InternationalJournalof MolecularSciences
Review
TransplantableMelanomasinHamstersandGerbilsa sModelsforHuman Melanoma.S e n s i t i z a t i o n in MelanomaR a d i o t h e r a p y —
F r o m A n i m a l M o d e l s t o ClinicalTrials
MartynaS
´niegocka1,*I D,EwaPodgórska1,*,PrzemysławM.Płonka1ID,MartynaElas1ID,Boz˙enaRomanowska- Dixon2,MałgorzataSzczygieł1,MichałA.Z˙mijewski3,
MirosławaCichorek4,AnnaMarkiewicz2,AnnaA.Broz˙yna5,6,AndrzejT.Słominski6,7,*I D andKrystynaUrban´ska1,*
1 DepartmentofBiophysics,FacultyofBiochemistry,BiophysicsandBiotechnology, JagiellonianUniversityinKraków,31-
007Kraków,Poland;przemyslaw.plonka@uj.edu.pl(P.M.P.);m a r t y n a . e l a s @ u j . e d u . p l ( M . E . ) ; g o s i a . s z c z y g i e l @ u j . e d u . p l ( M . S . )
2 DepartmentofOphthalmologyandOcularOncology,MedicalCollegeofJagiellonianUniversityinKraków, 31-007Kraków,Poland;bozena.romanowska-dixon@uj.edu.pl(B.R.-D.);annamarkiewicz@interia.pl(A.M.)
3 DepartmentofHistology,MedicalUniversityofGdansk,80-210Gdan
´sk,Poland;mzmijewski@gumed.edu.pl
4 Departmento f Em bryology,M e d i c a l U n i ve r s i t y o f G d a n s k , 8 0 - 2 1 0 G d a n
´sk,Po l a n d ; c i c h o rek@gumed.edu.pl
5 DepartmentofTumorPathologyandPathomorphology,FacultyofHealthSciences,NicolausCopernicusUniversit yCollegiumMedicuminBydgoszcz,OncologyCentre-
Prof.Franciszek ŁukaszczykMemorialHospital,85-796Bydgoszcz,Poland;anna.brozyna@cm.umk.pl 6 DepartmentofDermatology,ComprehensiveCancerCenterCancerChemopreventionProgram,
UniversityofAlabamaatBirmingham,Birmingham,AL35294,USA 7 VAMedicalCenter,Birmingham,AL35294,USA
* Correspondence:martyna.sniegocka@doctoral.uj.edu.pl(M.S
´.);Ewa.podgorska@doctoral.uj.edu.pl(E.P.);aslominski@uabmc.edu(A.T.S.);krystyna.urbanska@uj.edu.pl(K.
U.);Tel.:+001-205-934-5188(A.T.S.);
+48-12-664-6153(K.U.)
Received:29December2017;Accepted:28March2018;Published:1April2018 checkforupdate s
Abstract:Thefocusofthepresentreviewistoinvestigatetheroleofmelaninintheradioprotectionofm e l a n o m a a n d a t t e m p t s t o s e n s i t i z e t u m o r s t o r a d i a t i o n b y i n h i b i t i n g m e l a n o g e n e s i s . E a r l y studiesshowedradicalscavenging,oxygenconsumptionandadsorptionasmechanismsofmelaninradioprotecti on.Experimentalmodelsofmelanomainhamstersandingerbilsaredescribedaswellastheiruseinbioche micalandradiobiologicalstudies,in clu d ing aspontaneouslymetastasizingocularmodel.S o me resultsfrominvitrostudiesontheinhibitionofmelanogenesisarepresentedaswellasradio- chelationtherapyinexperimentalandclinicalsettings.Incontrasttocutaneousmelanoma,u v e a l m e l a n o m a i s v e r y s u c c e s s f u l l y t reatedw i t h r a d i a t i o n , b o t h u s i n g p h o t o n a n d pr otonbeams.Wepointoutthatthepresenceorlackofmelaninpigmentationshouldbeconsidered,whenchoosingt herapeuticoptions,andthatboththeexperimentalandclinicaldatasuggestthatmelanincouldb eatargetforradiosensitizingmelanomacellstoincreaseefficacyofradiotherapyagainstmelanoma.
Keywords:m e l a n o m a ; m e l a n i n s ; B o m i r s k i hamstermelanoma;o c u l a r melanoma;h u m a n melanoma;X-rays;neutrons;protonbeamirradiation;radio-chelationtherapy;radio-photo- therapy;chemo-radiotherapy;tumorvessels
Int.J.Mol.Sci.2018,19,1048;doi:10.3390/ijms19041048 www.mdpi.com/journal/ijms
2of29
Int.J.Mol.Sci.2018,19,1048
1. Introduction
Malignantmelanomaisoneofthemostnotoriousanddeadlyhumantumors.Ithasbeenknownsinceatleastthefifthcen turyB.C.,whenitwasmentionedbyHippocratesofCos[1].I t s mostcharacteristicbiologicalfeatureist heabilitytoproduceandstoremelanin[1],whichinturnisalsoresponsibleforimportantfeaturesofthetumor,makingitr esistanttomanymodesoftherapy[2].
Therearenumerouspapersexploringthemodelsofmurinemelanomas. T h e presentreview intendstosummarizetheworkonlesspopularanimalmodelsinmelanomaresearch,i.e., Syria ngoldenhamsters(MesocricetusauratusWaterhouse1839)andMongoliangerbils(MerionesunguiculatusMilne- Edwards1867).T h e s e laboratoryanimalsandtheirmelanomasrevealquiteuniquefeatureswhichin someaspectsmakethemakintohumanmelanomas.Theresearchhasbeencarriedoutforover50yearsinseveralr esearchcenters,mainlyinPolandandUSA,largelyusingmodelsdevelopedlocallyinGdanskandKrakow(Bomirskiha mstermelanomaBHM[3–
6]),andgerbilmelanomaZemanUJ90[7,8],whichmakesthiscompilationofresearchquiteunique.Aparticula remphasishasbeenplacedonradiotherapyandmelaninasfactorsdeterminingtheeffectivenessofmelanomat herapy.
2. MelaninFunctionsandTheirRadioprotectiveRoleinMelanoma
Melanomaoriginatesfrommelanocytes,whichareresponsibleforgeneratingmelanin[9].
Thispolymerisproducedinmelanosomes,organellescomingfromlysosomallineage,andtransferredtoothercellsinth emammalianskin,mainlytokeratinocytes[10].T hi s transfercanbedisturbedinmelanomas,whichmay eitherstorelargeamountsofmelanininthecytoplasmorsecreteittotheenvironment[8,11,12].C o n s e q u e n t l y,i t leadstointra-
orextracellular accumulationofmelaninoritsprecursors,whichcanbetoxic[13,14].Duringthepro cessofmelanogenesis,suchintermediatesaregenerated,whichmayacceleratetheinductionofsecondarymutationsin melanomacells[15].Thiscanbeoneofthereasonsforwhichamelanoticphenotypeinlate-
stagemelanomaisconnectedwithpoorprognosis[16].Moreover,thesignalingpathwaysresponsibleformel aninsynthesisarepartiallycommonwiththoseinvolvedinneoplastictransformation[17–
20].Asaresult,inamelanogenicallyactivecell,everymutationingenescodingregulatoryfactors,sucha sthecAMPresponseelement-bindingprotein(CREB)[21],PI3K/p70[22],c-KIT[23],
(neuroblastomaRASviraloncogenehomolog)(NRAS)[24,25],or(serine/threonine-proteinkinaseB-raf) (BRAF),becomesautomaticallyexpressed[26–
28],manifestingitselfinthepigmentphenotype,andingainingneoplasticfeaturesatthesametime[29].
Ont h e o t h e r h a n d , t h e g e n e r a t i o n o f m e l a n i n i t s e l f i s p rotectivef o r t h e b o d y a n d t h e c e l l . Theproductionofmelaninisofaclearlyadaptivevalue.Thus,itprovidesanimalcamouflage,anditp rotectsthebodyagainstdifferenttypeofradiationincludingUVprotectionspectrum[30,31].Beingamacro moleculeofamorphousstructure,itoftenrevealspropertiesofinorganicratherthanorganicsubstances[32].I tsspectrumofabsorptionresemblesinorganicmaterial[33],asdevoidofcharacteristicmaximaofabsorption,andmonotonously decreasesfromUVtowardslongerwaves,andforinfra-
redirradiationmelaninbecomesalmosttransparent[33,34].Thisbroadeningofspectrumisresponsibleforit sbrowntoblackcoloration,andalsomakesitagoodphotoprotectorbecauseofahighefficiencyofconversionoft heabsorbedirradiationintoheat[32,34].M e l a n i n containsnumerouscarboxylicgroupsofvari ousdegreesofprotonation,andquinone/hydroquinonegroupsofvariousdegreesofoxidation,containin galsosemiquinonesresponsibleforitsparamagneticproperties[32,35,36].Allthatmakesmelaninapowerfulbuffer abletoreversiblyprotonate,andaredox-
bufferabletoreversiblyoxidateandrecombinewithexternalfreeradicals(e.g.,producedbyradio-
orphotolysisofwaterinthecytoplasm[37,38]).Thecomplicated,irregularinnerstructuremakesmelaninanefficiention- exchangeresinabletoreversiblyadsorbmetalcations(zinc,calcium,iron,copper,manganese,etc.),organ iccompounds,gases,andwater[32,36,39,40].
