Synthesis and Analysis of Mammary Gland Organoids

The mammary gland is an important model system for several reasons but principal among them is that structural and signaling motifs found in the mammary gland recur throughout other organ systems and that due to surgical discard material, human tissue is readily available for study. The mammary gland is composed of a bilayered, tubular epithelial tree embedded in stroma. This system has been leveraged in-vitro in three-dimensional (3D) cell culture to model the impact of stromal-epithelial interactions at the scale of organoids. To better understand cells interacting with their microenvironment, we developed DNA-based chemical tools to control the three-dimensional position and adhesion of cells. We optimized a bipartite system to label cell membranes with fatty acid modified, single strand DNA (ssDNA) in order to encode specific binding properties unto the cells. We demonstrate that this approach is synthetically simple, produces more robust labeling than comparable chemistries, and results in labeling of primary cells. We optimized the kinetics of this ssDNA labeling system and developed a capping strategy to effectively quench the reactivity of residual ssDNA on cell surfaces. We combined this ssDNA system with photolithographically defined microwells to rapidly cast multicellular structures of arbitrary shape, size and throughput. Finally, we studied primary organoids from multiple reduction mammoplasty samples with single cell RNA sequencing (scRNA seq) in order to enumerate the cell types and heterogeneity within the epithelial compartment of the mammary gland. Greater understanding of the populations of cells within the mammary epithelial system and how those cells signal to their environment are likely to yield important insights for mammary pathologies such as breast cancer and guide future treatment regimes