Differential capacity of CD90+ cells in autophagy activation following chemotherapy in hepatocellular carcinoma

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ContentslistsavailableatScienceDirect

Annals

of

Hepatology

j o ur na l h o me p a g e : w w w . e l s e v i e r . e s / a n n a l s o f h e p a t o l o g y

Original

article

Differential

capacity

of

CD90+

cells

in

autophagy

activation

following

chemotherapy

in

hepatocellular

carcinoma

Huy

Q

Do

a,b,1

_,

_An

_B

_Luong

a,c,1

_,

_Deborah

_Bonazza

d

_,

_Cristina

_Bottin

e

_,

_Thao

_PT

_Doan

f

_,

Long

DC

Tran

g

_,

_Nhung

_H

_Truong

b

_,

_Gianluca

_Tell

h

_,

_Hoa

_LT

_Pham

g

_,

_Claudio

_Tiribelli

a

_,

Caecilia

HC

Sukowati

a,h,∗

a_Fondazione_Italiana_Fegato_-_ONLUS,_AREA_Science_Park,_Basovizza,_Trieste,_Italy

b_Laboratory_of_Stem_Cell_Research_and_Application,_{VNUHCM-University}_of_Science,_Ho_Chi_Minh,_Vietnam c_Center_for_Molecular_Biomedicine,_University_of_Medicine_and_Pharmacy_at_Ho_Chi_Minh,_Vietnam

d_Surgical_Pathology_Unit,_Cattinara_Hospital,_Azienda_Sanitaria_{Universitaria}_Giuliana_Isontina_(ASUGI),_Trieste,_Italy e_Department_of_Medical,_Surgical_and_Health_Sciences,_University_of_Trieste,_Trieste,_Italy

f_Department_of_Pathology,_University_of_Medicine_and_Pharmacy_at_Ho_Chi_Minh,_Vietnam g_University_Medical_Center,_University_of_Medicine_and_Pharmacy_at_Ho_Chi_Minh,_Vietnam

h_Laboratory_of_Molecular_Biology_and_DNA_repair,_Department_of_Medicine_(DAME),_University_of_Udine,_Udine,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received26June2020 Accepted11July2020 Availableonlinexxx Keywords: hepatocellularcarcinoma autophagy

cancerstemcells CD90

chemoresistance

a

b

s

t

r

a

c

t

IntroductionandObjectives.Analysisofcancerbiomarkersisanimportanttoolindeveloping targeted-therapyandinmodulatingchemoresistance.Here,weanalyzetherelevanceofCD90,amarkerofcancer stemcells(CSC)inhepatocellularcarcinoma(HCC)anditscorrelationwithautophagy.Materialsand Methods.Forinvivostudy,86specimenswerecollectedfrom43patientsundergoingliverresections.In eachpatient,HCCnodule(HCC)andsurroundingnon-tumor(SNT)werecollected.Forinvitrostudy,HCC cellsJHH6subpopulationsexpressingCD90+andCD90-wereisolatedusingmagnetic-sorterand con-firmedbyflow-cytometry.Upondoxorubicintreatment,autophagyturn-overwasanalyzedbyRTqPCR formRNAexpression,Westernblotforproteinexpression,andautophagosomestainingfor autophagy-flux.CytotoxicitytestwasperformedbyMTTassay.Geneandproteinanalysiswereperformedinclinical samplestogetherwithimmunohistostaining.Results.CD90mRNAexpressionwashigherinHCCthan inSNTfor8-fold(p<0.001).LC3-IIproteinwasup-regulatedintheHCCincomparisonwiththeSNT (p<0.05).InvitromodelshowedthatCD90+andCD90-cellshaddiverseexpressionsof autophagy-relatedgenes.Upondoxorubicintreatment,autophagywasactivatedinbothcellsbyincreasingLC3-II proteinexpression,autophagicvacuoles,anddysregulationofautophagy-relatedmRNAs.Adifferential autophagiccapacitywasnoticedbetweentwosubpopulationsanditwascorrelatedwithcellular toxic-ityassay.Conclusions.WedemonstratedtherelevanceofdifferentialautophagycapacityofCD90+cells inHCC.Autophagywasinvolvedincancer-defensemechanismagainstdoxorubicin.Cancerpromoting functionofautophagyinCD90+cellswasalsorelatedtocancerenvironment.

