3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2014 Sep 24

PMID 25245692

Citations 87

Authors

Kim-Vy Nguyen-Ngoc

Eliah R Shamir

Robert J Huebner

Jennifer N Beck

Kevin J Cheung

Andrew J Ewald

Affiliations

Soon will be listed here.

Abstract

Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called "organoids," to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems.

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References

Griffith L, Swartz M . Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol. 2006; 7(3):211-24. DOI: 10.1038/nrm1858. View

Gudjonsson T, Ronnov-Jessen L, Villadsen R, Bissell M, Petersen O . To create the correct microenvironment: three-dimensional heterotypic collagen assays for human breast epithelial morphogenesis and neoplasia. Methods. 2003; 30(3):247-55. PMC: 2933212. DOI: 10.1016/s1046-2023(03)00031-8. View

Hogg N, Harrison C, Tickle C . Lumen formation in the developing mouse mammary gland. J Embryol Exp Morphol. 1983; 73:39-57. View

Nelson C, Bissell M . Modeling dynamic reciprocity: engineering three-dimensional culture models of breast architecture, function, and neoplastic transformation. Semin Cancer Biol. 2005; 15(5):342-52. PMC: 2933210. DOI: 10.1016/j.semcancer.2005.05.001. View

Nguyen-Ngoc K, Ewald A . Mammary ductal elongation and myoepithelial migration are regulated by the composition of the extracellular matrix. J Microsc. 2013; 251(3):212-23. PMC: 3978143. DOI: 10.1111/jmi.12017. View

Conklin M, Eickhoff J, Riching K, Pehlke C, Eliceiri K, Provenzano P . Aligned collagen is a prognostic signature for survival in human breast carcinoma. Am J Pathol. 2011; 178(3):1221-32. PMC: 3070581. DOI: 10.1016/j.ajpath.2010.11.076. View

Provenzano P, Inman D, Eliceiri K, Knittel J, Yan L, Rueden C . Collagen density promotes mammary tumor initiation and progression. BMC Med. 2008; 6:11. PMC: 2386807. DOI: 10.1186/1741-7015-6-11. View

Sternlicht M, Kouros-Mehr H, Lu P, Werb Z . Hormonal and local control of mammary branching morphogenesis. Differentiation. 2006; 74(7):365-81. PMC: 2580831. DOI: 10.1111/j.1432-0436.2006.00105.x. View

Simian M, Hirai Y, Navre M, Werb Z, Lochter A, Bissell M . The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development. 2001; 128(16):3117-31. PMC: 2785713. DOI: 10.1242/dev.128.16.3117. View

10.

Levental K, Yu H, Kass L, Lakins J, Egeblad M, Erler J . Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell. 2009; 139(5):891-906. PMC: 2788004. DOI: 10.1016/j.cell.2009.10.027. View

11.

Sternlicht M . Key stages in mammary gland development: the cues that regulate ductal branching morphogenesis. Breast Cancer Res. 2006; 8(1):201. PMC: 1413974. DOI: 10.1186/bcr1368. View

12.

Hinck L, Silberstein G . Key stages in mammary gland development: the mammary end bud as a motile organ. Breast Cancer Res. 2005; 7(6):245-51. PMC: 1410762. DOI: 10.1186/bcr1331. View

13.

Provenzano P, Eliceiri K, Inman D, Keely P . Engineering three-dimensional collagen matrices to provide contact guidance during 3D cell migration. Curr Protoc Cell Biol. 2010; Chapter 10:Unit 10.17. PMC: 3905986. DOI: 10.1002/0471143030.cb1017s47. View

14.

Cheung K, Gabrielson E, Werb Z, Ewald A . Collective invasion in breast cancer requires a conserved basal epithelial program. Cell. 2013; 155(7):1639-51. PMC: 3941206. DOI: 10.1016/j.cell.2013.11.029. View

15.

Ewald A, Brenot A, Duong M, Chan B, Werb Z . Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev Cell. 2008; 14(4):570-81. PMC: 2773823. DOI: 10.1016/j.devcel.2008.03.003. View

16.

Debnath J, Muthuswamy S, Brugge J . Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods. 2003; 30(3):256-68. DOI: 10.1016/s1046-2023(03)00032-x. View

17.

Huebner R, Lechler T, Ewald A . Developmental stratification of the mammary epithelium occurs through symmetry-breaking vertical divisions of apically positioned luminal cells. Development. 2014; 141(5):1085-94. PMC: 3929409. DOI: 10.1242/dev.103333. View

18.

Wolf K, Alexander S, Schacht V, Coussens L, von Andrian U, van Rheenen J . Collagen-based cell migration models in vitro and in vivo. Semin Cell Dev Biol. 2009; 20(8):931-41. PMC: 4021709. DOI: 10.1016/j.semcdb.2009.08.005. View

19.

Provenzano P, Eliceiri K, Campbell J, Inman D, White J, Keely P . Collagen reorganization at the tumor-stromal interface facilitates local invasion. BMC Med. 2006; 4(1):38. PMC: 1781458. DOI: 10.1186/1741-7015-4-38. View

20.

Nguyen-Ngoc K, Cheung K, Brenot A, Shamir E, Gray R, Hines W . ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc Natl Acad Sci U S A. 2012; 109(39):E2595-604. PMC: 3465416. DOI: 10.1073/pnas.1212834109. View