Mechanochemical Regulation of Cell Adhesion by Incorporation of Synthetic Polymers to Plasma Membranes - Kaizuka, Machida, Ota - 2021 -

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pubs.acs.org/LangmuirArticleMechanochemicalRegulationofCellAdhesionbyIncorporationofSyntheticPolymerstoPlasmaMembranesYoshihisaKaizuka,*RikaMachida,andYoshihikoOtaCiteThis:Langmuir2021,37,366−375ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Chemicalcontrolofcell−cellinteractionsusingsyntheticmaterialsisusefulforawiderangeofbiomedicalapplications.Herein,wereportamethodtoregulatecelladhesionanddispersionbyintroducingrepulsiveforcestolivecellmembranes.Toinducerepulsion,wetetheredamphiphilicpolymers,suchascholesterol-modifiedpoly(ethyleneglycol)(PEG-CLS),tocellmembranes.Wefoundthattherepulsiveforcesintroducedbythesetetheredpolymersinducedcelldetachmentfromasubstrateandallowedcelldispersioninasuspension,modulatedthespeedofcellmigration,andimprovedtheseparationofcellsfromtissues.Ouranalysesshowedthatcoatingthecellswithtetheredpolymersmostlikelygeneratedtwodistinctrepulsiveforces,lateraltensionandstericrepulsion,onthesurface,whichweretunedbyalteringthepolymersizeanddensity.Wemodeledhowthesetwoforcesaregeneratedinkineticallydistinctivemannerstoexplainthevariousresponsesofcellstothecoating.Collectively,ourobservationsdemonstratemechanochemicalregulationofcelladhesionanddispersionbysimplyaddingpolymerstocellswithoutgeneticmanipulationorchemicalsynthesisinthecells,whichmaycontributetotheoptimizationofchemicalcoatingstrategiestoregulatevarioustypesofcell−cellinteractingsystems.■INTRODUCTIONamphiphilicpolymerisverydifferentfromthecelladhesionCelladhesion,interaction,anddispersionregulateawiderangemediatedbynaturallyexpressedadhesiveproteins.Addition-ofinvitroandinvivoprocesses.1−6Bothtwo-dimensionalandally,thesepolymersrestrictedspreadingoftheareaofcellthree-dimensionalinvitrocellculturesrequirethecontrolofadhesionandeffectivelyinducearepulsiveforceonthecellcell−cellorcell−substrateinteractions.Collectingcellsfrommembrane,whichareintrinsicdifficultiesofartificiallytissuesandpreparingcellsforflowcytometricandsingle-cellachievingcelladhesionbymodifyingcellmembranes.geneticanalysesrequirecelldispersionatthesingle-celllevel.However,thechemicallyinducedrepulsiveforceexertedbyAdsorptionofcellstothesurfaceofbiomaterialsmustbepolymersoncellmembranessuggeststhatthissystemcanmanaged,andcell−cellinteractionsandcellularcommunica-potentiallybeusedforcelldispersion,whichmaybeantionsareessentialfortheformationofspheroidsandalternativetocurrentcelldispersionmethodsthatinvolveorganoids.Invivoprocessesinvolvecomplicatedcell−cellpartialandirreversibledisruptionofsamplesmechanicallyorDownloadedviaUNIVOFCALIFORNIASANTABARBARAonMay16,2021at09:14:13(UTC).Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.interactions,andthefine-tuningofsuchcell−cellcommuni-enzymatically.cationhasenabledcancerimmunologytherapiesusingHere,wereportamethodforchemicalcontrolofcellcheckpointinhibitors.Inadditiontomanybindingproteinsadhesionanddispersion.Byintroducingrepulsiveforcesonandtheextracellularmatrix,themechanicalpropertiesofcellcellmembranesusingamphiphilicpolymers,wesucceededinmembranesandthecytoskeletonalsomodulatecell−cellchemicalcontrolofcells.Thismethoddoesnotinvolveany7−9interactions.geneticmanipulation,fabricationofsubstrates,orchemicalPrecisecontrolofcelladhesionanddispersionbysyntheticsynthesisinvolvingthecellsandthesyntheticcholesterol-materialsisapromisingapproachforfuturetherapeutics,butitmodifiedamphiphilicpolymersaresimplyaddedtothecells.10−16remainsachallenge.Forexample,multiplestrategiesWithgoodwatersolubility,theseamphiphilicpolymerscaninvolvingmodificationsoftheplasmamembraneofcellshavebeendevelopedbuteachmethodhasitsadvantagesanddisadvantages.DNAhybridizationhasbeenusedtocontrolReceived:October9,2020specificcell−cellinteractions,buttheapplicationsarelimitedRevised:December12,2020becauseofdifficultiesinmolecularcontrolandstability.17WePublished:December28,2020foundthatpolymersmodifiedwithmultiplecholesterolmoietiescanbindtocellmembranesandfunctionasadhesive18molecules.However,thecelladhesionmediatedbysuchan©2021AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.langmuir.0c02955366Langmuir2021,37,366−375