Despitei t s resistancet o a c i d h y d rolysis,m e l a n i n u n d e rgoesd e g r a d a t i o n i n t h e b o d y,pr obablywiththeengagementofthereducednicotinamideadeninedinucleotidephosphate(NAD PH)-dependentoxidoreductases(NOX),and/orthevisiblelightandUV[41,42].Thesubstances
adsorbedinthepastmaybereleasedbackintheprocessofmelanindegradation,and madeacti vewithadelay,someofthem(e.g.,ironorcopper)beingphotochemicallyorredox-
activethemselves.T h i s isresponsibleformelanintoxicityandphototoxicity,strongerforpheomelanin thanforeumelanin[19,38,43–45].
Thehighaccumulationofparamagneticcentersinthemoleculesofmelaninwassuggestedtoenablethispolymertointe ractwithradiation-
inducedfreeradicalspeciesasaradicalscavenger.Thecomparativestudiesonthesensitivityofanimalcellstolow-
linearenergytransfer(LET)radiationsupporttheviewthatmelanincontentofthecellcanaffectitsradiationresponse[46].
Polymethylmethacrylate(PMM)mixedwithmelaninindifferentconcentrationswasirradiatedwith75Gy tostudytheX-ray-generatedfreeradicalsignal.Itwasdemonstratedthatoxygen-
centeredpolymersignalwasdecreasingfasterinthepresenceofincreasingconcentrationsofsyntheticmelanin[47],dire ctlyshowingtheprotectiveroleofmelaninaddition(seeFigure1).Similarreactionscanbefoundifdrycollagenisusedasamatrixi nsteadofPMM[47].
Figure1.Thepresenceofmelanininpolymethylmethacrylate(PMM)inhibitsthedecayoffreeradicalsignal,inducedbyX- rays.A —450mgofpurePMM,B—450mgofPMMwith2mgofmelanin,C—
450mgofPMMwith4mgofmelanin,D—
450mgofPMMwith6mgofmelanin.Figurebasedon[47].Copyright1975IAEA.
Inadditiontotheinteractionofmelaninwithradiolyticproducts,anotherpurelychemicalprocesswhichma y c o n t ri b u t e to t h e ove ra ll l e ve l o f ce l lu la r rad io resistancew a s reported[48].Ith as t h e natureofnon enzymaticbindingofmolecularoxygenbyalltypesofnaturalandsyntheticmelanins,andnotofphysicals orptionbythesepolymers,ascanbededucedfromthetemperaturedependenceoftheprocess.Inmelanomacells,mela ninisenclosedinmelanosomes,andseparatedfromtherestofthecell.Oxygenisawell-
knownradiosensitizer.Thus,anyprocesswhichsequestratesitinthecell,anddecreasesitsconcentrationinthedir ectneighborhoodoforganellessensitivetoradiationdamage(mitochondria,thenucleus),shouldbeassociatedwithane nhancementofradioresistance.
Therearethreemainmechanismsofradioprotectionofcellsbymelanin:
1. Melaninisabletoabsorbtheradiationitself(e.g.,theUVirradiation[49,50],ionizingradiation[51,52])du etothebroadenedspectrumofabsorptionandefficientinternalconversionoftheabsorbedenergy[32, 34];
2. Melaninrecombineswithproductsofradio-
andphotolysisofwater[37],andothercompoundsofmelanoticcells[32,53];
3. Melaninc o n s u m e s a n d a d s o r b s o x y g e n , a n d t h e p rocesso f m e l a n i n s y n t h e s i s c o n s u m e s significanta m o u n t o f t h e c o - s u b s t r a t e —
o x y g e n ( e . g . , f o r t y rosinase[54],o r p h e n y l a l a n i n e hydroxylase[55],etc.).
Asthesynthesisofnewmelanosomesiscontinuousinpigmentedcells[8,19]thismakesthes tationaryintracellularoxygenlevelslow,thusrenderingthewholecelllessvulnerabletoirradiation(asoxyg enisanefficientradio-andphotosensitizer[56]).
Despitethemanymechanismsofprotection,thepotentialtoxicityofmelaninmayinsomecasesoutweightheb enefitsofitspresenceinthecellasaradioprotector[57].Forexample,melanintoxicityissynergicwithmanganese(II)io nsandfacilitatesDNAdamageinneurons[58].Recently,ithasbeenshownthatthehigh-
symmetrymanganouscomplexesareresponsibleforradioresistanceinmanyorganisms,whilecompl exesoflowsymmetrymayradiosensitizethecells[59].Asthelatterarecreatedinactivecentersofenzymes(suchassuper oxidedismutases),radiationresistancemaynotdependonthegeneticallyencodedabilitytominimizeoxida tivestress,butontheactualnutritionalstatusofthecell[59].Thismay,tosomeextent,explainthesynergisticeffe ctofmelaninandmanganese(II)incellradiosensitization,becausemelaninhasbeenconvincinglyproventosequestrateM n2+cationsbymeansofcreatinglow-symmetrycomplexes[40].
3. HamsterasaModelofSpontaneouslyOccurringMelanoma
Thefullhistoryofthediscovery,description,anddomesticationoftheSyriangoldenhamst er(MesocricetusauratusWaterhouse1839)forlaboratorypurposes,andaspets,isexcitingandworthas e p a r a t e p a p e r ( s u p p l e m e n t e d w i t h t h e n e w e s t d i s c o v e r i e s ,
[60]).C a u g h t n e a r A l e p p o , S y r i a , in1930[60],thegoldenhamstersturnedouttobeperfectlabor atoryrodentsfortwomainreasons:easyandinstanttamingofthewildanimals,andtheirextremeresi stancetoinbreddespiteahighgeneticdriftandfoundereffect[60].Asaresult,thelaboratorypopulationrev ealslowpolymorphismint h e h i s t o c o m p a tibilityl o c i [9,61,62].C o n s e q u e n t l y,t h e a n i m a l s a r ea b l e t o m a i n t a i n a l l o g e n i c transplants,a n d inparticulartheones oftumortissues,i n ahigh erpercentageofcasesthanthewildpopulation.Anotherconsequenceisaparticularsusceptibilityofl aboratoryhamsterstohumanviralinfections[61].T h e spectrumofapplicationofthoseanimalsasl aboratorymodelsofhumandiseasesisthusunexpectedlywide.HelenValentineetal.
[63]describedatleast17typesofhumanpathologies,i n c l u d i n g c h ronico b s t ructivep u l m o n a r y d i s e a s e ( C O P D ) , a t h e rosclerosis,d i a b e t e s mellitus,oramyloidosis,whichhavebeenstudi edusingtheSyrianhamstermodel.H a m s t e r s arealsousedtostudytheEbolavirus[64].I t iswort hremindingthattheprimaryreasonforacceptingtheg o l d e n h a m s t e r a s a l a b o r a torya n i m a l w a s i t s p a r t i c u l a r l y h i g h s e n s i t i v i t y t o i n f e c t i o n s w i t h Salmonellatyphimurium,whichraisedres earchershopeofdevelopingavaccine[65].O f note,eventhoughtheworksareonholdnow,asthemicrobeisn owexploredaspartofanalternativestrategyinc a n c e r t reatment[66].Anotheri m p o r t a n t f e a t u re o f t h e S y r i a n h a m s t e r s i s t h e v a r i a b i l i t y i n hair-
coatcolorationphenotypes,withnumerouscolormutants[67],whichmakesthemparticularlyinte restingobjectsofgeneticstudiesandstudiesontheinfluenceofhaircolorphenotypesonmelanomadevelopment[68].
Asmodelsforhumanmelanoma,theSyrianhamstersturnedouttodevelopspontaneousand chemically-
inducedmelanomaseasily,andwithparticularmetastaticpotential,especiallyinthefirstgroup[69].Thea nimalstreatedwithcarcinogenswerefoundtodevelopbothnestsofextrafollicularmelanocytesarou ndselectedpilo-
sebaceousunits[70]andbenignpigmentedneviadjacenttothebasementmembraneoftheepithelium[69].Theyalsoreveal edamelanoticmelanocytesatthedermo-
epidermal junctionoftheskin[71].A l l oftheseatypicalgroupsofmelanocytes,aswellasdermal melanocytesmightbeabletotransformintomelanomas[69,70,72,73].Therefore,theactualoriginofvarioustypesoftumorsisnotyetfully understood.Interestingly,thehamstersareresistanttoUV-
inducedmelanomagenesis[69,74].Severalinvivoandinvitromodelsofmelanomaingoldenhamsterhavebeendeveloped.Th emostimportantspontaneousmelanomasareGreenemelanomamodels[75]
(usede.g.asxenotransplantstotheanteriorchamberoftherabbiteyeto
studytheeffectivenessofhyperthermia[76]andphotodynamictherapy[77]),Fortner’shamstermelano ma[71,78]
(knowntopresentasascitesintheperitonealcavity[79,80]),andBomirskimelanoma[3],describedi ndetailsinthenextchapters.
