Abstract
Culturing mammary epithelial cells in laminin-rich extracellular matrices (three dimensional or 3D culture) offers significant advantages over that in the conventional two-dimensional (2D) tissue culture system in that it takes into considetation the impact of extracellular matrix (ECM) microenvironment on the proliferation, survival, and differentiation of mammary epithelial cells. When grown in the 3D culture, untransformed mammary epithelial cells undergo morphogenesis to form a multicellular and polarized acini-like structure that functionally mimics the differentiated alveoli in the pregnancy mammary gland. This process is subjected to regulation by many growth factors and cytokines. The transforming growth factor-ß (TGFß) is a multipotent cytokine that regulates multiple aspects of development and tumorigenesis. In addition to its effects on epithelial cell proliferation, survival, and differentiation, it is also a potent regulator of the cell–matrix interaction. Thus, the 3D culture model may recapitulate the complex in vivo epithelial cell microenvironment and allow us to fully evaluate the role of TGFß signaling in multiple aspects of normal and cancerous cell behavior. In this chapter we provide detailed protocols for growing mammary epithelial cells in the 3D Matrigel for analysis of signaling pathways.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Emerman JT, Pitelka DR (1977) Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13(5):316–328
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(19):9064–9068
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(2):223–235
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(1):136–140
Emerman JT, Bartley JC, Bissell MJ (1981) Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells. Exp Cell Res 134(1):241–250
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(5):1419–1423
Weigelt B, Bissell MJ (2008) Unraveling the microenvironmental influences on the normal mammary gland and breast cancer. Semin Cancer Biol 18(5):311–321
Rizki A, Weaver VM, Lee SY, Rozenberg GI, Chin K, Myers CA et al (2008) A human breast cell model of preinvasive to invasive transition. Cancer Res 68(5):1378–1387
Wang F, Weaver VM, Petersen OW, Larabell CA, Dedhar S, Briand P et al (1998) Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology. Proc Natl Acad Sci U S A 95(25):14821–14826
Weaver VM, Petersen OW, Wang F, Larabell CA, Briand P, Damsky C et al (1997) Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol 137(1):231–245
Shay JW, Tomlinson G, Piatyszek MA, Gollahon LS (1995) Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome. Mol Cell Biol 15(1):425–432
Soule HD, Maloney TM, Wolman SR, Peterson WD Jr, Brenz R, McGrath CM et al (1990) Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 50(18):6075–6086
Niemann C, Brinkmann V, Spitzer E, Hartmann G, Sachs M, Naundorf H et al (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(2):533–545
Soriano JV, Pepper MS, Nakamura T, Orci L, Montesano R (1995) Hepatocyte growth factor stimulates extensive development of branching duct-like structures by cloned mammary gland epithelial cells. J Cell Sci 108(Pt 2):413–430
Lee GY, Kenny PA, Lee EH, Bissell MJ (2007) Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4(4):359–365
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(Pt 1):39–50
Anders M, Hansen R, Ding RX, Rauen KA, Bissell MJ, Korn WM (2003) Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor. Proc Natl Acad Sci U S A 100(4):1943–1948
Kenny PA, Lee GY, Myers CA, Neve RM, Semeiks JR, Spellman PT et al (2007) The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol Oncol 1(1):84–96
Moses H, Barcellos-Hoff MH (2011) TGF-beta biology in mammary development and breast cancer. Cold Spring Harb Perspect Biol. 3(1):a003277
Derynck R, Akhurst RJ, Balmain A (2001) TGF-beta signaling in tumor suppression and cancer progression. Nat Genet 29(2):117–129
Wu MY, Hill CS (2009) Tgf-beta superfamily signaling in embryonic development and homeostasis. Dev Cell 16(3):329–343
Heldin CH, Landstrom M, Moustakas A (2009) Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Curr Opin Cell Biol 21(2):166–176
Massague J (2008) TGFbeta in cancer. Cell 134(2):215–230
Akhurst RJ (2004) TGF beta signaling in health and disease. Nat Genet 36(8):790–792
Feng XH, Derynck R (2005) Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol 21:659–693
Park CC, Henshall-Powell RL, Erickson AC, Talhouk R, Parvin B, Bissell MJ et al (2003) Ionizing radiation induces heritable disruption of epithelial cell interactions. Proc Natl Acad Sci U S A 100(19):10728–10733