Abbreviations: CSC,cancerstemcells;HCC,hepatocellularcarcinoma;SNT, surroundingnon-tumor;RTqPCR,reversetranscriptionquantitativepolymerase chain reaction; MTT, (4,5-dimethyl thiazolyl-2)-2,5 diphenyltetrazolium; IHC, immunohistochemistry;WB,Westernblot;ESgrade,EdmonsonSteinergrade; HBV,HepatitisBvirus;HCV,HepatitisCVirus;CTP,Child-Turcotte-Pugh;ALT, alanine transaminase; AST, aspartate transaminase; AFP, alpha fetoprotein; ATG8/GABARAPL1,GABAtypeAreceptorassociatedproteinlike1.

∗ Correspondingauthor.

E-mailaddress:caecilia.sukowati@fegato.it(C.H.Sukowati). 1 _The_authors_equally_contributed_to_the_study.

1. Introduction

RecentepidemiologydataGlobocan2018predictedthatliver cancer,mainlydominatedbyhepatocellularcarcinoma(HCC),tobe thesixthmostcommonlydiagnosedcancerandthefourthleading causeofcancer-relatedmortalityworldwide.Inthemale popula-tion,itsincidenceandmortalityare2to3timeshighercompared to female,thus it ranks ﬁfthin terms of globalcases and sec-ondintermsofdeaths[1].PoorclinicaloutcomeofHCCpatients isattributedtothelatediagnosis,theresistanceofHCCcells to currentlyavailabletherapies,andtumorrecurrenceaftercurative therapies[2].

https://doi.org/10.1016/j.aohep.2020.07.007

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Hepaticcancerstemcells(CSC)arewidelyacceptedasamajor originofHCC[3,4].These cellspopulationsareknownfortheir resistancetoradio-andchemotherapies,leadingtotherapyfailure andmetastasis[5,6].Variousproteinmarkershadbeenproposed forthedetectionandtheclassificationofhepaticCSC[7].TheCD90 (Thy-1)isoneofthemostcommonhepaticCSCusedinmultiple invitroand invivostudies[8–10].It is a 25-37kDa glycophos-phatidylinositol(GPI)-anchoredproteinexpressedinvariouscells. BesidesTcellactivation,CD90hasnumerous non-immunologic biologicalprocesses,asanimportantregulatorofthecelltocelland celltomatrixinteraction,apoptosis,adhesion,migration,cancer, andfibrosis[11,12].Basedonageneontologyanalysis,the over-expressedgenesin CD90+cells fromHCCwereassociatedwith inflammation,drugresistance,andlipidmetabolismcomparedto CD90+fromnontumoralliver[13].

Autophagy,aself-digestionmechanismfortheeliminationof cytoplasmicmaterials,cellularcomponents(lipid,proteins),and organelles,isoneofthekeyfactorsforcellhomeostasisinnormal physiology.Itinvolvestheformationof double-membrane vesi-cles(autophagosomes)engulﬁngthecargostothelysosomes.The outermembraneoftheautophagosomethenfuseswiththe lyso-somalmembrane,andtheinnervesicle,togetherwithitscargo,is degraded[14].

Incancer,autophagyisacknowledgedasadouble-edgedsword asitfunctionsbothascancer-suppressorandcancerpromoter[15]. Cancersuppressivefunctionofautophagyiswidelycharacterized tobeinvolvedinpreventionofcellsinﬂammation[16],whilethe cancer-promotingfactorsarerelatedtohypoxia[17–20]and exten-sivereactiveoxygenspecies[21–25].However,inHCC,thisdual roleofautophagyisevenmorecomplicated,beingattributedby thecomplexity ofcellularheterogeneity,variousetiologies, and multiplefactors(lifestyle,treatmentchoice,gender,etc.)during long-termdiseasedevelopment.

Asrecently reviewed, dysregulation of autophagy is known in CSC populations of several cancers [15]. As for HCC, how-ever, information on autophagy preference to hepatic CSC is still unclear and is mainly limited to the CD133+ phenotype. AutophagywasinvolvedinthemaintenanceofCD133+CSCunder theoxygen-andnutrient-deprivedconditionsthataretypicalofthe HCCmicroenvironment[26,27].Autophagywasalsoactivatedin CD133+cellsuponaninsufﬁcientradiofrequencyablation[28]and immunotherapyusinginterferon-gamma[29].Recently,an inter-estinglineage-tracingstudyintransgenicratlivercirrhosisshowed thathepaticCSCAxin2/EGFP+CD90+cellsweregenerated exclu-sivelyincirrhoticliverswithaberrantautophagyandpromoted hepatocarcinogenesis[30].

