Proton beam irradiation inhibits the migration of melanoma cells

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PLOSONE|https://doi.o r g/10.13 7 1/journal.p o ne.0186 0 0 2 Octobe r10,2017 1^/16

OPENACCESS Citation:Jasińska-

KoniorK,PochylczukK,CzajkaE,MichalikM,Romano wska-DixonB,SwakońJ,etal.

(2017)Protonbeamirradiationinhibitsthemigration ofmelanomacells.PLoSONE12(10):e0186002.htt ps://

d oi.org/10.1371/ j ournal. pone.0186002 Editor:RobertoAmendola,ENEACentroRicercheC asaccia,ITALY

Konioretal.Thisisanopenaccessarticledistributed underthetermsoftheCreativeCommons Attributi o n License,whichpermitsunrestricteduse,distribution ,andreproductioninanymedium,providedtheorigin alauthorandsourcearecredited.

DataAvailabilityStatement:Allrelevantdataare withinthepaperanditsSupportingInformationfiles.

Funding:Thisprojectwassupportedbygrant2012/

07/B/NZ4/01657fromNationalScienceCentre,Cra cow,Poland(https://ww w .ncn.gov.p l /?

language=en).FacultyofBiochemistry,Biophysics andBiotechnologyofJagiellonianUniversityisapart neroftheLeadingNationalResearchCenter(KNOW )supportedbytheMinistryofScienceand

RESEARCHARTICLE

Protonbeamirradiationinhibitsthemigrationof melanomacells

KatarzynaJasińska-

Konior¹,KatarzynaPochylczuk¹,ElżbietaCzajka¹,MartaMichalik²,BożenaRomanowska- Dixon³,JanSwakoń⁴,KrystynaUrbańska¹,MartynaElas¹*

1 DepartmentofBiophysics,FacultyofBiochemistry,BiophysicsandBiotechnology,Cracow,Poland, 2 DepartmentofCellBiology,FacultyofBiochemistry,BiophysicsandBiotechnology,Cracow,Poland,

3 DepartmentofOphthalmologyandOphthalmicOncology,JagiellonianUniversityMedicalCollege,Cracow,Pol and,4InstituteofNuclearPhysics,PAS,Cracow,Poland

*martyna.el a s@uj.e d u.pl

Abstract

Purpose

Inrecentyearsexperimentaldatahaveindicatedthatlow-

energyprotonbeamradiationmightinduceadifferenceincellularmigrationincomparisontoph otons.WethereforesetouttocomparetheeffectofprotonbeamirradiationandX-

raysonthesurvivalandlong-

termmigratorypropertiesoftwocelllines:uvealmelanomaMel270andskinmelanomaBLM.

Materialsandmethods

CellstreatedwitheitherprotonbeamorX-rayswereanalyzedfortheirsurvivalusingclono- genicassayandMTTtest.Long-termmigratorypropertieswereassessedwithtime-

lapsemonitoringofindividualcellmovements,woundtestandtransporemigration,whiletheexp ressionoftherelatedproteinswasmeasuredwithwesternblot.

Results

ExposuretoprotonbeamandX-

raysledtosimilarsurvivalbutthequalityofthecellcolonieswasmarkedlydifferent.Moreparaclone swithalowproliferativeactivityandfewerhighly-

proliferativeholocloneswerefoundafterprotonbeamirradiationincomparisontoX- rays.At20or40dayspost-

irradiation,migratorycapacitywasdecreasedmorebyprotonbeamthanbyX-rays.Thebeta-1- integrinlevelwasdecreasedinMel270cellsafterbothtypesofradia-

tion,whilevimentin,amarkerofEMT,wasincreasedinBLMcellsonly.

Conclusions

Weconcludethatprotonbeamirradiationinducedlong-

terminhibitionofcellularmotility,aswellaschangesinthelevelofbeta-

1integrinandvimentin.Ifconfirmed,thechangeinthequality,butnotinthenumberofcoloniesafte rprotonbeamirradiationmightfavortumorgrowthinhibitionafterfractionatedprotontherapy.

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PLOSONE|https://doi.o r g/10.13 7 1/journal.p o ne.0186 0 0 2 Octobe r10,2017 2^/16

HigherEducation.Thefundershadnoroleinstudy

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Protonbeamirradiationandmelanomacellmigration

design,datacollectionandanalysis,decisiontop ublish,orpreparationofthemanuscript.

Competinginterests:Theauthorshavedeclaredth atnocompetinginterestsexist.

Introduction

Protonbeamradiationisusedtotreatmalignanciesbecauseofitssuperiorbiophysicalproper- tiesconcerningdosedepositionintissuescomparedtophotonradiation[1].Incontrasttothewidel yacceptedview,thatthetwotypesofradiationexertsimilarbiologicaleffectsintissues,withtherela tivebiologicaleffectivenessof1.1,severalintriguingdifferencesbetweenlow-

energyprotonbeamandphotonirradiatedtumorcellshavebeenreported.Forexample,homolog ousrecombinationwasmoresignificantforprotonbeaminducedDNAdamage[2].High-

LETprotonbeamirradiationcausedclusterDNAdamagewithhighercomplexitywithincreasingL ET[3],butlow-LETprotonbeamcausedsimilarDNAdamagetophotonirradia-

tion[4].Otherdifferenceswerefoundintheleveloftheproductionoffreeradicals,cellcycleinhibitio nandapoptoticsignaling[5].Invitrotreatmentoftumorcellswithaprotonbeamresultedinahigherp ercentageofapoptoticcellswhencomparedtophotonradiation[6].Additionally,differenceswere observedincellcycleregulation:ahigh-

LETprotonradiationinducedaG2phasearrestwhichwasnoticeablylongerandhardertoresolvei ncomparisontosimilardosesofphotonradiation[7].Thiswasnotseenforlow-

LETprotonradiation[8].

