UC Berkeley

In characterizing the interface between the mammary epithelium and the microenvironment, it is necessary to first study specific components individually before interpreting the results in a larger context. With this in mind, the cell-cell component of the interface was first studied with respect to self-organization. Since initial experiments showed significant variance in size and morphology, a unique methodology was employed combining a micropatterning approach that confined cells to a cylindrical geometry with an algorithm to quantify changes of cellular distribution over time in order to measure the ability of different cell types to self-organize relative to each other. Using normal human mammary epithelial cells enriched into pools of the two principal lineages, luminal and myoepithelial cells, experiments demonstrated that bilayered organization in mammary epithelium was driven mainly by lineage-specific differential E-cadherin expression, but that P-cadherin contributed specifically to organization of the myoepithelial layer. Disruption of the actomyosin network or of adherens junction proteins resulted in either prevention of bilayer formation or loss of preformed bilayers, consistent with continual sampling of the local microenvironment by cadherins. Together these data show that self-organization is an innate and reversible property of communities of normal adult human mammary epithelial cells.

Next, the cell-extracellular matrix (ECM) component was considered, focusing on the role of extracellular stiffness in both functional differentiation as well as branching morphogenesis. Since functional differentiation of the mammary epithelium involves the production of milk proteins such as caseins, a fluorescent reporter construct with the mouse beta-casein promoter was used as a metric for functional differentiation. This system was used for studies in which extracellular stiffness was tightly controlled and cellular stiffness measured and demonstrated a strong association between extra- and intracellular elasticity as well as functional differentiation in mammary epithelial cells (MECs). A benchmark for biomimetic intracellular elasticity was empirically determined via atomic force microscopy (AFM) measurements on normal mammary epithelium and used in subsequent experiments as a marker for the range of normal intracellular elasticity. The results established that maintenance of beta-casein expression required both laminin signaling and a `soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signaling, led to the loss of beta-casein expression and non-biomimetic intracellular elasticity. Extracellular stiffness was further plummeted with respect to branching morphogenesis by utilizing a three-dimensional culture model composed of type collagen-I. AFM was used to measure local stiffness of matrices either at invasive fronts or away from cell clusters and found the average stiffness at invasive fronts was nearly half the average measurement far from cell clusters. Further mechanistic studies demonstrated the local softening was MMP-dependent and necessary at sufficient extracellular stiffness to permit branching. Taken together, these results demonstrate that extracellular stiffness is a critical cue of the cell-ECM interface that can regulate various processes.

Finally, from studying extracellular stiffness at the cell-ECM interface, it became apparent that matrix organization was critical during branching in collagen I matrices. Initial time course studies demonstrated that local, cellular contractions via the actomyosin machinery generated aligned collagen I tracks at invasive fronts that the multicellular branch co-orient with. Further work, using a novel approach to aligning three-dimensional matrices, demonstrated that aligned collagen I matrices provided a strong directional cue during branching and that cellular machinery for co-orientation was independent from the actomyosin machinery previously mentioned. Orientation analysis in extracted mammary glands provided strong evidence that pre-oriented collagen I tracks exist in the fat pad prior to onset of branching morphogenesis. Moreover, the orientation of the mammary epithelial tree formed during branching morphogenesis is uni-axially biased and co-oriented with the collagen I tracks previously described. Taken together, this study strongly implies the possibility that the fat pad encodes the orientation of the epithelial tree well before branching morphogenesis. Combining the findings from both cell-cell and cell-ECM centric studies gives a greater appreciation for the complexity of decision-making taken on by the epithelium as well as how it must query and alter its interface with the microenvironment during development.