3.1. BomirskiH a ms ter M el an o ma
TheBomirskihamstermelanoma(BHM)modelconsistsoftwobasicmelanomalines:melanoticMa(black)anda melanoticAb(white),withthelatterbeinganexampleoftumorprogression[5,6,81,82].TheoriginalmelanoticMalinewasderive dfromaspontaneousmelanomaoftheskin(locatednearthenose)thathadappearedinamaleSyrian(golden)hamsterin1959, andwhichhasbeenmaintainedbyserialtransplantationamongrandom-
bredanimals.Theamelanoticmelanomaline(Ab)originatedin1963fromtheMaformbyspontaneousalteration,whichi ncludedlossoftheabilitytoproducemelaninpigment,markedincreaseingrowthrate,lossoftheabilitytometastasizeand decreaseofthesurvivaltime[4,5].Onceestablished,thesemelanomaspossessedaconsiderabledegreeofphenotypicstabilityoverd ecadesofpassaging[4,5].T h e twolineshavebeensuccessfullytransferredallogenicallyinhamste rsforoverfiftyyears.AsaresultofthefastergrowthoftheAbline,itstransplantationintervalisshorterthanthatofthe nativeMaline,althoughwithyearsofpassagingithasbeendecreasingforbothlines[5],asfurtherdocumentedbya shortertransplantationandlatencyperiodanddecreaseofminimaltumorcellsrequiredfortransplantabilit y[6,83,84].T h e subcutaneousgrowthoftumorsiscomparedinFigure2a.
Figure2.
(a)ThekineticsofBomirskihamstermelanoma (BHM)AbandMatumorsg rowthaftersubcutan eousimplantationinSyrianHamsters.Whitedotsindicateamelanotictumorsgrowthintime,whereasblackdotsindic atemelanotictumorsgrowthintime.Amelanotictumorsbegintogrow3daysafterimplantation,whereasmelanotict umorsstartgrowing12daysafterimplantation.(b)ThegrowthkineticsofBHMAb[O](n=9)andBHMMa[]
(n=14)inthehamstereye.Eachpointrepresentsthemean±SEM[84].
Metastasesof t h e Ma li n e t o lun g s a n d regionallymp h n o d e sh a ve be en o b s e r ve d f romt he beginning,whiletheAblineformedmetastasesinkidneysandliveronlytwelveyearsafterthefirsttranspl antation[5].
TheA b a me la n o t i c me la no ma l o st t h e a b ilit y t o s yn t h e si ze me la n in a sa resulto f a b lo ck i n melanosomesb i o g e n e s i s , b u t h a s retainedt h e t y rosinasea c t i v i t y,a l t h o u g h a t a m a r k e d l y l o w e r levelt h a n t h a t i n t h e M a l i n e [4–
6].T h e d e t e c t a b l e t y rosinasea c t i v i t y i s u n i q u e a m o n g a n i m a l amelanoticmelanomasmaki ngtheAblinesimilartohumanamelanoticformsexpressingtyrosinase
activity.Thelossofmelaninsynthesiswasaccompaniedbychangesinmanybiologicalfeatu re s,includingafastertumorg rowthrate, shorte ranimalsurviva l, and changesintheultrast ructur eofcells.Thelossofabilitytoproducemelaninpigmentwasreversible,sincethecellsstartedtoproducemelanind enovowhenincubatedinmediahighintyrosineorothermelaninprecursors[85–
90],withcomplexphenotypicchangesdependentonthetypeandconcentrationofthemelanin precursorsused[81,82,87,91–102].
ThehistologicalandultrastructureanalysisshowedthatAbmelanomacells,besidestheabsenceofp remelanosome s,h a v e a n e x t e n s i v e G o l g i a p p a r a t u s , a b u n d a n t r i b o s o m e s , a n d t h e i r p l a s m a membr anestructureandcontentofDNAischangedincomparisontoMamelanoma(seedetailsinTable1).Inadd ition,MaandAbdifferentiallyinfluencetheimmunologicalsystemthroughmodifiedantigenicity,immuno genicity,andcytokinesecretion(detailsinTable1).
Apartf romt h e h i g h e r p roportiono f c e l l s i n S / G 2 / M p h a s e s ( Table1),t h e A b l i n e a l s o h a s adecreasedabilitytoundergospontaneousapoptosisincomparisontotheMaline[103,104].However,theAblineisverysens itivetocamptothecin-
induceddeath[103,104]andshowedsignificantlyhigherradiosensitivityincomparisontomelanoticMamelano ma[105].
Basicm e t a b o l i c p a r a m e t e r s i n d i c a t e t h a t t h e A b l i n e i s d i fferenti n t e r m s o f t h e t y p e o f energy-
yieldingmetabolism,includingglycolysisandmitochondrialoxidation,andothermetabolicparameters,i n c l u d i n g t h e p e n t o s e p h o s p h a t e p a t h w a y,f romt h e n a t i v e m e l a n o t i c m e l a n o m a line[84,92,105–
108].ThenativemelanoticMamelanomacellshavehigheroxygenconsumptionthantheamelanoticAbline[106 ],whileamelanoticmelanomahasahigherrateofaerobicandanaerobicglycolysis[84],andhigherbasalmit ochondrialtransmembranepotential∆ΨincomparisontoMalinecells[109]
(detailsinTable1[6,84,106,107]).Thesemetabolicdifferences,definedbythepresenceorabsenceofmelaninpigmenta tion,werefurthermechanisticallysubstantiatedbyanalyzingthemduringinductionorstimulationofmelanogenesisincellcultureorinisolat edcells[84,92,108].
SincetheestablishmentoftheBHMlines,eachBHMmelanomalinehasmaintainedphenotypicalstability(melaninpro duction,growthrate,morphology) foroverfiftyyearsoftransplantation.Thecomparativebiologicalchar acteristicsofmelanoticMaandamelanoticAbBHMarepresentedinTable1.
Table1 . P a t h o b i o l o g i c a l p a r a m e t e r s t r a n s p l a n t a b l e m e l a n o t i c ( M a ) a n d a m e l a notic( A b ) l i n e s o f Bomirskihamstermelanomamodel.
MelanoticMelanomaMa AmelanoticMelanomaAb Reference
Origin
Developedasaspontaneousmalign antmelanomao fthes kin .
Developedasaspontaneousalteration
ofMamelanoticmelanoma. [4,5]
Yearoforigin 1959 1963
[4]Amountoftissueneededfor
100%transplantability(mg) 200 50 [4,5]
Transplantationinterval(days) 21±3
12±2Survivaltimeofimplanted
hamster(days) 81±5.8 27±1.5 [5]
Mostfrequentlocations
ofmetastases Lungs,lymphnodes Kidneys,liver,lymphnodes [4]
Histologicaland
ultrastructuralfeatures [4,110]
Epitheloidalcells Polygonalc ells
Melanosomesandpremelanosomes Lackofmelaninandmelanosomes GolgiareaismoreextensivethaninMa;
Golgiareaismoderatelydeveloped Productso f t y rosinasea c t i v i t y accumulateinthevesiclesofthetra ns-GA
Moderateamountofribosomes Abundantribosomes
Table1.Cont.
Plasmamembranestructure:
1. Carbohydratesc o n t e n t
MelanoticMelanomaMa AmelanoticMelanomaAb Reference Mitosisisrare
Mitosisi s f requentRERandSERaremoderately
developed RERandSERareverywelldeveloped
Somemitochondria Somemitochondria
(nmol/mgofprotein) 1702 631 [111]
2.Heterogeneity 3proteinfractions 6glycoproteinfractions 3. Membranefluidityand
1proteinfraction
8glycoproteinfractions [112]
Lowerdegreeoforderinthe molecularmobilityinthep
lasmaticm e m b r a n e 4. ExpressionofPglycoprotein
phospholipidbilayer;increaseinme mbranef lu i d i t y
[113]
(Pgp) 70%ofcellsPgppositive 10%ofcellsPgppositive [114]
5. Gangliosidecontent HighlevelofGM3
LowlevelofGD3and9-O-acetyl-GD3 LowlevelofGM3
HighlevelofGD3and9-O-acetyl-GD3 [115,116]
6. Neutralglycolipidcontent HighlevelofGL1 HighlevelofGb3,Gb4,Gb5
[117]Antigenicity Low IncreasedincomparisontoMa [118]
Immunogenicity Low IncreasedincomparisontoMa [119,120]
Cytokinesecretion AlteredsecretionofIL-6,IL-10,TNF-α [121]
DNAploidy 4n 3n [5,122]
Radiosensitivity Low High
[105]Lowendogenousapoptosisbuthighly Abilityforapoptosis Highpropensityforspontaneous
apoptosis sensitivetocamptothecin-
inducedapoptosis
[103,104]
Cellcycleanalysis 30%inS+G2/Mphase 40%inS+G2/Mphase
[104]Mainbi o c h e mi c al fe a tu res
1. Tyrosinaseactivity Hightyrosinaseactivity Lowtyrosinaseactivity [123,124]
2. Glycolysis Highaerobicandanaerobicglycolysis [84]
3. Antioxidantenzymes
Higha ct iv it y o f dismu tase/pe
roxidase
Relativelylowactivityofdismutases/peroxidas
e [125]
4. Mitochondrial
transmembranepotential∆Ψ Relativelylow Relativelyhigh [109]
5. Oxygenconsumption Relativelyhigh Relativelylow [106]
6. Enzymeactivities
Relativelyh i g h a c t i v i t i e s o f c i t r a t e synthase,succinatedehydrogenase, malatedehydrogenasehigherthani nA b
Relativelyhighactivitiesof NAD-dependentglycerol-3-
phosphatedehydrogenasehigherthanMa [106]
3.2. MIMelanomaandAb-455
AthirdvariantoftransplantableBomirskimelanomawasestablishedin1976fromapartiallydepigmentedpas sage104ofMamelanoma,andthentransplantedsubcutaneouslyinhamsters[6,126].ItdifferedfromtheparentalMamelanomai nitshighertyrosinaseactivity,lowerpigmentationlevel,abilitytoproducepheomelanin,andslightlyslowergrowthrate,with otherparameterssimilartotheMamelanoma[126].SelectionformorepigmentedtissueduringtransplantationofMI melanomageneratedanothervariantthatdifferedfromtheMImelanomaonlyintermsofitshigherpigmentationlevel[127].Additio nallineAb-
455,transplantableinhamsters,wasderivedfromtheinvitrocelllineoriginatingfromtheprimarycultureofAbmelanom a[88].Thatamelanotictumorwastyrosinasenegative,grewsignificantlyslowerthantheparentalAbmelanoma, andhadadifferentmetastasispatternthatwassimilartotheMamelanoma.I n t e restingly,duringserialtrans plantation,arapidaccelerationofAb-455growthoccurred,renderingitsimilartotheoriginalAbmelanoma[88].