Radiationmayalsoaffecttheformationofmetastasis,includingcelldetachmentfromtheprimarytu mor,migrationalongtheextra-

cellularmatrix(ECM),degradationofthebasementmembrane,andintravasationintothebloodorlymp haticvessels[9].Tumorcell-

migrationitselfisamultistageprocesswhichdependsonvariousfactorssuchasproteinaseactivity[10, 11],thecytoskeletonorganizationofthemigratingcells[12]andadhesiontotheECMmediatedbyrecep torssuchasintegrins.Radiationmayaffectmanyofthesesteps,andadiffer-

entialinfluenceofprotonandphotonradiationhasbeensuggested[5].

Asprotonbeamtherapyaswellasradio-activeplaquetherapyaremainstaysinthetreat-

mentofuvealmelanoma,wewonderedhowthesedifferentapproachesaffectedmelanomacells.Weth ereforestudiedthelong-

termeffectsofsublethaldosesofprotonbeamirradiationandofphotontreatmentonthemigratoryprope rtiesofuvealmelanomaandmetastatichumanmelanomaskincells.Wetestedcellularsurvival,motility andthelevelofβ1-

integrinandvimentinafterprotonbeamandphotonirradiationandshowedthatprotonbeam,butnotpho tonirradiation,inhibitedcellularrectilinearmotilityandchangedheterogeneityofcol-

onies.Theseeffectswereobservedatlong-termaftertreatment.

Materials&methods Cellculture

WeusedMel270,aprimaryhumanuvealmelanomacellline[13],andBLM,acelllinederivedfromt helungmetastasesofskinmelanoma[14].Bothcelllineswereculturedat37˚C,5%CO2inRPMIme dia(Sigma-

Aldrich,St.Louis,MO),supplementedwith10%fetalbovineserum(BiologicalIndustries,Cromw ell,CT)andpenicillin/streptomycin(Polpharma,Poland).TheMel270cellswereagiftfromprof.M.

JagerfromLeidenUniversity(TheNether-

lands)andBLMcellsfromDrG.N.P.vanMuijen,DepartmentofPathology,RadboudUniver- sityNijmegenMedicalCentre,Nijmegen(TheNetherlands).Thecellswerepassagedat70–

80%ofconfluenceevery5–6days,sothatthe4^thpassagewasatday20post- radiationandthe7^thpassagewasatday40.

Irradiation

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Protonbeamirradiationandmelanomacellmigration

CellsirradiationswithX-

raysandhighenergyprotonswerep erformedattheInstituteofNuclear Physics,PolishAcademyofScienc es(IFJPAN),Cracow,Poland.For X-rayirradia-tionPhillipsMCN- 323tubeatthevoltageof250kVpan dthedoserateof1.8Gy/minwas

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applied.Thebeamfilteredwith4mmberylliumandadditional1mmofbrass.Dosimetrywasperformed usingthePTWTM31013ionizationchamberwithPTWUNIDOSelectrometer,calibratedinthesecond arystandardlaboratoryattheCentralOfficeofMeasuresinWarsaw,Poland.Cellcultureswereirradiat edinEppendorftubesplacedonthesurfaceofthePMMAphantom,Eppendorf’swallthicknesswassuffi cienttocompensateforthebuild-upeffect.Pro-

tonbeamirradiationtookplaceattheCyclotronCentreBronowiceatIFJPAN.The230MeVprotonbea mproducedattheIBAProteusC-

235cyclotron[15]anddegradedattheenergyselectortothe70MeVwasdeliveredtotheeyetreatmentr oomandmechanicallyformedusingasetofscaterersandenergymodulatorattheeyeirradiationunit.F ullymodulatedpro-

tonbeamwithenergy61MeV(31.5mmrangeinwater)collimatedtothe40mmlateraldiam-

eterhasbeenusedforirradiation.Duringtheirradiationthedoses1,3,or5Gyhavebeendeliveredwithd oserateof1Gy/min,2Gy/minand6.6Gy/min,respectively.Atthecenterofcellcontainerpositioni.e.att hedepths15.8mmoftheSOBPthecalculatedContinuesSlow-

ingDownApproximation(CSDA)doseaveragedLETdwas2.8keV/μm.Beamdosimetrywasperforme daccordingtotheTRS-

398protocolrecommendedbyInternationalAtomicEnergyAgency[16]usingareferencedosimeterc onsistingofaPTWTM31010semiflexionizationchamberandaPTWUNIDOSWeblineelectrometer(

PTW,Freiburg,Germany).Thedosim-

etersetwascalibratedattheIFJPANatTheratron780⁶⁰Cotreatmentunit.Cellswereirradi-

atedintheEppendorftubespositionedorthogonallytothedirectionoftheprotonbeamusingthededicat edphantommadeoutofPMMA.Cellsweretransportedonicebetweenthefacili-

ties,includingtheuntreatedcontrol.

