T.

Hernandez-Boussard,C.Livasy,D.Cowan,L.Dressler,L.A.Akslen,J.Ragaz, A.M.Gown,C.B.Gilks,M.vandeRijn,C.M.Perou,Immunohistochemicaland clinicalcharacterizationofthebasal-likesubtypeofinvasivebreast carcinoma,Clin.CancerRes.10(2004)5367–5374.

[2]M.C.Cheang,D.Voduc,C.Bajdik,S.Leung,S.McKinney,S.K.Chia,C.M.Perou, T.O.Nielsen,Basal-likebreastcancerdeﬁnedbyﬁvebiomarkershassuperior prognosticvaluethantriple-negativephenotype,Clin.CancerRes.14(2008) 1368–1376.

[3]F.Bertucci,P.Finetti,D.Birnbaum,Basalbreastcancer:acomplexanddeadly molecularsubtype,Curr.Mol.Med.12(2012)96–110.

[4]W.D.Foulkes,I.E.Smith,J.S.Reis-Filho,Triple-negativebreastcancer,N.Engl. J.Med.363(20)(2010)1938–1948.

[5]L.Kelland,Theresurgenceofplatinum-basedcancerchemotherapy,Nat.Rev. Cancer7(2007)573–584.

[6]M.J.Piccart,H.Lamb,J.B.Vermorken,Currentandfuturepotentialrolesofthe platinumdrugsinthetreatmentofovariancancer,Ann.Oncol.12(2001) 1195–1203.

[7]M.Tian,Y.Zhong,F.Zhou,C.Xie,Y.Zhou,Z.Liao,Platinum-basedtherapyfor triple-negativebreastcancertreatment:ameta-analysis,Mol.Clin.Oncol.3 (2015)720–724.

[8]D.P.Silver,A.L.Richardson,A.C.Eklund,Z.C.Wang,Z.Szallasi,Q.Li,N.Juul, C.O.Leong,D.Calogrias,A.Buraimoh,A.Fatima,R.S.Gelman,P.D.Ryan,N.M. Tung,A.DeNicolo,S.Ganesan,A.Miron,C.Colin,D.C.Sgroi,L.W.Ellisen,E.P. Winer,J.E.Garber,Efﬁcacyofneoadjuvantcisplatinintriple-negativebreast cancer,J.Clin.Oncol.28(2010)1145–1153.

[9]N.J.Wheate,S.Walker,G.E.Craig,R.Oun,Thestatusofplatinumanticancer drugsintheclinicandinclinicaltrials,DaltonTrans.39(2010)8113–8127.

[10]F.Muggia,BRCA-deﬁcientanimalmodelsandcisplatinresistance,Gynecol. Oncol.112(2009)275–281.

[11]J.Liu,X.Chen,T.Ward,M.Pegram,K.Shen,Combinedniclosamidewith cisplatininhibitsepithelial-mesenchymaltransitionandtumorgrowthin cisplatin-resistanttriple-negativebreastcancer,TumourBiol.(2016)1–11.

[12]L.Kelland,Rutheniumantimetastaticagents,Nat.Rev.Cancer7(2007) 573–584.

[13]N.P.Barry,P.J.Sadler,Explorationofthemedicalperiodictable:towardsnew targets,Chem.Commun.49(2013)5106–5131.

[14]A.L.Noffke,A.Habtemariam,M.A.Pizarro,P.J.Sadler,Designing organometalliccompoundsforcatalysisandtherapy,Chem.Commun.48 (2012)5219–5246.

[15]G.Gasser,I.Ott,N.Metzler-Nolte,Organometallicanticancercompounds,J. Med.Chem.54(2011)3–25.

[16]G.S.Smith,B.Therrien,Targetedandmultifunctionalareneruthenium chemotherapeutics,DaltonTrans.40(2011)10793–10800.

[17]G.Sava,A.Bergamo,P.J.Dyson,Metal-basedantitumourdrugsinthe post-genomicera:whatcomesnext?DaltonTrans.40(2011)9069–9075.