3.3. RadiosensitivityofBHMGrowingintheSkin 3.3.1.EffectsofLow-LETRadiation
Astrikingdifferencebetweentheradiosensitivityofthepigmented(BHMMa)andnonpigmentedBomirskihams termelanoma(BHMAb)wasobservedintheearly70’s(unpublished).Subcutaneouslygrowingtumorswereirradiatedwit h48GyoffractionatedX-
rays(twotimes7Gyandtwotimes5Gyevery24h,thenrepeatedafter6days,50kV,25mA,Al1mm,4.98Gy/min).Th egrowthofbothBHMMaandBHMAbtumorswasinhibited,butamelanotictumorsdisappearedmarkedlyf aster[128].
Thehigherradioresistanceofpigmentedcellswasverifiedlaterinamoreelaborateexperimentinvolvingi rradiatingmelanomacellsinvitro,anddeterminingthesurvivalfractioninvivo.That stepwasdesignedin ordertocheckifthehigherradioresistanceofpigmentedlinesoriginatedatthetissueoratthecellularlevel.
Cellswereirrad iat edin vitro,andimmed iate lyafte rim plantedsub cutan eouslyin tohams te rs,alwaysusingthesamenumber,106cells.Theaveragerateoftumorgrowthwasdeterminedforeachdose.Th esurvivalfractionofirradiatedcellswascalculatedfromasetoftumorgrowthcurves,wherethetumorwasinitiatedwith variouscellnumbers.Figure3showsthatpigmentedcellswere2.4timesmoreradioresistantthanunpigmentedB HMAb.Themeanlethaldosewas4.8GyforBHMMaand
2.0forBHMAb[129].
Figure3.Pigmented cellsweremoreresistanttoX-
raysthanunpigmentedAbcells.Thecellswereirradiatedinvitro,andimmediatelyafterimplantedsubcut aneouslyintohamsters,alwaysusingthesamenumberofcells(106).Theaveragerateoftumorgrowthwasdet erminedforeachdose,andthesurvivalfractionofirradiatedcellswascalculatedfromasetoftumorgrowthcurves, wherethetumorwasinitiatedwithvariouscellnumbers[129].Copyright1984Gurbiel,R.
3.3.2.Radio-ChelationTherapy Radio-
chelationt h e r a p y i s b a s e d o n t h e c o m b i n a t i o n o f r a d i o t h e r a p y w i t h a p a r a l l e l u s e o f c helatingdrugsasradiosensitizers.Thelattermayexertnooncostaticeffectbythemselves.ThechelatorEdathamilcalcium- disodium(ECD) wascombinedwith20 GyofX-
rays,d e li ve redas4Gyevery5days.Thehighestconcentrationofthechelatorinthetumortissuecouldbe achievedupontopicalapplicationofa10%ointmentofECDoversubcutaneously(s.c.)implantedhamstermelanoma.Tum orgrowthwasinhibitedfor33daysandtumorvolumeatday33wasthreetimessmallerthanthecontrol,i.e.,vehicleplu sradiation(Lukiewiczetal.,datanotpublished).
3.3.3.EffectsofNeutrons
Thenextstepwastochecktheradiosensitivityinthesameinvitro-invivomodelagainsthigh-
LETradiation,i . e . , n e u t rons.A s t h e rei s n o o x y g e n e ffecti n h i g h - L E T r a d i a t i o n , b o t h s u b - l i n e s w e reexpectedtoexhibitthesameradiosensitivity.Indeed,experimentsdemonstratedthatthethenstriking
differenceinradiosensitivitybetweenMaandAblines,clearlyvisibleforlow-LETradiation(X- rays),disappearedfortheirradiationofthetwotestedsub-lineswith5.5MeWneutrons(Figure4).
Figure4.N o differenceinradiosensitivitybetweenAbandMacellstreatedwithneutrons[130].Copyright200 0Urbanska,K.
3.4. RadiosensitivityofBHMTumorsTransplantedintheEye
Severala n i m a l m o d e l s o f o c u l a r m e l a n o m a w e rep roposed,i n c l u d i n g i m p l a n t a t i o n o f s k i n melanomaintotheeye,suchastheGreenemelanoma,B16[131],orhumanuvealmelanomai nthenudemouseeye[117,132–
135].H u successfullyestablishedmousexenograftsinthechoroidofanimmunosuppressedrabbitusingB 16F10cellline[134].O u r oculartumormodelofmelanomawasobtainedbyimplantingsmallpiecesofBHM,fr eshlyexcisedfromthecutaneoustissue,intotheeyeoftheSyrianhamster(Mesocricetusauratus)
[135].Tumorfragmentssized0.4–
1.0mmwereimplantedintotheanteriorchamber(AC)oftheeye.Duringthefirst2to3days,disappearanceoftheimplant swasobserved,followedbytheappearanceofiristumorsafter4–6daysinthecaseofBHMAb,andafter8–
10daysinthecaseofBHMMa.WhentheACwascompletelyfilledwiththetumormass,theeyewasenucleated, andtheanimalswereobservedformetastases,developingwithin20–
30daysinthelung.M e l a n o m a ce llsg rowingin th e i ri s in th e fo rmo f n o d u le s inf ilt ra t ed a llsu r r oundingt issue s, andtheciliarybodyinparticular,andalwaysremainedpigmented.Thedistantmetastases,asse ssedmacroscopically,wereencounteredinthelungs(after48daysin100%ofanimals),andsometimesinthekidney s(after48daysonlysporadically)aswellasintheregionallymphnodeswhichwerealsoclearlyenlarged[5,13 6].
Thev a s c u l a t u reo f B H M t u m o r s g rowingi n t h e e y e w a s m a i n l y i n d u c e d f romt h e a n t e r i o r capillaryandantero-
venularlayersoftheiris.Asitwasrevealedbyscanningelectronmicroscopyofvascularcorrosioncasts(Figure 5),thetumorvasculaturewascharacterizedbypronouncedtortuouscoursesofthebloodvesselswithunevenc ontoursandvariablediameters.A l l vesselswerehighlyirregularandheterogeneous,withmanyemb olizations,fenestrations,andsprouting.Venulesandsinusoidalcapillaries,exhibitingheterogeneou sintra-
tumordensity,wereintensivelyinterconnected.Avascularareaswerealsoseen.Thepresenceofnumerousno dularoutgrowths,varyinginsize,onhesurfaceofdilatedvenulesandvenousvesselsrepresentsmorphologicalevid enceforthecontinuousremodelingoftumorvasculatu re.Theobservedfeatu resofthevascularsys temseemtop rovideapathwayforfurthertumorexpansion[137].
AlthoughBHMisacutaneousmelanoma,thedevelopmentofspontaneousmetastasesisan advantageofthemodel[136].Also,unliketherabbitmodel,BHMmelanomaisallotransplantedwithhamstersbeingbothth edonorandtherecipientofthegraft.T hi s eliminatesimmunologicalcomplicationssuchasgraftrejection,whi chcanoccurafterthetransplantationofhamstermelanoma(Greenemelanoma)intotherabbiteye.
Figure5.
(A)Tumorvasculaturecastrevealingamissingvesselhierarchyandheterogenousvasculardensity.St ronglydila tedvenousvessels(fullwhitecircles)andcapillaries(c)predominateoverfewarterialfeeders(a)Nodul ar,nest-
like,avascularareas,surroundedbytuftsofcapillarieswithshortterminalbranches,aremarkedwit hadashedline.T h e externalperimeteroftumorvasculatureisindicatedwitharrowheads.E x t r a v a s a t i o n ofresinisalsovisible(e)Bar=500µm.
