Figure 4.1.

Identification of mammary epithelial tumor–promoting functions. A novel mouse mammary epithelial cell model for investigating the impact of oncogene activation and tumor suppressor inactivation on mammary tumorigenesis is summarized here. Primary mouse mammary epithelial cells (MMECs) are isolated from wild-type or mutant mice and immortalized (iMMECs) by concurrent inactivation of the Rb and p53 pathways. iMMECs can be grown and studied in standard two-dimensional (2D) in vitro culture. The functional properties of iMMECs can also be interrogated in a 3D morphogenesis assay, where iMMECs from wild-type mice initially form a solid acinus, as seen by β-catenin staining (green) to outline the cellperiphery and Dapi counterstaining (blue) for DNA. With time (14 to 21days), cell death in the acinar center creates a hollow lumen, closely mimicking duct formation in mammary epithelium in vivo. Oncogenic events perturb 3D morphogenesis, as illustrated in the phase-contrast images of the structures generated by Her2/neu-expressing iMMECs (larger, but hollow, acini) and by activated Ras-expressing iMMECs (nonacinar structures invading the basement membrane). iMMECs from wild-type mice are poorly, clonally tumorigenic on orthotopic transplantation into the mammary fat pad. Tumors formed by wild-type iMMECs are distinctly adenocarcinomas (right) compared with normal mouse mammary tissue (left), as indicated by histologic analysis of H&E–stained sections.