1Langmuirpubs.acs.org/LangmuirArticlemixwithcellsinanaqueoussolutionandbindtocellCellDetachmentandDispersionAssays.Inthecelldetach-membraneswithrelativelyhighaffinity.Weshowthatcoatingmentassay,HEK293cellsorHeLacellswereincubatedat37°CincellswiththeseamphiphilicpolymersatadensityofonlyaDMEMthatcontainedtheindicatedconcentrationsofpolymersandfractionofthetotaldensityofsurfacemembrane-expressedimagedimmediatelyaftertheadditionofpolymers.Thisassaywasproteinsmodulatestheadhesionanddispersionofcells.dependentonthecellcultureandadhesionstates.Thus,tocompareexperimentsdirectly,weusedcellsculturedinafour-wellglass-■bottomdishinwhichallwellssharedthesamesubstrate(glassEXPERIMENTALSECTIONcoverslipcoatedwithadhesivepolymers)(CELLview,Greiner,CellsandChemicals.HEK293,HeLa,Jurkat,andNIH3T3cellsTokyo,Japan).Cellculturewasstartedat48hpriortothewereobtainedfromtheJCRBCellBank(Osaka,Japan).MC-38anddetachmentexperiment.Theareaofcelladhesion,whichwasdarkinK562cellswereobtainedfromtheCellResourceCenterforreflectioninterferencecontrastmicroscopy(RICM)images,wasBiomedicalResearch,InstituteofDevelopment,AgingandCancer,analyzedbyImageJusingbackgroundsubtractionandthresholdingtoTohokuUniversity(Sendai,Japan).Cholesterol-modifiedpoly-obtainabinaryimageforadhesionsites.Thesummedareaof(ethyleneglycol)(PEG-CLS)waspurchasedfromBiochempegadhesionsitesateachtimepointwasnormalizedtothatatthestarting(Watertown,MA).Cholesterol-modifiedbovineserumalbuminpointandplotted.Fluorescence,bright-field(BF),andreflection(BSA-CLS)wassynthesizedbybioconjugationofBSA(Merck,Tokyo,Japan)andNHS-PEG1kcholesterol(Nanocs,NewYork,NY)interferencemicroscopyimagingwereperformedusinganAF6000LXbyincubating100mg/mLofBSA(∼150μM)with1mMNHS-microscope(Leica,Solms,Germany)equippedwitha100×1.46NAPEG1kcholesterolatpH8.5for8hatroomtemperatureandthenoilimmersionobjectiveandCascadeIIEMCCDcamera(Roper,removingexcessPEGbygelfiltration.Gelatin-CLSwassynthesizedasTuscon,AZ).ForRICMimaging,afiltercubeconsistingofanarrow-18describedpreviously.Calcein-AMandcellcountingkit-8(CCK-8)bandpathfilter(543−553nm)andabeamsplitterwereusedwitharcwerepurchasedfromDojindo(Kumamoto,Japan).Collagenasetypelampillumination.IandDispaseIIwereobtainedfromWakoFujifilm(Tokyo,Japan),Inthecell−celldispersionassay,intrinsicallyaggregatingK562cellsandDNaseIwasfromFunakoshi(Tokyo,Japan).RedbloodcelllysisinRPMI1640mediumwerethoroughlypipettedoncetodispersethebufferwasobtainedfromBayBioscience(Kobe,Japan).A70μmcellcellsandthenmixedwith1or10μM3.4kPEG-CLSor3.4kmPEG.strainerwasobtainedfromCorning(Tokyo,Japan).AntibodiesusedThecellswerethenincubatedat37°Cfor1hbeforeobtaininginthisstudywereanti-CD45(fluoresceinisothiocyanate(FITC),images.Cellscoatedwith10μM3.4kPEG-CLSwerewashedinthecatalogno.130-102-997),anti-CD11b(phycoerythrin(PE),catalogmediumtwicefor15minandthenfor2hata10-foldlowercellno.130-113-806),andanti-CD11c(FITC,catalogno.130-102-798)concentrationwithslowrotation.Animagewasthenobtainedagain(MiltenyiBiotecTokyo,Japan).afterthecellshadsettled.ThenumberofcellsinclusterswascountedCellSwellingAssay.Cellswellingwasassessedbyimaging1.5×6inimages.10cells/mLofJurkatcellsstainedwith1μg/mLofcalcein-AMinRoswellParkMemorialInstitute(RPMI)1640mediaand10μM2kCellMigrationAssay.Cellmigrationassayswereperformedor20kPEG-CLSsfor1hat37°C.Toobservetheeffectsofwashout,usingatwo-wellsiliconeinsertwithadefinedcell-freegap(Ibidi,thecellswerewashedwithamediumtwicefor15minandthenfor2Grafel̈fing,Germany).Polymers(5μMunmodifiedgelatinorgel-hata10-foldlowercellconcentrationwithrotation.Afterwashing,CLS)wereaddedtothecellculturemediumimmediatelyafterreleasethecellswereresuspendedinmediaandimagedbyepifluorescenceofthesiliconeinserttoinducecellmigration.Thepolymerswerekeptmicroscopy.CellmorphologywasanalyzedusingImageJ(NIH)byinthemediumduringtheexperiment.Thenumberofmigratedcellsthresholdingcalcein-positivepixelstoobtainabinaryimageandingapregionswerecountedinimagesofvariousgapregions(widthcalculatingtheroundnessofcellsas4×area/(π×major-axis2).P-500μm,insideofthetwoblack-dottedlinesinimages)intwovaluesweredeterminedusingtheStudent’st-test.Thesumoftheduplicatesamplesforeachcondition.ThelengthofthelongeraxisinsectionareawasalsoanalyzedinthebinaryimagesthatweremigratingcellsattheleadingedgewasmeasuredusingImageJ.P-generatedbythesameimageprocessingfor1.5×106cells/mLvaluesweredeterminedbytheStudent’st-test.HEK293cellsthatwerestainedandtreatedwiththesameAnimalExperiments.Experimentsinvolvinganimalscompliedconcentrationsofcalcein-AMandPEG-CLSs.ToobtaintheareawithprotocolsapprovedbytheAnimalCareandUseCommitteeofpercell,thesumsofsectionareasobtainedfromthebinaryimagestheNationalInstituteforMaterialsScience.Age-matched6−8-week-weredividedbythenumberofcellscountedinimages.oldfemaleC57BL/6mice(JapanSLC,Inc.,Hamamatsu,Japan)wereCytotoxicityandProliferationAssays.Acellviabilityassaywasimplantedsubcutaneouslyintherightflankwith∼1×105conductedbyincubating1.5×106cells/mLHEK293cellsfor1hatMC-38tumorcells.Isolatedtumors(∼1cm3each)weredissected.Equal37°CinDulbecco’smodifiedEagle’smedium(DMEM)with25μM5kmPEGor2−20kPEG-CLS,followedbytrypanbluestainingandweightofthedissectedtumorwasincubatedinRPMI1640mediumcountinglivecells.Threesampleswerepreparedseparatelyforeachcontainingpolymersorenzymes.Fourmicewereusedforonecondition,andtheaverageviabilitywasplottedwithstandardexperiment.Threesampleswerepreparedforeachofsixdifferent19deviation.CellproliferationwasmeasuredusingtheCCK-8kit.Theconditions(mediumonly,500μM3.4kmPEG,100and500μM3.4kcellproliferationwasevaluatedastheactivityofdehydrogenasesinPEG-CLS,100unit/mLcollagenaseIand40unit/mLDNAseI,orlivecellsconvertedtotheabsorbanceofWST-8formazanat460nm,100unit/mLDispaseII).Thus,∼4cm3tumorsfromfourmice(∼4×whichwasproducedbyreductionfromWST-8substrateinsolution109ofcancercellsandimmunecells)20weredividedinto18samplesviacoupledreactionsincludingintracellulardehydrogenases;1.5×andeachsamplewasincubatedin0.5mLofmedium,resultingin4.4610cells/mLHEK293cellswereincubatedinamediumcontaining×108cells/mL,whichwasa>100-foldhigherconcentrationthanthat25μM2−20kPEG-CLSor5kmPEGfor2hat37°Candthenintheinvitrotoxicityassay.Afterthetumorswereincubatedinthewashedwiththemediumtwicefor15minata10-foldlowercellmediumat37°Cfor30minwithoccasionalvortexing,thecellswereconcentrationwithrotation.Thecellsweretransferredto48-wellcentrifugedat5gfor10mintwicetoremovedebris.Thesuspensionmicrowellplatesforassaysatlatertimepointsorsubjectedtoanwasthenpassedthrougha70μmcellstrainer.Afterfurtherimmediateassay.Theexperimentalconditionwas10000cellspercentrifugation,redbloodcelllysisbufferwasappliedtothecellsfor3wellin48-wellmicroplatesbyassumingthatcelllosswasminimalduringpreparation.TheabsorbanceofWST-8formazanwasminandthenwashedoutbycentrifugation.Thecellsweremeasuredinaccordancewiththemanufacturer’sprotocolusingaresuspendedinRPMI1640mediumandsubjectedtoantibodymicroplatereader(HitachiHigh-Tech,Tokyo,Japan).ThreesamplesstainingwithFITC-anti-CD45,PE-anti-CD11b,andFITC-anti-werepreparedseparatelyforeachcondition,andtheaverageCD11cantibodies.ThestainedcellswereanalyzedbyanSH800absorbancewasplotted.cellsorter(Sony,Tokyo,Japan).367https://dx.doi.org/10.1021/acs.langmuir.0c02955Langmuir2021,37,366−375

2Langmuir■pubs.acs.org/LangmuirArticleRESULTSANDDISCUSSIONWeevaluatedthebindingofcholesterol-modifiedpoly-(ethyleneglycol)(PEG-CLS)asamodelamphiphiliccoatingmaterialtolivecellmembranes.PEG-CLSwasincorporatedintothemembranesofHEK293cells,ashasbeenpreviously18shownforcholesterol-modifiedgelatin,whereasunmodifiedsolublePEGmethylether(mPEG)barelyinteractedwithcellsurfaces(Figure1A).IncorporationofPEG-CLSintothecellmembraneswasquantifiedbyflowcytometry(Figure1B).ThemoleculardensityofthetetheredPEG-CLSinthecell18membraneswascalibratedasreportedpreviously.Thecellmembranescontained5000−10000PEG-CLSmolecules/μm2whenincubatedwith10−100μMPEG-CLS.ThedensityoftheboundPEG-CLSwasindependentofthesizeofPEGmoleculesandsimilartothedensityofcholesterol-modifiedgelatin(gel-CLS)incorporatedintocellmembranes(2500−2187500/μm).Thisdensityrangewasapproximately12−33%ofthatofmembraneproteinsnativelyexpressedinlivecell221membranes(30000−40000/μm),whichcorrespondedto<0.7%oftotalphospholipidsina1μm2area.Thus,theexclusionvolumecreatedbythetetheredPEG-CLSmoleculescouldbealtered,whichdependedonthetethereddensityandsizeofPEGmolecules,butwasstillcomparablewithorlessthantheexclusionvolumecreatedbynativemembraneproteinsexpressedataseveralfoldhigherdensity.WealsoanalyzedthekineticsofPEG-CLSbindingtocellmembranes.ThebindingofPEG-CLStocellswasmeasuredatdifferenttimepoints.Next,wecalculatedkobsasthetimeconstantoffittingcurvestoapproximatefirst-orderbindingkineticsandtherateconstantsforassociationanddissociationas0.22min−1μM−1and0.25min−1fromtheplotofk(Figureobs1C,D).WeobservedthatsucharangeofassociationanddissociationratesfacilitatedrapidexchangeofPEG-CLSmoleculesbetweenplasmamembranesandthesolutionbycompetitivebindinganalysisinwhichmorethan80%ofFigure1.CoatingofthecellswithPEG-CLS.(A,B)Imagingandmoleculeswereexchangedin1hasshownpreviouslyforgel-flowcytometryanalysisofHEK293cellscoatedwithPEG-CLS.HEK293cells(1.5×106cells/mL)insuspensionwerestainedfor1hCLS(FigureS1A,B).TheseresultssuggestedthatPEG-CLSswitheither25μM5kmPEGand5kPEG-CLScontaining10%FITC-dissociatedfromcellmembranesinthetimescaleofhourswithlabeledmolecules(A)orwitharangeofPEG-CLSmoleculeswithappropriatewashoutprocesses.variousPEGsizesandvariousconcentrationscontaining5%FITC-Whencoatingcellmembranes,theamphiphilicpolymerslabeledmolecules(B),andwereimagedandanalyzedafterwashingweretetheredtolipidbilayersviacholesterol.Therefore,theseoncewithphosphate-bufferedsaline(PBS).(C)Incorporationofpolymersmayfacilitatecreationofapolymerbrushstructurevariousconcentrationsof3.4kPEG-CLSthatcontained10%FITC-onthemembranesurface,potentiallyincombinationwithlabeled3.4kPEG-CLSintocellmembraneswasmeasuredatdifferentnativelyexpressedproteins.22,23Comparedwithdenselytimepointsbyflowcytometryandcalibratedtothedensityonsurfacepackedpolymersthatgraftonthesurfaceandformanidealasin(B).TimecoursesofPEG-CLSbindingdensitieswereplottedtruebrushregime,suchasthatwith>0.065chains/nm2ofandkobsaswereobtainedastimeconstantsofbest-fitexponential24curves.Theerrorbarsin(B)and(C)representthestandarderrorof17000molecularweight(MW)polyacrylamide,thedensitymean.(D)kobsobtainedin(C)wereplottedalongwiththevariousofthetetheredPEG-CLSmoleculesonthecellmembranesconcentrationsofPEG-CLS,andkonandkoffwereobtainedfromthewasonly7−15%.Morespecifically,measurementofPEGonalinearfit.