( B)Fragmentofintratumorv a s c u l a t u res h o w i n g stronglyd i l a t e d v e n o u s v e s s e l s ( f u l l w h i t e c i r cles)i n t e rconnectedwithloopsformedbywidesinusoidalcapillaries(c)Tuftsofcapillarieswithshortt erminalbranchesareindicated(asterisks)andshowninhighermagnification(inset).Note alsothepos teriorvascularlayeroftheiris(arrow).Bar=100gmand50gm,respectively.
(C)Vascularsprouts(arrowheads)andglobularoutgrowths(whitecircles)ontheproliferating,dilatedtu morcapillaries.N o t e alsothetinyholes(arrows)typicaloftheintussusceptiveangiogenesis.Bar=500µm.Re producedwithpermissionfromAnnalsofAnatomy[137].Copyright2001Elas,M.
3.4.1.EffectsofLow-LETRadiation
Since2000,thehamstermodelofmelanomalocatedintheeyehasbeenappliedtostudydistantmetastasesstudie s[130].Twosub-
linesdifferingintheirmelanincontentwerecomparedwithregardtotheirradiosensitivitytoruthenium- 106(106Ru)radiation.Tumorsgrowingintheirisweretreatedwith3 , 6 , o r 1 0 G y o f1 0 6Rua d m i n i s t e r eda s a s i n g l e d o s e o r i n f o u r f r a c t i o n s a t 2 4 h i n t e r v a l s .
Dose-
dependentdelayoftumorgrowthwasobservedinbothmelanomas.Fo llo win g thetreatmentwitha d o s e o f 6 G y,t h e a m e l a n o t i c ( B H M A b ) t u m o r s g rew2 . 6 t i m e s s l o w e r,a n d t h e m e l a n o t i c (BHM Ma)tumors1.4timesslowerthantheuntreatedones.Exposuretoβ-
radiationfrom106Rudidnotsignificantlyaffecteitherthenumberorthesizeofmetastases,exceptatadoseof10 Gy,whereastatisticallysignificantdecreaseinthenumberofmetastaseswasfoundinthemelanoticsub -line(BHMMa)
[138].Histologicalanalysisshowedsignsoftumorbloodvesseldamagesuchasendothelialcellswelling,erythrocyteextr avasation,andtumornecrosis.T h e s e signsincreasedwiththerisingdoseofβ-
radiation.Changeoffractionationfromfourequaldosestoaboostdoseof4Gy,followedby3×2Gy,causedacompletei nhibitionofmetastasesfor70days(unpublisheddata).
3.4.2.RadiotherapyUsingProtonBeamIrradiation
Asingledoseof10GyofprotonbeamirradiationdelayedthegrowthofBHMMamelanomaint h e h a m s t e r e y e b y 1 0 d a y s [138].Albeitt h e i n h i b i t i o n o f t h e i m p l a n t e d t u m o r g rowth w a s moderate,protontherapynoticeablyreducedthemassofthemetastasesinthelungincomparisonwit hu n t reatedt u m o r s ( F i g u re6).O n a v e r a g e , 1 0 G y o f p rotoni r r a d i a t i o n d i m i n i s h e d t h e m a s s ofmetastases4.35times,e ve n thoughtherewasasignificantspreadbetweenindividualan imals(Figure6).Theseresultsareinagreementwithdatapresentedforosteosarcoma[139].Likewise,protonbeami rradiationdecreasedcellmigrationandinvasioninadose-
dependentmannerandstronglyinhibitedmatrixmetalloproteinase2(MM-
2)activityinhighlyaggressiveHT1080humanfibrosarcomacellsinvitro[139].Similarly,i t wassho wn thatinvit romodels,t h e adhesion,mi g r a t io n , i n va s i o n , andthelevelofexpressionoractivityofmoleculesrelate dtometastases,suchasαVβ3,β1i n t e g r i n , andMMP-
2,werealldecreased,evenaftertreatmentwithsmalldosesofprotonbeam[140].
Figure6.
(A)InhibitionofBHMmelanomatumorgrowinginthehamstereye,irradiatedwithaprotonbeamatasingledoseof 10Gy(n=7,blacksquare),ascomparedwiththeuntreatedcontrol(n=6,blackdiamond).
(B)Themassoflungmetastasesdecreased4.35timesasaresultoftheprotonbeamirradiation(10Gy)ofBHMmelan omatumorgrowinginthehamstereye(p=0.0052).AveragemasswithSEMisshown.Thenumberofcontrolanim alswassix,andthenumberofirradiatedanimalswasseven.Representativeisolatedlungswithmetastasesfromu ntreated(C)andirradiated(D)animals.Reproducedwithpermissionfrom[138].
3.4.3.Radio-Phototherapy
Studiesontheeffectsofphotodynamictherapy(PDT)combinedwithγ-
radiationontheBHMtransplantedintotheeye wereespeciallyimportantasbothof themethods arenon-
invasiveandthuspreventsurgicalintervention.Themainadvantageofionizingradiationisthedeeppen etrationofr a d i a t i o n i n t o t h e t is s ue,w h e rei t i n h i b i t s ce l ld iv is io n . PDT,o n t h e o t h erh a n d , t a rg etsma in ly tumorcells,butvisibleirradiation,usingthepreferredexcitationwavelengthfortheMC540sensitizer,p enetratesonlypartlyintothetumortissue.CombiningPDTwithaverylow-doserateofγ-
irradiationwasfoundtoleadtotumorinhibition[141].A l t h o u g h thehistologicaldamagewasseve reforbothlines,thenon-
pigmentedBHMAblinewasmoresensitiveandrespondedbetterthanthepigmentedmelanoma.T h e most significantinhibitionwasobtainedwhenboththeγ-
radiationandPDTweredeliveredindosesspreadovertime.S u c h treatmentresultedin6weeksofinhibi tion,afargreaterlengthoftimethantheinhibitionperiodobservedafterasingletreatment(2daysfornon- pigmentedcellsand4daysforthepigmentedones).
Thesignificantincreaseineffectivenesswiththefourdividedcombinedtreatmentdosesmaybed u e t o r a d i a t i o n -
i n d u c e d d e p l e t i o n o f t h e v i a b l e s t e m c e l l s . Thatf i n d i n g a g reesw i t h t h e resultsobtainedi ntherabbiteyeforGreenemelanoma[94].ThecumulativeresultsindicatethatMC540-
mediatedPDTincombinationwithionizingradiationhassignificanteffectsontherapidlygrowingmelanomaintheey e[142].
4. GerbilsasAnimalModelsforChemically-InducedMelanomas
TheMongoliangerbilbelongstothefamilyMuridae,subfamilyGerbillinae,orderRodentia[143].Gerbilsaresmallroden tsthatoccurnaturallyinthedesertregionsofNortheastChina,EasternMongoliaandthesteppesofRussia[
144],livinginsmallcoloniesinextendedburrowsystems[145].Theyhaveseveralphasesoftwenty-four-
houractivity;thetwomostactiveperiodsarejustafterdawn,andarounddusk,butmanygerbilsremainactivethroughouttheday[1 46].
Gerbilshavebeenusedforscientificpurposessincethe1880s,beginningwiththeresearchontuberculo sis;theyalsoplayedasignificantroleinbilharziaresearchduringthe1950s–
1960s[147].Duetoitscharacteristicbehavioralandphysiologicalfeatures,gerbilswereusedinawidespectrumofresear ch,coveringavarietyofresearchfields,includingbehavioralinvestigations[148],biologicalandbehavioralprocessesofag ing[149],epilepsy[150],infectiousdiseases[151–
153],dermatitis,neurologyresearch,audiometryandsoundeffect,coatcolorgenes[154],melaninandtyrosinasea ctivity[155],andothers.Thegerbilisanimportantlaboratoryanimalinoncologyresearch[8,156–158].
4.1. ZemanUJ90Melanoma
GerbilshavealsoplayedaspecialpartinthehistoryofresearchcarriedoutattheDepartmentofB i o p h y s i c s o f t h e J a g i e l l o n i a n U n i v e r s i t y i n K r a k o w.I n t h e 1 9 9 0 s , i n t h e a n i m a l f a c i l i t y o f t h e D epartmentofBiophysicsoftheJagiellonianUniversityinKrakow,amelanotictumorwasfoundontheearofo neanimalfromagroupofMongoliangerbils,previouslytreatedwithN-ethyl-N-
nitrosourea.Thiscompoundhasbeendescribedintheliteratureasacarcinogenwithaparticularaffinitytocel lsofneuralorigin[159],showingapreferenceforplacesrichinpigment,hairyareas,andthoseoftenexpos edtothesun(paws,ears,andtail).Itcaninducetumorsin40%ofgerbilsexposedtothatcarcinog en[160].Thetumorwastransplantedintootherrelatedanimalsofthebreedingstock,whichhadbeenmaintained byinbredcrossing.Thus,anewtransplantableZemanUJ90melanomalineingerbilswasstabilized[7].This t ransplantablemelanomalinewasusedtocarryoutinvivoElectronParamagneticResonance(EPR)experi ments.IncontrasttothegoldenSyrianhamsters,gerbilshavealongtailthatcanbeplacedinsidearesonantcavity,similarto mousetailsinoculatedwithmelanoma.Thatobservationwasagoodstartingpointforfurtherextensiveresea rch,mostofwhichhasbeenpublished,andisbrieflyreportedbelow.