[18]A.F.Peacock,P.J.Sadler,Medicinalorganometallicchemistry:designingmetal arenecomplexesasanticanceragents,Chem.AsianJ.3(2008)1890–1899.

[19]E.Alessio,G.Mestroni,A.Bergamo,G.Sava,Rutheniumanticancerdrugs, Curr.Top.Med.Chem.4(2004)1525–1535.

[20]R.Trondl,P.Heffeter,C.R.Kowol,M.A.Jakupec,W.Berger,B.K.Keppler, NKP-1339,theﬁrstruthenium-basedanticancerdrugontheedgetoclinical application,Chem.Sci.5(2014)2925–2932.

[21]B.Lippert,Z.Cisplat,Chemistrybiochemistryofaleadinganticancerdrug.The mechanismofactionofcisplatin:fromadductstoapoptosis,4,111–134.Ed.; VerlagHelveticaChimicaActa:Zurich;Wiley-VCH:Weinheim,Germany (1999).

[22]Z.Adhireksan,G.E.Davey,P.Campomanes,M.Groessl,C.M.Clavel,H.Yu,A.A. Nazarov,H.F.Yeo,W.H.Ang,P.Droge,U.Rothlisberger,P.J.Dyson,C.A.Davey, Ligandsubstitutionsbetweenruthenium-cymenecompoundscancontrol proteinversusDNAtargetingandanticanceractivity,Nat.Commun.5(2014) 3462.

[23]A.Casini,C.G.Hartinger,A.A.Nazarov,P.J.Dyson,Organometallicantitumour agentswithalternativemodesofaction,Top.Organomet.Chem.32(2010) 57–80.

[24]R.E.Aird,J.Cummings,A.A.Ritchie,M.Muir,R.E.Morris,H.Chen,P.J.Sadler, D.I.Jodrell,Invitroandinvivoactivityandcrossresistanceproﬁlesofnovel ruthenium(II)organometallicarenecomplexesinhumanovariancancer,Br.J. Cancer86(2002)1652–1657.

[25]C.Scolaro,A.Bergamo,L.Brescacin,R.Delﬁno,M.Cocchietto,G.Laurenczy, T.J.Geldbach,G.Sava,P.J.Dyson,Invitroandinvivoevaluationof ruthenium(II)-arenePTAcomplexes,J.Med.Chem.48(2005)4161–4171.

[26]F.Marchetti,C.Pettinari,R.Pettinari,A.Cerquetella,C.DiNicola,A.Macchioni, D.Zuccaccia,M.Monari,F.Piccinelli,Synthesisandintramolecularand interionicstructuralcharacterizationofhalf-sandwich(arene)ruthenium(II) derivativesofbis(pyrazolyl)alkanes,Inorg.Chem.47(2008)11593–11603.

[27]G.Mestroni,E.Alessio,G.Sava,PCTInt.Appl.WO980004311998. [28]M.Galiè,C.Sorrentino,M.Montani,L.Micossi,E.DiCarlo,T.D’Antuono,L.

Calderan,P.Marzola,D.Benati,F.Merigo,F.Orlando,A.Smorlesi,C.Marchini, A.Amici,A.Sbarbati,Mammarycarcinomaprovideshighlytumourigenicand invasivereactivestromalcells,Carcinogenesis26(2005)1868–1878.

[29]M.Galiè,G.Konstantinidou,D.Peroni,I.Scambi,C.Marchini,V.Lisi,M. Krampera,P.Magnani,F.Merigo,M.Montani,F.Boschi,P.Marzola,R.Orrù,P. Farace,A.Sbarbati,A.Amici,Mesenchymalstemcellssharemolecular

(9)

Ramadori,F.Gabrielli,A.Baruzzi,G.Berton,F.Merigo,S.Fin,M.Iezzi,B. Bisaro,A.Sbarbati,M.Zerani,M.Galiè,A.Amici,Mesenchymal/stromalgene expressionsignaturerelatestobasal-likebreastcancers,identiﬁesbone metastasisandpredictsresistancetotherapies,PLoSOne5(2010)e14131.

