Abstract
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 stem/progenitor cells, however, 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 differentiated cell state. Importantly, we uncover a minority population of Il33-expressing epithelial cells that express both luminal and basal markers (i.e. hybrid), which accumulate in aged nulliparous mice but are significantly reduced in aged parous mice. Functionally, IL33 treatment of mammary epithelial cells from young mice phenocopies aged nulliparous epithelial cells, induces proliferation and promotes formation of organoids with Trp53 knockdown. Collectively, our study demonstrates that pregnancy blocks the age-associated imbalances in lineage integrity in the basal layer, including a decrease in Il33+ hybrid cells, that could potentially contribute to pregnancy-induced breast cancer protection.
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Data generated or analyzed during this study are included in this published article (and its supplemental information files). Data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplemental Materials. Single-cell RNA-sequencing data generated in this study has been deposited in the Gene Expression Omnibus (GEO) with the primary accession code GSE272932 and can be explored at https://aging-mouse-pregnancy.cells.ucsc.edu. The published single-cell RNA-sequencing data used in this study are available in the GEO database under accession codes GSE216542, GSE205573, GSM2967054, and GSE195665, and in the ArrayExpress database under the accession code E-MTAB-13664. Source data are provided with this paper.
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Acknowledgements
We thank Bari Nazario and Patricia Lovelace for their help with flow cytometry. The FACS Aria instrument was funded by NIH grant 1S10RR02933801 and Cytoflex RRID SCR_021149 and grant NIH S10OD030423. We thank Benjamin Abrams, UCSC Life Sciences Microscopy Center, RRID: SCR_021135 for technical support during image acquisition and processing. We thank Gunsagar Singh Gulati for advice on data integration and Bryce Manso for flow cytometry analysis of immune cells. We also thank the animal facility core members for animal maintenance. We thank Camilla Forsberg, Lindsay Hinck, Aaron Newman, and members of the Sikandar lab for critical feedback on the manuscript. The authors declare no competing interests. This work was supported by the Hellman Fellows Award and startup funds (to S.S.S), and NIH T-32 (5T32GM133391-04) and NIH/NCI F31 (1F31CA294932-01A1) (to A.O). S.S.S is also supported by the NIH/NCI (R37CA269754).
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S.S.S. and A.O. conceived and designed the study. A.O. performed most of the experiments and analyzed the data with assistance from V.H.A, M.D., and S.K., and under the supervision of S.S.S. V.H.A. collected samples for single-cell RNA sequencing and performed experiments with human cells. P.M. performed all bioinformatic analysis under supervision of S.S.S. A.O., and S.S.S. wrote the manuscript with contributions from P.M. and V.H.A. All authors commented on the manuscript.
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Olander, A., Medina, P., Haro Acosta, V. et al. Divergent aging of nulliparous and parous mammary glands reveals IL33+ hybrid epithelial cells. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68611-0
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DOI: https://doi.org/10.1038/s41467-026-68611-0
