Three-dimensional culture models of normal and malignant breast epithelial cells

ABSTRACT Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype1. Important signals are lost when cells are cultured _ex vivo_ on two-dimensional plastic

substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM)2. These 3D culture assays allow

phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form

disorganized, proliferative and nonpolar colonies3. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to

basement membrane–like gels4,5,6,7. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype _ex vivo_. We describe

here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for

molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel8 (Fig. 1). By lrECM, we refer to the solubilized

extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells9. For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in

which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 (Fig. 1 and Fig. 2). Access through your institution Buy or

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DETERMINISTIC CULTURING OF SINGLE CELLS IN 3D Article Open access 02 July 2020 MAMMARY EPITHELIAL MORPHOGENESIS IN 3D COMBINATORIAL MICROENVIRONMENTS Article Open access 10 December 2020

COMPRESSIVE STRESS-MEDIATED P38 ACTIVATION REQUIRED FOR ERΑ + PHENOTYPE IN BREAST CANCER Article Open access 29 November 2021 REFERENCES * Bissell, M.J., Radisky, D.C., Rizki, A., Weaver,

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work of many members of the Bissell laboratory over many years. We apologize to those whose work could not be cited owing to space limitations and have cited reviews where possible. This

work was supported by grants from the Office of Biological and Environmental Research of the US Department of Energy (DE-AC03-76SF00098 and a Distinguished Fellow Award to M.J.B.), the US

National Cancer Institute (CA64786 to M.J.B.; CA57621 to Zena Werb and M.J.B.) and the Breast Cancer Research Program of the US Department of Defense (Innovator Award DAMD17-02-1-438 to

M.J.B). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Life Sciences Division, Lawrence Berkeley National Laboratory, MS 977R225A, Berkeley, 94720, California, USA Genee Y Lee, Paraic A

Kenny, Eva H Lee & Mina J Bissell Authors * Genee Y Lee View author publications You can also search for this author inPubMed Google Scholar * Paraic A Kenny View author publications You

can also search for this author inPubMed Google Scholar * Eva H Lee View author publications You can also search for this author inPubMed Google Scholar * Mina J Bissell View author

publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Mina J Bissell. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY VIDEO 1 Time-lapse movies of HMT-3522 T4-2 cells treated with vehicle and cultured in the 3D on-top assay for 5 d.

Phase contrast images were taken at 1-h intervals. (MOV 1420 kb) SUPPLEMENTARY VIDEO 2 Time-lapse movies of HMT-3522 T4-2 cells treated with an EGFR inhibitor, AG1478, and cultured in the 3D

on-top assay for 5 d. Phase contrast images were taken at 1-h intervals. (MOV 1193 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Lee, G., Kenny,

P., Lee, E. _et al._ Three-dimensional culture models of normal and malignant breast epithelial cells. _Nat Methods_ 4, 359–365 (2007). https://doi.org/10.1038/nmeth1015 Download citation *

Published: 29 March 2007 * Issue Date: April 2007 * DOI: https://doi.org/10.1038/nmeth1015 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get

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