[35]B.Bisaro,M.Montani,G.Konstantinidou,C.Marchini,L.Pietrella,M.Iezzi,M. Galiè,F.Orso,A.Camporeale,S.M.Colombo,P.DiStefano,G.Tornillo,M.P. Camacho-Leal,E.Turco,D.Taverna,S.Cabodi,A.Amici,P.Deﬁlippi, p130Cas/Cyclooxygenase-2axisinthecontrolofmesenchymalplasticityof breastcancercells,BreastCancerRes.14(2012)R137.

[36]G.Singh-Ranger,M.Salhab,K.Mokbel,Theroleofcyclooxygenase-2inbreast cancer:review,BreastCancerRes.Treat.109(2008)189–198.

[37]L.Zitvogel,L.Galluzzi,M.J.Smyth,G.Kroemer,Mechanismofactionof conventionalandtargetedanticancertherapies:reinstating immunosurveillance,Immunity39(2013)74–88.

[38]S.Ladoire,L.Arnould,L.Apetoh,B.Coudert,F.Martin,B.Chauffert,P. Fumoleau,F.Ghiringhelli,Pathologiccompleteresponsetoneoadjuvant chemotherapyofbreastcarcinomaisassociatedwiththedisappearanceof tumor-inﬁltratingfoxp3+regulatoryTcells,Clin.CancerRes.14(2008) 2413–2420.

[39]S.Demaria,M.D.Volm,R.L.Shapiro,H.T.Yee,R.Oratz,S.C.Formenti,F. Muggia,W.F.Symmans,Developmentoftumor-inﬁltratinglymphocytesin breastcancerafterneoadjuvantpaclitaxelchemotherapy,Clin.CancerRes.7 (2001)3025–3030.

[40]C.Denkert,G.vonMinckwitz,J.C.Brase,B.V.Sinn,S.Gade,R.Kronenwett,B.M. Pﬁtzner,C.Salat,S.Loi,W.D.Schmitt,C.Schem,K.Fisch,S.Darb-Esfahani,K. Mehta,C.Sotiriou,S.Wienert,P.Klare,F.André,F.Klauschen,J.U.Blohmer,K. Krappmann,M.Schmidt,H.Tesch,S.Kümmel,P.Sinn,C.Jackisch,M.Dietel,T. Reimer,M.Untch,S.Loibl,Tumor-inﬁltratinglymphocytesandresponseto neoadjuvantchemotherapywithorwithoutcarboplatininhumanepidermal growthfactorreceptor2-positiveandtriple-negativeprimarybreastcancers, J.Clin.Oncol.33(2015)983–991.

[41]S.Sakaguchi,M.Ono,R.Setoguchi,H.Yagi,S.Hori,Z.Fehervari,J.Shimizu,T. Takahashi,T.Nomura,Foxp3+CD25+CD4+naturalregulatoryTcellsin dominantself-toleranceandautoimmunedisease,Immunol.Rev.212(2006) 8–27.

[42]T.J.Curiel,G.Coukos,L.Zou,X.Alvarez,P.Cheng,P.Mottram,M. Evdemon-Hogan,J.R.Conejo-Garcia,L.Zhang,M.Burow,Y.Zhu,S.Wei,I. Kryczek,B.Daniel,A.Gordon,L.Myers,A.Lackner,M.L.Disis,K.L.Knutson,L. Chen,W.Zou,SpeciﬁcrecruitmentofregulatoryTcellsinovariancarcinoma fostersimmuneprivilegeandpredictsreducedsurvival,Nat.Med.10(2004) 942–949.