Asadesertanimal,theMongoliangerbilcomesfromamicrobiologicallypureenvironmentandrev ealsanimpairedimmunologicalreactivitytosomeimmunologicalstimuli.Itismanifestedbyaweakresponseoft heanimal’smacrophagestolatexparticlechallenge[161],andalsobyweakgraft-versus-
host[162]andmixedlymphocyteresponsesfromallogenicmixedcultures[163].Thosefeaturesmayadditionallysuggesta weakreactivityoftheNKcells,andalsoalowhistocompatibilityvariability,resultingfromahighinbreedinglevelintheirbree dingstocks.ItturnedoutthattheanimalsrevealedaweakresponseofiNOS(induciblenitricoxidesynthase),manifestedinE PRstudiesbyshowingalowlevelsofnitricoxideandtheirhemoglobincomplexes(nitroso-
hemoglobin,HbNO)intumorsgrowinginsitu.Thatwasentirelydifferentfromotheranimaltumorsstudiedbyus,butsimil artohumantumors[164,165].AssolidtumorsofBHMrevealedtheHbNOsignal,itslackofexpressioningerbiltumorsmust havebeenaresultoflowiNOSactivityratherthanthelowpolymorphismofhistocompatibilitygenes.TheEPRsig nalofHbNOinZemanUJ90tumorscouldbeinducedonlybyastrongimmunologicalstimulus—lipopolysaccharide(LPS)
[166,167].Theexistenceofamelanoticsub-line(seefurther)madeitpossibletodemonstratethetime- anddosedependenceoftheHbNOEPRsignalintensityonLPS,anditsinhibitionbyanalogsofL-
Arginine,thesubstrateforNOsynthesisbyiNOS[168].Thatextraordinarypropertyofthoselaboratoryanimalscouldalso bedemonstratedbyaslowerandlowerresponseofgerbilstoxenotransplantsofrathearttissues,andaweakerdependenceonpre- sensitizationwithdonorsplenocytes[169].TheabilitytoinduceNOsynthesisbyLPSservedtodemonstrateaninvivoprodu ctionofNObyspin-trappingatS-
band,whichwasfollowedbyinvivoobservationsofNOproductioninmelanomainsitu.Thesefindingswereobserv edinanimal(gerbilandmouse)tailsplacedinsidetheresonantcavity[170].
Initially,theZemanUJ90linewasheavilypigmentedandgrewslowly.Afterthefourthpassage,arapidaccelerationofgrowt hwasobservedandtheFGM(FastGrowingMelanoma)sub-
lineemerged.Duringtheseventhpassage,apartofthegrowingtumorwascompletelydevoidofpigment.Asecondamelanotics ub-line(A-FGM)wasderivedfromthatfragment(Figure7).InthecaseoftheZemanUJ90melanoma,thenon-
pigmentedsub-
linehasagreatercapacitytocreatemetastases(in80%oftheimplantedanimals)incomparisontothepigmentedsub- line(33%animals)
[7].TheobservedphenomenonstandsincontrastwiththecaseofBomirskihamstermelanoma,whereahighermetastaticcapacit ywasreportedfortheheavilypigmentedBHMMaline[5].C o mpa r i s o n oftheregressionlineslopesreve aledthattherateofgrowthoftheA-FGMsub-
linewasaboutthreetimeslowerthanthatofthemelanoticM-FGMsub-
line,whichwasconsistentwiththeobserveddifferenceintumorsizemeasuredaftertheexperime nt.Thischangetowardsaslowergrowthrateofthenon-
pigmentedamelanoticlinewasalsounexpected,asexperimentalamelanotictumorstendtogrowfaster(lessdiffer entiated),beingbetteroxygenatedandnourished(betterblood-
supply)thanmelanoticones[6,127,171,172].However,thisisnotalwaysthecase,asshownforthehypomelanoticBM HMI,whichgrowsslowerthanthemorepigmentedMamelanoma[126].SelectionofBHMMIforlessandmorepigmentedv ariantsgeneratedsub-
lineswithsignificantlydifferentlevelsofmelaninandtyrosinase,butwithoutanysignificanteffectongrowthrate[127].
Figure7.Electronparamagneticresonance(EPR)analysisofmelanotic(leftEPRspectrum)andamelanotic tumors(right)ofZemanUJ90melanomas,correspondingtotheblackandwhitetumors(photographsinthemiddlerow).T hewhitetumorwasobtainedfromthewhitepartsofthetwo-
colortumorwhichappearedinpassage7(upperphotograph).DPPH:thepositionofafreeradicalsignal(g=2.0037).Reprinted withpermissionfromCopyright2003JohnWiley&SonsLtd.[8].
4.2. IrradiationofZemanUJ90Melanoma
ThemelanoticandamelanoticversionsofZemanUJ90melanomaturnedouttobeinterestingmodelstodeterminetum orsensitivitytoXradiation,andtheywereexposedtolowradiationdoses.Theresponsetothedosesof5and10Gywasweak, whichwasnotsurprising.Radiationhasaninhibitoryeffectontumorgrowth:boththepigmentedandthenon- pigmentedformsgrewslowerafterirradiationthaninthecontrolarm.TheeffectivenessofXradiationincreasedlinearlyw iththedoseapplied,whichwasexpected.Unexpectedly,melanindidplayaradioprotectiveroleforthetumor,un likeinthecaseofhamstertumors.However,thatwasnotauniqueorisolatedphenomenon[57].
Ther a d i o l o g i c a l p h e n o m e n a a s s o c i a t e d w i t h Z e m a n U J 9 0 m e l a n o m a s a r ec o n g ruentw i t h earlierobse rva tions on age ne ra lra d io resistanceofMo ng o lia ng e rb ils [173 –
176].A l l th at ma ke s theM o n g o l i a n g e r b i l a reallyu n i q u e l a b o r a t o r y a n i m a l i n t h e c o n t e x t o f m e l a n o m a r a d i o t h e r a p y studies.Inthiscontext,onecanconcludethattheactualradioprotectionofm elaninstronglydependsonthemodelinvolved,andinclinicalpracticeontheparticularcaseinquestionandthestageoftum ordevelopment[57,177].
5. HumanMelanoma
5.1. RadiosensitivityofHumanSkinMelanoma/WhyIsRadiationNotUsedintheTreatmentofHumanSkinMelano ma
Prevention,earlydiagnosis,andsurgicalexcisionofthetumor,whenthediseaseislocalizedtotheskin,r epresentthegoldenstandardsofmelanomamanagement[178,179].R e c e n t advancesinmelanomather apyhaveledtoasuccessfuluseoftargetedtherapyortherapybasedonmodulationsofimmuneresponses,whilem anagingstageIII–IVdisease[180–
182].Althoughsuchstrategiesareassociatedwithadverseeffects,financialcosts,anddevelopmentoft umorresistancemechanismsresultinginrecurrentdiseaseandultimatedeath(discussedin[82,182–
186]),systemicstrategiesarepreferredinmostclinicalsettings.
Thepastreluctancetoapplyradiotherapyinmelanomatreatmentwassecondarytotheopinionthatm e l a n o m a s i n g e n e r a l w e reresistantt o r a d i a t i o n [187].However,s i g n i f i c a n t e v i d e n c e h a s be enaccumulated,indicatingthatmelanomashaveawiderangeofsensitivitytoradiation[187,188].Currently,radio therapyisusedinselectedpatientswithlentigomalignamelanoma,andasanadjuvantorpalliativeapproachinselected patientswithregionalorsystemicmetastaticdisease[189–
194].Itmustbenotedthatsomeauthorsrecommendedcautionintheuseofadjuvantradiotherapythat shouldbereservedforhigh-
riskpatients,becau seofitsnegativeimpactonoverallsurvival[192].Interestingly,b e n e f i c i a l e ffectso f a d ju va n t rad io th e ra p y h a ve b e e n do cu me n te d f o r d e s mo p la s t i c, lentigomaligna, a nd mucosalmelanomas[192–
201].Theuseofradiotherapyinthetreatmentoflentigomalignamayrequiresomeselectivity,becauseofthe superiorityofthesurgicalapproach[202]andanattractivealternative,i.e.,topicaltreatmentwithimiquimod[203].
Themostpromisingaretheeffectsofradiotherapyinadjuvanttreatmentofdesmoplasticmelanomas[193,19 5,196,204]thataredevoidofmelaninpigment[179,202].
Therefore,itcanbesafelyconcludedthatradiationshouldbeconsideredasanadjuvanttherapy,dependingonth econtext.Also,thepresenceorlackofmelaninpigmentationshouldbeconsidered,whenselectingdifferent therapeuticoptions,becausemelanogenesismayaffectthebehaviorandmetabolicstatusofmel anomacells[15,19,92,108,205,206].Th elatterconsiderationisinagreementwithrecentclinicalandpatholo gicalstudiesthathavedemonstratedthatthepresenceofmelanininmetastaticmelanomasattenuatedthepo sitiveoutcomeofradiotherapy[177].