However,untilnow,thereislimitedinformationonthe rele-vanceofautophagyinCD90HCCcellularsubpopulations.Here,we providenewevidenceofautophagypreferenceinCD90+cells,both ininvitroand clinicalHCCtissues,includingitsactivationafter chemotherapy.Thisﬁndingrevealsnewprospectsonthe develop-mentoffuturestrategiestomanageHCC.

2. MaterialsandMethods 2.1. Samples

2.1.1. Humantissuesamples

Eighty-six specimens were collected from 43 selected HCC patientsundergoingliverresectionsintheUniversityMedical Cen-ter Ho Chi Minh City, Vietnam between2014 and 2017. From each patient,HCC nodule(HCC) and the mostdistant tissue of theresectedHCC(surroundingnon-tumoral;SNT)werecollected. FreshtissueswerekeptinRNAlaterStabilizationSolution (Invit-rogen,ThermoFisherScientiﬁc,Waltham, MA,USA)and stored

in−80◦_C_before_further_processing._The_diagnosis_of_patients_was establishedoninternationalcriteriatogetherwithitsEdmondson Steiner(ES)HCCgrading,tumorparameters,laboratoryresults,and otherclinicalﬁndings.Informedconsenttoparticipateinthestudy wasobtainedfromeachpatientorbyalegalrepresentativeandthe protocolwasapprovedbytheethicalcommitteeoftheUMPHoChi MinhCityno.240/DHYD-HDDD.

2.1.2. Cellline

HumanHCCcelllineJHH6wasusedasinvitromodel. JHH6 subpopulations expressing CSCmarker CD90+ and its negative counterpartCD90-wereisolatedbyusingMACSmagneticcell sort-ing(MiltenyiBiotecGmbH,BergischGladbach,Germany)according tomanufacturer’sinstruction.Thepurity andthepercentage of CD90+cellswerevalidatedbyﬂowcytometry(FACSCalibur,BD Bio-sciences,NJ,USA).Detailsoncellseparationandsubpopulations’ characteristicswerereportedpreviously[31]. Cellsweregrown inWilliams’Emediumsupplementedwith10%(v/v)FBS,1% L-glutamineand1%antibiotics.Theyweremaintainedat37◦Cina humidiﬁed5%CO2incubatorandwereroutinelyexpandedby0.05% trypsindetachment.

2.2. Histologicalanalysis

Immediatelyaftersurgery,livertissueswerefixedinformalin andincludedinaparaffinblock.Thefixedslicesweresubjected tohematoxylin &eosin andimmunostaining.Twoindependent pathologists(UMCHCM Cityand ASUGI)performedhistological analysisandfinaldiagnosis.

2.3. Immunostaining

Immunohistochemistry(IHC)analysiswasperformed onthe paraffinatedtissuessectionusedforhistologicalanalysis.Paired HCCandSNTtissuesweresubjectedtoimmunostainingforLC3 protein.Briefly, hepaticslices werede-paraffinizedwithxylene andrehydratedwitha gradualconcentrationofethanol.Tissues wereincubatedwithantibodyagainstLC3(PA1-16930, Thermo Scientific,Rockford,IL,USA)followedbyincubationwithprimary universalantibodyenhancerandHRPpolymer(ThermoFisher Sci-entific,Cheshire,UK).ThecomplexwasvisualizedwiththeDBA peroxidasesubstratekit(VectorLaboratories,UK)andthenucleus was stained withhematoxylin. IHC imageswere generated by microscopicalsystemsLeicaDM2000(Leica,Wetzlar,Germany). 2.4. Treatmentinvitro

E64D(SC201280,SantaCruzBiotechnology,Dallas,Texas,USA), aninhibitoroflysosomalproteases,wasdissolvedinDMSO.JHH6 CD90+andCD90-cellswereplatedwithconcentrationof30,000 cells/cm2_for₂₄_hours._Cells_then_were_exposed_for₂₄_hours_to₅_␮M doxorubicin(Pﬁzer)withandwithoutthepresenceof50␮ME64D. DMSOconcentrationwascalculatedtobe0.3%ineachtreatment. Cellviabilitywasdeterminedby3(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium(MTT,SigmaAldrich,StLouis,MO,USA)assay. TotalproteinandRNAextractswerecollectedforproteinandgene analysis,respectively.Inparallel,MTTassaywasalsoperformedfor autophagyinducerrapamycinandautophagyinhibitorchloroquine (EnzoLifeSciences,Farmingdale,NY,USA)

2.5. Assessmentofautophagyﬂux

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Table1

ClinicalandpathologicalcharacteristicsoftheHCCpatients.

