A new tool for differentiating hepatocellular cancer cells: patterned carbon nanotube arrays

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ContentslistsavailableatScienceDirect

Applied

Surface

Science

j o ur na l ho me pa g e :w w w . e l s e v i e r . c o m / l o c a t e / a p s u s c

A

new

tool

for

differentiating

hepatocellular

cancer

cells:

Patterned

carbon

nanotube

arrays

Gokce

Kucukayan-Dogu

a,1

_,

_Damla

_Gozen

b,1

_,

_Verda

_Bitirim

b

_,

_Kamil

_Can

_Akcali

c

_,

Erman

Bengu

d,∗

a_Materials_Science_and_{Nanotechnology}_Graduate_Program,_Bilkent_University,_Turkey

b_Department_of_Molecular_Biology_and_Genetics,_Bilkent_University,_Turkey

c_Department_of_Biophysics,_Faculty_of_Medicine,_Ankara_University,_Turkey

d_Department_of_Chemistry,_Bilkent_University,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15February2015

Receivedinrevisedform6May2015

Accepted9May2015

Availableonline18May2015

Keywords: Carbonnanotube Cancercell Differentiation Collagen Patterning Toxicity

a

b

s

t

r

a

c

t

Weaimedtodevelopanewapproachtodetecttheinvasivenessandmetastaticdegreeofhepatocellular

carcinomacells(HCC)basedontheirepithelialmesenchymaltransition(EMT)statusbyusingpatterned

carbonnanotubes(CNT)withoutanyfurthersurfacefunctionalization.WeusedwelldifferentiatedHUH7

andpoorlydifferentiatedSNU182cellstoexamineandcomparetheiradhesivefeaturesonpatterned

CNTs.WefoundthatthewelldifferentiatedHUH7cellsattachedsigniﬁcantlymoreonthepatterned

CNTsthanthepoorlydifferentiatedSNU182cellsduetothedifferenceinepithelialandmesenchymal

phenotypesofthesecells.CollagencoatedpatternedCNTshavinglessroughnessresultedinadecreasein

thenumberofattachedcellscomparedtonon-coatedpatternedsurfacesindicatingthatsurface

topog-raphyplayingalsoavitalroleonthecellattachment.LDHtestingindicatednoadverse,orthereoftoxic

effectofcollagencoatedornon-coatedpatternedsurfacesontheHCCcells.Theresultsofthisstudy

clearlysuggestthatpatternedCNTsurfacescanbeusedasadiagnostictooltodeterminethe

invasive-nessandmetastaticlevelofHCCs.Hence,CNTscouldbeconsideredasapromisingdiagnostictoolforthe

detectionofdifferentiationandinvasivenessoftheHCCcells.

1. Introduction

Cancerisadevastatingdiseaseandresponsibleforhigh mor-talityrates[1].Theaggressivenessofacancercelldependsonits interactionwithneighboringcellularstructure,andthe metasta-sisofthesecells todistantorgansis oneofthemain causesof cancermortality[2,3].Hence,thefactorsthatregulatethe attach-mentofcancercellsareoneofthemostheavilystudiedareasof biology.Epithelial-mesenchymaltransition(EMT)isoneofthese processesthathasimportantrolesintheregulationoftheadhesion ofcancercellsleadingtoanincreaseininvasivenessandmetastatic potentials[4].

During development, EMT is a normal physiological and requiredprocessandhascriticalrolesinembryogenesis.Epithelial and mesenchymal cells are two different cell types with dis-tinctcellmorphologiesandfunctions[4].Thecellswithepithelial

∗ Correspondingauthor.Tel.:+903122902153;fax:+903122664068.

E-mailaddresses:bengu@fen.bilkent.edu.tr,erbengu@gmail.com(E.Bengu).

1_Equal_{contributions}_of_authors.

characteristic arepolarized immobile cells which interactwith basement membrane.On theotherhand,cellsin mesenchymal phenotypebecomemoremigratoryandinvasivewithdistinct mor-phology,proteinexpressionandgenesignatures.EMTisinitiated byseveralstepsincluding;activationoftranscriptionfactorssuch asTwistandSnail,expressionofspeciﬁcproteinsandmicroRNAs, reorganizationofcytoskeletonandproductionofcertainenzymes asaresultofwhichanepithelialcellgainmesenchymal character-istic[5,6].