5.2. RadiosensitizationofMelanoma CellsthroughInhibition ofMelanoma Pigmentation Experimental,cellculture-
basedstudiesshowedradioresistanceofmelanomacells,butthefirstresultsrelatedtothesensitivityofhuma nmelanomacelllineswithdifferentpigmentationlevelstoionizingradiationwerecontradictory.WhileKi nnaertetal.[207]foundthatnon-
pigmentedmelanomacellshadasignificantlylowerresistancetoXradiationthanthepigmentedones,Barrancoetal.
[208]observedahighradioresistanceofmelanomacells,independentoftheirpigmentationlevels,inthreediffere ntm e l a n o m a c e l l l i n e s . H o w e v e r,thosed i s c repanciesc o u l d h a v e a r i s e n f romd i ff erentgenotypesofthemelanomacelllinesunderinvestigation.O u r study,usingonelineofmela nomacellsS K M e l -
1 8 8 , w i t h d i fferentm e l a n o g e n e s i s d e p e n d e n t o n t h e m e l a n i n p recursorl e v e l s i n t h e medi um[209],e l i m i n a t e d t h e i m p a c t o f g e n o m i c d i fferenceso n t h e r a d i o s e n s i t i v i t y o f m e l a n o m a cells[210].Th ehumanmelanomacelllineSKMel-
188ischaracterizedbyinduciblemelanogenesis.Undertheconditionsoflowlevelsofmelaninprecursorsi nculturemediummelanomacellsremainamelanotic,whereaswhenculturedinthepresenceofhighle velsofmelaninprecursors,thecellsbecomemelanotic[92,108,206,209,211].PigmentedSKMel-
188melanomacellsshowedhigherviabilityaftergammairradiationandincreasedmelanogenicactivitywaspositivelycorrelat edwithmelanomacellviabilityafterirradiationwith15Gygammaradiation(r=0.8,p<0.0001).Tyro sinaseactivityinhibitionwithN-phenylthioureaorcopper-chelatingagent,D-
penicillamine,resultedinanincreasedsensitivitytogammaradiation,anddecreasedsurvivalafterirradiatio n[210].Interestingly,thesameapproachmightpotentiallysensitizemelanomacellstochemo-
orimmunotherapy[15,82,206,212].
Ourrecentc l i n i c a l -
b a s e d s t u d y s h o w e d t h a t m e l a n o g e n e s i s i n h u m a n c u t a n e o u s p r i m a r y metastasizingmelanomas(stagesIIIandIV),andinlymphnodemelanomametastases,wasrelatedtoashorteroverallanddiseas e-
freesurvival[16].Asubsequentanalysisrevealedthatmelanomapatientswithamelanoticmetastatictumorsshowedsignifi cantlylongersurvivalafterradiotherapy,andlongeroveralls u r v i v a l t i m e t h a n p a t i e n t s w i t h p i g m e n t e d t u m o r s , w h o receivede i t h e r r a d i o t h e r a p y o r chemotherapyandradiotherapy[177].Addit ionally,Shieldsandco-
authorsobservedthatthepresenceofmelaninwasfoundtobeanunfavorablemarkerofmetastasisanddeathinamultivari ableanalysisofciliarybodyandchoroidalmelanomas[213,214].
Thus,theexperimentalandclinicaldataindicatethatinhibitionofmelanogenesiscouldbeused f o r t h e r a d i o s e nsitizationo f m e l a n o m a c e l l s t o i o n i z i n g r a d i a t i o n t o i m p rovem e l a n o m a radiotherapyefficacy.
5.3. RadiosensitivityofUveal Mel anoma Tumors 5.3.1.BrachytherapyinClinicalPractice
Humanm e l a n o m a s a rel e s s r a d i osensitivea s c o m p a redw i t h s o m e o t h e r n e o p l a s m s d u e t o asl o we rc e l lt u r n o ve r,b u t m o s t o f t h e u ve a l m e l a n o m a s ( UM )s h o ws a t is f a c t o r y regress iona f t e r radiotherapy.R a d i o t h e r a p y ofintraocularmelanomaisatherapeuticmethodusedne xttosurgery.Bothe x t e r n a l b e a m a n d b r a c h y t h e r a p y ( p l a q u e s ) a reu s e d . P l a q u e b r a c h y t h e r a p y ( rutheniumo r iodine)i s t h e m o s t c o m m o n co n se r v a t i v e t reatmenti n t h e m a n a g e m e n t o f c h o roidalm e l a n o m a s , followedb y p rotonb e a m r a d i o t h e r a p y.Brachytherapyh a s b e e n u se d t o t reati n t r a o cu l a r t u m o r s since1930.C o n s e c u t i v e publicationsreport60Co,106Ru,125I,103Pd,90S r,and130Cssources[215].ThemostcommonlyusedareRu(betaemitter,recommendedforsmallandme dium-
sizedtumors)andIplaques(gammairradiationwithdeeperpenetration).T h e prescriptiondoserangeis 70–
100Gy[216].T h e COMS(CollaborativeOcularMelanomaStudy)studycomparedenucleationto1 2 5Ib r a c h y t h e r a p y i n m edium-sizet u m o r s . T h e rew a s n o d i fferencei n m e l a n o m a -
a s s o c i a t e d andoverall-
causemortalitybetweenthetwotreatmentmodalities.Th e COMSstudywasrestrictedtotheuseof125Ip laques[217].Thefive-yearlocalcontrolratesafterbrachytherapyaveragedat89.5%(range69.9–
97.9%).Therecurrenceratefollowing106Rubrachytherapywas3–
16%invariousstudies[217].However,brachytherapyalsoaffectstheintraocularstructures,sclera,ocularmusc les,conjunctiva,cornealsurfaceintegrity,tearproduction,eyelashes,andeyelids.Withintheeye,radiationcancausecatara ct,retinopathy,opticneuropathy,hemorrhage,retinaldetachment,neovascularization,andsecondaryglaucoma.Th esideeffectsinvolvedmayresultinseveredeteriorationorthelossofv i s i o n . R e s u l t s o f v a r i o u s s t u d i e s [218]u s i n g d i fferenta n a l y t i c a l t e c h n i q u e s a n d v i s u a l a c u i t y endpoints,h a v e i n d i c a t e d t h a t v i s u a l a c u i t y i s g e nerallyp reservedi n p a t i e n tsw i t h s m a l l e r u v e a l melanomasituate dfartherfromtheopticdiscandfovea. At10years’follow-
up,6 8 % ofpatientsdemonstratedpoorvisualacuity[218].
5.3.2.ProtonBeamRadiotherapy(PBRT)ofUvealMelanoma
Teleradiotherapyi s t h e s e c o n d m e t h o d o f r a d i o t h e r a p y,n e x t t o b r a c h y t h e r a p y,d e d i c a t e d t o uvealmelanomapatients. P B RThasbeenthemostcommonlyusedoption among s t alltypesof teleradiotherapy,sincethe1970s.Chargedparticletherapyofuvealmelanomaissuccessfullyappliedinm anyclinicalcentersa roundtheworld.PBRTischaracterized byaveryprecisedispersionof rad iationthatenablesdestructionofthetargetedneoplastictissueatvariousdepthsinthebody.
Protonbeamradiotherapyisparticularlydedicatedtolesionslocatedclosetotheopticdiscandmacula[21 6–
222].I n intraocularneoplasms,protonbeamradiotherapyhasthesameeffectonthesurvivalrateasbrachy therapy[216–222].
Vermaetal.s u m m a r i z e d fourteenoriginalinv es tig ations from10di fferentinstitutions ,c o n d uc te dfrom2000to2015.Inthatanalysis,five-
yearlocalcontrolratesexceeded90%,whichpersistedat10and15y e a r s . F i v e -
y e a r o v e r a l l s u r v i v a l r a t e s r a n g e d f rom7 0 % t o 8 5 % , f i v e - y e a r m e t a s t a s i s - f rees u r v i v a l
anddisease-
specificsurvivalrangedfrom75%to90%,withamorerecentseriesreportinghighervalues.Withtheremovalofs mallerstudies,five-yearenucleationrateswereconsistentlybetween7%and10%.Manypatients(60–70%)showedapost- PBRTvisualacuitydecrease,butstillretainedpurposefulvision(>20/200)[223].
Theaboveresultsarecomparablewithouroutcomes.Weobserveda93.3%localcontrolrate,anddete riorationofvisualacuityin60%ofpatients.Complicationswereobservedin31.5%ofcases(dryeyesyndrome,gla ucoma,cataract,retinopathy,maculopathy,andneuropathy).Enucleationwasperformedin2.8%ofthecases ,duetoamassivemelanomarelapseorneovascularglaucomawithamassivevitreoushemorrhage[221,224].
Protonbeamradiotherapyenablesaveryhighlocaltumorcontrol,andpreservationoftheeyeballinmanycases,wit hvisualacuitydependingonthetumorsizeandlocation.