(E,F)Fluorescentimagesofcalcein-AM-loadedJurkatcellssupportedlipidbilayershowedthatthetransitionfromauncoatedorcoatedwith10μM2kand20kPEG-CLS((E)left,andmushroomregimetoabrushregimeoccurredatadensityofnotshown)andJurkatcellscoatedwith10μM2kand20kPEG-CLS4−8%PEGlipidsinthemembranes,whichwassignificantlyat2hafterrepeatedwashout((E)right,andnotshown)werehigherthantheobserved0.7%incorporationofPEG-CLSinanalyzedtocalculateroundnessofthecells(n=28−58cells),which256/5areplottedin(F).Statisticalsignificanceisgivenby***P<10−3;ns,cells.Theconventionalmodel,Ng>1/ρg,alsopredictednonsignificant.thatthemeasureddensityofPEG-CLSinoursystemwas1−3ordersofmagnitudelessthanthedensityrequiredtoforma26truebrushofMW1000−20000PEGmolecules.Therefore,graftedonasubstrate.ThetetheredpolymersinsertinthelipidthetetheredPEG-CLSmoleculesinthecellmembraneswerebilayersandmaythusalterthemolecularpackingandthree-likelytoformamushroomregimeandbehaveastwo-dimensionalelasticityofmembranes.dimensionallyfluidmoleculesinthelipidbilayerswithoutTheeffectoftheincorporationofamphiphilicpolymersinto25,27,28gelation.Suchfluidityisafeatureofpolymerstetheredcellmembraneswasanalyzedfurther.Weobservedswellingoftolipidbilayers,whichisverydifferentfromfixedpolymersJurkatcellsinsuspensionaftercoatingwithPEG-CLS(Figure368https://dx.doi.org/10.1021/acs.langmuir.0c02955Langmuir2021,37,366−375

3Langmuirpubs.acs.org/LangmuirArticle1E).TherewasaconsistentincreaseintheroundnessofcellsstrongertendencyofshorterPEGlipidstoinducemicelle37coatedwithasaturatedamountofeither2000or20000MWformation.However,theproliferationratewasrecoveredPEG-CLS(Figures1FandS1C).TwoPEGswithveryquicklyaftercultureundernormalconditionsinallcasesdifferentsizeswereusedtoexaminetheeffectofpolymersize,(FigureS1G).butthedifferencesintheroundnessofcellsboundwitheitherPreviousobservationshaveindicatedthatinsertionof2kor20kPEGswerenotsignificant.Uponswelling,thecholesterol-modifiedpolymersinducesarepulsiveinteraction18plasmamembranesofcoatedcellswerestretchedandmostbetweencellsandopposingsurfaces.Here,weevaluatedthislikelytensionedmorestronglycomparedwiththoseofrepulsiveforcebymeasuringtheadhesionofcellscoatedwithuncoatedcells.SuchchangescanbeexplainedbytheclassicalPEG-CLSmoleculesandotheramphiphilicpolymers.Bytime-29−31theoryofmembranespontaneouscurvature.Membraneslapseimagingofcelladhesiontothesubstrateusingbright-haveanaturalcurvaturethatresultsinafree-energyminimum,fieldandreflectioninterferencecontrastmicroscopy(BFandandmodulationofthisspontaneouscurvaturehasbeenRICM,respectively),weobservedrapidinductionoftheobservedbyvariousexamplesofmacromolecularinsertiondetachmentofHEK293cellscoatedwith50μM3.4kPEG-29,32−34intolipidbilayers.Inparticular,suchartificialchangesCLS,5μMgel-CLS,and5μMcholesterol-modifiedbovine35incurvaturegeneratelateraltension.Drasticstructuralserumalbumin(BSA-CLS)(Figure2A,B).Additionoftransitionofbuddinghasevenbeenobservedinhighlyunmodifiedpolymersdidnotaltertheadhesionstate.ThetensionedlipidbilayersandincellsexpressingalargeamountareaofadhesionwasmeasuredinRICMimageswherethe29,32−34,3618ofhighlybulkyglycocalyx.Expansionofpackedlipidsadheredareaappeareddark(Figure2A).Ata10-foldlowerinbilayershasbeenobservedfollowingPEGlipidinsertionbyconcentrationthanPEG-CLS,thelargegel-CLSandBSA-CLSelectronspinresonance,whichmaybethereasonforthemoleculesinducedmorerapiddetachment(MWsofBSAand37increaseinthemembranespontaneouscurvature.Inthegelatinusedinthisexperimentwere∼66and∼70kDa,presentstudy,buddingofcellmembraneswasobservedmostlyrespectively).Thisresultsuggestedthatlargemoleculeshadainthestretchedendofcellmembranes,whichwereflattenedstrongereffectontheinductionofrepulsiveforce.andadheredtothesubstrateandmaybeenrichedwithfocalAnalysisofcellscoatedwithPEG-CLSrevealedtwodistinctadhesion,orcellsburstabruptlywhentheywereincubatedrepulsiveforcesgeneratedbythetetheredpolymers,namely,withahighconcentrationofPEG-CLSs(1mM)(FigureS2C).stericrepulsionandlateraltension(Figure1).TheStablebuddingformationfromthecellbodydidnotoccurmechanismsofcelldetachmentreflecthowthesepolymersoften.Thisisprobablybecausethecellmembranesinitiallygeneratedrepulsiveforces.Stericrepulsioniscausedbythehadalowvolume-to-arearatioandaccommodatedalargepolymerexclusionvolume.Thus,itsstrengthdependsonbothincreaseinmembranetensionorbecausethecellmembranesthesizeanddensityofthetetheredpolymers.However,thehadsufficientstiffness,whichwassupportedbythecellswellingwasindependentofthesizeofPEGmolecules,ascytoskeletonandboundmolecules,toresisttheformationofshowninFigure1F,whichsuggestedthatthemembranelargebudding.Suchincreasesinmembranebendingrigiditytensionwasgovernedprimarilybythedensityofthetetheredandstiffnesshavebeenobservedinlipidbilayersandcellspolymers.Toinvestigatehowthecellsrespondedtothese38,39boundwithPEGorproteins.Additionally,amembrane-forcesinmoredetail,wecompareddetachmentofPEG-CLS-condensingeffect,whichispossiblyinducedbyinsertedcoatedHEK293cellsusingvariouslysizedPEGmolecules,1−40cholesterol,mightplayaroleandcellscoatedwithPEG-CLS3.4k(averagevalues).WeassumedthatthesePEG-CLSsmayactivelyadjusttheircellularvolumesinresponsetotheboundtocellswithsimilarkinetics,whereasPEG-CLSswithchangeinspontaneouscurvature.However,thevolumechangemuchlargerPEGsmaybetransferredslowlytothecell−ofeachcellwasnotclearlyresolvedinsuspendedJurkatcells,substrateinterfacebecauseofsterichindrance.ThePEGalthoughwedetectedanincreaseinthesumofthesectionareamoleculesinthisMWrangecorrespondto1.9−3.9nmin42ofmanycellsinducedbythebindingofPEG-CLStoHEK293Florylength,andthus,theexclusionvolumesof1kand3.4k39cellsthatarelargerthanJurkatcells(FigureS1D,E).PEGmoleculesdifferedbyapproximately8-fold.WeCellswellingcouldbereversedafterrepeatedwashoutfor2observedthatcellscoatedwithPEG-CLSmoleculesofvarioush(Figure1E,F).SuchareversingeffectlikelyoccurredwhensizesandatdifferentconcentrationsdetachedwithdistinctivethePEG-CLSdensityincellmembranesbecamelowerthankinetics.Forexample,ataconcentrationof10μM,wethethresholddensitytotriggerswelling.DissociationofPEG-observedasignificantPEGsize-dependentdifferenceintheCLScontributedtosuchachange(FigureS1A,B),whilekineticsofcelldetachment(Figure2C−E).Incontrast,atinternalizationofPEG-CLSalsocontributed,whichwasshownconcentrationshigherthan100μM,cellscoatedwitheven41tobesignificantincellsculturedformorethan5h.Theshort-chainPEGmoleculesdetachedrapidly(Figure2D,E),toxicityofPEG-CLShasbeenexaminedpreviously,whichandatalowerconcentrationof1μM,evenlong-chainPEGshowedthatculturingadherentcellswith2kPEG-CLSatmoleculeswerenoteffectivetoinducedetachmentafter60concentrationsof<50μMhadonlyasmalleffectoncellmin(Figure2E),whichsuggestedPEGdensity-dependent41viability.Here,weconfirmedthatthetoxicityofPEG-CLSinregulation.Short-chain1kPEGwillcreateanexclusionvolumesuspendedcellsforashortperiodofculture(1h)wasnotveryandstericrepulsiveforce.Thus,stericrepulsionandtensionsignificantat25μMPEG-CLSwithamaximumof∼7%lossinwerenotcompletelydecoupledinourexperimentalsystem.viability(FigureS1F).Thus,thetoxicityofPEG-CLSintheseHowever,ourobservationssuggestedthatthemembranemeasurementsforbindingkineticsandcellswellingexperimenttensionprimarilyregulatedthedetachmentofHEK293cellswasminimal.Treatmentofcellsuspensionswith25μMPEG-fromthesubstrateoverawiderangeofPEG-CLSCLSforlongerthan2hhadvariouseffectsoncellproliferationconcentrations,whereasatthemiddleofthetestedPEG-rates,whichdependedonthesizeofPEGs.Specifically,thereCLSconcentrationrange,stericrepulsionalsobecameawasaminimaleffectforlongerPEG(20k),but∼30%decreasecriticalfactorindeterminingthekineticsofdetachment.NoteinproliferationforshorterPEG(2k),whichmightreflectthethathigherPEG-CLSconcentrationsthanthecellviability369https://dx.doi.org/10.1021/acs.langmuir.0c02955Langmuir2021,37,366−375

4Langmuirpubs.acs.org/LangmuirArticleFigure2.continuedCLS(G)and5μMBSA-cholesterol(H)at0and30min.(I)Amodelofhowtwodistinctrepulsiveforcesaregeneratedinthecellscoatedwithamphiphilicpolymers.Thecellsinsuspensionandadherentcellscouldexperiencebothmembranelateraltensionandsurfacestericrepulsion.(J)Amodelofthedifferentkineticsbetweentensionandstericrepulsion.assay(FigureS1F)wereusedtocomparetheresultstounderstandthemechanismsofregulations.Insomecases(1mM2kPEG-CLS,FigureS2C),weobservedruptureeventsofcells,butallrupturesoccurredafterthecellshaddetachedfromthesubstrate.WespeculatethatthePEG-CLSdensitythatinducedcelldetachmentwaslowerthanthattodamagethecellsanddetachmentoccurredfasterthancellrupture.Thisalsoindicatesthatthetoxicityandkineticsofinductionoftherepulsiveforcemustbecarefullybalancedwhenemployingthistechnologyforcertainapplicationstocollectcells.WealsotestedthedetachmentofHeLacells.Interestingly,HeLacellsrespondedmorestronglytoahighconcentrationofPEG-CLSthantoalowconcentrationofBSA-CLS,whichwasincontrasttotheresultsforHEK293cells(Figure2B,F).Theseresultssuggestedthat,atleastoverashorttimerangeof30−60min,thestrongstericrepulsioncreatedbythelargeBSAmoleculeswasnotaseffectiveinHeLacellsasinHEK293cells.Conversely,themembranetensioncreatedbyahighconcentrationofPEG-CLSresultedindetachmentofHeLaandHEK293cellsatsimilarrates.Ourobservationssuggestedthatbothtensionandstericrepulsioncontributedtothecelldetachment,buttheeffectswereslightlydifferentindifferentadhesionstructures.Tensionisaneffectiveforcefordetachmentofalltypesofadhesions,includingspecializedadhesivestructuressuchasfocaladhesions,whichareformedmoreinHeLacellsthaninHEK293cells.Incontrast,stericrepulsionisalsoeffectiveinalltypesofadhesions,butdependsmoreonthekineticscomparedwithtension.Stericrepulsionmustfunctionlocally.Thus,largepolymersneedtobedistributedtothesiteofadhesion.However,theaccessofsuchlargemoleculestoadhesionsitesislikelyobstructedstronglyintightandcondensedjunctionssuchasfocaladhesions.Incontrast,membranetensionfunctionsmoreglobally,whichdependsprimarilyonthetotalnumberofamphiphilicpolymerstetheredtoacell.Thesekineticdifferencesinhowthetwoforcesaregeneratedmayexplainthedifferentresponsesofthetwocelllines.Asanotherexample,inHEK293cells,thegradualdetachmentofindividualcellsinducedbyPEG-CLSindicatedthattensionactedtodetachcellsfromboththeFigure2.Detachmentofcoatedcellsfromsubstrate.