Abstract PS3-03-19: Single Cell Proteomic Phenotyping Reveals Mitigation of Abrupt Involution-Induced Alteration in Mammary Epithelium Upon Tamoxifen Treatment

Abstract Background: Epidemiological studies link lack of or short-term breast feeding to increased risk of breast cancer (BC), including triple negative breast cancer (TNBC). However, few models allow interrogation of the underlying biology. To decipher this link, we developed a model of short-term breastfeeding (BF) leading to abrupt involution (AI) versus long-term BF leading to gradual involution (GI) in mice. In our prior work, we showed that AI led to significant increase in estrogen signaling and precancerous changes (hyperplasia, squamous metaplasia) in mammary glands (MG) (Basree, et al Br Can Res 2019). Further, AI glands are characterized by a notable increase in alveolar progenitor (AP) population, the cells of origin of TNBC. In this study, we addressed the hypothesis that the anti-estrogen tamoxifen (TAM) could revert the adverse effects of abrupt involution/lack of breast feeding by deep interrogation of shifts in cellular phenotypes through CyTOF single cell proteomics. Methods: FVB/N female mice (∼8 week old) were bred and allowed to nurse six pups/dam at partum. All pups were removed on postpartum (PP) day 7 to induce AI. On PP day 8, 5mg-60day sustained release tamoxifen pellet or placebo pellet (PLAC) was implanted in the subscapular region of the AI mice. MGs were harvested on PP day 28 and 120 from three groups: 1) TAM-treated AI; 2) PLAC-treated AI; 3) age matched nulliparous mice. FFPE sections were used for histology and immunohistochemistry. Immune cell composition and epithelial subpopulations from PP day 28 and day 120 MGs were evaluated by FACS and subjected to CyTOF using an established 40-antibody murine mammary targeted proteome panel (Gray, et al Cell Rep 2024). The Ingest algorithm was used to integrate and analyze mass cytometry datasets. Results: Overall, TAM treatment for 60 days completely abrogated the hyperplastic changes induced by AI in MGs, including reduction in proliferation index, collagen deposition, macrophages, and alveolar AP cells in the AI glands. As seen in our prior work, at day 120, abrupt involution-placebo glands (relative to nulliparous MGs) demonstrated marked enrichment of AP (1.5-fold) and basal (BA 1.6-fold) cells at the expense of hormone sensing (HS) cells. Analyses of MGs from the abrupt involution-tamoxifen group at day 120 revealed a significant increase (3-fold) in hormone sensing (HS) epithelial cells and an attendant decrease in AP cells compared to placebo. Further, abrupt involution-placebo glands show upregulation of AP markers (CD61, TSPAN8, SSEA1, CD14) and basal markers (CD54, CD47, CD104, CD49F) in the AP cells and higher expression of BA markers and reduced expression of HS markers in the HS cells. TAM treatment reversed these aberrant changes in abrupt involution-placebo glands in fact inducing a shift in HS cells to an involution-like state. Integration of the AI-PLAC/TAM data with lineage trajectory of these three cell types across the gestation-lactation-involution cycle revealed that AI-AP cells exhibited a mix of post-involutional and gestational states at day 120. TAM led to a 4.1-fold higher proportion of post-involutional-like AP cells and a 10.2-fold lower proportion of the aberrant gestational-like cells at day 120. Similar changes were observed in BA cells but not in HS cells upon TAM treatment. Conclusion: Further understanding of the biological impact of short/no breast feeding is critical and this work suggests that TAM treatment offers marked protection against AI-induced precancerous changes in a murine model. TAM restored long-term lineage balance after AI, altering AP differentiation towards a typical post-involution state as opposed to AI-induced hyperplasia. The corrective effect of TAM provides a rationale for short-term TAM treatment for women who are unable to BF to reduce risk of BC. Citation Format: S. Majumder, K. G. Gray, N. Shinde, R. Mawalkar, A. Zhang, M. Basree, K. Ormiston, X. Zhang, R. Ganju, G. Sizemore, B. Ramaswamy, D. G. Stover. Single Cell Proteomic Phenotyping Reveals Mitigation of Abrupt Involution-Induced Alteration in Mammary Epithelium Upon Tamoxifen Treatment abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-03-19.