Abstract
The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our laboratory—are cultured in relevant 3D microenvironments. We focus on the design of functional assays that enable us to understand the intricate signaling events underlying mammary gland biology, and address the advantages and limitations of the different culture settings. Finally we also discuss how advances in bioengineering tools may help towards the ultimate goal of building tissues and organs in culture for basic research and clinical studies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Babinet C (2000) Transgenic mice: an irreplaceable tool for the study of mammalian development and biology. J Am Soc Nephrol 11(Suppl 16):S88–S94
Proia DA, Kuperwasser C (2006) Reconstruction of human mammary tissues in a mouse model. Nat Protoc 1:206–214
Ronnov-Jessen L, Petersen OW, Bissell MJ (1996) Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction. Physiol Rev 76:69–125
Schmeichel KL, Weaver VM, Bissell MJ (1998) Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial cell phenotype. J Mammary Gland Biol Neoplasia 3:201–213
Richert MM, Schwertfeger KL, Ryder JW, Anderson SM (2000) An atlas of mouse mammary gland development. J Mammary Gland Biol Neoplasia 5:227–241
Bolander FF Jr (1990) Differential characteristics of the thoracic and abdominal mammary glands from mice. Exp Cell Res 189:142–144
Fendrick JL, Raafat AM, Haslam SZ (1998) Mammary gland growth and development from the postnatal period to postmenopause: ovarian steroid receptor ontogeny and regulation in the mouse. J Mammary Gland Biol Neoplasia 3:7–22
Wiseman BS, Sternlicht MD, Lund LR, Alexander CM, Mott J, Bissell MJ, Soloway P, Itohara S, Werb Z (2003) Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis. J Cell Biol 162:1123–1133
Silberstein GB (2001) Postnatal mammary gland morphogenesis. Microsc Res Tech 52:155–162
Daniel CW, Robinson S, Silberstein GB (1996) The role of TGF-beta in patterning and growth of the mammary ductal tree. J Mammary Gland Biol Neoplasia 1:331–341
Nandi S (1958) Endocrine control of mammarygland development and function in the C3H/He Crgl mouse. J Natl Cancer Inst 21:1039–1063
Ramanathan P, Martin I, Thomson P, Taylor R, Moran C, Williamson P (2007) Genomewide analysis of secretory activation in mouse models. J Mammary Gland Biol Neoplasia 12:305–314
Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ (1989) Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 105:223–235
Lee EY, Lee WH, Kaetzel CS, Parry G, Bissell MJ (1985) Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion. Proc Natl Acad Sci U S A 82:1419–1423
Howlett AR, Bissell MJ (1993) The influence of tissue microenvironment (stroma and extracellular matrix) on the development and function of mammary epithelium. Epithelial Cell Biol 2:79–89
Rosen JM, Wyszomierski SL, Hadsell D (1999) Regulation of milk protein gene expression. Annu Rev Nutr 19:407–436
Li M, Liu X, Robinson G, Bar-Peled U, Wagner KU, Young WS, Hennighausen L, Furth PA (1997) Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution. Proc Natl Acad Sci U S A 94:3425–3430
Furth PA, Bar-Peled U, Li M (1997) Apoptosis and mammary gland involution: reviewing the process. Apoptosis 2:19–24
Quarrie LH, Addey CV, Wilde CJ (1995) Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling. Cell Tissue Res 281:413–419
Li M, Hu J, Heermeier K, Hennighausen L, Furth PA (1996) Apoptosis and remodeling of mammary gland tissue during involution proceeds through p53-independent pathways. Cell Growth Differ 7:13–20
Daniel CW, Deome KB (1965) Growth of mouse mammary glands in vivo after monolayer culture. Science 149:634–636
