Matrix compositions and the development of breast acini and ducts in 3D cultures

Abstract

Breast epithelial cells develop into polarized and highly organized acinar and ductal structures in response to stromal cues, including extracellular matrix composition and density, which can in part be reproduced in 3D culture conditions. Here, we present the effects of various 3D in vitro stroma compositions (termed "matrices" or "substrates") on the ability of heterotypic cultures of epithelial and mesenchymal stem cells to organize into acinar and tubular structures. Normal murine mammary gland (NMuMG) cells were cultured, either alone or in combination (30:70) with mouse mesenchymal stem cells (D1), in 3D matrices generated by agarose, collagen, and Matrigel alone or by a combination thereof. After 3-5 d in culture, cell distribution, organization, and the presence of acinus-like and tubule-like structures were determined. The number of acinar structures was significantly higher in cultures grown in combination matrices of agarose with Matrigel or collagen I when compared with cultures grown in Matrigel or collagen I alone (p < 0.05). No tubular structures were formed when agarose was included in the matrix, regardless of the combination. In Matrigel, but not in collagen I/Matrigel microenvironment, the number of tubular structures was significantly increased in NMuMG/D1 coculture when compared with culture of NMuMG cells alone (p < 0.05). By immunohistochemical analysis, NMuMG cells cocultured with D1 cells were shown to form acinar structures with the NMuMG epithelial cells surrounding a lumen composed of dead cells while the D1 cells were mostly peripheral. Immunostaining for laminin indicated the presence of basement membrane when NMuMG cells were grown in Matrigel alone or cocultured with D1 cells in a combination of Matrigel and collagen I. These results indicate that the physical and biochemical properties of the matrix and cellular composition alter the organization of the mammary gland.

IGFBP-5 enhances epithelial cell adhesion and protects epithelial cells from TGFβ1-induced mesenchymal invasion

“…NMuMG cells are able to form luminal, polarised structures similar to the normal mammary alveolar structure, (Swamydas et al , 2010). Our studies confirmed previous findings (Maeda, Johnson, 2005) that NMuMG cells can be separated into stable clones with either epithelial or mesenchymal characteristics.…”

Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases

“…45,46 Our data confirm that D1 mesenchymal stem cells secrete and/or shed many different molecules including the CCL5 and CCL9 chemokines at concentrations much higher than mammary epithelial and tumor cells and that CCL5 and CCL9 in particular promoted 4T1 mammary cell invasion. Those chemokines, CCL5 especially, have been shown to play key roles in breast cancer cell migration as major regulators of cell trafficking and adhesion including of tumor cells, immune cells, and stem cells.…”

“…[43][44][45] Furthermore, normal murine mammary gland (NMuMG) cells formed acini only in the presence of murine D1 mesenchymal stem cells whereas 4T1 murine tumor cells did not. 46 To determine whether chemokines present within the breast tumor microenvironment, especially CCL5 and CCL9, promoted the invasion of mammary tumor cells, we first determined the effects of molecules secreted by epithelial (NMuMG), tumor (4T1) cells, and mesenchymal D1 stem cells on NMuMG and 4T1 cell invasions using 2D and 3D culture conditions. Further, we identified the predominant role of CCL5 and CCL9 in the invasion of 4T1 tumor cells through CCR1 and CCR5 receptor activation and the secretion of MMPs.…”

“…5,14,47,48 For example, D1 mesenchymal stem cells in culture with NMuMG cells promoted the generation of 3D acinus and duct-like structures whereas NMuMG mammary epithelial cells alone did not. 46 Here we investigated the effects of stroma mesenchymal D1 stem cells' secretions especially the chemokines their cell surface compared with 4T1 cells incubated in control conditions (Fig. 5B).…”

The ubiquitin ligase Smurf2 suppresses TGFβ-induced epithelial–mesenchymal transition in a sumoylation-regulated manner

“…33,36,37 Growing evidence suggests that culturing epithelial cells in the context of an extracellular support system such as Matrigel provides a three-dimensional (3D) environment for cells resembling the stromal basement membrane under more 'physiological' conditions. [38][39][40] Accordingly, glandular-derived epithelial cells in a 3D matrix form acini with hollow centers similar to the morphology of these structures in vivo. Importantly, EMT disrupts the acinar morphology that manifests as filling of the hollow centers, outward invasiveness, and dysregulation of the abundance or localization of the epithelial marker protein E-cadherin.…”