Bielecka-2017-Three-dimensional cell culture m.pdf

Biol.Rev.(2017),92,pp.15051520.1505doi:10.1111/brv.12293Three-dimensional cell culture modelutilization in cancer stem cell researchZofia F.Bielecka1,2,Kamila Maliszewska-Olejniczak1,3,Ilan J.Safir4,Cezary Szczylik1and Anna M.Czarnecka1,1Department of Oncology with Laboratory of Molecular Oncology,Military Institute of Medicine,Szaser ow 128,04-141,Warsaw,Poland2Postgraduate School of Molecular Medicine,Medical University of Warsaw,Zwirki i Wigury 61,02-109,Warsaw,Poland3Laboratory of DNA Sequencing and Oligonucleotides Synthesis,Institute of Biochemistry and Biophysics,Polish Academy of Sciences,Pawinskiego5a,02-106 Warsaw,Poland4Department of Urology,Emory University School of Medicine,Atlanta,GA 30322,U.S.A.ABSTRACTThree-dimensional(3D)cell culture models are becoming increasingly popular in contemporary cancer research anddrug resistance studies.Recently,scientists have begun incorporating cancer stem cells(CSCs)into 3D models andmodifying culture components in order to mimic in vivo conditions better.Currently,the global cell culture marketis primarily focused on either 3D cancer cell cultures or stem cell cultures,with less focus on CSCs.This is evidentin the low product availability officially indicated for 3D CSC model research.This review discusses the currentlyavailable commercial products for CSC 3D culture model research.Additionally,we discuss different culture media andcomponents that result in higher levels of stem cell subpopulations while better recreating the tumor microenvironment.In summary,although progress has been made applying 3D technology to CSC research,this technology could befurther utilized and a greater number of 3D kits dedicated specifically to CSCs should be implemented.Key words:three-dimensional systems,cancer stem cells(CSCs),biomimic cultures,tumor microenvironment,cancerniche.CONTENTSI.Introduction.1506II.Cancer stem cells.1507(1)Cancer stem cell model.1507(2)Cancer stem cells and carcinogenesis.1508III.Gold-standard methods in characterizing cancer stem cells.1509IV.Three-dimensional cell culture models and cancer stem cells.1509(1)Starting material:cells lines or primary tissue.1510V.Modifying three-dimensional cancer stem cell models.1510(1)Proposed culture media.1511(2)Proposed growth factors.1511(3)Emerging role of hormones in CSCs and 3D CSC models.1511(4)Presence or absence of serum in(3D)CSC models.1512(5)Extracellular matrix components and their impact on the relevance of 3D CSC models.1512(6)Comparison of in vitro culture conditions to the known in vivo CSC niche.1513(7)Hypoxia or normoxia/physioxia in 3D CSC models seeking the optimal solution.1513*Address for correspondence(Tel:+48 261 817 172;E-mail:)Biological Reviews 92(2017)15051520 2016 The Authors.Biological Reviews published by John Wiley&Sons Ltd on behalf of Cambridge Philosophical Society.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License,which permits use and distribution in any medium,providedthe original work is properly cited,the use is non-commercial and no modifications or adaptations are made.1506Zofia F.Bielecka and others(8)Co-cultures in 3D models:seeding cells in the presence of stromal components.1513(9)Culture vessels and 3D CSC models.1513(10)Natural scaffolds.1514(11)Synthetic scaffolds.1514(12)Commercial ready-to-use three-dimensional kits.1515VI.Three-dimensional cancer stem cell culture systems.1515(1)The multicellular tumor spheroid model.1516(2)Suspension bioreactors.1516(3)Rotating wall vessel technology.1516(4)Hydrodynamic focusing bioreactor.1516(5)Biomimetic nano-cilia and microfluidics.1516VII.Recent advances in three-dimensional cancer stem cell technologies.1516VIII.Conclusions.1517IX.Acknowledgement.1518X.References.1518I.INTRODUCTIONThree-dimensional(3D)assays are known to stimulate in vivocellular conditions better in comparison with traditionaltwo-dimensional(2D)cell culture systems and influencethe formation of a subpopulation of cancer cells withstem cell-like properties,providing new insights into cancertreatment and cancer stem cell(CSC)research(Chen etal.,2012).Recent studies have identified the presence of asmall subpopulation of cells within a tumor containing stemcell-like properties.CSCs share some cellular characteristicswith non-malignant stem cells,however,they also possessuniquephysiologicalattributes.Non-malignantstemcellsarevital during development and are present in all tissues withhigh regenerative abilities;stem cells are able to self-renewand are responsible for sustaining tissue homeostasis andtissue repair following injury(Trumpp&Wiestler,2008;Rojas-Ros&Gonz alez-Reyes,2014).CSCs,originallydescribedinleukemiaresearch,represent a rare subpopulation of cancer cells withunlimited self-renewal capacity and the ability to initiateand sustain tumor growth(Chen etal.,2007;Rojas-Ros&Gonz alez-Reyes,2014).Recentstudiesoncancerresearch demonstrate that CSCs strongly influence tumorgrowth and the