Clonogeniccellsurvivalassay

Thenumberofcellsseededinto6cmdiameterdisheswasadjustedforeachdosetoachievetheopti malnumberofcoloniesafterradiation.Theexperimentwasperformedthreetimesandthreereplic ateplateswereseededforeachgroupineveryrepetition.ForBLMandMel270cellsalike,100(cont rol),300(1Gy),500(3Gy)and700(5Gy)cellswereusedinthecaseofprotonbeamradiationandth enumbersofseededcellsforX-

rayswere100(control),200(1Gy),500(3Gy)and700cells(5Gy).Theplateswereincubatedfor2w eeksandthenthecellswerefixedandstainedwithGiemsastain(Sigma-

Aldrich,St.Louis,MO).Thecoloniesformedwerecounted,withminimum50cells/colony,andPE(

platingefficiency)andSF(survivingfraction)wereevaluated.Thecoloniesweredividedintothree groupsaccordingtotheirsizedeterminedasholo-,mero-,andparaclones,asdescribedearlier[17 ].Thenumberofcellsincolonieswere:(i)2500–6000forholoclones,(ii)500–

2500formeroclonesand(iii)<500forparaclones.

PE¼ Numberofcoloniescounted

Numberofcellsplated x100 ð1Þ

SF¼ PEoftreatedsample

PEofcontrol x100 ð2Þ

RBE(RelativeBiologicalEffectiveness)wascalculatedastheratiooftheabsorbeddoseofreferencer adiation(X-

rays)totheabsorbeddoseofradiationbeingresearched(protonbeam)whichcausesthesamebiological effect(37%ofcellsthatsurvivedtreatment).

MTTassay

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ThemetabolicactivityofMel270andBLMcellswasmeasuredwithtetrazoliumdyeMTT(3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)assay.Thecellswereseededinto

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24-

wellplates(10^4cellsperwell),andcellnumbersweredeterminedeachdayduringthefirstfivedaysdirectl yafterirradiationandat20and40dayspost-treatment.Cellsweresupple-

mentedwith10%ofMTT(Sigma-

Aldrich,St.Louis,MO)stocksolution(0.5mg/ml)andincubatedfor2.5hrs.TheMTTformazancrystalst hatformedweredissolvedinDMSO(Avantor,Poland)andmethanol(Avantor,Poland)solution(1:1).A bsorbancewasmeasuredatawavelengthof560nmwiththeTecanGENiosPlusplatereader(Tecan,S witzerland).

Cellmigration

Time-

lapsemonitoringofindividualcellmovementswasusedasanindicatorofcellularmigrationproperties.

Theindividualtrajectoriesofcellswereassessed20days(4^thpassage)and40days(7^thpassage)afterirradiati oninbothcelllines.Cellswereplatedatadensityof72cells/mm².After48hoursthemigrationofcellswa srecordedat37˚Cfor10h,at10mininter-

vals.Thetrajectoriesofindividualcellswereevaluatedfromthechangesincellcentroidloca- tion,asdescribedpreviously[18].Foreachcell,thefollowingvariablesweredetermined[19]:

(i)averagespeedofcellmovement,i.e.thetotallengthofcelltrajectory/timeofrecording;

(ii)thetotallengthofcelldisplacement(μm),i.e.thedistancefromthestartingpointdirecttothecell’

sfinalposition.ThevalueofCME(CoefficientofMovementEfficiency)wascalculatedastheratio ofthetotalcelldisplacementtothetotallengthofcelltrajectory.Foreachvalue,50cellswereanalyz edfrom3differentwells.

Woundhealingassay

Thecellswereplatedonto6-

wellplates(2.5x10^4cellsperwell)at20(4^thpassage)and40(7^thpassage)daysandtheassaywasperformedont hethirddayafterseeding.Awound(scratch)wasmadewithasteriletip.Picturesofthewoundweretaken attimepoint0hand9h.ThewoundareawasanalyzedusingImageJv.1.43U(WayneRasband,National InstituteofHealth,USA)andthepercentageofwoundhealingwascalculated.

Invasionassay

CellinvasionwasassessedwithBoydenChambers(8.0μmporesize,Falcon,NY,USA)in

24- wellplates.Cells(10⁴)wereputontotheuppersurfaceofchambers.Chamberswereincu- batedat37˚Cfor48h,atwhichtimethenumberofcellsatthebottomofthewellswerecountedandthepe rcentageofcellsthatinvadedthroughthemembranewasestablished.

Westernblot

Cellmonolayerswerelysedinlysingbuffercontaining1MTris-

HCl(pH7.5),3MNaCl,NP4O,distilledwater,aproteaseinhibitorcocktail(Roche,Switzerland),P MSFandsodiumorthovanadate.Cellswerecentrifugedat13000RPM,4˚C.Theamountofprotei nwasmea-suredusingtheBradfordassay[20]andstoredat-

80˚Cuntilused.Equalamountsofprotein(20μg)wererunonBolt1Bis- TrisPlusgels(Invitrogen,ThermoFisherScientific)andtrans-

ferredtoanitrocellulosemembraneusingiBlot1DryBlottingSystem(Invitrogen,ThermoFisherS cientific).Themembraneswereblockedwith5%skimmilkinaTBSbufferwith1%ofTween20for1 handincubatedwithprimaryantibodiesagainstvimentin(D21H3)(CellSig-

nalingTechnology,MA,USA)andβ1integrin(CellSignalingTechnology,MA,USA)at4˚Covernig ht.Membraneswerewashed3timesinTBSandincubatedwithsuitablesecondaryantibodiesandt

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henwashed3timesinTBS.SignalsweredetectedusingLumiGLO1chemilu- minescentsubstrate(CellSignallingTechnology,Danveers,MA).