Parameter n Parameter n

n 43

Age(mean,95%CI) 58(55-61) Serummarkers

Sex(M/F) 35/8 ALT(U/ml)[mean(95%CI)] 58(32-84)

Etiology AST(U/ml)[mean(95%CI)] 74(46-102)

HBV 28 GGT(U/ml)[mean(95%CI)] 95(53-137)

HCV 6 Alphafetoprotein

Metabolic/Alcohol 9 <20ng/mL 14

Diseasescores 20-400ng/mL 14

CTPA/B/C 36/2/nd >400ng/mL 9

Tumorparameters

Number(single/multiple) 27/8 Biggestlesionsize

Diameter<2cm 5

Diameter>2and<5cm 16

Large 17

Vascularinvasion(yes/no) 6/37 ESgrade(ES1/ES2/ES3-4) 15/11/11

detectionkit(EnzoLifeSciences,Farmingdale,NY,USA)wasused todetectacidautophagicvacuoles.Briefly,JHH6CD90+and CD90-cellswereseeded ina 96-welldarkfluorescenceplatefor24h. Cellsthenwereexposed for24hto5␮Mdoxorubicinwithand without the presence of 50␮M E64D. 100␮M chloroquine, an autophagyfluxinhibitor,wasusedasa positive controlforthe detectionofautophagicvacuoles,basedonthemanufacturer’s sug-gestion.DMSOconcentrationwascalculated tobe0.3%in each treatment.Aftertreatment,cellswerestainedwithCyto-IDGreen and Hoechst 33342 nuclearstain dyes for 30minutes at 37◦C, followedbywashing.Thefluorescentsignalwasmeasuredbya fluorescentmicroplatereader(PerkinElmer,EnSpireMultimode Plate Reader,Hamburg, Germany). Simultaneous detectionwas performedfor theCyto-IDGreen and Hoechst33342 ona FITC filter(Excitation∼480nm,Emission∼530nm)andblueDAPI fil-ter(Excitation∼340nm,Emission∼480nm).Thegreenautophagy signalwasthennormalizedwiththebluesignalandtheresults expressedasrelativetocontrolcells.

2.6. Proteinisolationandquantiﬁcation

24hoursaftertreatment,totalproteinsfromcellswerecollected usingcelllysisbuffer(CellSignalingTechnology,Danvers,MA,USA) andhomogenizedbyscrappingandvortexing.Theextractwas cen-trifugatedfor10minutesat14,000xginarefrigeratedmicrofuge. Attheend,supernatantswererecoveredandproteinconcentration wasdeterminedbythebicinchonicacidproteinassay.

2.7. WesternBlotanalysis

LC3proteinanalysiswasperformedbyusingWesternblot(WB) ontotalproteinextractobtainedfromhumanlivertissuesandJHH6 CD90+andCD90-fractions.Proteins(20␮g)weresize-separated bySDS–PAGEon12%polyacrylamidegel.Electro-transferredgel onPVDFmembranewasimmunoblottedwiththeLC3antibody whileactin(A2066,Sigma)wasusedashousekeepingprotein.The peroxidasereactionwasobtainedbyexposureofmembraneinthe ECL-PlusWBdetectionsystemsolutions(ECLPlusWesternBlotting DetectionReagents,GE Healthcare,Italy).Proteinquantiﬁcation wasperformedafterthedensitometricanalysisofbandsvs.actin ineachsample(ImageStudioTM_,_LI-COR,_Lincoln,_Nebraska,_USA). 2.8. ReverseTranscription–quantitativeReal-TimePCR(RT– qPCR)

Reverse Transcription (RT) was performed to obtain cDNA from1␮gofpuriﬁedRNAwiththeHighCapacitycDNAReverse

TranscriptionKits(AppliedBiosystem,USA)accordingtothe man-ufacture’sprotocol.Real-timePCRwasperformedaccordingtothe SYBRGreenSupermixprotocol(Bio-RadLaboratories,Hercules,CA, USA).PCRamplificationwascarriedoutin15␮Lreactionvolume containing25ngcDNA,1xSYBRGreenSupermix–composedby 100nMKCl,40nMTris-HCl,pH8.4,0.4mMeachdNTP,40U/mL iTaqDNApolymerase,6mMMgCl2,SYBRGreenI,20mM fluores-cein,andstabilizers(Bio-Rad),and250nMofgene-specificforward andreverseprimers.ThereactionwasruninCFX9600real-time PCRsystem(Bio-Rad).Theprimersequencesarelistedin Supple-mentaryDataTableS1.

