Functional delineation of the luminal epithelial microenvironment in breast using cell-based screening in combinatorial microenvironments

Microenvironment signals are potent determinants of cell fate and arbiters of tissue homeostasis, however un-derstanding how different microenvironment factors coordinately regulate cellular phenotype has been experi-mentally challenging. Here we used a high-throughput microenvironment microarray comprised of 2640 unique pairwise signals to identify factors that support proliferation and maintenance of primary human mammary luminal epithelial cells. Multiple microenvironment factors that modulated luminal cell number were identified, including: HGF, NRG1, BMP2, CXCL1, TGFB1, FGF2, PDGFB, RANKL, WNT3A, SPP1, HA, VTN, and OMD. All of these factors were previously shown to modulate luminal cell numbers in painstaking mouse genetics experi-ments, or were shown to have a role in breast cancer, demonstrating the relevance and power of our high-dimensional approach to dissect key microenvironmental signals. RNA-sequencing of primary epithelial and stromal cell lineages identified the cell types that express these signals and the cognate receptors in vivo. Cell-based functional studies confirmed which effects from microenvironment factors were reproducible and robust to individual variation. Hepatocyte growth factor (HGF) was the factor most robust to individual variation and drove expansion of luminal cells via cKit+ progenitor cells, which expressed abundant MET receptor. Luminal cells from women who are genetically high risk for breast cancer had significantly more MET receptor and may explain the characteristic expansion of the luminal lineage in those women. In ensemble, our approach provides proof of principle that microenvironment signals that control specific cellular states can be dissected with high-dimensional cell-based approaches.

Automated summary

Microenvironment signals have a significant impact on cell fate and tissue homeostasis. Understanding how different microenvironment factors regulate cellular phenotype has been challenging. In this study, a high-throughput microenvironment microarray was used to identify factors that support the proliferation and maintenance of primary human mammary luminal epithelial cells. Multiple factors that modulate luminal cell number were identified and their effects were confirmed using RNA sequencing and cell-based functional studies. Hepatocyte growth factor (HGF) was found to be robust to individual variation and played a role in expanding luminal cells. Our approach demonstrates the power of high-dimensional cell-based approaches in dissecting microenvironmental signals.