Cellular Microenvironment Influences the Ability of Mammary Epithelia to Undergo Cell Cycle

The use of cell culture models is a principal and fundamental technology used in understanding how mammalian cells work. However, for some cell types such as mammary epithelia, the lines selected for extended culture are often transformed or have chromosomal abnormalities, while primary cultures have such a curtailed lifespan that their use is restricted. For example, mammary luminal epithelial cells (MECs) are used to study mechanisms of breast cancer, but the proliferation of primary cell cultures is highly limited. Here we describe the establishment of a new culture system to allow extended analysis of cultures of primary mouse MECs. In 2D monolayer culture, primary MECs showed a burst of proliferation 2–3 days post isolation, after which cell cycle decreased substantially. Addition of mammary epithelial growth factors, such as Epidermal Growth Factor, Fibroblast Growth Factor-2, Hepatocyte Growth Factor, and Receptor Activator for Nuclear Factor kB Ligand, or extracellular matrix proteins did not maintain their proliferation potential, neither did replating the cells to increase the mitogenic response. However, culturing MECs directly after tissue extraction in a 3D microenvironment consisting of basement membrane proteins, extended the time in culture in which the cells could proliferate. Our data reveal that the cellular microenvironment has profound effects on the proliferative properties of the mammary epithelia and is dominant over growth factors. Moreover, manipulating the cellular environment using this novel method can maintain the proliferative potential of primary MECs, thus enabling cell cycle to be studied as an endpoint after gene transfer or gen

3D culture maintains the potential of MECs to proliferate when they are subsequently returned to conventional 2D culture.

<p>(a) MECs were plated directly onto 3D BM-matrix, treated with EdU each day, and confocal projection images were obtained. (b) The percentage of EdU positive nuclei was determined in comparison to the total number of DAPI-staining cells, each day over 6-d. (c) 3D acini were isolated after 2-d culture in 3D BM-matrix by washing in PBS-EDTA to dissolve the matrix followed by centrifugation to recover the acini, which were replated onto 2D collagen I. (d) The cells proliferated and emigrated from the acini. Proliferation in the 2D cultures was determined by EdU incorporation each day after replating. (e) Proliferation was determined each day in 3D culture and then in the resulting 2D cultures. Cells were cultured in 3D for (i) 2-d, (ii) 3-d, or (iii) 7-d before the acini were isolated and replated onto 2D collagen I. Statistical significance determined by ANOVA is indicated: *** = p<0.001. Here we have only included the statistical differences in the %EdU positive cells between the last day of culture in 3D and the first 2 days of culture on 2D.</p

3D culture retains, but does not reset, the ability of cells to proliferate subsequently in 2D.

<p>MECs cultured in 2D for (a) 2-d, (b) 3-d, or (c) 5-d were replated onto 3D BM-matrix for 2-d, the acini were then isolated using PBS-EDTA and subsequently replated back onto 2D collagen-coated dishes. Proliferation was assessed each day. Statistical differences (ANOVA) in the %EdU positive cells between the last day of culture in 3D and the first day of culture on 2D are indicated.</p

Primary MECs display limited proliferation potential in 2D culture.

<p>(a) MECs were isolated from pregnant mice and plated onto collagen I coated coverslips in complete media. (i) The percentage of proliferating cells was determined by EdU incorporation on each day for 6-d after isolation. Statistical significance determined by ANOVA is indicated: *** = p<0.001. (ii) Cells were co-stained with keratin 5 to detect the myoepithelial cells. Scale bar: 13 µm. (b) MECs from pregnancy days 12–14 (mid-grey) and 10 week old virgin (light-grey) mice were isolated and treated as above, and their proliferation compared to the cells isolated at pregnancy days 16–18 (black) by EdU incorporation. There were no significant differences in proliferation between MECs from ducts and alveoli within each time point (not shown on the graph).</p

Specific β-containing integrins exert differential control on proliferation and two-dimensional collective cell migration in mammary epithelial cells

Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration

An integrin-ILK-microtubule network orients cell polarity and lumen formation in glandular epithelium

The extracellular matrix has a crucial role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues, however the underlying mechanisms remain elusive. By using Cre-Lox deletion we show that β1-integrins are required for normal mammary gland morphogenesis and lumen formation, both in vivo and in a 3D primary culture model where epithelial cells directly contact basement membrane. Downstream of basement membrane-β1-integrins, Rac1 is not involved, however ILK is needed to polarize microtubule plus ends at the basolateral membrane and disrupting each of these components prevents lumen formation. The integrin-microtubule axis is necessary for the endocytic removal of apical proteins from the basement membrane-cell interface and for internal Golgi positioning. We propose that this integrin-signalling network controls the delivery of apical components to the correct surface and thereby governs the orientation of polarity and development of lumens