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Statisticalanalysis

StatisticalanalysiswascarriedoutusingStatisticav12(StatSoft.Inc.).Sincewecomparedmorethanth reeexperimentalgroups,significancewasdeterminedbyone-wayanalysisofvar-

iance(ANOVA)afterevaluationofhomogeneityofvarianceswithLevene’sTest.Thediffer-

enceswereconsideredtobestatisticallysignificantatprobabilitylevelsofp<0.05,p<0.01andp<0.001.

Results

Cellsurvivalafterprotonandphotonirradiation

Tocomparetheeffectsofradiationbetweenuvealandskinmelanomacells,theirradiosensi-

tivitywasdeterminedusingaclonogenictestafterthetworadiationqualities.Asimilarpatternofdosede pendenceofsurvivalfraction(SF)intheclonogenictestwasseenforbothcelllinestested(Fig1Aand 1B ) .TheRelativeBiologicalEffectiveness(RBE)calculatedfor37%ofSFwasveryclose,1.1forMel270an d1.13forBLM.Thisisexactlythesamevalueasthatacceptedforclinicalprotonradiotherapy[21].

Fig 1. ClonogenicassayofcellsurvivalofMel270(A)andBLM(B)cells,treatedwithprotonbeam(■)orXray s(●).RepresentativeimagesofcoloniesarepresentedatS1Fig.Cellwereseededimmediatelyafter

radiation.MeanvalueswithSEM,*p<0.05;**p<0.001.RBEvaluesweredeterminedfromalinear- quadraticmodelandwere1.10forMel270,and1.13forBLMcells.(C–

E)ThreetypesofcoloniesformedbyMel270(C)andBLM(D)cellsintwoweeksafterirradiationwith1–

5GyofprotonbeamorX-

rays,determinedasthepercentageofthetotalnumberofcolonies.Mel270andBLMcellsformthreetypesofcolonie sdescribedasholo-,mero-

andparaclones(E).Holoclonesarelarge,packedcoloniesdisplayingheterogeneity,whicharebelievedtobederiv edfromcancerinitiatingcells;meroclonesareputativelyderivedfromtransit-

amplifyingcellsandparaclonesarelooselypackedcells,derivedfromdifferentiatedcells[17].

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ToassesscellviabilityMTTassaywasperformedtoevaluatethemetabolicactivityofcellsdirectlyaft erprotonbeamandX-rayradiation,aswellas20and40dayspost-irradiation.Thelong-termtime- pointswerethesameasinthemigrationactivitytests.Mel270showedhighersensitivitytoprotonbeamr adiationthantoX-raysinthefirstfivedaysdirectlyaftertreatment(S2 Fig ).Atthelatertime-

pointsof20and40daysthemetabolicactivityofcellswasnotaffected(S3 Fig ).

Irradiationchangedheterogeneityofcellularcolonies

Accordingtotheliterature[17]wedividedthemintothreegroups:holo-,mero-,andparaclones(Fig1C–1E).Thelargestholoclones,containingbetween2500–

6000cells,aredescribedasdisplayingheterogeneityandasderivedfromcancer-

initiatingcells,middlesizedmero-clones(500–2500cells)areprobablyderivedfromtransit- amplifyingcellsandthesmallestparaclones(<500cells)arelooselypackedcells,derivedfromdiff erentiatedcells[17].Asthegrowthtimeofthesecolonieswas14days,weestimatedtheaveragedo ublingtimeforeachtypeofcolonies.Forholoclonesthedoublingtimewas26–

29h,meroclones29–

33h,andforparaclones38h,whichreflectedsubstantialdifferencesintheirproliferativecapacity.

Non-

treatedprimaryMel270cellsformedsimilarnumbersofholo-,andmerocloneswithasmallnumberofpa raclones(7.6%)(Fig1C).Incontrast,BLMcellsdevelopedasimilarnum-

berofallclonetypes(Fig1D).BothprotonbeamandX-raysreducedthenumberofholo-

clonesinbothcelllinessubstantially.However,protonbeamcausedadecreaseinthenumberofholoclo nesfromapprox.45%to4%inMel270(alldoses)andfrom35%toalmostnoneinBLMcells(0.38%for1G y),formingparaclonesinstead.Incontrast,X-

rayirradiationresultedinconsiderableformationofholoclonesafter1and3GyinMel270(from45%to34

%and24.6%,respectively)aswellasinBLMcells(from35%to15.6%and5.6%,respectively).Thenum berofparaclonesincreasedinadose-

dependentmannerupto67.5%forMel270and78%forBLMcells.Theseresultsshowtheheterogeneity ofbothcelllinepopulationsandsuggestthatbothtypesofirradiationshifttheformationofcoloniesfromh oloclones(highlyprolifer-

ative)toparaclones(lessproliferativeactivity),eventhoughtheoverallresultingsurvivingfraction,calc ulatedfromthetotalnumberofcolonies,issimilarforbothtypesofradiation.

Protonbeamradiationinhibitedthemotilityofcellsandchangedtheirdirection ofmovement

Long-

termirradiationeffectswerecomparedforbothradiationqualitiesinMel270andBLMcells.Ananalysis ofMel270time-

lapserecordings(S4 Fig )revealedinhibitionofmotilityat20daysafterprotonbeamradiation(Fig2),atr educedvaluesofSpeed(71–79%)andDisplace-ment(47–

68%).TheCME(coefficientofmovementefficiency)valueswerealsoreducedforalldoses.Reduced CMEsuggestsachangeinthedirectionofmovement.ThelowertheCME,themorecellsmoveincircles ratherthaninastraightline.ThemotilityofMel270cells,at40daysaftertreatmentwithprotonbeamradi ation,wasinhibitedto71.2%ofcontrol,withastrongereffectafter5Gy(Speed35.3%;Displacement52

%),whichtranslatesintoanincreaseinCMEfor5Gy.Therefore,theoverallmotilityofuvealmelanomac ellswasinhibitedat40daysaswell,butwithouttheimpactonthedirectionofcellmovement.

