APPLICATIONNOTE
ArtiCYT3DTumorModels
KeyFeatures:
• Matrixembedded3Dco-culture
oftumorcellsandstromalcells
inArtiCYTBioactiveBasal
Membrane(BM)3DMatrix
• 3Dtumormodeltakinginto
accountcell-cellandcellextracellualrmatrixinteractions
ArtiCYTBioactiveBasal
MembraneKitfor3D
TumorCo-culture
Background
Cancersarecomplexandheterogeneous
pathological“organs”indynamic
interplaywiththeirenvironment.Traditionalinvitro2D
monoculturemodelsfailtocapturemanyimportantaspectsofthis
complexity,includingthe3Dorganizationandthedynamicand
reciprocalinteractionsofthetumorwithstromalcellsandwiththe
extracellularmatrix(ECM).1
3Dspheroidshavebecomeanimportantplatformin
biopharmaceuticaldrugdiscovery,astheycanmodelthe3D
aspectsoftumors.2Inthecaseofco-culturewithstromalcells,
paracrineinteractionsintumorsarecapturedaswell.Yet,theystill
missanessentialdynamicregulatoroftumorbiology,theECM.3
ArtiCYTisafullysynthetic,biomimeticmatrixdisplayingcellular
bindingsitesmimickingtheimportantECMcomponentslaminin,
fibronectin,andcollagenIV.Thiswell-definedmatrixcanprovide
signalingcuesviatransmembranereceptorsandthecytoskeleton
tothenuclearmatrixinamorecontrolledwaythanlaminin-rich
basalmembraneextractgelsofmurineoriginandcouldallowfor
cellstobeanalyzedforcellinvasionandmigrationupontreatment
withcompounds.
Application
ThegrowthofPC346cwasfollowedonan
multiple3Dmulticellulartumorgrowthassays
OperaHighContentImagingSystemusingthe
havebeendevelopedtobettermimicthein
increaseinredfluorescencefromthelabeled
vivosituationoftumorsbyorganizinga
cancercells.Growthcurvesfor21daysare
mixtureoftumorcellsandstromalcellsina
showninFigure1.Bothmatrixtypesallowfor
lamininrich3Dextracellularproteingel.
crosstalkbetweenthetwocelltypes,as
Thesemodelscanbeoptimizedand
PC346cproliferationincreaseswithincreased
translatedintoscreenableassays.
CAFconcentration.InArtiCYTBM3DMatrix,
thecellsproliferatebetter,andcellscanbe
Here,wepresentacoculturemodelofthe
keptincultureforalongertime,asevidenced
PC346cprostatecancercelllinelabeledwith
bytheabsenceofadecreaseinfluorescence
RFPandCancerAssociatedFibroblasts(CAFs)
inthelastweekforallbutthehighest
labeledwithGFP.Varyingconcentrationsof
concentrationofcells.Thehighest
CAFswereusedtoinvestigatethecrosstalk
concentrationofCAFsappearstohighforlong
betweencancerandstromalcells.Both
termcultivationundertheseconditions
fluorescentlabelsarestablyexpressed.
Confocalimagestakenafter21daysinculture
ArtiCYTisafullysynthetic,biomimeticmatrix
(Figure2)showthatPC346ccellsandCAFsare
displayingcellularbindingsitesmimicking
bothstillpresent.ThePC346ccellsform
importantECMcomponentspresentin
tumorspheroids,whiletheCAFsare
lamininrichECMgels,therebyofferinga
interspersedwiththePC346cclusters.Atthe
moredefinedalternative.
higherconcentrations,someclusteringof
CAFswithPC346ccellsoccursinBME.
Cellswereculturedforthreeweeksin96well
platesusing100µLofeitherArtiCYTBM3D
After21daysinculture,cellswerestained
MatrixorCultrexBME(Trevigen)asacontrol.
withHoechst(nuclei),5-ethynyl-2’RPMIsupplementedwith2%serumwasused
deoxyuridine(EdU)cellproliferationassay,
astheculturemedium.InthecaseofArtiCYT, Growth curve of PC346C in BME
Growth
curve of PC346C in BM
themediumwasfurthersupplementedwith
multiplegrowthfactorstooptimizethegelfor
tumorgrowth.
A
B
Figure1:RelativegrowthcurvesofPC346cprostatecancercells(20kstartingcellcount)inmonoculture( ⎯)
orin20:1(⎯),2:1( ⎯),or1:5( ⎯)ratioswithCAFs,asdeterminedfromtheincreaseinfluorescence,inA)
28
ArtiCYTBM3DMatrixandB)BMEmatrix.
Figure2:LivefluorescenceimagesofPC346candCAFcellsculturedfor21daysinArtiCYT(upper
panel)orBME(lowerpanel).
andNucView488Caspase-3apoptosisassay
andimagesweretakenintheOperasystem.
TheimagesinFigure3showthatafterthree
weeks,proliferatingcellsarestillpresentin
thetumorspheroidsinallsamples.In
addition,largerspheroidscontainapoptotic
regions,demonstratingthatthetumor
spheroidsaredenselypacked.
Figure3:FluorescenceimageofPC346candCAFcellsculturedfor21daysinArtiCYT(upperpanel)or
BME(lowerpanel)andstainedwithHoechst(blue,nuclei),EdU(red,cellproliferation),andNucView
488(green,apoptosis).
Conclusion
Inthisstudy,wecomparedthegrowthof3D
tumormicrotissuesinBMEgelsandArtiCYT
BM3DMatrix.PC346Cprostatetumorcells
appeartoproliferatebetterinArtiCYTBM3D
Matrixandthehighcellnumberscanbe
bettersustained.
ArtiCYTBM3DMatrixallowsforsignaling
betweenmultiplecelltypes,asdemonstrated
bytheincreasedproliferationofPC346Cin
thepresenceofCAFs.
ArtiCYTBM3DMatrixallowsforthe
optimizationofthemediumforspecific
purposesbyfine-tuningofthegrowthfactor
composition.
TheOperasystemisanidealplatformto
followthegrowthoffluorescentlylabeled
1
Hickman,J.A.;Graeser,R.;deHoogt,R.;
Vidic,S.;Brito,C.;Gutekunst,M.;vander
Kuip,H.Biotechnol.J.2014,9,1115–1128.
cellsgrowninArtiCYTBM3DMatrixusinglive
imagingtechniques.
Insummary,ArtiCYTBM3DMatrixallowsa
newgenerationofmatrixembeddedcell3D
cellmodelscompatiblewithhighcontent
screeningapplications.
Authors
MennodeJong
AnnemarieTuin
NanoFiberMatricesB.V.
Groningen,NL
Cellculturedataobtainedbypartner
2
Thoma,C.R.;Zimmermann,M.;Agarkova,I.;
Kelm,J.M.;Krek,W.;Adv.DrugDeliv.Rev.
2014,69–70,29–41.
3
Weigelt,B.;Ghajar,C.M.;Bissell,M.J.Adv.
DrugDeliv.Rev.2014,69–70,42–51.