A spatially resolved single-cell genomic atlas of the adult human breast.

The adult human breast is comprised of an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue1-3. Although most previous studies have focused on the breast epithelial system4-6, many of the non-epithelial cell types remain understudied. Here we constructed the comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics study profiled 714,331 cells from 126 women, and 117,346 nuclei from 20 women, identifying 12 major cell types and 58 biological cell states. These data reveal abundant perivascular, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Spatial mapping using four different technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide a reference of the adult normal breast tissue for studying mammary biology and diseases such as breast cancer.

Redefining normal breast cell populations using long noncoding RNAs.

Single-cell RNAseq has allowed unprecedented insight into gene expression across different cell populations in normal tissue and disease states. However, almost all studies rely on annotated gene sets to capture gene expression levels and sequencing reads that do not align to known genes are discarded. Here, we discover thousands of long noncoding RNAs (lncRNAs) expressed in human mammary epithelial cells and analyze their expression in individual cells of the normal breast. We show that lncRNA expression alone can discriminate between luminal and basal cell types and define subpopulations of both compartments. Clustering cells based on lncRNA expression identified additional basal subpopulations, compared to clustering based on annotated gene expression, suggesting that lncRNAs can provide an additional layer of information to better distinguish breast cell subpopulations. In contrast, these breast-specific lncRNAs poorly distinguish brain cell populations, highlighting the need to annotate tissue-specific lncRNAs prior to expression analyses. We also identified a panel of 100 breast lncRNAs that could discern breast cancer subtypes better than protein-coding markers. Overall, our results suggest that lncRNAs are an unexplored resource for new biomarker and therapeutic target discovery in the normal breast and breast cancer subtypes.

The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition.

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor ß (TGFß) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFß, including TGFß auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFß signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFß type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFß family members.

Lineage Tracing Methods to Study Mammary Epithelial Hierarchies In Vivo.

Lineage tracing is now considered the gold standard approach to study cellular hierarchies and cell fate in vivo (McKenna and Gagnon, Development 146:dev169730, 2019; Kretzschmar and Watt, Cell 148:33-45, 2012). This type of clonal analysis consists of genetically labeling defined cells and following their destiny and progeny in vivo and in situ.Here we will describe different existing in vivo systems to clonally trace targeted cells and will discuss their respective advantages and inconveniences; we will then provide stepwise instructions for setting up and evaluate lineage tracing experiments, listing the most common downstream analyses and read-out assays.

The WID-BC-index identifies women with primary poor prognostic breast cancer based on DNA methylation in cervical samples.

Genetic and non-genetic factors contribute to breast cancer development. An epigenome-based signature capturing these components in easily accessible samples could identify women at risk. Here, we analyse the DNA methylome in 2,818 cervical, 357 and 227 matched buccal and blood samples respectively, and 42 breast tissue samples from women with and without breast cancer. Utilising cervical liquid-based cytology samples, we develop the DNA methylation-based Women's risk IDentification for Breast Cancer index (WID-BC-index) that identifies women with breast cancer with an AUROC (Area Under the Receiver Operator Characteristic) of 0.84 (95% CI: 0.80-0.88) and 0.81 (95% CI: 0.76-0.86) in internal and external validation sets, respectively. CpGs at progesterone receptor binding sites hypomethylated in normal breast tissue of women with breast cancer or in BRCA mutation carriers are also hypomethylated in cervical samples of women with poor prognostic breast cancer. Our data indicate that a systemic epigenetic programming defect is highly prevalent in women who develop breast cancer. Further studies validating the WID-BC-index may enable clinical implementation for monitoring breast cancer risk.

Transcriptional changes in the mammary gland during lactation revealed by single cell sequencing of cells from human milk.

Under normal conditions, the most significant expansion and differentiation of the adult mammary gland occurs in response to systemic reproductive hormones during pregnancy and lactation to enable milk synthesis and secretion to sustain the offspring. However, human mammary tissue remodelling that takes place during pregnancy and lactation remains poorly understood due to the challenge of acquiring samples. We report here single-cell transcriptomic analysis of 110,744 viable breast cells isolated from human milk or non-lactating breast tissue, isolated from nine and seven donors, respectively. We found that human milk largely contains epithelial cells belonging to the luminal lineage and a repertoire of immune cells. Further transcriptomic analysis of the milk cells identified two distinct secretory cell types that shared similarities with luminal progenitors, but no populations comparable to hormone-responsive cells. Taken together, our data offers a reference map and a window into the cellular dynamics that occur during human lactation and may provide further insights on the interplay between pregnancy, lactation and breast cancer.

