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We thank C. Thai, A. Sanchez, T. Chevez, A. Nunez and M. V. Robles for their help with tissue collection and clinical information from the women participating in our study. We also thank M. Miyano for assistance with immunofluorescence images, M. Basam for help with coding, and our patient advocates S. Samson and S. Preto for providing much needed context. Funding was provided by: DOD CDMRP (BC141351 Era of Hope Scholar Award and BC181737), Hilton-Ludwig Foundation and City of Hope Center for Cancer and Aging to M.A.L.; National Institutes of Health/National Cancer Institute (NIH/NCI) grants (nos. R01CA237602, U01CA244109, R33AG059206 and R01EB024989 to M.A.L. and nos. R01CA170851, P20CA24619, R01CA192914 and U01CA189283 to V.E.S.) and NCI Cancer Metabolism Training Program Postdoctoral Fellowship T32CA221709 to R.W.S. Research reported in this publication included work performed in the Integrative Genomics and Bioinformatics, Analytical Cytometry, and Biomarker Analysis and Validation Cores supported by the NCI of the NIH under grant no. P30CA033572. M.R.S. and J.C.G. are supported by US Department of Energy under contract no. DE-AC02-05CH11231. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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.

During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are susceptible to cancer initiation. In the present study, we show that histologically normal breast tissue from younger women who are susceptible to breast cancer, as a result of harboring a germline mutation in

Age is the major risk factor in most carcinomas, yet, little is known about the specific reasons aging increases cancer susceptibility. In the mammary gland, luminal epithelial cells rank high as the putative breast cancer cell of origin. Dysregulation of keratin intermediate filament proteins exemplifies a hallmark age-dependent change in luminal cells, which implicates mechanical states unique to cancer susceptible cells. We implemented mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell analysis that simultaneously measures cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We demonstrated that the epithelial lineages, chronological ages, and stages of cancer progression of primary human mammary epithelial cells (HMEC) exhibited discrete mechanical phenotypes. We trained a machine learning model that accurately predicted the chronological age of average risk HMEC cells based exclusively on mechanical properties. Application of the model to cells from women who are germline carriers of high-risk cancer-causing mutations showed that they are mechanically old irrespective of their chronological age, suggesting that mechanical states could be a window into detection and prevention of cancer susceptible states. Indeed, this mechano-age model detected high-risk women with >90% accuracy. Mass spectrometry and cell-based functional assays in mammary epithelia revealed that cytoskeleton related proteins keratin 14 (KRT14) and pseudopodium enriched atypical kinase 1 (PEAK1) were key drivers of age-dependent mechanical signatures. Pharmacological and gene silencing approaches that targeted KRT14 and PEAK1 modulated the mechanical age of HMEC and, in the case of PEAK1 modulation, ablated luminal epithelial cells in an age- and lineage- dependent manner. We define an intersection between mechanical phenotypes and novel age-dependent changes in cytoskeleton-related proteins that we hypothesize can be exploited to assess an individual’s breast cancer susceptibility and provide new targets for cancer-prevention strategies. Citation Format: Stefan Hinz, Masaru Miyano, Antigoni Manousopoulou, Rosalyn W. Sayaman, Kristina Y. Aguilera, Michael E. Todhunter, Jennifer C. Lopez, Leo D. Wang, Lydia L. Sohn, Mark A. LaBarge. Aging-dependent emergent mechanical properties of single epithelial cells exploited for detection of breast cancer susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A016.

Background : 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. Methods : 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 analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary organoid preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Results : Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm 2 ) 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:M1 macrophage ratio increased with age and was accompanied by an increased 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. Conclusions : 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 .

Summary Age is the major risk factor in most carcinomas, yet little is known about how proteomes change with age in any human epithelium. We present comprehensive proteomes comprised of >9,000 total proteins and >15,000 phosphopeptides from normal primary human mammary epithelia at lineage resolution from ten women ranging in age from 19 to 68 years. Data were quality controlled and results were biologically validated with cell-based assays. Age-dependent protein signatures were identified using differential expression analyses and weighted protein co-expression network analyses. Upregulation of basal markers in luminal cells, including KRT14 and AXL, were a prominent consequence of aging. PEAK1 was identified as an age-dependent signaling kinase in luminal cells, which revealed a potential age-dependent vulnerability for targeted ablation. Correlation analyses between transcriptome and proteome revealed age-associated loss of proteostasis regulation. Age-dependent proteome changes in the breast epithelium identified heretofore unknown potential therapeutic targets for reducing breast cancer susceptibility., Graphical abstract, Highlights • Age-dependent proteomic profiling of HMECs at lineage resolution • Loss of proteostasis is unique to the luminal epithelial lineage with age • Identification of co-regulated proteins with age in luminal cells including PEAK1 • PEAK1 inhibition selectively ablates older luminal cells – cancer cell of origin, Developmental biology; Complex system biology; Proteomics

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.