Sankar S, Mahooti-Brooks N, Bensen L, McCarthy TL, Centrella M, Madri JA (1996) Modulation of transforming growth factor beta receptor levels on microvascular endothelial cells during in vitro angiogenesis. J Clin Invest 97(6):1436–1446
Henry LA, Johnson DA, Sarrio D, Lee S, Quinlan PR, Crook T et al (2010) Endoglin expression in breast tumor cells suppresses invasion and metastasis and correlates with improved clinical outcome. Oncogene 30(9):1046–1058
Wendt MK, Taylor MA, Schiemann BJ, Schiemann WP (2011) Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer. Mol Biol Cell 22(14):2423–2435
Ganapathy V, Ge R, Grazioli A, Xie W, Banach-Petrosky W, Kang Y et al (2010) Targeting the Transforming growth factor-beta pathway inhibits human basal-like breast cancer metastasis. Mol Cancer 9:122
Sempere LF, Gunn JR, Korc M (2011) A novel 3-dimensional culture system uncovers growth stimulatory actions by TGFbeta in pancreatic cancer cells. Cancer Biol Ther 12(3):198–207
Jahchan NS, Wang D, Bissell MJ, Luo K (2012) SnoN regulates mammary gland alveologenesis and onset of lactation by promoting prolactin/Stat5 signaling. Development 139(17):3147–3156
Krakowski AR, Laboureau J, Mauviel A, Bissell MJ, Luo K (2005) Cytoplasmic SnoN in normal tissues and nonmalignant cells antagonizes TGF-beta signaling by sequestration of the Smad proteins. Proc Natl Acad Sci U S A 102(35):12437–12442
Merlo GR, Basolo F, Fiore L, Duboc L, Hynes NE (1995) p53-dependent and p53-independent activation of apoptosis in mammary epithelial cells reveals a survival function of EGF and insulin. J Cell Biol 128(6):1185–1196
Yaswen P, Stampfer MR (2002) Molecular changes accompanying senescence and immortalization of cultured human mammary epithelial cells. Int J Biochem Cell Biol 34(11):1382–1394
Debnath J, Muthuswamy SK, Brugge JS (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30(3):256–268
Muthuswamy SK, Li D, Lelievre S, Bissell MJ, Brugge JS (2001) ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Nat Cell Biol 3(9):785–792
Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS (2002) The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell 111(1):29–40
Debnath J, Brugge JS (2005) Modelling glandular epithelial cancers in three-dimensional cultures. Nat Rev 5(9):675–688
Godde NJ, Galea RC, Elsum IA, Humbert PO (2010) Cell polarity in motion: redefining mammary tissue organization through EMT and cell polarity transitions. J Mammary Gland Biol Neoplasia 15(2):149–168
Santner SJ, Dawson PJ, Tait L, Soule HD, Eliason J, Mohamed AN et al (2001) Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells. Breast Cancer Res Treat 65(2):101–110
Kleinman HK, McGarvey ML, Hassell JR, Star VL, Cannon FB, Laurie GW et al (1986) Basement membrane complexes with biological activity. Biochemistry 25(2):312–318
Han J, Chang H, Giricz O, Lee GY, Baehner FL, Gray JW et al (2010) Molecular predictors of 3D morphogenesis by breast cancer cell lines in 3D culture. PLoS Comput Biol 6(2):e1000684
Wang F, Hansen RK, Radisky D, Yoneda T, Barcellos-Hoff MH, Petersen OW et al (2002) Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts. J Natl Cancer Inst 94(19):1494–1503
Blatchford DR, Quarrie LH, Tonner E, McCarthy C, Flint DJ, Wilde CJ (1999) Influence of microenvironment on mammary epithelial cell survival in primary culture. J Cell Physiol 181(2):304–311
Coucouvanis EC, Martin GR, Nadeau JH (1995) Genetic approaches for studying programmed cell death during development of the laboratory mouse. Methods Cell Biol 46:387–440
Boudreau N, Sympson CJ, Werb Z, Bissell MJ (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 267(5199):891–893
Frisch SM, Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124(4):619–626
Acknowledgement
We would like to thank members of Dr. Mina Bissell’s group for their assistance. The work is supported by NIH RO1 CA101891 and RO1 DK090347 to K. Luo. J. Rashidian is supported by the Susan G. Komen For The Cure KG101263.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Rashidian, J., Luo, K. (2016). Three-dimensional Mammary Epithelial Cell Morphogenesis Model for Analysis of TGFß Signaling. In: Feng, XH., Xu, P., Lin, X. (eds) TGF-β Signaling. Methods in Molecular Biology, vol 1344. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2966-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2966-5_7
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2965-8
Online ISBN: 978-1-4939-2966-5
eBook Packages: Springer Protocols