[43]G.J.Bates,S.B.Fox,C.Han,R.D.Leek,J.F.Garcia,A.L.Harris,A.H.Banham, QuantiﬁcationofregulatoryTcellsenablestheidentiﬁcationofhigh-risk

Rello-Varona,C.Locher,V.Poirier-Colame,M.Talbot,A.Valent,F.Berardinelli, A.Antoccia,F.Ciccosanti,G.M.Fimia,M.Piacentini,A.Fueyo,N.L.Messina,M. Li,C.J.Chan,V.Sigl,G.Pourcher,C.Ruckenstuhl,D.Carmona-Gutierrez,V. Lazar,J.M.Penninger,F.Madeo,C.López-Otín,M.J.Smyth,L.Zitvogel,M. Castedo,G.Kroemer,Animmunosurveillancemechanismcontrolscancercell ploidy,Science337(2012)1678–1684.

[49]E.Sato,S.H.Olson,J.Ahn,B.Bundy,H.Nishikawa,F.Qian,A.A.Jungbluth,D. Frosina,S.Gnjatic,C.Ambrosone,J.Kepner,T.Odunsi,G.Ritter,S.Lele,Y.T. Chen,H.Ohtani,L.J.Old,K.Odunsi,IntraepithelialCD8+tumor-inﬁltrating lymphocytesandahighCD8+/regulatoryTcellratioareassociatedwith favorableprognosisinovariancancer,Proc.Natl.Acad.Sci.U.S.A.102(2005) 18538–18543.

[50]B.A.Pockaj,G.D.Basu,L.B.Pathangey,R.J.Gray,J.L.Hernandez,S.J.Gendler,P. Mukherjee,ReducedT-cellanddendriticcellfunctionisrelatedto cyclooxygenase-2overexpressionandprostaglandinE2secretioninpatients withbreastcancer,Ann.Surg.Oncol.11(2004)328–339.

[51]J.Karavitis,L.M.Hix,Y.H.Shi,R.F.Schultz,K.Khazaie,M.Zhang,Regulationof COX2expressioninmousemammarytumorcellscontrolsbonemetastasis andPGE2-inductionofregulatoryTcellmigration,PLoSOne7(9)(2012) e46342.

[52]C.Garulli,C.Kalogris,L.Pietrella,C.Bartolacci,C.Andreani,M.Falconi,C. Marchini,C.Amici,Dorsomorphinreversesthemesenchymalphenotypeof breastcancerinitiatingcellsbyinhibitionofbonemorphogeneticprotein signaling,Cell.Signal.26(2014)352–362.

[53]C.Kalogris,C.Garulli,L.Pietrella,V.Gambini,S.Pucciarelli,C.Lucci,M.Tilio, M.E.Zabaleta,C.Bartolacci,C.Andreani,M.Giangrossi,M.Iezzi,B.Belletti,C. Marchini,A.Amici,Sanguinarinesuppressesbasal-likebreastcancergrowth throughdihydrofolatereductaseinhibition,Biochem.Pharmacol.90(3) (2014)226–234,http://dx.doi.org/10.1016/j.bcp.2014.05.014.

[54]S.Cui,J.S.Reichner,R.B.Mateo,J.E.Albina,Activatedmurinemacrophages induceapoptosisintumorcellsthroughnitricoxide-dependentor -independentmechanisms,CancerRes.54(1994)2462–2467.

[55]J.H.Martin,S.W.Edwards,Changesinmechanismsof

monocyte/macrophage-mediatedcytotoxicityduringcultureReactiveoxygen intermediatesareinvolvedinmonocyte-mediatedcytotoxicity,whereas reactivenitrogenintermediatesareemployedbymacrophagesintumorcell killing,J.Immunol.150(1993)3478–3486.

[56]J.Forssell,A.Oberg,M.L.Henriksson,R.Stenling,A.Jung,R.Palmqvist,High macrophageinﬁltrationalongthetumorfrontcorrelateswithimproved survivalincoloncancer,Clin.CancerRes.13(2007)1472–1479.

[57]C.Jochems,J.Schlom,Tumor-inﬁltratingimmunecellsandprognosis:the potentiallinkbetweenconventionalcancertherapyandimmunity,Exp.Biol. Med.236(2011)567–579.