To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1 +/tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatmentnaive primary tumors, including estrogen receptor (ER) + , HER2 + , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1 +/tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8 + T cells characterized triple-negative and HER2 + cancers but not ER + tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER + tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a highresolution map of cell diversity in normal and cancerous human breast.

E2F transcription factors are major regulators of cell proliferation. The diversity of the E2F family suggests that individual members perform distinct functions in cell cycle control. E2F4 and E2F5 constitute a defined subset of the family. Until now, there has been little understanding of their individual biochemical and biological functions. Here, we report that simultaneous inactivation of E2F4 and E2F5 in mice results in neonatal lethality, suggesting that they perform overlapping functions during mouse development. Embryonic fibroblasts isolated from these mice proliferated normally and reentered from Go with normal kinetics compared to wild-type cells. However, they failed to arrest in G1 in response to p16INK4a. Thus, E2F4 and E2F5 are dispensable for cell cycle progression but necessary for pocket protein-mediated G1 arrest of cycling cells.

The mammary gland is a highly dynamic organ that undergoes profound changes within its epithelium during puberty and the reproductive cycle. These changes are fueled by dedicated stem and progenitor cells. Both short- and long-lived lineage-restricted progenitors have been identified in adult tissue as well as a small pool of multipotent mammary stem cells (MaSCs), reflecting intrinsic complexity within the epithelial hierarchy. While unipotent progenitor cells predominantly execute day-to-day homeostasis and postnatal morphogenesis during puberty and pregnancy, multipotent MaSCs have been implicated in coordinating alveologenesis and long-term ductal maintenance. Nonetheless, the multipotency of stem cells in the adult remains controversial. The advent of large-scale single-cell molecular profiling has revealed striking changes in the gene expression landscape through ontogeny and the presence of transient intermediate populations. An increasing number of lineage cell-fate determination factors and potential niche regulators have now been mapped along the hierarchy, with many implicated in breast carcinogenesis. The emerging diversity among stem and progenitor populations of the mammary epithelium is likely to underpin the heterogeneity that characterizes breast cancer.