2.9. Statisticalanalysis

Student’s t-test was performed for statistical comparison between groups using software GraphPad Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA). For human tissue samples,continuousvariables ofmRNAdistributionbyRT-qPCR werecalculatedusingt-testfollowingnormalitytestandvalues wererepresentedasmean(95%CI).Datainvitrowereobtained fromatleastthreeindependentexperimentsandareexpressedas mean±SD. Statisticalsigniﬁcance wassettop-value<0.05and reportedasindicatedhere:*p<0.05,**p<0.01,and***p<0.001. 3. Results

3.1. BaselinecharacteristicsofHCCclinicalsamples

Thedemographic features ofthe HCCpatientsareshown in Table1.Theparticipantswerepredominantlymale(35malevs8 female)withameanageof58±10yearold.Theaetiologyofchronic liverdiseasewas63%HepatitisBvirus(HBV),14%HepatitisCVirus (HCV),and21%othercauses(metabolic/alcohol).

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0 50 100 150 0 2 1 3 4 relative mRNA e xpression relative mRNA e xpression LC3-II pr otein / Actin LC3 e x pression

CD90/THY-1

SNT

HCC

_SNT

_HCC

A

C

E

B

D

ATG8/GABARAPL1

Paired LC3-II protein

LC3 protein HCC 1 SNT HCC SNT HCC SNT HCC SNT HCC SNT HCC SNT HCC HCC 2 _{HCC 3} _{HCC 4} 0 0 2 4 6 8 1 2 3 4 5 6 7 8 9 10 11 4 8 12 16 MW 21 17 42 LC3-I LC3-II LC3-I LC3-II Actin

Fig.1.RelevanceofautophagyinHCCtissues.

A.mRNAexpressionofCSCmarkerCD90/THY-1.B.mRNAexpressionofautophagymarkerATG8/GABARAPL1C.RepresentativeofLC3blottingofpairedHCCandits correspondingsurroundingnon-tumoraltissue(SNT)showedthepresenceofLC3-I(21kDa)andLC3-II(17kDa).Actinwasusedashousekeepingprotein.D.RelativeLC3-II expressionofelevenpairedHCCandSNT.DensitometricanalysiswasperformedbynormalizingLC3-IIsignaltoactin.E.MeanvalueofLC3-IandLC3-IIproteinexpression. TargetgenewasnormalizedtotworeferencegenesACTBand18sRNA.Theexpressionofanormalsamplewasconsideredas1.0.Student’st-test:*p<0.05,***p<0.001

3.2. HighLC3proteinexpressioninHCCtissues

Ourpreviousstudyshowedahighup-regulationofCSCmarker CD90/THY-1 in HCC tissues in European cohort [31]. Here we checkeditsexpressioninthisVietnamesecohortbyquantitative RT-PCR.WeanalyzedmRNAexpressionfromatotalof86tissue samplespecimensbyRT-qPCRandexpressedinarbitraryunit(au) withasampleconsideredas1.0au.AsigniﬁcantincreaseofCD90 wasobservedin HCC compared toitspaired surrounding non-tumoraltissue(SNT),withthemeanvalueexpressed(95%CI)of 23.2(14.2-32.4)and 2.7(1.0-4.5)forHCC andSNT,respectively (p<0.001)(Fig.1A).

UsingthesamesetofsamplesasforCD90,autophagymarkersof theATGfamily,theGABAtypeAreceptorassociatedproteinlike1

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Table2

CorrelationbetweenATG8mRNAandclinicalparameters.

