Next day 3D cultures were overlaid with 2% of BM-matrix in DMEM/F12 media. the cellular microenvironment. Breast epithelia can therefore maintain a proliferative plasticity that correlates with nuclear remodelling. However, the changes in nuclear architecture are cell lineage-specific and do not occur in fibroblasts, and moreover they are overcome in breast malignancy cells. Keywords: breast cancer, breast mammary gland, cell cycle, cell senescence, extracellular matrix, integrin, nuclear structure Introduction Interphase nuclei are sophisticated organelles that contain a number of compartments involved with determining transcript profiles and cell fates. Within the interphase cell, higher order nuclear organization has widespread effects on tissue-specific gene expression, and structural remodeling of the nucleus has a key influence on cell phenotype.1 Several nuclear compartments including nucleoli, nuclear speckles and transcription centers have been characterized, and chromosomes are partitioned into discrete territories.2-6 However, little is known about the mechanisms that determine the number of nuclear compartments, or how their sub-nuclear distributions and dynamic properties are controlled.7-9 In addition, the extent to which spatial nuclear organization defines cell fate decisions is not well established.10 Understanding how the internal structure of nuclei is regulated is important 2-MPPA because defects in nuclear organization contribute to diseases such as cancer.11 Cells in vivo function in 3-dimensional tissues. However, the experimental analysis of mechanisms controlling intracellular processes, including nuclear business, usually involves planar 2-dimensional cultures of cells on plastic dishes. Contemporary opinion now indicates that this 3D microenvironment within tissues has a profound influence on cell phenotype, by controlling gene expression.12,13 This cellular niche includes the extracellular matrix (ECM), soluble factors and other cells, and all of these, together with the dimensionality of the niche itself, determine the fate and phenotype of cells.14-18 We therefore hypothesized that one mechanism to explain the link between the microenvironment 2-MPPA of a cell and its fate is via a control on the number and function of nuclear compartments.19 Here we address this hypothesis using breast epithelia, a paradigm for understanding the molecular basis of cellular differentiation and cancer progression. Using this cell model, we demonstrate that this cellular microenvironment controls the internal architecture of nuclei, and that the mechanism is via a novel form of cell cycle arrest. Moreover, while the link between matrix dimensionality, cell cycle arrest and nuclear architecture operates in normal epithelia, it is uncoupled in breast cancer. Results Cellular microenvironment dictates the nuclear complexity of breast epithelia To determine mechanisms controlling nuclear architecture, we compared the distribution and number of nuclear sub-compartments of breast epithelia cultured on planar 2-dimensional substrata (2D culture) and 3-dimensional laminin-rich ECM MLLT3 gels (LrECM) (3D culture). In 2D culture, human MCF10A breast epithelia proliferated to form linens of cells, which contained multiple fibrillarin-containing nucleoli (Fig.?1A-B). The number of these sub-nuclear compartments was impartial of either cell confluence or the type of ECM substrata used (Fig.?S1). In contrast to planar culture, cells in 3D culture formed multicellular acini resembling in vivo alveoli (Fig.?1C).14 Under these conditions the spatial organization 2-MPPA of nuclear compartments became simplified, with the number of nucleoli reducing to one in most cells, by 14C21?days in 3D culture (Fig.?1B-C). Primary mammary epithelial cells isolated directly from mice (MECs), also contained fewer nucleoli in 3D culture than on planar substrata, particularly after 6?days in culture (Fig.?1D-E). These results suggest that the cellular microenvironment determines the internal spatial arrangement of nuclei. Open in a separate window Physique 1. Cellular microenvironment dictates the nucleolar complexity of breast epithelia (A-C) MCF10A. Representative low and high power views of cells in 2D (A) and 3D (C) stained with lamin-B1 (green) and fibrillarin (red); upper images are maximum imaging projections and lower images are high magnification views of confocal slices. The areas enlarged are shown by dotted lines and nucleoli indicated by arrows. The percentage of cells made up of 1, 2, 3, 4, or >5 nucleoli in planar culture (2D n = 192 nuclei for this representative experiment from at least triplicates); or after 7, 14 and 21-d on 3D culture on LrECM (n = 172, 177, 205 respectively) are shown (B). (D, E) Primary.