IrradiationofcutaneousmelanomaBLMcellsledtoslightlydifferentresults(Fig3).Fol- lowingprotonbeamirradiationadecreaseinSpeedvalueswasseen,withlowercellDisplace- ment.ThiswasreflectedinCME,whichdecreasedto79–87%at20daysand64–79%at40dayspost-

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treatmentandindicatedlessrectilinearmovement.Thedirectionofcellmovementwasnotchangedaft erX-rays.

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Fig2.CellularmigrationpropertiesofMel270cellstreatedwithprotonbeamradiationorXrays.Individual cellmovementswereevaluatedat20daysafterirradiation(A,B,C)andat40daysafterirradiation(D,E,F)andthre eparameterswerecalculated:‘Speed’,i.e.averagespeedofcellmovement;‘Displacement’,i.e.thetotallinearle ngthofthecelldisplacementfromthestartingpoint(μm)andCME

(coefficientofmovementefficiency),i.e.theratioofcelldisplacementtothecelltrajectorylength.Meanvalues presentedaspercentofcontrol;*p<0.05,**p<0.01,***p<0.001.

Insummary,protonbeamirradiationinhibitedmotileactivityinbothcelllines.Intheuvealmelanoma cellmovementwasreduced,whereasinametastaticcutaneousBLMcelllinecellmovementwasmore random.

Bothtypesofradiationsloweddownwoundclosure

Bothtypesofradiationdecreasedthewoundregrowthrate,althoughatdifferenttimescaleinthetwomela nomacelltypes.Theinhibitionofwoundclosureat40–80%ofcontrolwas

Fig3.CellularmigrationpropertiesofBLMcellstreatedwithprotonbeamradiationorXrays.Individualcell movementswereevaluatedat20daysafterirradiation(A,B,C)andat40daysafterirradiation(D,E,F)andwereeval uatedintermsof‘Speed’,i.e.averagespeedofcellmovement;‘Displacement’,i.e.thetotallinearlengthofthecelldis placementfromthestartingpoint(μm)andCME(coefficientofmovementefficiency),i.e.theratioofcelldisplaceme nttothecelltrajectorylength.Meanvaluespresentedaspercentofcontrol;*p<0.05,**p<0.01,***p<0.001.

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Fig 4. WoundhealingassayconductedforMel270(A)andBLM(B)cells,andcellinvasionassayforMel270(

C)andBLM(D).Bothassayswereperformed20and40daysposttreatmentwithprotonbeamorXrays.Forwoundh ealingtestcellconfluentmonolayerswerewoundedwithapipettetipandimagesofthewoundclosureweretakenafte r9hoursofincubation.Foreachoftwotime-points15–

20imageswerecaptured.Barspresentthewoundhealingpercentagenormalizedtocontrol.*p<0.05;**p<0.01,***p

<0.001.CellinvasionwasassessedbymeasuringthetransporemigrationwithBoydenchamberassay(notcoated, poresize8μm).Meanvaluesineachgroupshowthepercentageofcellsthatmigratedthroughthemembraneinrelati ontocontrolseededinthewellwithoutamembrane.Valuespresentedasthe%ofcontrol.Mean±SEM;*p<0.05.

observedinMel270cellsat40daysandinBLMcellsat20dayspost-

irradiation(Fig4Aand4B).SomeinhibitionwasalsoseenforBLMcellstreatedwith3and5GyofX- rays.Whatismore,thedoublingtimeforthecellpopulationofeachexperimentalgroupwasatleast30hr s,thereforetheassayresultswerenotaffectedbyproliferation.

Cellinvasion

ThetransmigrationofcellsthroughBoydenchambers(pore8μm)wasrelatedtothecontrolseede dwithoutmembraneandpresentedasthepercentageofthenon-

treatedcontrolcellsinFig4 C fo rMel270andinFig 4 D fo rBLM.Inbothprotonbeamtreatedcelllines, adecreaseintransmigrationwasseen,especiallyat40dayspost-

irradiation.Incontrast,alowernumberofX-

rayirradiatedcellswasonlyobservedat20daysforMel270cells,butthenumberswerenotstatistic allysignificant.

Irradiationdecreasedthelevelofβ1-integrin

WeperformedWBanalysisforintegrinβ1,whichisinvolvedinmetastasizingandknowntodeclineaft erirradiation.Bothtypesofirradiationstronglysuppressedintegrinβ1inMel270cells(Fig5A)at20da yspost-treatment,thevaluesrangedbetween18%–40%and10%–12%forprotonbeamandX- raysrespectively.At40daysthecellsshowedanincreaseintheir

β1-integrinlevel,althoughitstillremainedbelowthecontrollevel(50%- 69%).Incontrast,inBLMcells(Fig5B)theloweringofβ1-

integrinwasseenonlyafter5Gyat20days.