Daniel CW, Young LJ, Medina D, DeOme KB (1971) The influence of mammogenic hormones on serially transplanted mouse mammary gland. Exp Gerontol 6:95–101
Emerman JT, Burwen SJ, Pitelka DR (1979) Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture. Tissue Cell 11:109–119
Emerman JT, Pitelka DR (1977) Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13:316–328
Michalopoulos G, Pitot HC (1975) Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations. Exp Cell Res 94:70–78
Emerman JT, Enami J, Pitelka DR, Nandi S (1977) Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc Natl Acad Sci U S A 74:4466–4470
Lee EY, Parry G, Bissell MJ (1984) Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata. J Cell Biol 98:146–155
Aggeler J, Ward J, Blackie LM, Barcellos-Hoff MH, Streuli CH, Bissell MJ (1991) Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo. J Cell Sci 99(Pt 2):407–417
Chen LH, Bissell MJ (1989) A novel regulatory mechanism for whey acidic protein gene expression. Cell Regul 1:45–54
Streuli CH, Bailey N, Bissell MJ (1991) Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity. J Cell Biol 115:1383–1395
Xu R, Nelson CM, Muschler JL, Veiseh M, Vonderhaar BK, Bissell MJ (2009) Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function. J Cell Biol 184:57–66
Gudjonsson T, Ronnov-Jessen L, Villadsen R, Rank F, Bissell MJ, Petersen OW (2002) Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. J Cell Sci 115:39–50
Guo W, Giancotti FG (2004) Integrin signalling during tumour progression. Nat Rev Mol Cell Biol 5:816–826
Kalluri R (2003) Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer 3:422–433
Mott JD, Werb Z (2004) Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 16:558–564
Streuli CH, Schmidhauser C, Bailey N, Yurchenco P, Skubitz AP, Roskelley C, Bissell MJ (1995) Laminin mediates tissue-specific gene expression in mammary epithelia. J Cell Biol 129:591–603
Li ML, Aggeler J, Farson DA, Hatier C, Hassell J, Bissell MJ (1987) Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proc Natl Acad Sci U S A 84:136–140
Wicha MS, Lowrie G, Kohn E, Bagavandoss P, Mahn T (1982) Extracellular matrix promotes mammary epithelial growth and differentiation in vitro. Proc Natl Acad Sci U S A 79:3213–3217
Medina D, Li ML, Oborn CJ, Bissell MJ (1987) Casein gene expression in mouse mammary epithelial cell lines: dependence upon extracellular matrix and cell type. Exp Cell Res 172:192–203
Daniel CW, Berger JJ, Strickland P, Garcia R (1984) Similar growth pattern of mouse mammary epithelium cultivated in collagen matrix in vivo and in vitro. Dev Biol 104:57–64
Montesano R, Soriano JV, Fialka I, Orci L (1998) Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties. In Vitro Cell Dev Biol Anim 34:468–477
Niemann C, Brinkmann V, Spitzer E, Hartmann G, Sachs M, Naundorf H, Birchmeier W (1998) Reconstitution of mammary gland development in vitro: requirement of c-met and c-erbB2 signaling for branching and alveolar morphogenesis. J Cell Biol 143:533–545
Ruoslahti E, Pierschbacher MD (1986) Arg-Gly-Asp: a versatile cell recognition signal. Cell 44:517–518
Hynes RO (1987) Integrins: a family of cell surface receptors. Cell 48:549–554
Alcaraz J, Xu R, Mori H, Nelson CM, Mroue R, Spencer VA, Brownfield D, Radisky DC, Bustamante C, Bissell MJ (2008) Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia. EMBO J 27:2829–2838
Talhouk RS, Elble RC, Bassam R, Daher M, Sfeir A, Mosleh LA, El-Khoury H, Hamoui S, Pauli BU, El-Sabban ME (2005) Developmental expression patterns and regulation of connexins in the mouse mammary gland: expression of connexin30 in lactogenesis. Cell Tissue Res 319:49–59