The cells undergoing EMT typically lose the expression of epithelial cellmarkers suchasE-cadherin, and gain mesenchy-malmarkerexpressions, like␣-SMA, N-Cadherin,vimentinand ﬁbronectin[6].Thesecellslosetheiradhesivefeaturesto neighbor-ingcellsortoasurfaceorsubstratesuchasextracellularmatrix. Adhesion occurs fromthe action of family proteins, called cell adhesionmolecules(CAMs)whichcadherinisamemberofthis family.ThetumorsinearlystagesundergoingEMTprocessbecome aggressivemalignancies withincreasedinvasiveness,metastasis andsurvivalabilities[7,8].CancercellsundergoingEMTlosetheir adhesiveproperties.Thus,therelationbetweenthetumor aggres-sivenessandEMTwasclearlyshowninhepatocellularcarcinoma http://dx.doi.org/10.1016/j.apsusc.2015.05.054

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Fig.1.(a)SideviewSEMimagesofVA-CNTs.InsetshowsTEMimageofCNTs.(b)TopviewSEMimageofpatternedCNTsurfacewiththeinsetshowingahighmagniﬁed

imagefromthewallofcavity.(c)SchematicrepresentationofCNTsurfacesafterpatterningandcollagencoating.

(HCC)whichisthethirdleadingcauseofworld’scancerrelated mortality[9].HCCisanepithelialcancerwithfourstagesincluding welldifferentiated, moderately differentiated, poorly differenti-atedandundifferentiatedtumors.Thepoorlydifferentiatedcells gainmesenchymalphenotypethroughEMTtobecomemore inde-pendentfromtheunderlyingtissue, withincreased capacity to invadeandmetastase[10].

Novelstrategieshavebeendevelopedtoclassifyand character-izethestagesofcancercells.Thenanostructuredmaterialssuch asnanowires[11],metalnanoparticles[12,13]andquantumdots [14]havebeenwidelyinvestigatedbothforcancertreatmentand diagnosisapplications.Theserecentadvancesinthefieldof bio-materialsalsotriggeredresearchoncarbonnanotubes(CNTs)for theinvestigationofvarious cellularinteractions [15].Thereare numberofstudiesreportingtheuseofCNTsasanano-fibrous scaf-foldsforlivingcells[16–21].Furthermore,theuniqueproperties ofCNTspromptedtheirapplicationasapotentialnewtoolforthe detectionofdifferenttypesofcancercells(oral,prostateandlung cancers)notonlybytakingadvantageoftheirfunctionalization (e.g.bindingspecificmarkersonCNTs)[22–27]butalsothrough exploitingtheirrigidsurfacepropertiesforentrappingcancercells [28,29].Forinstance,improvementinthesensitivityanddetection limitsofcellshavebeenachievedbytheintegrationofCNTsin immunosensors[22,24,25].

Inthisstudy,wehaveemployedpatternedverticallyaligned CNTs(VA-CNTs)asatooltoassesstheinvasivenessandmetastatic degreeofcancercellsbasedontheirEMTproperties.Ourresults showedthat welldifferentiatedHCCcells attachedmoreonthe patterned CNTs compared to the poor differentiated HCC cells due to their EMT characteristics without displaying any cyto-toxic effects. Hence,ourresultssuggest thatVA-CNTscouldbe

developedintoapromisingtoolforthedetectionofdifferentiation andinvasivenessoftheHCCcells.

2. Materials

2.1. SynthesisandpatterningofVA-CNTs

VA-CNTs were grown by alcohol catalyzed chemical vapor deposition (ACCVD) method on oxidized Si (100) surfaces as describedbeforeinourpreviousstudy[18].Sandwichcatalyst lay-ers(Al/Co/Al)werepreparedforthegrowthofVA-CNTsbyusing theelectronbeamandthermalevaporationtechniques[18].Si sub-strateswiththeaforementionedlayerswereintroducedintothe ACCVDfurnaceforthegrowthofVA-CNTsthroughreductionand reactionstepsusing ethanol asa carbonsourceattemperature of625◦CunderﬂowingH2 andArgases(20sccmand100sccm,

respectively).Afterthegrowth,patterningwasinducedtothe VA-CNTsbyusing adropperﬁlled withdeionizedwater. Following thisstep,someofthepatternedCNTsweretreatedwith1␮g/␮l sterilized collagensolution for every cm2 _{(approximately} _10:1

weightratioofcollagentoCNT)resultingintwoseparategroups ofpatternedCNTarrays;onenon-coatedandtheothercollagen coated.

