Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Mulu Geletu; Victoria Hoskin; Blerta Starova; Maximilian Niit; Hanad Adan; Bruce Elliott; Patrick Gunning; Leda Raptis

doi:10.3791/60147

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Method Article

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

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DOI:

10.3791/60147

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February 27th, 2020

Mulu Geletu^*¹^,² , Victoria Hoskin^*¹ , Blerta Starova^*¹^,³ , Maximilian Niit¹^,⁴ , Hanad Adan¹ , Bruce Elliott¹ , Patrick Gunning² , Leda Raptis¹

¹Department of Biomedical and Molecular Sciences and Department of Pathology and Molecular Medicine, Queen's University, Kingston, ²Department of Chemical and Physical Sciences, University of Toronto, Mississauga, ³Department of Laboratory Medicine, Brampton Civic Hospital Campus, William Osler Health System, ⁴Latner Thoracic Surgery Research Laboratories

^* These authors contributed equally

Summary

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Abstract

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.

The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins β-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes".

The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce β-casein and WAP.

Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated Rac^V12 increase differentiation, high Rac^V12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of Rac^V12.

Introduction

In normal tissues or tumors, cells have extensive opportunities for adhesion to their neighbors in a three-dimensional organization, and this is mimicked in culture by high density cell growth. Cell-to-cell adhesion is mediated mainly through cadherin receptors, which define cell and tissue architecture. Interestingly, it was recently demonstrated that cadherins also play a powerful role in signal transduction, especially in survival signaling¹. Paradoxically, some of these cell-to-cell adhesion signals emanating from cadherins were recently found to be shared by both differentiation and neoplasia². Here, we describe met....