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BLMcellsshowahigherexpressionofvimentinafterirradiation

Nosignificantdifferencesinvimentin,oneofthemarkersforepithelial-mesenchymaltransi-

tion(EMT),werefoundinMel270cells(Fig5C)afterirradiation.However,BLMcells(Fig5D)treatedwit haprotonbeamdisplayedanincreaseintheproteinlevelofbetween143and162%at20daysandbetw een163and214%at40daysafterirradiation.TheincreasewasalsoobservedafterX-

raysat20daysfollowingtreatment,especiallyinthecaseof3Gy(263%).Nevertheless,at40daysthevi mentinexpressiondecreasedapproximatelytothecontrollevel.

Discussion

Thecomparisonoftheeffectsoftworadiationqualitiesonmelanomacellsshoweddistinctdif- ferencesinthecoloniesgeneratedaswellaslong-

termmigratoryproperties.Despitethefactthatthetwomelanomalinestestedwereofadifferentori gin(uvealandcutaneous),inbothofthemtheprotonbeam,butnotphotonradiation,causedtheinhi bitionofactivelyproliferatingcellsandlong-termmotilityinhibition.Aswecomparedlong-

termeffects,weusedsublethaldosesoftwotypesofradiation.Thesesublethaldosesmaybesignifi cantinfractionatedther-

apyorincombinationtherapiesinvolvingradiationtreatment.Whatismore,wehaveto

Fig 5. Integrinβ1(A,B)andvimentin(C,D)proteinexpressionassessedwithWesternBlotinMel270(A,C)a ndBLM(B,D)cellstreatedwithdifferentdoses(1,3,5Gy)ofprotonbeamorXrays.Cellswerelysed20and40d aysafterirradiation.

(E)20μgofproteinwasappliedperwell.Controlofuntreatedcellswassettoa100%.Chemiluminescentevaluationo f3independentWesternblotsofcelllysateswasshownasameanofthepercentageofthecontrolandSEM.*p<0.05;

**p<0.01;***p<0.001.

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considermarginofradiationduringtreatment.Inspiteofrestrainedareaofthemargininthecaseof protonbeamirradiation,thetumorcellslocatedwithinmaycontributetoformationofmetastaticlesi ons.

Low-LETprotonbeamirradiationinhibitedactivelyproliferatingcells

Ourstudyshowedthatat2weekspost-treatment,overallclonogenicsurvivalofbothmela- nomacellsinvitro(Fig1Aand 1B )treatedwitheitherX-

raysorprotonbeamirradiationisverysimilarwithfairlycloseRBEvalues.However,whenwetakeintoc onsiderationthesubpopulationsofclonesfeatured(Fig1)weobservedashiftinthecoloniesformedaft erirra-diation.Thiswasmanifestedbyadramaticincreaseinthenumberoflessactiveclones(para- clones).Thesecellswerephysicallypresentintheirradiatedpopulationandyetmayhavelosttheircapa cityforsustainedproliferation.Suchastatemaybecalledareproductivedeath,i.e.acellmaybephysica llyintactbuthaslostitsabilitytodivide.Bothkindsofirradiation,butespe-

ciallyprotonbeamirradiationdrasticallydiminishedthenumberofholoclones(containingcancerstem -likecells[17])inbothcelllines.Suchadrasticdecreaseinthenumberofholo-

clones,containingactivelyproliferatingcells,withaconcomitantincreaseinlessactivepara-

clonesmighthaveaprofoundeffectinvivo.Consequently,fractionatedprotonbeamradiationmayres ultinastrongerinhibitionofthetumorgrowthbydecreasingactivelytheproliferatingcellpopulation.Thi shypothesisrequiresfurtherinvestigation.

Ithastobepointedoutthatthecellsstudiedwerenotsynchronizedinthecellcycle,sotheresultsobserv edareaveragedoverallcellsinthepopulation.Whatismore,weobservedhighlyheterogeneouscellpopu lationsinbothcelllines.

MelanomametastaticcelllineHTB140responsetoprotonbeamirradiationwasintensivelystudi ed[22,23].Itwasshownthatincomparisontogamma-

rays,protonbeamradiationinducedmoreapoptoticcells,fordosesrangingfrom8to24Gy[23].Ino urstudy,wehavefocusedonsublethaldosesofradiation1–

5Gyandobservedslightlylowermetabolicactivityanddecreasednumberofhighlyproliferativeclo nes(S2Fi g an dFig1).

Relativebiologicaleffectivenessofprotonbeamwaswidelystudieddependingonsuchfac- torsascellsensitivity,dose,LET,initialenergyofthebeamorthedepthinSOBP[21,24,25].RBEincreas eslinearlywithLET[26,27]andwithαcoefficientanddecreaseswithincreasing(α/β)photons[28].RBEval uesvaryfrom1.1to1.7for2Gyperfraction[29,30]andcanreacheven2.84atthedistalendofSOBPforv eryradioresistantcells[31].NeverthelessusuallyinvivostudiesshowRBEatmid-

SOBPapproximately1.1rangingfrom0.7to1.6[27].SuchincreaseinRBEissignificant,forexample,a pplicationofvariableRBEresultedinanincreaseofRBEweighteddoseintheSOBPplateaubyapproxi mately18%forbothnormalandtumorhumancells[32].SeveralmodelsforpredictingtheRBEforproto nbeamweredeveloped[24,33,34]andmanyauthorspostulateusingRBE-

weightedprotonbeammodulation,orLET-paintingintheclinic[26,30,32,35].

Low-LETprotonbeamirradiationinhibitedcellularmotility

Atbothlong-termtime-

pointsaneffectoncellularmotilitywasseen.InMel270cellsweobservedamajorinfluenceonthedirecti onofmovement,whichwasalsoparalleledinthedecreasedabilityofMel270cellstoinvade.InBLMcell sthedirectionofcellularmovementwasinhibitedbytheprotonbeamat40days,whichalsowasaccomp aniedbynumerousfilo-podiageneratedbythecells.IncontrasttoZhengetal.

