A 3D Fibroblast-Epithelium Co-culture Model for Understanding Microenvironmental Role in Branching Morphogenesis of the Mammary Gland

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1501)

Abstract

The mammary gland consists of numerous tissue compartments, including mammary epithelium, an array of stromal cells, and the extracellular matrix (ECM). Bidirectional interactions between the epithelium and its surrounding stroma are essential for proper mammary gland development and homeostasis, whereas their deregulation leads to developmental abnormalities and cancer. To study the relationships between the epithelium and the stroma, development of models that could recapitulate essential aspects of these interacting systems in vitro has become necessary. Here we describe a three-dimensional (3D) co-culture assay and show that the addition of fibroblasts to mammary organoid cultures promotes the epithelium to undergo branching morphogenesis, thus allowing the role of the stromal microenvironment to be examined in this essential developmental process.

Key words

3D culture Branching morphogenesis Extracellular matrix Fibroblasts Mammary epithelium Matrigel Organoids Organotypic assay Paracrine signaling 

Notes

Acknowledgements

This work was supported by grants from the National Institutes of Health (R03 HD060807), Breast Cancer Now, and ShanghaiTech University (to P.L.), and by the project “Employment of Best Young Scientists for International Cooperation Empowerment“(CZ.1.07/2.3.00/30.0037) co-financed from European Social Fund and the state budget of the Czech Republic and by the grant “Junior investigator 2015“ from Faculty of Medicine, Masaryk University (to Z.K.). The Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, was supported by core funding from the Wellcome Trust (088785/Z/09/Z).

References

  1. 1.
    Polyak K, Kalluri R (2010) The role of the microenvironment in mammary gland development and cancer. Cold Spring Harb Perspect Biol 2:a003244. doi: 10.1101/cshperspect.a003244 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Krause S, Maffini MV, Soto AM et al (2008) A novel 3D in vitro culture model to study stromal-epithelial interactions in the mammary gland. Tissue Eng Part C Methods 14:261–271CrossRefPubMedGoogle Scholar
  3. 3.
    Wang F, Weaver VM, Petersen OW 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 USA 95:14821–14826CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lee GY, Kenny PA, Lee EH et al (2007) Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4:359–365CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kass L, Erler JT, Dembo M et al (2007) Mammary epithelial cell: influence of extracellular matrix composition and organization during development and tumorigenesis. Int J Biochem Cell Biol 39:1987–1994CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Barnes C, Speroni L, Quinn KP et al (2014) From single cells to tissues: interactions between the matrix and human breast cells in real time. PLoS One 9, e93325. doi: 10.1371/journal.pone.0093325 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Basham KJ, Kieffer C, Shelton DN et al (2013) Chemical genetic screen reveals a role for desmosomal adhesion in mammary branching morphogenesis. J Biol Chem 288:2261–2270CrossRefPubMedGoogle Scholar
  8. 8.
    Heneweer M, Muusse M, Dingemans M et al (2005) Co-culture of primary human mammary fibroblasts and MCF-7 cells as an in vitro breast cancer model. Toxicol Sci 83:257–263CrossRefPubMedGoogle Scholar
  9. 9.
    Chhetri RK, Phillips ZF, Troester MA et al (2012) Longitudinal study of mammary epithelial and fibroblast co-cultures using optical coherence tomography reveals morphological hallmarks of pre-malignancy. PLoS One 7, e49148. doi: 10.1371/journal.pone.0049148 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lühr I, Friedl A, Overath T et al (2012) Mammary fibroblasts regulate morphogenesis of normal and tumorigenic breast epithelial cells by mechanical and paracrine signals. Cancer Lett 325:175–188CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhong A, Wang G, Yang J et al (2014) Stromal-epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands. J Cell Mol Med. doi: 10.1111/jcmm.12275 Google Scholar
  12. 12.
    Schedin P, Keely PJ (2011) Mammary gland ECM remodeling, stiffness, and mechanosignaling in normal development and tumor progression. Cold Spring Harb Perspect Biol 3:a003228. doi: 10.1101/cshperspect.a003228 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Wellcome Trust Centre for Cell Matrix Research, Faculty of Life SciencesUniversity of ManchesterManchesterUK
  2. 2.Department of Histology and Embryology, Faculty of MedicineMasaryk UniversityBrnoCzech Republic
  3. 3.Department of Anatomy and Program in Developmental and Stem Cell BiologyUniversity of CaliforniaSan FranciscoUSA
  4. 4.School of Life Science and TechnologyShanghai Tech UniversityShanghaiChina

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