development of resistance to conventionalradiotherapies and chemotherapies.A better understandingof the physiology of CSCs may explain different patientresponses to antineoplastic therapies,as well as provid-ing improved insight into the life cycle of tumor cells.CSCs typically possess efficient DNA repair mechanisms,overexpression of anti-apoptotic proteins,expression ofmultidrug-resistance-typeATP-bindingcassette(ABC)membrane transporters,and resistance to hypoxic environ-ments.These attributes may explain the resistance of CSCsto conventional anti-tumor therapies.CSCs,also describedas tumor-initiating cells(TICs),differ significantly fromnon-malignant stem cells.Although the terms CSC and TICare used interchangeably within the literature,it should benoted that TICs are preferably described as cells containingoncogenic mutations prior to developing into CSCs duringtumorigenesis(Gao,2008).Additional important CSC char-acteristics include low proliferation rates,high self-renewalrates,a tendency to differentiate into actively proliferatingcancer cells,and possessing natural resistance to currentanti-cancer therapies(Neuzil etal.,2007).Given their stemcell-like properties,CSCs are able to undergo asymmetricdivision and differentiation into different cell lineages.Furthermore,CSCs are categorized as a side population(a subpopulation of cells distinct from the main populationin terms of specific markers)due to their ability to effluxfluorescent DNA-staining dyes via ABC transporters(Huangetal.,2013).The concept of CSCs was first postulatedwhen evaluating tumorigenesis in immunodeficient mousemodels(Huang etal.,2013),and the current concept ofCSCs is summarized in Fig.1.The recent discoveryof experimentally defined CSC precursors from murinelymphoma models,called precancerous stem cells(pCSCs),demonstrates a further tumor cell subset.Depending onmicroenvironmental signals,pCSCs possess the potentialfor both benign and malignant differentiation(Chenetal.,2007).Herein we highlight recent advances in the applicationof 3D assays in cancer research.CSCs cultured using 3Dassays can better mimic tumor growth in vitro,as they aremore able to overexpress pro-angiogenic growth factorsand stemness genes(Smith etal.,2011;Chen etal.,2012).Further utilization of 3D assay models in CSC researchmay uncover a range of novel anti-neoplastic therapies.Itwas recently demonstrated that 3D CSC models show amore realistic drug response,thus allowing for improveddrug-resistance studies.This review summarizes 3D cellcultures,along with natural and synthetic scaffolds availablein cancer research as well as their roles in understandingthe concept of CSCs.Currently available,ready-to use 3Dsystems,aswellasthemediacomponentsmostsuitablefor3DculturingofCSCsarediscussed.Additionally,theglobalstemcell market is summarized.Future studies targeting uniqueCSC molecular characteristics utilizing available 3D assaysmay provide new oncological treatment targets and therapymodalities.Biological Reviews 92(2017)15051520 2016 The Authors.Biological Reviews published by John Wiley&Sons Ltd on behalf of Cambridge Philosophical Society.3D cell culture models in CSCs research1507Fig.1.Scheme summarizing terms related to current understanding of cancer stem cells(CSCs),their origin and differences.TIC,tumor-initiating cell;SP,side population;pCSC,precancerous stem cell.Oncogenic mutations are marked by a lightning arrow.II.CANCER STEM CELLS(1)Cancer stem cell modelIn recent years,several models of cancer developmenthave been proposed,including the stochastic,hierarchical,and clonal evolution(CSC hypothesis)models(Fig.2)(Gao,2008).The stochastic model,now only of historicalsignificance,proposes that all cancer cells within a tumorare homogenous but can reproduce to give phenotypicallyheterogeneous cell types.The stochastic model does notexplain the observed heterogeneity of cancer cells.Thehierarchical model,by contrast,proposes that a smallsubpopulation of cancer cells has the capacity to generatephenotypically heterogeneous cell lines,while other cellswithin the tumor population possess partial proliferativeability.Accordingtothehierarchicalmodel,asubsetofCSCscan only be maintained by cells possessing CSC potential,with their progeny containing partial proliferative ability(Nguyen etal.,2012).The hierarchical model gives rise tonovel cancer treatment concepts,suggesting that eliminationof all CSCs may inhibit tumor growth and relapse.Theclonal evolution model,consistent with the CSC hypothesis,proposes that malignant cells can mutate and generateabnormalprogenywhichalsohavethepotentialtotransform,potentially becoming a dominant cell-line population,leadingtotheformationofagroupofgeneticallytransformedclones all possessing proliferative abilities(Takebe&Ivy,2010).According to recent studies,the most accurate cancermodel is likely a combination of both the hierarchical andclonal evolution models(Ricci-Vitiani etal.,2007;Takebe&Ivy,2010).TherevisedCSChypothesismodel,incorporatingboth