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The advent of targeted therapies for cancer has provoked interest in experimental models for the systematic study of oncogene dependence. To that end, we developed a three-dimensional (3D) culture system to analyze the responses of primary mouse mammary epithelial cells to the induction and deinduction of oncogenes. Mammary cells derived from normal virgin mice, or from tritransgenic mice ( TetO-MYC;TetO-Kras G12D ;MMTV-rtTA)  in which  MYC  and mutant  Kras  can be regulated by doxycycline, develop from single cells into polarized acini. Lumen formation occurs without apparent apoptosis, and the hollow spheres of cells enlarge by division, with metaphase plates oriented perpendicularly to the apical surface. When  MYC  and  Kras G12D   are induced, the acini enlarge and form solid, depolarized spheres. Upon deinduction of  MYC  and  Kras G12D   the solid structures regress, leaving a repolarized monolayer of viable cells. These cells display a phenotype consistent with progenitors of mammary epithelium: They exclude Hoechst dye 33342, and reform acini in 3D cultures and repopulate mammary fat pads more efficiently than cells harvested from uninduced acini. Moreover, cells in the surviving spheres retain the ability to respond to reinduction and thus may represent the type of cells that give rise to recurrent tumors.

genes and development

Regulation of transgenes in three-dimensional cultures of primary mouse mammary cells demonstrates oncogene dependence and identifies cells that survive deinduction