Parameter n ATG8mRNAexpression(mean+/-SD)

HCC p SNT p Age <=58 21 0.66±0.73 ns 0.51±0.48 ns >58 22 0.83±1.00 0.71±0.74 Sex M 35 0.79±0.97 ns 0.54±0.61 ns F 8 0.51±0.40 0.98±0.61 Etiology HBV 28 1.15±1.42 <0.05 0.72±0.71 <0.05 HCV 6 0.59±0.75 0.48±0.53 Metabolic 9 0.96±0.91 1.08±0.80 HCCsize <5cm 21 0.61±0.61 ns 0.51±0.49 ns >5cm 17 0.82±1.10 0.59±0.72 ESgrade ES1 15 0.48±0.58 ns 0.44±0.56 ns ES2 11 0.84±1.24 0.75±0.51 ES3-4 11 1.01±1.05 0.58±0.81 ALT <59(U/mL) 31 0.57±0.69 <0.05 0.43±0.44 <0.01 >59(U/mL) 12 1.32±1.20 1.09±0.82 AST <75(U/mL) 33 0.72±0.88 ns 0.53±0.52 ns >75(U/mL) 10 0.87±0.98 0.82±0.81 AFP <20ng/mL 14 0.87±0.86 ns 0.54±0.60 ns 20-400ng/mL 14 0.67±0.89 0.38±0.24 >400ng/mL 9 0.34±0.44 0.40±0.38

TherelativeexpressionofLC3proteininpairedHCCandSNT tissueswasthendeterminedbyWesternblot(Fig.1C).Asshownin Fig.1E,bothLC3bands,theLC3-I(21kDa),andtheLC3-II(17kDa) werehighlyexpressedinHCCtissues(p<0.05).Densitometric anal-ysisinarepresentativeelevenpairedHCCanditscorresponding SNTshowedthatLC3-II,theautophagyturnovermarker,was sig-niﬁcantlyup-regulatedinHCCforaround5-fold(Fig.1D). 3.3. Differentialexpressionofautophagymarkersinisolated CD90+cells

SincetheCD90isamarkerfordifferentcelltypes,weexplored thefunctionofCD90+cellsinHCCbyusinganinvitromodelusinga HCCcellline.CD90+cellsfromJHH6wereisolatedbymagneticflow sortingandconfirmedbyflowcytometryaspreviouslydescribed [31].ThepurityofCD90+subpopulationsusedintheanalysiswas chosenhigherthan70%ofpositivity(upto5th_passage_after_sorting) (Fig.2A).Asexpected,atbasallevel,theCD90mRNAwasaround 60-foldhigherinisolatedCD90+subpopulationsthanits CD90-counterpart(p<0.001)(Fig.2A-B).Wecheckedalsoseveral mark-erssuchashepatocytegrowthfactor(HGF)andAXIN2whichwere previouslyreportedtoberelatedtoautophagyinCD90,showing up-regulationinthiscellsubpopulation(Fig.2B).

TheexpressionsofautophagygenesandbasalLC3proteinin JHH6 CD90+and CD90- cells were then determined by mRNA expression(RTq-PCR)andWesternblot,respectively. Autophagy-relatedgenes ATG8(p<0.05)and ATG18werelower expressed inCD90+comparedtoitsCD90-counterpartforaround50%and 80%,respectively,whileREDD1expressionwascomparable.The mRNAexpressionresultwasthen conﬁrmedbyprotein expres-sionusingLC3blotting.Atbasallevel(ctrlandDMSO),bothcells expressedLC3-II(17kDa)band.However,bothLC3-IIband(17kDa) andLC3-I(21kDa)werehigher intheCD90-cells comparedto CD90+(p<0.05)inlinewiththeATG8andATG18mRNAexpression data(Fig.2C-D).

InordertocheckLC3-IIturnoveruponautophagystimulation, bothcellsweretreatedwithE64D,aninhibitoroflysosomal pro-teases,atconcentrationsrangingfrom5to200␮Mfor24hours. DMSOconcentrationwascalculatedbeing0.3%ineachtreatment. MTTtestperformedtocheckcellviabilityupontreatmentdidnot showanytoxicityforthechosenconcentrationof50␮M(Fig.2E). UpontreatmentwithE64DandvehicleDMSO,theextentof LC3-IIaccumulationwasmorenoticeableinCD90+cells(about6-fold and2-fold,forE64DandDMSO,respectively)comparedto

CD90-cells (about3-foldforE64D)(Fig.2D-E).Thisdatamaysuggest thatCD90cellsresultmoresensitivetoautophagystimulationeven thoughthissensitivitywasnotreﬂectedbycellviability.Cell via-bilitytestsforrapamycin,anm-TORinhibitor,andchloroquine,a lysosomeproteasomalinhibitor,bothinvolvedinautophagy path-way,werealsoperformed,showinghigherviabilitiesfortheCD90+ cells(Fig.2E).