El-Sabban ME, Sfeir AJ, Daher MH, Kalaany NY, Bassam RA, Talhouk RS (2003) ECM-induced gap junctional communication enhances mammary epithelial cell differentiation. J Cell Sci 116:3531–3541
Nguyen DA, Neville MC (1998) Tight junction regulation in the mammary gland. J Mammary Gland Biol Neoplasia 3:233–246
Talhouk RS, Mroue R, Mokalled M, Abi-Mosleh L, Nehme R, Ismail A, Khalil A, Zaatari M, El-Sabban ME (2008) Heterocellular interaction enhances recruitment of alpha and beta-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells. Exp Cell Res 314:3275–3291
Wang X, Sun L, Maffini MV, Soto A, Sonnenschein C, Kaplan DL (2010) A complex 3D human tissue culture system based on mammary stromal cells and silk scaffolds for modeling breast morphogenesis and function. Biomaterials 31:3920–3929
Ingthorsson S, Sigurdsson V, Fridriksdottir AJ, Jonasson JG, Kjartansson J, Magnusson MK, Gudjonsson T (2010) Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture. BMC Res Note 3184
Chen A, Cuevas I, Kenny PA, Miyake H, Mace K, Ghajar C, Boudreau A, Bissell M, Boudreau N (2009) Endothelial cell migration and vascular endothelial growth factor expression are the result of loss of breast tissue polarity. Cancer Res 69:6721–6729
Petersen OW, Nielsen HL, Gudjonsson T, Villadsen R, Rank F, Niebuhr E, Bissell MJ, Ronnov-Jessen L (2003) Epithelial to mesenchymal transition in human breast cancer can provide a nonmalignant stroma. Am J Pathol 162:391–402
Ip MM, Darcy KM (1996) Three-dimensional mammary primary culture model systems. J Mammary Gland Biol Neoplasia 1:91–110
Hamamoto S, Imagawa W, Yang J, Nandi S (1988) Morphogenesis of mouse mammary epithelial cells growing within collagen gels: ultrastructural and immunocytochemical characterization. Cell Differ 22:191–201
Bissell MJ (1981) The differentiated state of normal and malignant cells or how to define a “normal” cell in culture. Int Rev Cytol 70:27–100
Petersen OW, Ronnov-Jessen L, Howlett AR, Bissell MJ (1992) Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci U S A 89:9064–9068
Lin CQ, Bissell MJ (1993) Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J 7:737–743
Streuli CH, Bissell MJ (1990) Expression of extracellular matrix components is regulated by substratum. J Cell Biol 110:1405–1415
Roskelley CD, Desprez PY, Bissell MJ (1994) Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction. Proc Natl Acad Sci U S A 91:12378–12382
Faraldo MM, Deugnier MA, Lukashev M, Thiery JP, Glukhova MA (1998) Perturbation of beta1-integrin function alters the development of murine mammary gland. EMBO J 17:2139–2147
Muschler J, Lochter A, Roskelley CD, Yurchenco P, Bissell MJ (1999) Division of labor among the alpha6beta4 integrin, beta1 integrins, and an E3 laminin receptor to signal morphogenesis and beta-casein expression in mammary epithelial cells. Mol Biol Cell 10:2817–2828
Weir ML, Oppizzi ML, Henry MD, Onishi A, Campbell KP, Bissell MJ, Muschler JL (2006) Dystroglycan loss disrupts polarity and beta-casein induction in mammary epithelial cells by perturbing laminin anchoring. J Cell Sci 119:4047–4058
Lin CQ, Dempsey PJ, Coffey RJ, Bissell MJ (1995) Extracellular matrix regulates whey acidic protein gene expression by suppression of TGF-alpha in mouse mammary epithelial cells: studies in culture and in transgenic mice. J Cell Biol 129:1115–1126
Zhang Y, Sif S, DeWille J (2007) The mouse C/EBPdelta gene promoter is regulated by STAT3 and Sp1 transcriptional activators, chromatin remodeling and c-Myc repression. J Cell Biochem 102:1256–1270
Spencer VA, Xu R, Bissell MJ (2010) Gene expression in the third dimension: the ECM-nucleus connection. J Mammary Gland Biol Neoplasia 15:65–71
