Pregnancy Restricts an Age-Driven Accumulation of Hybrid Cells in the Mammary Gland [bulk RNA-Seq]
Aging increases breast cancer risk while an early first pregnancy reduces a woman's life-long risk. Several studies have explored the effect of either aging or pregnancy on mammary epithelial cells (MECs), but the combined effect of both remains unclear. Here, we interrogate the functional and transcriptomic changes at single cell resolution in the mammary gland of aged nulliparous and parous mice to discover that pregnancy normalizes age-related imbalances in lineage composition, while also inducing a permanently differentiated cell state. Importantly, we uncover a minority population of Il33-expressing hybrid cells with high cellular potency that accumulate in aged nulliparous mice but is significantly reduced in aged parous mice. Functionally, IL33 treatment of basal, but not luminal, epithelial cells from young mice phenocopies aged nulliparous MECs and promotes formation of organoids with Tp53 knockdown. Collectively, our study demonstrates that pregnancy blocks the age-associated loss of lineage integrity in the basal layer through a decrease in Il33+ hybrid cells, potentially contributing to pregnancy-induced breast cancer protection. Overall design: Bulk RNA sequencing was performed and analyzed by Novogene on sorted basal (CD49fhi/EPCAMlow-med) and luminal (CD49flow/EPCAMhi) populations from 3m NP, 18m NP and 18m P mice (n=3 mice/group). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library80. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.