3.4. EffectofdoxorubicinonautophagyinCD90+cells

Autophagyisassociatedwithacelldefensemechanismagainst chemotherapy.HerewetreatedbothCD90+andCD90-cellswith doxorubicin,ananti-neoplasticdrugcommonlyusedtotreatHCC intheearly-intermediatestage.Concentrationof5␮Mwaschosen basedonourpreviousdataontheLC50ofdoxorubicininseveral HCCcelllines,includingintheJHH6[32].

Upondoxorubicintreatmentfor24hours,bothLC3bandswere increasedinCD90+andCD90-subpopulations.TheincreaseofLC3-I levelwasalsoapparent,alsoincomparisonwithE64D,indicatedan activationofautophagy-translatedgenesafterdoxorubicin expo-sure.Afterthetreatment,theextentofLC3-IIup-regulationwas higherinCD90+comparedtoCD90-cells(p<0.05)(Fig.3A).The patternofgenemodulationsbetweenthetwocellpopulationswas alsodissimilar.TheincreaseofATG8wasnoticedonlyinCD90+ cells(Fig.3B).

Thepresenceofautophagicvacuoleswasdetectedby ﬂuores-cencereading.Greenautophagysignalwasthennormalizedwith thebluenucleussignalandthedatawerepresentedasrelativeto controlofeachsubpopulation.Thepresenceofautophagicvacuoles afterE64DwasslightlynoticedforCD90+comparedtoCD90-cells (Fig.3C).Tocomparedatadoxorubicintreatmentinvitroin clini-calsample,wecheckedalsotwoHCCsampleswhosepatienthad receivedapreviouslocoregionaltherapybydoxorubicin,showing positivityofLC3onthetissues(Fig.3D).

4. Discussion

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Fig.2.DifferentialbasalautophagyactivitybetweenCD90+andCD90-subpopulationofJHH6.

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A

B

C

D

CD90-CD90+ CELLS

CD90+ CELLS

CD90+ H&E staining LC3 SNT HCC ctrl DMSO E64d E64d + Doxo Doxo

E64d + Doxo + ChloroQ ChloroQ CD90-300 200 100 0 fluorescence signal (%) 2.5 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 REDD1 ATG18 ATG8 REDD1 ctrl 0.3% dmso 5μM doxo ATG18 ATG8

*

***

*

* *

fold mRNA expression fold mRNA expression LC3-I LC3-II LC3-II Actin MW 17 21 42

CD90+

CD90-CTRL DMSO E64D DOX

4 3 2 1 0 CD90+ ctrl 0.3% dmso 50 u M E64 D 5 uM Do xo 5 uM Doxo E6 4D + Doxo ctrl 0.3 % dmso 50 uM E6 4D 5 u M Doxo 5 uM Do xo E64 D + Do xo

Fig.3. AutophagyactivationafterdoxorubicininCD90+andCD90-subpopulationofJHH6.

A.LC-3blottingofdoxorubicin-treatedcellstogetherwithitsrelativeLC3-IIproteinexpression.DensitometricanalysiswasperformedbynormalizingLC3-IIsignalto actin.CD90-DMSOwasindicatedas1.0.Redboxshowedcellstreatedwithdoxorubicin.B.mRNAexpressionofautophagymarkerATG8/GABARAPL1,ATG18/WIPI1,and REDD1/DDIT4.TargetgenewasnormalizedtotworeferencegenesACTBand18sRNA.Theexpressionofnon-treatedinCD90-cellswasconsideredas1.0.C.Determination ofautophagicvacuolesalsointhepresenceoflysosomalproteosomalinhibitorchloroquine.Untreatedcontrolcellsforeachcellsubpopulationswastakenas100%.Student’s t-test*p<0.05vsCTRLofeachsubpopulation.D.Upperpanel:ArepresentativeofHEandLC3staininginaHCCnodulewithanadministrationofdoxorubicin(arrow).Lower panel:LC3expressioninpairedSNTandHCCtissue.