5.3.3.ProteomicStudyofHumanSkinMelanomaCells(BLM)TreatedwithProtonBeamIrradiationProteomicanalysisof theBLMmelanomacelllineirradiatedwithalowdoseof3Gyofproton
beamshowsasignificant(morethan1.5×change)upregulationof13proteinsanddownregulationoffourprotein s[225].T h e s e proteinsmightberoughlygroupedintofourcategoriesbytheirfunction:
(i)DNArepairandRNAregulation(VCP,MVP,STRAP,FAB-2,LamineA/CGAPDH);
(ii)cellsurvivalandstressresponse(STRAP,MCM7,A nn e xin 7,MV P,Caprin- 1,P DCD6 , V CP,HSP70);
( i i i ) cellmetabolism( T I M , G A P D H , V C P ) ; a n d ( i v ) c y t o s k e l e t o n a n d m o t i l i t y ( M o e s i n , A c t i n i n 4 , FAB-
2,Vimentin,Annexin7,LamineA/C,LamineB).Ofparticularinterestisthesubstantialdecrease(2.3×)inviment in,amarkerofEMTandofthemetastaticpropertiesofmelanoma[226].Futureworkswillincludeothercance rlines,suchasuvealmelanomaorprostatecancer,bothofwhichrespondwelltoprotonbeamtherapy.
5.3.4.Radio-PhototherapyofUvealMelanoma
Indocyaninegreen(ICG)photodynamictherapyadministeredwithbrachytherapywastestedinaclinicalsetting,involving3 8patients[227].ThebaselineICGstudyshowedpathologicalintrinsicvasculatureinallexaminedcases.S i x month saftertheindocyanine-
PDTtreatment,changesinmicrocirculationweredetectedinallcasesaswellasasignificantdecreas eintumorthicknessinultrasonography(mean38%).Acompleteregressionofintrinsicvesselswasdemonstra tedbyindocyaninegreenangiographyin26cases,andpartialregressionofpathologicalvascularizationwasfoundin 12patients.IntheCampagnolistudy,involvingfivepatientswithamelanoticchoroidalmelanomatreatedwithPDT,fourpa tientsdidnotrespondtotreatment[228].Theyconcludedthatradiotherapywasthemaindamagingagent,withphot otherapyeffectsconsiderednegligible.Contrarytotheabove,verteporfin-
PDTasprimarytreatmentinsmallchoroidalmelanomasresultedinan80%rateoflocaltumorcontrol[229],whichslightlylowert haninclassicalbrachytherapy.
PDTt reatmento f s k i n m e l a n o m a w a s revieweda n d i t s e fficacyw a s d e p e n d e n t o n t h e photosensitizerused,andmoderateeffectswereseenwhenPDTwasusedincombinationwithimmun otherapy,butnotwithradiation[230].
5.3.5.Radio-ChelationTherapyinClinicalTrials
CuprenilandChelaton(Polfa)wereusedasradiosensitizersinpatientstreatedwith60Cogammarays( b r a c h y t h e r a p y ) f o r c h o roidalm e l a n o m a i n a p i l o t s t u d y.Chelatorsw e rea d m i n i s t e redf o r 7–
10daysbefore,and9–
14daysduringthebrachytherapy.Tumorsizewasdetermined8weeksand16weeksaftertreatment.Tumorsizevolum edecreasedapproximately45%at16weeksafterCupreniltreatment.T h e sensitizingactionofchelatorsma ybeduetotheinhibitionofoxygenconsumptioninm e l a n o t i c c e l l s b y t h o se c o m p o u n d s [231],a n d i n h i b i t io n o f m e l a n o g e n e s i s a s s h o w n ino t h e r models[206,210].
6. Conclusions
Sincemalignantmelanomasareresponsibleforthehighestmortalityrateamongpatientswithskincancers,andexhibit ahighincidencerateinthewhitepopulation,itisofutmostimportancetodevelopandtestmultipletherapeuticstrategies,usin gappropriateanimalmodels.Thisiscrucialforadvancedmelanomasattheverticalgrowthphaseormetastaticdisease.An impressiveprogresshasbeenmadeinthedevelopmentofnewstrategiesintargetedtherapyandimmu notherapyandothertreatmentmodalities.However,t h e reisalackofop timism, wi t h respecttolo n g -te rm survivalof melanomapatients,becauseofthepre-
existingoracquiredresistancedevelopingtotheappliedtherapies.
Radiotherapyisusedintheintraocularmelanomaswithsatisfactoryoutcomes.B o t h extern albeamandbrachytherapyareutilizedinthetreatmentofuvealmelanomas.Forbrachytherapy,60Co,106Ru,
125I,103Pd,90Sr,and130Csaretheavailableradiationsources,withthemostcommonlyusedbetaemitterbeing106Ru,andgam mairradiation(125I)preferredfortumorswithdeeperpenetration.Protonbeamradiotherapyconstitutesthesecond methodofradiotherapythatenablesefficientlocaltumorcontrolwithrelativepreservationoftheeyeball, depending ondiseaseprogression.R a d i o t h e r a p y israrelyusedincutaneousmelanomas,e xceptforapalliativeapproachinselectedpatients,likelyduetothelong-
heldopinionthatmelanomasareresistanttoradiation.Theresistancemayinpartbeexplainedbytheradioprotect ivepropertiesofmelanin.Interestingly,desmoplasticmelanomas,whichareamelanotic,areresponsivetoradiother apy.Therefore,radiotherapyrepresentsaviablealternativeinthetreatmentofmelanomas,dependingonthetumor phenotypeandlocation.
Thediscussedmodelsofrodentmelanoma, n a m e l y theBomirskiandZemanmelano malinestransplantableinhamstersandgerbils,respectively,constituteremarkableanimalmodels tostudyandtestdifferentradiotherapeuticapproachesbeforeclinicaltreatmentofstage3and4dis eas e. S p e c i f i c a l l y,n o n -
p i g m e n t e d BH Mmel an omais responsivetoradio therap y,e i t h e r aftersubcutaneoustrans plantationorimplantationintotheeye.S i m i l a r l y,Zemanmelanomaisradiosensitive,butindepende ntlyonpigmentation,whilepigmentedBHMmelanomasaremarkedlymoreresistanttoXirradiationthannon-
pigmentedones.Moreover,high-
LETradiation(fastneutrons)iseffectiveintheinhibitionofBHMtumorgrowthindependentlyonitspigmentation.
Thus,BomirskihamsterandZemangerbilmelanomasrepresentacomprehensivesetofpreclinicalmodels—
resistantandsensitivetotherapies,allowingtodefineoptimalconditionsforradiotherapysuchasinh ibitionofmelanogenesisinX-
raytherapyorselectionofproperradiation,whichwouldefficientlytreattumorsindependentlyofth elevelofpigmentation.
Acknowledgments:Thisreviewincludesworkperformedovermorethan40years.Sourcesoffundingarementionedint herespectivecitedoriginalmanuscripts.KUwasarecipientofgrantnoUMO-
2012/07/B/NZ4/01657fromPolishNationalScienceCentre.F a c u l t y ofBiochemistry,Biophysics,andBiotechnologyo ftheJagiellonianUniversityisapartneroftheLeadingNationalResearchCenter(KNOW)supportedbytheMinistryofScienceandHigh erEducation(GrantKNOW35p/10/2015toPMP).Partialsupportfromgrants1R01AR056666-01A2,R21AR066505,and1R01AR071189–
01A1toASfromNIHand2014/15/B/NZ4/00751toAABfromNationalScienceCentre,Poland,isalsoacknowledged.Thegraphicalabstractc ontainsanillustrationelementmadebyServierMedicalArt(https://smart.servier.com/).AuthorsaregratefultoDr.RadomirSlominskiforpro ofreadingthemanuscript.
AuthorContributions: Martyna S
´niegockap reparedi l l u s t r a t i o n s , andp e r f o r m e d l i t e r a t u rere search,
EwaPodgórskamanagedthereferences,andperformedliteratureresearch,PrzemysławM.Płonkadescribedtherol eo f m e l a n i n s a n d h a m s tera s a m o d e l a n i m a l , MartynaE l a s e d i t e d a n d revisedt h e t e x t , Boz˙enaRo manowska-
DixondescribedtheradiosensitivityofUMtumors,MałgorzataSzczygiełdescribedgerbilsasmodelanimals,ZemanUJ90melanomares ults,andpreparedagraphicalabstract,MichałA.Z˙mijewskiandMirosławaC i c h o rekd e s c r i b e d B o m i r s k i h a m s t e r m e l a n o m a , A n n a M a r k i e w i c z d e s c r i b e d U M b r a c h y t h e r a p y andp rotonb e a m t h e r a p y,AnnaA . B roz˙ynad e s c r i b e d theroleofi n h i b i t i o n o f melanomap i g m e n t a t i o n , AndrzejT.Słominskidelineatedthe conceptofthepaper,anddescribedclinicalaspectsofhumanskinmelanomaandcontributedtothesectiononrodentmelanomas,Kry stynaUrban
´skawasresponsiblefortheconceptofthepaper,describedtheradiophototherapy,radiosensitivityofBHM,andthepr oteomicstudy.
ConflictsofInterest:Theauthorsdeclarenoconflictsofinterest.
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