(A)Examplesofsurfaceandneighboringcells(Figure2G,fluorescenceimagingthecelldetachmentassay.ImagesofHEK293cellsadheredtotheisshowninFigureS2A).Incontrast,BSA-CLSfrequentlysubstratebybrightfieldandRICM,beforeand60minaftertheinducedabruptdetachmentofagroupofHEK293cellsmixingwith10μM3.4kPEG-CLS.(B)Timecourseofthe(Figure2H),whichmayreflectmorerapidentryoflargeBSAnormalizedadhesionareaofHEK293cellstreatedwithvarioustypesmoleculesandrapidinductionofstericrepulsionatthecell−ofamphiphilicpolymers(50μM3.4kPEG-CLS,5μMgelatin-substrateinterfaceratherthanthecell−cellinterface.Thetwocholesterol,5μMBSA-cholesterol,and5μMcontrolgelatinwithouttypesofrepulsiveforcesactingonthecellmembranesaremodification),measuredbyanalysisofRICMimages.(C,D)TimesummarizedinFigure2I,J.courseofthenormalizedadhesionareaofHEK293cellscoatedwithWealsoevaluatedinductionofrepulsiveforcesbetweencells10μM(C)or100μM(D)ofdifferent-sizedPEG-CLSmolecules.foruseincelldispersion.WetreatedK562cellswith3.4k(E)NormalizedadhesionareaofHEK293cells60minaftercoatingwithdifferentconcentrationsofPEG-CLSmoleculesofvarioussizes.PEG-CLSthatshowedthehighestdetachmentactivityamong(F)TimecourseofthenormalizedadhesionareaofHeLacellsthetestedPEG-CLSsinFigure2.Aggregatedcellsinsolutiontreatedwith50μM3.4kPEG-CLSand5μMBSA-cholesterol.(G,H)weredispersedoncebypipettingandthencoatedwithPEG-Bright-fieldimagesofHEK293cellstreatedwith50μM3.4kPEG-CLSatconcentrationslowerthanthatintheviabilityassay370https://dx.doi.org/10.1021/acs.langmuir.0c02955Langmuir2021,37,366−375

5Langmuirpubs.acs.org/LangmuirArticle(FigureS1F).WefoundthatcellsremaineddispersedonlyIncontrasttopreviousstudiesthatusedsyntheticsubstrates,whencoatedwithPEG-CLS,whichwasdependentontheweusedsolublematerialstomodulatecellmigration.WeusedPEG-CLSconcentration,whereasuncoatedcellsrevertedtoacellmigrationassayinwhichthenumberandshapeoftheaggregatedform(Figures3A−CandS2B).WhenNIH3T3cellsthatmigratedtowardapreparedgapregiononthesubstratecouldbedetermined.Weused5μMgel-CLSforthisexperiment,tolowerthetoxicitybuttomaintainahighrepulsiveforce.Migrationassaysrequirealong-timecellculture.Thus,toxicitycanbeanissueanditisdesirabletouselowconcentrationsofpolymers.Theuseofalargemolecule(gelatin)compensatesforthelossofsurfacetensionbecauseoflowdensityofmembrane-incorporatedpolymersbymax-imizingthestericrepulsionasshowninthecelldetachmentassay(Figure2B).Ataconcentrationof5μMgel-CLS,detachmentofNIH3T3cellsfromthesubstratewasnotobserved.However,weobservedanappreciableeffectofthecellcoatingonmigration.Introducingunmodifiedgelatinaloneaffectedthemigrationspeed,andtherewereapproximately50%lesscellscoatedwithgel-CLSthatmigratedtothegapcomparedwiththecellsincubatedwithunmodifiedgelatin(Figure4A−C).Unmodifiedgelatinatthisconcen-18trationdoesnotinteractwithcellmembranes.Therefore,ourresultsindicatedthatcoatingcellswithamphiphilicpolymerssloweddownthecellmigration.Tofurtheranalyzetheeffectofcellcoating,wecomparedtheshapesofmigratingcells.Migratingcellsincubatedwitheitherunmodifiedgelatinorgel-CLSexhibitedshorterelongationthanthecellsincubatedwithoutgelatin(Figure4D−G).Wespeculatedthatthiswascausedbychangesintheinteractionsbetweencellsandthesubstrateduetogelatinbindingtothesubstratewherethecellshadmigrated,althoughFigure3.Dispersionsofcoatedcellsinsolution.(A,B)Imagesoftheremaybeunknowntoxiceffectsofgelatins(forbothK562cellscoatedwith10μM3.4kPEG-CLS(A)andat2hafterunmodifiedandcholesterol-modifiedtypes)thataffectedrepeatedwashout(B).(C)Normalizedhistogramofthenumberofmigrations.Incontrast,althoughtherewasadifferenceinK562cellspresentassinglecells(one)orinclustersofcells(twotothemigrationspeeds,wecouldnotresolvethechangesincellfourorfiveormorecells),countedfromtheimages.Numberofmorphologyinducedbythecellcoatingbycomparingtheclusterscountedineachsamplerangesfrom83to394,andthetotallengthofcellsincubatedwithunmodifiedgelatinorgel-CLSnumberofcellsinallclustersineachcasevariesfrom84to876.Dataarerepresentativeofthreeindependentexperimentsthatyielded(Figure4E−G).Theseresultssuggestedthatcoatingcellswithsimilarresults.alowconcentrationofpolymersdidnotappreciablyalterthemembraneelasticpropertiesortheinternalforcesofthecytoskeleton.Instead,wespeculatedthatthemembranerepeatedlywashed,thecellsthathadbeendispersedalsocoatingmaypartiallyinhibitandslowdowntheformationofrevertedtoanaggregatedstate.Similartothereverseofcellfocaladhesionsbyinducingstericrepulsionbetweenthecellsswelling(Figure1F,G),thecellsurfacedensityofPEG-CLSandsubstrates,whichmayslowdownthewholemigrationlikelybecamelowerthanthethresholdtoinducedispersionbyprocess,whilesubstrate-adsorbedgelatinsdidnotfacilitatedissociationandinternalizationofPEG-CLSs(Figure3B,C).brushstructuresandthusmodulatedthemigrationdifferently.ThissystemenabledtheformationofsimpleanddurablecellFurtheranalysesarerequiredtofullyunderstandthesedispersionswithouttheadditionofdigestingproteinstotheprocesses.cellsorremovalofaggregatedcells.Therefore,thismethodAdditionally,weinvestigatedwhethercoatingcellswithmaybeusefulforsingle-cellanalysisinflowcytometryandamphiphilicpolymersdispersedcellsintissuesinvivoasgenetics.observedinvitro.CelldispersioniscriticaltocollectandWenextdeterminedwhethercoatingcellswithamphiphilicseparatecellsfromtissues,andmanystrategies,including48−50polymersregulatedcellularprocessesthatoccurredoveralongenzymaticandphysicaldisruptions,havebeendeveloped.timescale,suchascellmigration.Inaconventionaltwo-Althoughtheexistingtechnologieshavebeenveryuseful,dimensionalmigrationassaysystem,cellsformanchoringmethodsforhigherqualityandmoreefficientcellseparationpointstoasubstrate,suchasfocaladhesions,andconvertthefromtissuesaredesirable,particularlyforsingle-cellgeneticinternalpushingforcegeneratedbypolymerizedactinanalyses.Single-cellgeneticshaveprovidedagreatamountoffilamentsagainstthecellmembranestothemotionofthenovelinformationregardingcellpopulationsinnormalandwholecell,whereassuchanchoringismorecomplicatedindiseasedtissuesaswellasdevelopinganimals,whichhave43,4451−54three-dimensionalcellmigrationinvivo.Thespeedofrevolutionizedtheseareasinrecentyears.migratingcellsnotonlydependsonthecytoskeletonandAsamodelsystem,wetestedourstrategyforisolationof45membranestructure,butalsothecell−substrateinteraction.infiltratingimmunecellsintumors.ItisimportanttoobtainThus,manydifferentsyntheticandfabricatedsubstrateshavehigh-qualitysamplesoftheseimmunecellsforanalysisbecause46,47beenusedtoregulatecellmigration.thesecellsgoverntheefficacyoftumorimmunological371https://dx.doi.org/10.1021/acs.langmuir.0c02955Langmuir2021,37,366−375

6Langmuirpubs.acs.org/LangmuirArticleFigure4.Cellcoatingcanregulatecellmigration.(A,B)ImagesofNIH3T3cellsinwoundhealingassaysat0and14.5h,wherethecellswereincubatedwith5μMunmodified(A)orcholesterol-modified(B)gelatin.(C)Numberofcellsthatmigratedtowardthegapregiononthesubstrateatdifferenttimepoints,eitheruntreatedortreatedwithunmodifiedorcholesterol-modifiedgelatin.Averagewascalculatedfrom10imagesingapregion(width,500μm;length,∼1mm)intwoduplicatesamplesforeachcondition.Theerrorbarsrepresentstandarddeviationsbetweenanalyzedimages.Magnifiedviewofmigratingcellsuntreated(D)ortreatedwithunmodified(E)orcholesterol-modified(F)gelatin.(G)Lengthofthelongeraxisofthesemigratingcellsattheleadingedgemeasuredfromimages(n=35−40cells).Statisticalsignificanceisgivenby**P<0.01;ns,nonsignificant.Dataarerepresentativeoftwoindependentexperimentsthatyieldedsimilarresults.Figure5.Celldispersionintissues.(A−C)Flowcytometryanalysisoftumor-infiltratedimmunecellsextractedfromdissectedMC-38tumorsthatwereeitheruntreatedortreatedwith500μMmPEG,100or500μMPEG-CLS,themixtureof100unit/mLcollagenaseIand40unit/mLDNAseI,or100unit/mLDispaseII.Thetumor-infiltratedimmunecellswerestainedwithantibodiesagainstCD45(A),CD11b(B),andCD11c(C).(D)Yieldsofcellswiththesemarkerproteinscomparedwiththeuntreatedcontrolsamplein(A)−(C)(PEG-CLS=500μMPEG-CLS).Thenumberofcellsmeasuredinflowcytometryanalysiswasmorethan10000cellsinallcases.Dataarerepresentativeoftwoindependentexperimentsthatyieldedsimilarresults.6therapiesincludingcheckpointinhibitordrugs.Forexample,foldlowerandthetoxicitieswerealsoeffectivelylower.Indeed,high-resolutionsingle-cellgeneticsdataofcellpopulationsinthecollectedcellshadsimilargoodviability(>90%)inalltumorshaverevealedunappreciatedimmunologicalnetworkssamplestreatedwithorwithoutPEG-CLS.Thesecollected52,53intumors.Asamodeltumorsystem,weinjectedmurinecellswerestainedwithantibodiesagainstmarkerproteinsof55colonadenocarcinomacelllineMC-38intoC57BL/6mice.immunecellsforflowcytometricanalysis.WedetectedAfterdissectionoftheisolatedtumors,both3.4kPEG-CLSincreasesinthenumbersofCD45-,CD11b-,andCD11c-andvariousenzymes(collagenaseI,DNaseI,andDispase)positivecellsobtainedfromPEG-CLS-treatedtumorswereaddedandtheimmunecellswereseparated.The3.4kcomparedwithcontrolsamplesandthedegreeoftheincreasePEGwasusedbecauseitwaseffectiveinthecelldispersionwascomparablewithtumorstreatedwithenzymes(Figureassayinvitro(Figure3).Weusedhigherconcentrationsof3.4k5A−D).ThemarkerproteinsusedareexpressedinvariousPEG-CLS,butthecellconcentrationinthisassaywas∼100-immunecells.CD45phosphataseisexpressedinallfoldhigherthanthatintheinvitrocytotoxicityassay(Figurelymphocytesandisweaklyexpressedinotherleukocytes.20S1F)becauseweincludedisolatedtissuesintesttubes.Thus,TheintegrinCD11bisexpressedinnaturalkillercells,thenumberofPEG-CLSmoleculespereachcellwas∼100-granulocytes,andmacrophages;andtheintegrinCD11cis372https://dx.doi.org/10.1021/acs.langmuir.0c02955Langmuir2021,37,366−375