[36]presentingthestimulatingeffectonthemigratoryandinvasivepotentialoftonguesquamouscellc arcinomaatonly24hourspostradiation,wehavenotdetectedanincreaseinthemotilityofmelanomac ellstreatedwithX-raysatlongtermafterirradiation.

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Despitetheinhibitedmotility,cellinvasionabilitywasonlyslightly,ornotatallaffectedbyradiation,th ereforeitcouldbesupposedthatcellelasticityincellmigrationandinvasionplaysarole.However,inour experimentalsetup,wedidnotuseanychemoattractantinthetrans-

migrationtestandonlycellsthatwentthroughthetransporeandmigratedtothewellwerecounted.Ther emayhavebeencellsthatwereattachedtotheothersideofthemembrane,sowemayhaveunderestim atedthenumberofcells.

Anotherevidencesupportingthenotionofprotonbeamradiationinhibitingcellularmigrationwa sshowninthestudies,whereprotonbeamirradiationoftumorcellsandnormalprimaryhumanlens cellsresultedindown-regulationofMMP-2andMMP-9[37,38],there-

foresuggestingsuppressionofthecellmigration.Incontrast,photonsledtotheup- regulationofMMP-2aswellasofMMP-

9[37].Hencetheprotonbeamwasthoughttorestraincellmigrationwhilephotonsmayhavestimula tedit.

Suspectedswitchofthephenotype

Anotherinterestingobservationisadramaticlossofβ1integrininMel270cellsatonly20dayspost- radiation.Consideringtheabsenceofsubstantialchangesincellmorphologyonemightspeculate thatthelossofβ1integrinmaybecompensatedforwithanincreaseinadif-

ferentintegrin.Ontheotherhand,wedidnotseeanychangesinvimentinlevelinMel270,whichma yindicatethatthecellsdidnotundergoanEMTafterirradiation.Radiation-

inducedEMTwasreportedforexampleforcolorectalcells[39].Therefore,wepostulatethatMel27 0didnotlosetheirepithelialphenotypeastheydisplayastrongconnectionwithneighboringcellsa ndEMTaltertheintegrityofcell-

celljunctions,whichresultsinthelossofcontactbetweencells.InBLMcells,however,weobserved onlyaslightlossofβ1integrinat20daysafterprotonbeamwithanincreaseofvimentinlevel,which maysuggestsomeshifttowardsEMTphenotype.Nevertheless,weneedtoconsiderthefactthatth esecelllinesareofadiffer-

entorigin.BLMcellsarederivedfromlungmetastasisofcutaneousmelanoma,thereforetheyhave alreadygonethroughanEMTprocess,whichisassociatedwithanincreaseinvimentinlevel.

Switchingphenotypesbetweenepithelialandmesenchymalwasrecentlyreportedinskinmelano ma[40].Itwasobservedthatmelanomacellsofbothproliferativeandinvasivepopula-

tionswereabletostartatumorinvivo,althoughthelatteronestookamuchlongertime.How-

ever,intheendthetumorswerecomparableanditwassuggestedthatthecellscouldswitchphenotypes inbothdirections[41].Despitethefact,thattheproliferatingpopulationofcellsmaybethemajorcontribu tortothegrowthofatumor,thephenotypeswitchingmechanismmaybeusedtoevadegrowtharrest,the reforetoacquireresistance,asinthecaseofNSCLC,wheretheauthorsobservedahighlevelofvimenti nandalsoZEB1andAXL1[42].Inthiscon-

text,ourresultsmightbeinterpretedinsuchawaythatsublethaldosesofradiationleadtoswitching,orsh iftingofthephenotypetowardsmoremesenchymal,asweobserved(i)pheno-typicheterogeneity, (ii)changesincellmigrationanddirectionofmovement,

(iii)differencesinβ1integrinandvimentinlevels.ForfurtherinvestigationEMT- associatedsignalingpath-

waysshouldbeexplored,especiallythetranscriptionfactorsregulatingtheactivityoftheE- cadherinpromoterandE-cadherinrepressorsandthereforetheactivityofthebeta- catenin/TCF4complex,MAP/ERKandJAK/STAT3pathways[40].

Furtherstudiesdirections

Neverthelessweneedtobeawareofthecomplexityofthepossiblemechanismsunderlyingthediff erencesinresponseofmelanomacellstoprotonbeamandphotonirradiation.ApartfromEMTcon nectedfactors,manypathwaysandprocessesmaybeengagedinpost-radiation

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changesinmelanomacells.Ascellmigrationrequiresacoordinatedadhesioncontactsbetweenc ellsaswellascell-

ECM(extracellularmatrix)interactions,thereisavarietyofintracellularandextracellularproteins suchasintegrins,cadherinsandcatenins,mayhavebeenaffected[43].Anotheraspectsarethesi gnalingpathwayssuchasMAPKandWNT.MAPKpathwaycoversmanykinasemodulesthatcon veyextracellularsignalstoproteinscontrollingessentialcellularprocessessuchascellgrowth,pr oliferation,differentiation,migrationandapoptosis[44,45]andWNTsignalingpathwayisrespon sibleforcellprolifera-

tion,migrationandpolarization[46].WorthpursuingarethemechanismsofDNAdamagerepair,a sitwasalreadyshownthathomologousrecombinationismoreimportantinrepairingprotonbeam inducedDNAdamage[2]andthatatthedistalendofSOBPanincreasedcom-

plexityofDNAlesionsandslowerrepairkineticswasobserved[4].Eventhoughthedirectconsequ encesofprotonbeamradiationarebeingbetterunderstoodeachyear,verylittleisknownaboutitsl ongtermeffects.Epigeneticchangesmayberesponsibleformaintaining

post-radiationphenotype[47],andthepresenceofcancerstemcellsmayimpactthepost-radi- ationlong-

termresponse[48].Thereforeinordertofullyexplainthemechanismsbehindofthedifferencebet weenprotonbeamandphotonradiationonmelanomacellsfurtherstudiesarerequired.

