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A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age. Supplementary Information The online version contains supplementary material available at 10.1007/s10911-021-09495-2.

A robust breast cancer prevention strategy requires risk assessment biomarkers for early detection. We show that expression of ELF5, a transcription factor critical for normal mammary development, is downregulated in mammary luminal epithelia with age. DNA methylation of the ELF5 promoter is negatively correlated with expression in an age-dependent manner. Both ELF5 methylation and gene expression were used to build biological clocks to estimate chronological ages of mammary epithelia. ELF5 clock-based estimates of biological age in luminal epithelia from average-risk women were within three years of chronological age. Biological ages of breast epithelia from BRCA1 or BRCA2 mutation carriers, who were high risk for developing breast cancer, suggested they were accelerated by two decades relative to chronological age. The ELF5 DNA methylation clock had better performance at predicting biological age in luminal epithelial cells as compared with two other epigenetic clocks based on whole tissues. We propose that the changes in ELF5 expression or ELF5-proximal DNA methylation in luminal epithelia are emergent properties of at-risk breast tissue and constitute breast-specific biological clocks. Prevention Relevance: ELF5 expression or DNA methylation level at the ELF5 promoter region can be used as breast-specific biological clocks to identify women at higher than average risk of breast cancer.

A robust breast cancer prevention strategy requires risk assessment biomarkers for early detection. We show that expression of ELF5, a transcription factor critical for normal mammary development, is downregulated in mammary luminal epithelia with age. DNA-methylation of the ELF5 promoter is negatively correlated with expression in an age-dependent manner. Both ELF5 methylation and gene expression were used to build biological clocks to estimate chronological ages of mammary epithelia. ELF5 clock-based estimates of biological age in luminal epithelia from average-risk women were within 3 years of chronological age. Biological ages of breast epithelia from BRCA1 or BRCA2 mutation carriers, who were high risk for developing breast cancer, suggested they were accelerated by two decades relative to chronological age. The ELF5 DNA methylation clock had better performance at predicting biological age in luminal epithelial cells as compared to two other epigenetic clocks based on whole tissues. We propose that the changes in ELF5 expression or ELF5-proximal DNA methylation in luminal epithelia are emergent properties of at-risk breast tissue and constitute breast-specific biological clocks.