2.2. Cellculture

We used two humanHCC lines (SNU182and HUH7) in our experiments.SNU182 ispoorlydifferentiated and HUH7is well differentiatedhepatocellular cancercellline.SNU182cells were culturedinRPMImedium(Lonza,Verviers,Belgium)supplemented with10%fetal bovineserum,1%non-essentialamino acidsand

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1%Penicillin/Streptomycinantibiotic.HUH7cellswereculturedin DMEMmedium(Lonza)supplementedwith10%fetalbovineserum and1%Penicillin/Streptomycinantibiotic.

3. Methods

3.1. TotalRNAisolationandreversetranscription

HCCcellsweretrypsinizedandtotalRNAswereextractedfrom theprecipitate by usingNucleoSpin RNA IIKit (MN Macherey-Nagel,Duren, Germany).ProtoScriptM-MuLVFirstStrandcDNA SynthesisKit(NewEnglandBiolabs,MA,USA)wasusedtoprepare cDNAsfromtotalRNAaccordingtothemanufacturer’sprotocol. 3.2. RT-PCR

AllPCRreactionswereperformedwith1␮lcDNA,for30cycles. TheampliﬁcationsforE-cadherin,Vimentin,Fibronectin,␣-SMA, N-Cadherin,Slug,Twist1,Sip1andGAPDHwereperformedusing TaqPCR Kit (NewEngland Biolabs, MA,USA). The primersand ampliconsizeswerelistedinTable1.Theinitialdenaturationstep wasperformedat94◦Cfor30s,followedby30cyclesof denatur-ationat94◦Cfor30s,annealingat60◦Cfor30sandextensionat 68◦Cfor30s.Theﬁnalextensionstepwasperformedat68◦Cfor 5min.Theproductswereanalyzedon1.5%agarosegel.

3.3. HCCsonpatternedCNTsurfaces

TheVA-CNTswereplaced in6-wellplatesand 3×105

num-bersofHUH7andSNU182cellswereculturedoneachwellin3ml medium.AnegativecontrolgroupwithVA-CNTexcludingany cul-turedcellswasalsoprepared.Atthe3rddayofcellculture,the cells werewashedwithPBSand preparedforimaging by scan-ningelectronmicroscope(SEM)atlowvacuummode(40bar)and acceleratingvoltageof10kV.

3.4. CytotoxicityofVA-CNTssurfacesonHCC

TheVA-CNTswereplaced in96-wellplatesintriplicatesand 2×104 _numbers _of _HUH7_and _SNU182 _cells _were_cultured _on

wellsin200␮lmedium.LDHCytotoxicityDetectionKit(Clontech®_,

MountainView,CA)wasusedtomeasurethecytotoxicity accord-ingtothemanufacturer’sprotocol.Thereadingswereperformed byusinganELISAreaderatthewavelengthvalueof490nm.Forthe calculations,theaveragevalueofbackgroundgroupreadingswas subtractedfromallofthesamplereadings.

3.5. Statisticalanalysis

AlltheresultswereanalyzedwithANOVA.Minitab®₁₅

Statis-ticalSoftwarewasusedtomakemultiplecomparisonsofcontrol toexperimentalgroups.Allthecomparisonswereperformedusing Fisher’stestwith95%conﬁdentialinterval.Thegraphsweredrawn bytheusageofGraphPad®_program.

4. Resultsanddiscussion 4.1. PreparationofVA-CNTs

VA-CNTsweresynthesizedthroughACCVDmethodonSi sub-stratesusingsandwichcatalystdesign(Al/Co/Al)aswereported previously [18].The sideviewSEM imageofVA-CNTsrevealed that CNTswere 10␮m in heightwhereas theiraverage diame-terswerearound 10nmshown byTEManalysis(Fig.1a).Prior tothe cellseeding step onCNTs, we createdasperities for the

Fig.2.EMTexpressionsofHUH7andSNU182celllinesatmRNAlevel.

growth.Aforementionedpatterningstepwasinducedbydropping deionized wateronthesesurfaces.Asa resultof watercontact ofVA-CNTs,thealignedCNTswerecollapsedandsurroundedby standingCNTsformingcavities(Fig.1b).Theaveragewidthofthese cavitieswas10␮m.Themechanismbehindtheformationof self-assembled patterningisbasedontheelasto-capillaryeffectand hydrophobicpropertyofalignedCNTsasexplainedbyusand oth-erspreviously[18,30].Ourexperimentaldesignofpatterningwas showninFig.1c.Followingthepatterningprocess,patternedCNTs wereeithertreatedwithcollagenornotandweredesignatedas patternedcollagencoatedandnon-coatedCNTsurfacesthereafter. 4.2. EMTmarkerexpressionsofHCC

