Pregnancy, lactation, and involution-induced remodeling and cell state changes in breast tissues of healthy women

Pregnancy, lactation, and involution have opposing effects on breast cancer incidence and tumor aggressiveness. While breast feeding and subsequent involution protects against breast cancers, pregnancy-associated breast cancers (breast cancers diagnosed during pregnancy and within 2 years postpartum) are associated with poor outcome. Clear understanding of these opposing effects of reproductive cycle needs single cell resolution of breasts during pregnancy, lactation and involution. We recently reported a reference single nucleus chromatin accessibility and transcriptome atlas of breast tissues of women of diverse genetic ancestry and reported 10 major cell types in the breasts. These include three epithelial cell types [luminal hormone sensing (LHS), luminal adaptive secretory precursors (LASP), and basal-myoepithelial cells (BM)], two adipocyte subtypes, two endothelial cell subtypes, T cells, macrophages, and fibroblasts. In this study, we generated single nucleus atlas of breast tissues of healthy women collected during pregnancy, lactation, and involution. Pregnancy, lactation, and involution are associated with dramatic changes in cell type proportions and cell state shifts within a cell type. LASP cell numbers increased from 24% in the normal reference breasts to 71% during pregnancy, 63% during lactation but returned to 25% during involution. There is also a dramatic shift in LASP cell states during pregnancy, lactation and involution. While the LASP cells in the normal reference breasts and breasts during pregnancy were in both LASP-basal-luminal (LASP-BL) and LASP-alveolar (LASP-AP) states, LASP-AP cells were dominant in lactating and involuting breasts. Increase in LASP cells during pregnancy and lactation is at the expense of BM cells, which reduced from 27% in the normal reference breasts to 7-9% during pregnancy and lactation. While T cell proportion was similar between normal reference and pregnant breasts (2-3%), it increased to 7-8% during lactation and involution, which a recent study has suggested to be responsible for breast feeding-associated protection against breast cancer. Increase in T cells during lactation and involution is likely due to expansion of tissue resident T (T RM ) cells as T cells of lactating and involuting breasts expressed higher levels of T RM cell markers CD69 and CXCR6. We present pregnancy, lactation and involution-associated chromatin accessibility and transcriptome changes at individual gene and cell type levels. Several biomarkers associated with these changes have been identified, which will be a useful resource to mechanistically evaluate evolution of breasts during reproductive history and breast cancer development.