Acesso e acessibilidade ao rastreamento de câncer em mulheres brasileiras com lesão medular / Acceso y accesibilidad al rastreo de cáncer en mujeres brasileñas con lesión medular / Access and accessibility to cancer screening for Brazilian women with spinal cord injury

Resumo Objetivo identificar e analisar a acessibilidade e o acesso de mulheres brasileiras com lesão medular para a realização de exames preventivos do câncer de mama e colo de útero. Método estudo quantitativo e transversal desenvolvido em plataforma virtual. Realizadas análises estatísticas descritivas e de associação entre as variáveis qualitativas por meio do teste exato de Fisher. Quando identificada a associação (p<0,05), foi realizada a regressão logística. Resultados participaram 120 mulheres brasileiras com lesão medular com idades entre 25 e 67 anos; 85,83% foram ao ginecologista após a lesão medular, 79,17% realizaram a citologia e 52,50%, a mamografia. Observou-se que as mulheres que utilizavam a saúde suplementar apresentaram maior probabilidade de terem ido ao ginecologista do que as usuárias do serviço público. Aquelas com companheiro e as de maior idade apresentaram maior probabilidade de terem realizado o exame de citologia. Para a mamografia, aquelas de maior idade e que utilizavam a saúde suplementar apresentaram maiores chances de terem realizado o exame de mamografia após a lesão medular. Conclusão mulheres com lesão medular buscam a realização de exames de rastreamento. Entretanto, encontram dificuldades relacionadas à estrutura física, aos equipamentos, transporte, profissionais da saúde, assim como dificuldades sociodemográficas e quanto ao serviço de saúde utilizado.

Resumen Objetivo este estudio tuvo como objetivo identificar y analizar la accesibilidad y el acceso de mujeres brasileñas con lesión medular para la realización de exámenes preventivos de cáncer de mama y de cuello uterino. Método se desarrolló un estudio cuantitativo y transversal, realizado en un entorno virtual. Los análisis estadísticos descriptivos y la asociación entre variables cualitativas se realizaron mediante la prueba exacta de Fisher, cuando se identificó una asociación se realizó una regresión logística. Resultados participaron 120 mujeres brasileñas con lesión medular, la edad de las participantes varió de 25 a 67 años. Con relación al rastreo, el 85,83% de las mujeres acudió al ginecólogo tras la LM, el 79,17% se sometió a citología y el 52,50% a mamografía. Se observó que las mujeres que utilizaban un seguro médico privado tenían más probabilidades de haber visto a un ginecólogo que las usuarias del servicio público. Las que tenían pareja y mayores tenían más probabilidades de someterse a citología oncótica. Para la mamografía, las que eran mayores y que usaban un seguro médico privado tenían más probabilidades de someterse al examen después de la LM. Conclusión las mujeres con LM buscan pruebas de detección. Sin embargo, enfrentan dificultades relacionadas con la estructura física, equipamientos, transporte, profesionales de la salud, así como dificultades sociodemográficas relacionadas con el tipo de servicio de salud utilizado.

Abstract Objective to identify and analyze the accessibility and accessibility of Brazilian women with spinal cord injury to preventive examinations for breast and cervical cancer. Method quantitative and cross-sectional study developed in a virtual platform. Descriptive statistical analysis was performed, as well as association analysis between qualitative variables using Fisher's exact test. When identified the association (p<0.05), logistic regression was performed. Results a total of 120 Brazilian women with spinal cord injury, aged between 25 and 67 years participated in the study; 85.83% visited a gynecologist after the spinal cord injury, 79.17% underwent cytology and 52.50% underwent mammography. It was observed that women who used the supplementary health plan were more likely to have visited a gynecologist than those who used the public service. Those who had a partner and were older were more likely to have undergone the cytology exam. For mammography, those who were older and who used supplementary health care were more likely to have had mammography exams after the spinal cord injury. Conclusion women with spinal cord injury seek screening tests. However, they encounter difficulties related to the physical structure, equipment, transportation, health professionals, as well as socio-demographic difficulties and difficulties regarding the health service used.

Breast tissue regeneration is driven by cell-matrix interactions coordinating multi-lineage stem cell differentiation through DDR1.

Mammary morphogenesis is an orchestrated process involving differentiation, proliferation and organization of cells to form a bi-layered epithelial network of ducts and lobules embedded in stromal tissue. We have engineered a 3D biomimetic human breast that makes it possible to study how stem cell fate decisions translate to tissue-level structure and function. Using this advancement, we describe the mechanism by which breast epithelial cells build a complex three-dimensional, multi-lineage tissue by signaling through a collagen receptor. Discoidin domain receptor tyrosine kinase 1 induces stem cells to differentiate into basal cells, which in turn stimulate luminal progenitor cells via Notch signaling to differentiate and form lobules. These findings demonstrate how human breast tissue regeneration is triggered by transmission of signals from the extracellular matrix through an epithelial bilayer to coordinate structural changes that lead to formation of a complex ductal-lobular network.

Diagnosis of bone metastases in breast cancer: Lesion-based sensitivity of dual-time-point FDG-PET/CT compared to low-dose CT and bone scintigraphy.