WemonitoredtheEMTmarkerexpressionprofilesoftwoHCC linesatmRNAlevel(Fig.2).OurresultsshowedthatSNU182cell linewaspositivefortheexpressionofallthemesenchymal mark-erstestedexceptSip1(Vimentin,Fibronectin,␣-SMA,N-Cad,Slug, Twist1). In contrary to SNU182, only three of the tested mes-enchymalmarkers(Vimentin,␣-SMAandN-Cad.)werefoundtobe expressedinHUH7cells.Hence,theexpressionprofilesofthesecell linesconfirmedthatSNU182cellsarepoorlydifferentiatedwhereas HUH7cellsarewelldifferentiatedHCCcellline[10].Inparallelto previousreports,ourresultsalsoindicatedthatHUH7hadamore epithelialcharacteristiccomparedtoSNU182cells[10].

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Table1

PrimersequencesusedinPCRampliﬁcations.

Genename Forwardprimer(5_–3₎ _Reverse_primer₍₅_–3₎

E-Cadherin GACTCGTAACGACGTTGCAC GGTCAGTATCAGCCGCTTTC

Vimentin GCAGGAGGAGATGCTTCAGA ATTCCACTTTGCGTTCAAGG

Fibronectin AATATCTCGGTGCCATTTGC CAGTAGTGCCTTCGGGACTG

␣-SMA TATCAGGGGGCACCACTATG GCTGGAAGGTGGACAGAGAG

N-Cadherin TCCAGACCCCAATTCAATTAATATTAC AAAATCACCATTAAGCCGAGTGA

Slug CTTTTTCTTGCCCTCACTGC AGCAGCCAGATTCCTCATGT

Twist1 GGAGTCCGCAGTCTTACGAG TCTGGAGGACCTGGTAGAGG

SIP1 TGTAGATGGTCCAGAAGAAATGAA TTGGCAAAGTATTCCTCAAAATCT

GAPDH GGCTGAGAACGGGAAGCTTGTCAT CAGCCTTCTCCATGGTGGTGAAGA

4.3. ProliferationandattachmentofHCCsonpatternedCNTs

AftertheCNTsurfacesweremodiﬁedandsterilizedbyUVfor

onehour,poor(SNU182)andwell(HUH7)differentiatedHCCcells

wereseededonbothnon-coatedandcollagencoatedpatterned

CNTsurfaces.Thecellswereincubatedfor3daysonthepatterned

CNTsurfaces and low vacuumSEM imaging wasperformed to

assesstherelationbetweentheirdifferentiationlevelsand

attach-mentability bothoncollagencoatedand non-coatedpatterned

CNTsurfaces(Fig.3).Fig.3aandbshowSEMimagesofattached

HUH7cellsoncollagencoatedandnon-coatedpatternedCNT sur-faces,respectively.Furthermore,attachedSNU182cellswerealso observedbothoncollagencoatedandnon-coatedpatternedCNT surfaces(Fig.3candd,respectively).Forcomparisonofthe num-berofattachedcellsonpatternedCNTs,wecountedthecellsunder SEMandaveragearealcelldensitywascalculateddividingthetotal cellnumberonthesurfacetototalsurfacearea.Thedensityplot given inFig.3eindicatesthattheaveragecellnumberfor well

Fig.3.LowvacuumSEMimagesofHUH7andSNU182cellsafterseededandculturedfor3dayson(aandc)collagencoatedand(bandd)non-coatedpatternedCNTsurfaces,

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Fig.4.LDHassayofcancercellsonnon-coatedandcollagencoatedCNTsurfaces.

Lowcontrol(LC)andhighcontrol(HC)groupsrepresentthecasewherethereisno

CNTsurface.

differentiatedcellswerehigherthanthenumberforpoor differ-entiatedones.Thedifferencebetweenthearealdensityofwelland poordifferentiatedcellswasstatisticallysignificant(significance value,p<0.05).ThissuggeststhatpatternedCNTsurfacescanbe usedfordefiningthedifferentiationlevelofHCCcells.

CollagenbyitselfhaspoormechanicalpropertieswhileCNTs have high elasticity and tensile strength in addition to their nanoscaledimensions[31,32].Hence,toenhancethemechanical andfunctionalpropertiesofcollagen,collagenblendedwithCNTs areusedascompositebiomaterials[33–35].Inourstudy,although collagenisusedasacoatingonthepatternedCNTs,asignificant effectofcollagenisobservedforthewelldifferentiatedcells.The numberofwelldifferentiatedcellsoncollagencoatedpatterned CNTswascomparablylowerthannon-coatedpatternedCNTs. Con-trarily,therewasnosignificanteffectofcollagencoatingonthe attachmentofpoordifferentiated cellswhichis likelyreflecting thealreadylowadhesivepropensityofthesecells(Fig.3e).