7Langmuirpubs.acs.org/LangmuirArticleexpressedinmonocytes,naturalkillercells,andmacrophages.NotesThus,increasesinthesignalsfromtheseproteinsindicatedanTheauthorsdeclarethefollowingcompetingfinancialoverallincreaseintheamountofisolatedimmunecells.interest(s):Y.K.istheinventoronpatentapplicationbasedTheseresultssuggestedthatthischemicalapproachforcellonpartofthiswork.dispersionmaybeusefultocollectcellsfromtissues.Coatingofcellmembraneswithamphiphilicpolymersisamildprocess,■ACKNOWLEDGMENTSasitcanbereversedasshownintheinvitroexperimentsandTheauthorsacknowledgetheuseofthesoftmateriallinedoesnotinvolveanychemicalmodificationordisruption.facilityinNIMSandthankthestaffofthefacilityfortheirAdditionally,thisstrategycanbecombinedwithotherphysicalassistance.TheyalsothankS.MatsuuraandY.Shiraiforandenzymatictissuedigestionprotocols.Thus,moreassistancewithanimalexperiments,andT.Minowa,S.Sakai,sophisticatedcombinationmethodsmaybedevelopedintheH.Nakao,T.Muir,andM.Aricofordiscussionsorhelpwithfuture.manuscriptpreparation.Thisworkwassupportedbyintra-■muralfundingfromNIMS.CONCLUSIONSWehavedevelopedamethodtochemicallycontrolcell■adhesionanddispersionbyintroducingcholesterol-modifiedREFERENCESpolymersascoatingmaterialsintocellmembranes.We(1)Huh,D.;Hamilton,G.A.;Ingber,D.E.From3Dcellculturetoorgans-on-chips.TrendsCellBiol.2011,21,745−754.concludedthatthesecoatingpolymersgeneratedtworepulsive(2)Jessell,T.M.Adhesionmoleculesandthehierarchyofneuralforces,tensionandstericrepulsion.Bothtensionandstericdevelopment.Neuron1988,1,3−13.repulsionareeffectiveforcelldetachment,butfurtheranalysis(3)Springer,T.A.;Dustin,M.L.;Kishimoto,T.K.;Marlin,S.D.isnecessarytofullyunderstandhowtherepulsiveforcesareThelymphocytefunction-associatedLFA-1,CD2,andLFA-3generated,whichwillfacilitateoptimizationofthechemicalmolecules:celladhesionreceptorsoftheimmunesystem.Annu.controlofcelladhesionversusdispersionoftargetcells.WeRev.Immunol.1987,5,223−252.investigatedcelldetachment,dispersion,andmigrationas(4)Debnath,S.;Greenblatt,M.B.SpecimenPreparationforSingle-potentialapplicationsforthecell-coatingmethod.However,CellSequencingAnalysisofSkeletalCells.MethodsMol.Biol.2021,therepulsiveforces,bothtensionandstericrepulsion,are2221,89−100.intrinsicincellsbecausecellsexpressmanyproteins,andsuch(5)Gjorevski,N.;Sachs,N.;Manfrin,A.;Giger,S.;Bragina,M.E.;Ordoń̃ez-Moran,P.;Clevers,H.;Lutolf,M.P.Designermatricesfoŕnativerepulsiveforcesmaybebalancedwithotherforcestointestinalstemcellandorganoidculture.Nature2016,539,560−564.integratecellularcommunications.Therefore,furtherstudyof(6)Yang,Y.Cancerimmunotherapy:harnessingtheimmunesystemthismechanochemicalcontrolmethodmaycontributetothetobattlecancer.J.Clin.Invest.2015,125,3335−3337.futuredevelopmentofnative-like,syntheticcellorganizational(7)Ladoux,B.;Mege,R.M.Mechanobiologyofcollectivecell̀systems.behaviours.Nat.Rev.Mol.CellBiol.2017,18,743−757.(8)Paszek,M.J.;DuFort,C.C.;Rossier,O.;Bainer,R.;Mouw,J.■ASSOCIATEDCONTENTK.;Godula,K.;Hudak,J.E.;Lakins,J.N.;Wijekoon,A.C.;*sıSupportingInformationCassereau,L.;Rubashkin,M.G.;Magbanua,M.J.;Thorn,K.S.;Davidson,M.W.;Rugo,H.S.;Park,J.W.;Hammer,D.A.;Giannone,TheSupportingInformationisavailablefreeofchargeatG.;Bertozzi,C.R.;Weaver,V.M.Thecancerglycocalyxmechanicallyhttps://pubs.acs.org/doi/10.1021/acs.langmuir.0c02955.primesintegrin-mediatedgrowthandsurvival.Nature2014,511,Incorporationandcompetitivebindingof25μM3.4k319−325.PEG-CLS,box-and-whiskerplotforFigure1F,imagesof(9)Blumenthal,D.;Chandra,V.;Avery,L.;Burkhardt,J.K.Mousecalcein-loadedHEK293cellsbeforeandafterTcellprimingisenhancedbymaturation-dependentstiffeningoftheincorporationofPEG-CLS,calculatedsectionareaperdendriticcellcortex.eLife2020,9,No.e55995.singlecellfromprocessedimagesofcalcein-loadedHEK(10)Griffith,L.G.;Naughton,G.Tissueengineering–currentchallengesandexpandingopportunities.Science2002,295,1009−293cells,viabilityofJurkatcells,andproliferationassay1014.ofHEK293cells(FigureS1)anddetachingofHEK293(11)Stevens,M.M.;George,J.H.Exploringandengineeringthecellsloadedwithcalcein,ruptureofdetachedHEK293cellsurfaceinterface.Science2005,310,1135−1138.cells,dispersionofK562cellsloadedwithcalcein,and(12)Cho,Y.;Yu,S.J.;Kim,J.;Ko,U.H.;Park,E.Y.;Choung,J.S.;box-and-whiskerplotforFigure4G(FigureS2)(PDF)Choi,G.;Kim,D.;Lee,E.;Im,S.G.;Shin,J.H.RemodelingofAdhesionNetworkwithinCancerSpheroidsviaCell−Polymer■Interaction.ACSBiomater.Sci.Eng.2020,6,5632−5644.AUTHORINFORMATION(13)Choi,M.;Yu,S.J.;Choi,Y.;Lee,H.R.;Lee,E.;Lee,E.;Lee,CorrespondingAuthorY.;Song,J.;Son,J.G.;Lee,T.G.;Kim,J.Y.;Kang,S.;Baek,J.;Lee,YoshihisaKaizuka−NationalInstituteforMaterialsScience,D.;Im,S.G.;Jon,S.PolymerThinFilm-InducedTumorSpheroidsTsukuba,Ibaraki305-0047,Japan;orcid.org/0000-AcquireCancerStemCell-likeProperties.CancerRes.2018,78,0002-8019-0873;Email:KAIZUKA.Yoshihisa@nims.go.jp6890−6902.(14)Woods,E.C.;Kai,F.;Barnes,J.M.;Pedram,K.;Pickup,M.W.;AuthorsHollander,M.J.;Weaver,V.M.;Bertozzi,C.R.AbulkyglycocalyxRikaMachida−NationalInstituteforMaterialsScience,fostersmetastasi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