Currently,chemotherapybytransarterialchemoembolization isaninternationally-approvedtreatmentoptionforHCCpatients [33].Doxorubicinisoneofthemostcommonchemotherapyagents usedtotreatHCC;however,HCCresistancetodoxorubicinisa com-monfeatureoftherapeuticregimens,thusreducingtheefﬁcacyof thetreatment.Furthermore,responsetodoxorubicinmightvary notonlyamongpatientsbutalsoinanindividualHCCcases. Dis-paratesensitivitiesmayberelatedtoHCCheterogeneity,including inthepresenceofvariouscellularpopulations.

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dif-ferentiation,inﬂammation,ABCtransporters,drugresistance,and lipidmetabolismcomparedtoCD90-cellsorCD90+cellsfrom non-tumoralliver[9,10,34].

Invitrostudieshadshownthatdoxorubicininducedcytotoxicity and alteration of cellularorganelles together withthe upregu-lationofLC3-IIproteinand theinvolvementof autophagy[35]. InthisstudyweshowedthatinJHH6,apoor-differentiatedHCC cellline, autophagyactivation extentby doxorubicin were dis-tinctbetweenCD90+andCD90-subpopulations,withthehigher capacityobservedinthepositivesubpopulation.FrombothmRNA (ATG8/LC3/GABARAPL1) and LC3 immunoblotting data, it was noticedthateventhoughtheCD90+subpopulationshadalower basalexpression,theyweremoresensitivetoautophagic induc-tionshownbythehigherup-regulationascomparedtoCD90-cells. ThisincreasewasnotonlyobservedindoxorubicinandE64D treat-ments,butasubstantialeffectwasalsonoticedupon0.3%DMSO asavehicle.

In HCC tissues, ATG8 mRNA distributions were compara-ble between HCC nodules (high CD90) and SNT (low CD90), whileinJHH6cellline,ATG8expressionwassigniﬁcantlylower in CD90+ cells. This data was in line with a previous report showing that a low level of ATG8 in HCC tissues was related to the bad prognosis of the patient [36], similarly to what observed for the high level of CD90. From clinical data, we observed that ATG8 expression is signiﬁcantly high in HBV-relatedHCCsandpatientswithhighALTlevels.Furthermore,ATG8 expressioninHCCnodulesprogressivelyincreasedmovingfrom well-differentiated(ES1),moderate-differentiated(ES2)to poor-differentiatedHCC(ES3/ES4).Aninversecorrelationwasfoundfor AFPlevel,alsoobserved forhighCD90+expression inprevious studies[8,31].

ToconfirmmRNAdata,weperformedLC3proteinexpression inlivertissues.Ourdatawasinlinewithpreviousstudies[37–39] showingahighLC3expressioninHCCcomparedtoSNTtissues. Fromimmunoblotting data ofpaired HCCtissues, we observed highvariabilityof LC3expression,withthemajoritybeinghigh intheHCCnodulecomparedtoitsSNT(72%).Itshouldbenoted thattheassociationbetweenautophagygenesandproteininHCC nodulesandtheclinicalvariablescouldbecontrasting.We con-sider that a direct quantification ofLC3 or autophagygenes in differentHCCspecimensmightberathercomplicatedsince nor-malcells have a basal autophagic flux that mayvary between individualsina physiologicmanner.Widecancerheterogeneity and cancer microenvironmentfurther complicate the data.For this reason,in-depth analysisof HCC subpopulations as shown inthis studywould beuseful tobetterunderstandthe mecha-nism.

Inconclusion,weconﬁrmthatHCCCD90+andCD90-cellshave differentautophagyactivation.Autophagyisinvolvedin cancer-defense mechanism against doxorubicin. Cancer promotingthe functionofautophagyinCD90+cellswasrelatedtoHCC environ-ment.

Conﬂictofinterest

Authorsdeclarenoconﬂictofinterest.

Financialsupport

ThisworkwasfundedbyagrantofInternationalCooperation withVietnamfromtheRegioneofFriuliVeneziaGiuliatothe Fon-dazioneItalianaFegato.CHCSwassupportedbythegrantfrom AssociazioneItalianaperlaRicercasulCancro(AIRC)(IG19862)to GT.

Acknowledgments

The authors thank Dr. C Bellarosa for the reagents on the autophagyinvitroexperimentsattheFondazioneItalianaFegato. TheauthorsalsothanktheVietnamNationalFoundationforScience and Technology Development (NAFOSTED; grant no. 108.05-2017.30)forreagentsontheautophagyexperimentinVietnam. AppendixA. Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,atdoi:https://doi.org/10.1016/j.aohep.2020.07. 007.

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