Itiswellknownthatsurfacetopographyandroughnesshasa keyroleontheattachmentofcells [36–38].Collagencoatingof CNTsmaybesmoothingtheroughnessbyinﬁltratingandﬁllingthe voidsbetweenCNTs.Thereby,lesssurfaceareawouldbeavailable forcellattachmentwhichcouldbethereasonfortheobservation oflessnumberofcellattachmentandgrowthforcollagencoated surfaces.SeveralSEMimagesdisplayingthesmoothing effectof thecollagencoatingareprovidedassupplementaryinformation. Theresultssummarizedabovesuggestthatthedifferenceinthe attachmentabilityofpoorandwelldifferentiatedHCCcelllines onthenon-coatedCNTsurfacescouldbeusedforevaluatingthe differentiationlevelandthusaggressivenessofHCCcelllines.

Toxicity of CNTs has also been a controversial subject for researchers since there are conﬂictingreports on theirtoxicity [17,39,40].Therefore,inordertoinvestigatethecytotoxiceffectof patternedCNTsurfacesonHCCcelllinesweperformedLDH cyto-toxicityanalysisbothoncollagencoatedandnon-coatedpatterned CNTs(Fig.4).InLDHtest,twocontrolgroupswereused;low con-trol(LC)andhighcontrol(HC)groupsinwhich HCCcells were seededonbareplatesurfaces.LCgroupshowstheminimumLDH releasewhileHCrepresentsthemaximumrelease.Thecloserthe absorbancereadingsofthesamplestoHC,themorecytotoxicthe conditionsareforthatsample.OurLDHresultsclearlyshowedthat HUH7andSNU182cellshadminimumLDHreleaseonthe non-coatedandcoatedpatternedCNTs.ThisresultindicatesthatCNTs arenotcytotoxicfortheseHCClines.Thisdataalsosuggeststhat

thedifferenceintheattachmentratesofthetwocelllinesaredue tocells’interactioncharacteristicswithCNTsurfaces;notbecause of thecytotoxiceffectsof theCNTsurfaces.Therefore, thewell differentiatedHCCline,HUH7hadgreatertendencytoattachon CNTsurfaceswhencomparedtothepoordifferentiatedHCCline, SNU182.

Thedifferenceinthecancercellsabilitytoattachtopatterned CNTarrayswillbeparticularlyanimportanttoolforthe diagno-sisoftheexacttumorcelltypeanddifferentiationstatusnotonly forhepatocellularcancerbutformanydifferentcancers.Molecular characterizationoftumorcellsisveryimportant,sinceits charac-terizationistheindicatoroftheclassiﬁcationoflivercancersimilar toproposedforlungcancer[41,42].Therefore,nanomedicaldevice fortheclassiﬁcationofhepatocellularcancerisimportantforthe possibletreatmentofthesetumorsinthefuture.

5. Conclusion

WehavedemonstratedthatpatternedCNTsurfaceshave poten-tialtodistinguishbetweenwelldifferentiatedHUH7andpoorly differentiated SNU182 cells. This is based on the differencein observedpropensityofadhesionofHCCsonthecollagencoatedand non-coatedpatternedCNTsurfacesaccordingtotheir aggressive-nessandmetastaticgrades.Ourresultsshowedthatsigniﬁcantly morenumberofwelldifferentiatedHUH7cellswereobservedon CNTsurfacesthanthepoorlydifferentiatedSNU182cells irrespec-tiveofwhethertheCNTsurfaceswerecoatedwithcollagenornot (p<0.05).However,thisdifferenceinthecelladhesionwasnotdue toanytoxiceffectofCNTs,sincetheattachedformofpatterned CNTsonarigidsurfacewasfoundtohavenotoxiceffectsonthe HCCcellsasdeterminedbyLDHtest.Furthermore,thenumberof attachedcellswasdoubledonthenon-coatedpatternedCNT sur-faceswhencomparedtocollagencoatedpatternedsurfaces,where collagencoatingactedasasmoothingagent.Hence,itcanbesaid thatphysicaltopographyofthesurfacesmayhaveanimportant role ontheHCCattachment.Through thistechnique metastatic gradeof cancercellscouldbedetectedinafastandfacileway, whichcouldbeusedeasilyappliedfordiagnosisinorderto deter-minethemosteffectivetherapystrategydependingonthetumor characteristics.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.apsusc.2015.05. 054

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