Lelievre S, Weaver VM, Bissell MJ (1996) Extracellular matrix signaling from the cellular membrane skeleton to the nuclear skeleton: a model of gene regulation. Recent Prog Horm Res 51:417–432
Xu R, Spencer VA, Bissell MJ (2007) Extracellular matrix-regulated gene expression requires cooperation of SWI/SNF and transcription factors. J Biol Chem 282:14992–14999
Schmidhauser C, Bissell MJ, Myers CA, Casperson GF (1990) Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5’ sequences in stably transfected mouse mammary cells. Proc Natl Acad Sci U S A 87:9118–9122
Bissell MJ, Hall HG, Parry G (1982) How does the extracellular matrix direct gene expression? J Theor Biol 99:31–68
Talhouk RS, Chin JR, Unemori EN, Werb Z, Bissell MJ (1991) Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture. Development 112:439–449
Talhouk RS, Bissell MJ, Werb Z (1992) Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution. J Cell Biol 118:1271–1282
Sympson CJ, Talhouk RS, Alexander CM, Chin JR, Clift SM, Bissell MJ, Werb Z (1994) Targeted expression of stromelysin-1 in mammary gland provides evidence for a role of proteinases in branching morphogenesis and the requirement for an intact basement membrane for tissue-specific gene expression. J Cell Biol 125:681–693
Simian M, Hirai Y, Navre M, Werb Z, Lochter A, Bissell MJ (2001) The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development 128:3117–3131
Nelson CM, Vanduijn MM, Inman JL, Fletcher DA, Bissell MJ (2006) Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures. Science 314:298–300
Fata JE, Mori H, Ewald AJ, Zhang H, Yao E, Werb Z, Bissell MJ (2007) The MAPK(ERK-1,2) pathway integrates distinct and antagonistic signals from TGFalpha and FGF7 in morphogenesis of mouse mammary epithelium. Dev Biol 306:193–207
Stoker AW, Streuli CH, Martins-Green M, Bissell MJ (1990) Designer microenvironments for the analysis of cell and tissue function. Curr Opin Cell Biol 2:864–874
Nelson CM, Bissell MJ (2006) Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu Rev Cell Dev Biol 22:287–309
Bissell MJ, Barcellos-Hoff MH (1987) The influence of extracellular matrix on gene expression: is structure the message? J Cell Sci Suppl 8:327–343
Reichmann E, Ball R, Groner B, Friis RR (1989) New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally. J Cell Biol 108:1127–1138
Reichmann E, Schwarz H, Deiner EM, Leitner I, Eilers M, Berger J, Busslinger M, Beug H (1992) Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion. Cell 71:1103–1116
Acknowledgments
We thank Dr. Joni Mott for critical reading of the manuscript and Dr. Hidetoshi Mori for providing images of cells in Fig. 2b, c. The work from MJB’s laboratory is supported by grants from the US Department of Energy, Office of Biological and Environmental Research, a Distinguished Fellow Award to MJB and Low Dose Radiation Program (contract no. DE-AC02-05CH1123); by National Cancer Institute (awards R37CA064786, U54CA126552, R01CA057621, U54CA112970, U54CA143836, U01CA143233); and by US Department of Defense (W81XWH0810736). RM is supported by US Department of Defense, BCRP fellowship (W81XWH0810481).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Mroue, R., Bissell, M.J. (2012). Three-Dimensional Cultures of Mouse Mammary Epithelial Cells. In: Randell, S., Fulcher, M. (eds) Epithelial Cell Culture Protocols. Methods in Molecular Biology, vol 945. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-125-7_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-125-7_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-124-0
Online ISBN: 978-1-62703-125-7
eBook Packages: Springer Protocols