paradigms,proposes the existence of primary CSCs,which after acquiring a subset of mutations are transformedinto a population of secondary CSCs,and then becomeFig.2.Schematic model of the current cancer stem cell(CSC)hypothesis combining hierarchical and clonal evolution models.ThismodelshowsasubsetofCSCswhichareabletoproliferate.These cells have self-renewal properties and are capable ofrecapitulating a tumor hierarchy.pCSC,precancerous stemcell.Oncogenic mutations are marked by lightning arrows.metastatic CSCs after acquiring further mutations with thepotential to form tumors at distant sites.This revised modelsuggests that a tumor mass contains only a small fraction ofmetastatic CSCs.The CSC combined model is supported bystudies on the hierarchical model of human acute myeloidleukemia(AML),demonstrating that leukemia originatesfrom a primitive hematopoietic stem cell with the potentialto differentiate into different cell lines(Bonnet&Dick,1997).The first CSC theories date back to the 19th century.In1855,Rudolph Virchow postulated a theory of embryonalrest,assuminganembryonicoriginfortumorcells(Moltzahnetal.,2008).In 1937,Furth and Kahn proposed the existenceof tumor stem cells when studying a leukemia mouseBiological Reviews 92(2017)15051520 2016 The Authors.Biological Reviews published by John Wiley&Sons Ltd on behalf of Cambridge Philosophical Society.1508Zofia F.Bielecka and othersmodel(Moltzahn etal.,2008).A recent version of theCSC hypothesis,postulated by Pierce&Speers(1988),characterized carcinomas as caricatures of tissue renewal,being a mixture of malignant stem cells with the abilityto proliferate and differentiate under normal homeostaticconditions(Bonnet&Dick,1997).In support of the CSChypothesis,Bonnet&Dick(1997)transplanted single AMLcells into mice with severe combined immunodeficiencydisease and observed subsequent development of AML inthe recipient.The leukemia-initiating cells were able toproliferate,differentiate,and self-renew(Bonnet&Dick,1997).Moreover,the isolated malignant cells overexpressedsurfacemarkersCD34+/CD38,representingahierarchicalcellular organization with molecular markers representativeof the normal hematopoietic developmental pathway.Theirfindings suggested that malignant AML cells originated froma primitive hematopoietic cell line.CSC research in solid cancers,specifically breast cancer,demonstrated that only a minor fraction of cells within abreast tumor have the potential for tumorigenesis whileother cells within the mass lack this ability(Al-Hajj etal.,2003).By utilizing an immunocompromised mouse model toinvestigate breast cancer,Al-Hajj etal.(2003)distinguishedsubpopulationsoftumorcellspossessingcellsurfacemarkers.Through flow cytometry,they demonstrated that only asubsetofcellswasheterogeneous,tumorigenic,andexpressedcell surface markers CD44+/CD24/low.CSC populationshave been identified in other solid-organ malignancies,including brain,prostate,ovarian,colorectal,pancreatic,head and neck,lung,liver and renal cancers(Bisignanietal.,1999;Chang etal.,2013).The majority of CSC studiesevaluated cell surface marker expression,including CD34and CD38(leukemia),CD44 and CD24(breast cancer),CD105(renal cancer),CD133(colon and brain cancer),andCD271(melanoma)(Clevers,2011).Interestingly,cellsurfaceantigen CD133,although initially considered a marker ofmalignant stem cells,was later identified in stem cells withinnormal tissues(Dean,Fojo&Bates,2005).(2)Cancer stem cells and carcinogenesisCSCs are increasingly recognized as having important rolesin tumorigenesis,relapse and metastasis(Huang etal.,2013).The earliest documented theory of metastasis was proposedin 1889 by Stephen Paget(Lin etal.,2008).He proposed thatmetastasis is dependent on communication between selectedcancer cells(seeds)and specific organ microenvironments(soil).His work thus was described as the Seed and Soiltheory.The link between the seeds CSCs/TICs andmetastasis lies primarily within the process of selecting forstress-resistant phenotypes which is externally influenced byspecificparacrinemicroenvironments(soil).Thishypothesiswas revisited subsequently,incorporating proposed stemcell subpopulations consisting of CSCs,mesenchymal stemcells,and circulating endothelial progenitors working in acoordinated multicellular complex(Lin etal.,2008).CSCs share many molecular properties with stemcells originally responsible for organogenesis and tissueregeneration(Moltzahn etal.,2008).Growing evidence hasshown that CSCs overexpress traditional stem cell genes,including octamer-binding transcription factor 4(OCT4),notch homolog 1(NOTCH1),aldehyde dehydrogenase 1(ALDH1),fibroblast growth factor receptor 1(FGFR1),and sex-determining region Y-box 1(SOX1)(Takebe&Ivy,2010).A major difference between normal stem cellsand CSCs is the stem cells dependence on the cellularmicroenvironment(niche)and growth factors that maintainstem cells by influencing their number,proliferation andfate(Li&Neave