External forces direct morphogenesis and tumorigenesis of the mammary gland

Breast epithelia exist in a constant state of interaction with their surrounding environment. Morphogenesis is the developmental process by which breast cells grow into their surrounding matrix and form the ducts and milk-producing lobules. When morphogenesis breaks down, breast cancer occurs. Traditionally, biologists think of cancer through the framework of genetic mutations. Significant work in the past few decades demonstrated that the mechanical environment plays a critical role in determining growth and malignancy of breast cancers independent of genetic mutations. For example, increasing the stiffness of the extracellular matrix drives phenotypic malignancy through cell-generated contraction. However, the role of forces felt by the tissue due to external causes remains unclear. This dissertation describes experiments that reveal a critical role for external forces in branching morphogenesis and tumorigenesis. The experiments make use of a simple method to apply external compression to mammary epithelial cells embedded in biologically relevant gels./nIn branching, compression mechanically aligned collagen fibers and directed multicellular branch growth along these fibers. Fiber alignment sensing required fascin activity, but did not require RhoA-mediated contraction. Contraction served a separate purpose of generating fiber alignment in collagen networks. These findings suggest that migrating cells sense fiber alignment through fascin-mediated filopodia formation rather than through RhoA-mediated contraction./nIn tumorigenesis, compression encouraged malignant cells to form normal-looking acini, a process called `phenotypic reversion.' A transient compressive force at the one-cell state was sufficient to induce reversion without genetic manipulations or pharmacological treatments. Time-lapse microscopy of the malignant cells revealed that compression restored coherent rotation of malignant cell doublets, a behavior associated with the formation of a phenotypically normal structure. Blocking E-Cadherin eliminated compression sensitivity, indicating that cell-cell communication was required for force-induced reversion./nAs external forces altered the structure of a growing multicellular colony, changes in multicellular structure could affect epithelial mechanics. The mechanical properties of multicellular epithelial structures were measured using an atomic force microscope. Hollow lumen structures were softer than filled lumen structure. The increased stiffness associated with lumen filling could contribute to malignancy during cancer progression./nTaken together, this work demonstrates the importance of external forces during morphogenesis and tumorigenesis of the mammary gland. External compression can direct multicellular migration and encourage malignant cells to re-enter the `normal' morphogenetic program. Cell-cell communication plays an important role in multicellular mechanosensing, contributing to both morphogenesis and mechanosensing in mammary epithelial structures. Further studies of multicellular mechanosensing incorporating the data and techniques presented here could identify new molecular targets for breast cancer treatment and prevention.

Advisor: Fletcher, Daniel A