Clinicalrelevance

Asmetastasisisthemainreasonofmortalityofpatientswithbothskinanduvealmelanoma,anytreatme ntinhibitingmigratorypropertiesofcellswouldbeofbenefitintheclinic.Protonbeamtherapyisusedfortr eatmentofuvealmelanomasince1975anditsresultsarecompara-

bletobrachytherapy,withthemeanlocalcontrolover95%,andrateofcomplications7.7%[49,50].Itmay bespeculatedthatifprotonbeamtherapyindeedinhibitsmetastaticpropertiesofcells,highersurvivalof uvealmelanomapatientstreatedwithprotonbeammightbeseen,atleastinthelongterm.However,itisn otpossiblefromthedatapresentedintheliterature,toconcludeunequivocallywhethersuchdifferencee xists.Laneetal.pointoutintheirrecentanalysisofalargecohortofUMpatientstreatedwithprotonbeamt hat25-yearcumulativeUM-

relatedmortalitywasapproximately30%forprotonbeamtherapyand50%afterenucle- ation.For¹²⁵Ibrachytherapy,theCOMSstudyreport12-

yearmortalityforolderpatientswithlargetumorsalsoat30%[51].

Ontheotherhand,onemayarguethattheeffectofradiationonthecellularmigratorypropertiesisexe rtedatthetimeoftreatment,whenmicrometastasesarealreadypresent[52],andthereforetheoveralls urvivalmightnotbeaffected.Perhapssomemorelightontotherolethemigratorypropertiesofuvealmel anomacellswillinthefuturecomefromstudiesshowingthemortalitydependenceonthegeneticstatus ofthetumor[53]andfromabetterunder-standingofthedevelopmentofUMmetastases[54].

Conclusions

Ourresultsindicatethatthereareseveraldistinctdifferencesbetweentheeffectofprotonbeamirradiati onandX-raysonthesurvivalmechanismsandmigratorypropertiesofmela-

nomacells.Protonbeamradiationinhibitedcellularrectilinearmotilityanddecreasedinvasivepotentia lincomparisontoX-

rays.β1integrinlevelwasdecreasedafterbothtypesofradiationinuvealmelanomacells,andthelevelo fvimentinincreasedinBLM,cutaneousmelanomacells.Animportantobservationisthechangetoward salessproliferativetypeofcoloniesgen-

eratedafterirradiationwithaprotonbeam.Ifconfirmedinaninvivosetting,thismighthaveprofoundimpl icationsfortheincreasedefficacyoffractionatedprotonbeamradiotherapy.

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Supportinginformation

S1Fig.RepresentativeimagesofcoloniesformedbyMel270andBLMcells.Thenumberofseede dcellsisshownintheupperrightcornerofeachplatephoto.

(TIF)

S2Fig.MTTtestshowingmetabolicactivityofMel270cells(A,B)andBLMcellline(C,D).Met abolicactivitywasestimatedduringthefirstfivedaysdirectlyaftertreatment(A,D)withprotonbea morXraysexpressedaspercentofcontrolforeachday.Meanvalues,withSEM,#p<0.05;^*p<0.01

;^**p<0.001.

(TIF)

S3Fig.MTTtestshowingmetabolicactivityofMel270(A)andBLM(B)celllineafter20daysand 40dayspostirradiation.Itwasexpressedforeachdayaspercentofcontrol.Meanvalues,withSEM,

#p<0.05;^*p<0.01;^**p<0.001.

(TIF)

S4Fig.Individualtrajectoriesof50non-dividingMel270cellsexpressedascirculardia- grams.Singlelinerepresentasinglecelltrajectorywithinitialpointofeachtrajectorysetatthe0pointoft hediagram.Cellswereseeded20daysafterirradiationwithprotonbeamorX-

rays.Cellmovementwasrecordedfor10hrs,with10minintervals.Arepresentativetransmit- tedlightimageofthecellsistotheright(magnification200x).

(TIF)

AuthorContributions

Conceptualization:KatarzynaJasińska-Konior,MartaMichalik,BożenaRomanowska-Dixon.

Datacuration:KatarzynaJasińska-Konior,KatarzynaPochylczuk,ElżbietaCzajka,MartynaElas.

Formalanalysis:KatarzynaJasińska-Konior,MartynaElas.

Fundingacquisition:KrystynaUrbańska,MartynaElas.

Investigation:KatarzynaJasińska-Konior,KatarzynaPochylczuk,ElżbietaCzajka.

Methodology:KatarzynaJasińska-

Konior,MartaMichalik,JanSwakoń.Projectadministration:KrystynaUrba ńska,MartynaElas.Supervision:MartynaElas.

Writing–originaldraft:KatarzynaJasińska-Konior,MartynaElas.

Writing–review&editing:KatarzynaJasińska-Konior,MartynaElas.

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