Introduction: Multiple epidemiological studies have shown that prolonged breastfeeding is associated with a reduced risk of developing triple-negative/basal-like breast cancers (TN/BLBC). However, no preclinical models that delineate the mechanism of this link exist. This understanding is critical not only to prevent TN/BLBC, but also to address disparity in breast cancer outcomes, as African-American women have a higher incidence of TN/BLBC and lower prevalence of breastfeeding. We present our data using mouse models that provides new insights into this link.

Experimental procedure: We modeled gradual involution (GI) and abrupt involution (AI) of mammary glands (MGs) in wild type FVB/N mice. Uniparous mice were assigned to AI or GI cohorts either by removal of all pups on day7 postpartum (AI) or allowing the pups to naturally wean (GI). MGs were harvested for analysis on postpartum day28, day56 and day120. We assessed MG morphology/histology using whole mounts and H&E stained sections, collagen deposition using Trichrome and PicroSirus red staining, inflammatory markers and immune cell infiltration using immunohistochemistry. Mammary epithelial cell hierarchy was analyzed by Fluorescence-Activated Cell Sorting of a single cell suspension prepared from the MGs. Gene expression was analyzed in mouse mammary epithelial cells using Affymatrix Gene ChIP Mouse Transcriptome array 1.0 and Gene Set Enrichment Analysis (GSEA) was used to analyze gene expression data.

Summary: Abruptly involuted MGs exhibited altered morphology including denser stroma, increased collagen deposition with higher levels of Type I collagen, increased inflammation (pStat3-Y705), increased immune cell infiltration and proliferation compared to GI glands. The mammary epithelial cell hierarchy was disrupted with marked expansion of the luminal progenitor (LP) population in the AI glands but not in the GI glands, a trend frequently observed in women heterozygous for BRCA1 mutation at higher risk of developing BLBC. Enrichment of an LP gene signature and Notch pathway was observed in mouse LP cells isolated from the AI glands. Most strikingly, the AI glands developed alveolar hyperplasia, and squamous metaplasia within 4 months of removal of the pups.

Conclusion: Involution leading to a pro-inflammatory milieu in the MG is well established. Whether involution that happens gradually after prolonged breastfeeding differs in its effect on MGs has not been well studied. Using novel animal modeling to study the differences in MGs following abrupt vs gradual involution, we report a distinct morphology in the mammary tissue favoring pro-carcinogenic changes following AI. Furthermore, we show for the first time, expansion of the LP population in AI glands, which is known to be cell of origin for TN/BLBC potentially linking the risk of TN/BLBC with lack of breastfeeding. Further studies are ongoing.

Citation Format: Mustafa M. Basree, Neelam Shinde, Christopher Koivisto, Maria Cuitino, Raleigh Kladney, Allen Zhang, Hee Kyung Kim, Anthony Trimboli, Jianying Zhang, Gustavo W. Leone, Gina M. Sizemore, Sarmila Majumder, Bhuvaneswari Ramaswamy. Breastfeeding protects against pro-tumorigenic changes in the mammary gland by limiting epithelial luminal progenitor cell expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2242.