We compared lesion-based sensitivity of dual-time-point FDG-PET/CT, bone scintigraphy (BS), and low-dose CT (LDCT) for detection of various types of bone metastases in patients with metastatic breast cancer. Prospectively, we included 18 patients with recurrent breast cancer who underwent dual-time-point FDG-PET/CT with LDCT and BS within a median time interval of three days. A total of 488 bone lesions were detected on any of the modalities and were categorized by the LDCT into osteolytic, osteosclerotic, mixed morphologic, and CT-negative lesions. Lesion-based sensitivity was 98.2% (95.4-99.3) and 98.8% (96.8-99.5) for early and delayed FDG-PET/CT, respectively, compared with 79.9% (51.1-93.8) for LDCT, 76.0% (36.3-94.6) for BS, and 98.6% (95.4-99.6) for the combined BS+LDCT. BS detected only 51.2% of osteolytic lesions which was significantly lower than other metastatic types. SUVs were significantly higher for all lesion types on delayed scans than on early scans (P<0.0001). Osteolytic and mixed-type lesions had higher SUVs than osteosclerotic and CT-negative metastases at both time-points. FDG-PET/CT had significantly higher lesion-based sensitivity than LDCT and BS, while a combination of the two yielded sensitivity comparable to that of FDG-PET/CT. Therefore, FDG-PET/CT could be considered as a sensitive one-stop-shop in case of clinical suspicion of bone metastases in breast cancer patients.

Simultaneous expression of MMB-FOXM1 complex components enables efficient bypass of senescence.

Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21WAF1/CIP1 and pRB/p16INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3EcoR) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence.

Expression of LGR5 in mammary myoepithelial cells and in triple-negative breast cancers.

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no ß-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted.

Desensitization of human breast progenitors by a transient exposure to pregnancy levels of estrogen.

Full term pregnancy at an early age is the only factor known to consistently protect against breast cancer. Because hormone receptor positive progenitors in the human breast relay endocrine signaling, we here sought to determine whether an experimental mimicry of the third trimester surge of hormones would change their susceptibility to growth stimulation. Hormone receptor positive, reduction mammoplasty-derived human breast epithelial progenitors were exposed to a short-term, pregnancy-level of estradiol, and their subsequent response to estradiol stimulation was analyzed. Exposure to pregnancy-level of estradiol results in subsequent lower sensitivity to estrogen-induced proliferation. Expression array and immunoblotting reveal upregulation of S100A7 and down-regulation of p27, both associated with parity and epithelial differentiation. Notably, we find that the epithelial differentiation is accompanied by upregulation of E-cadherin and down-regulation of vimentin as well as by diminished migration and more mature luminal epithelial differentiation in a mouse transplantation model. Our findings are in support of a de-sensitization mechanism for pregnancy-induced prevention against breast cancer.

From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion.

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM-ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM-ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.

Transcription factor enrichment analysis (TFEA) quantifies the activity of multiple transcription factors from a single experiment.

Detecting changes in the activity of a transcription factor (TF) in response to a perturbation provides insights into the underlying cellular process. Transcription Factor Enrichment Analysis (TFEA) is a robust and reliable computational method that detects positional motif enrichment associated with changes in transcription observed in response to a perturbation. TFEA detects positional motif enrichment within a list of ranked regions of interest (ROIs), typically sites of RNA polymerase initiation inferred from regulatory data such as nascent transcription. Therefore, we also introduce muMerge, a statistically principled method of generating a consensus list of ROIs from multiple replicates and conditions. TFEA is broadly applicable to data that informs on transcriptional regulation including nascent transcription (eg. PRO-Seq), CAGE, histone ChIP-Seq, and accessibility data (e.g., ATAC-Seq). TFEA not only identifies the key regulators responding to a perturbation, but also temporally unravels regulatory networks with time series data. Consequently, TFEA serves as a hypothesis-generating tool that provides an easy, rigorous, and cost-effective means to broadly assess TF activity yielding new biological insights.

Mammary cell gene expression atlas links epithelial cell remodeling events to breast carcinogenesis.

The female mammary epithelium undergoes reorganization during development, pregnancy, and menopause, linking higher risk with breast cancer development. To characterize these periods of complex remodeling, here we report integrated 50 K mouse and 24 K human mammary epithelial cell atlases obtained by single-cell RNA sequencing, which covers most lifetime stages. Our results indicate a putative trajectory that originates from embryonic mammary stem cells which differentiates into three epithelial lineages (basal, luminal hormone-sensing, and luminal alveolar), presumably arising from unipotent progenitors in postnatal glands. The lineage-specific genes infer cells of origin of breast cancer using The Cancer Genome Atlas data and single-cell RNA sequencing of human breast cancer, as well as the association of gland reorganization to different breast cancer subtypes. This comprehensive mammary cell gene expression atlas ( https://mouse-mammary-epithelium-integrated.cells.ucsc.edu ) presents insights into the impact of the internal and external stimuli on the mammary epithelium at an advanced resolution.

Targeting of GLUT5 for Transporter-Mediated Drug-Delivery Is Contingent upon Substrate Hydrophilicity.

Specific link between high fructose uptake and cancer development and progression highlighted fructose transporters as potential means to achieve GLUT-mediated discrimination between normal and cancer cells. The gained expression of fructose-specific transporter GLUT5 in various cancers offers a possibility for developing cancer-specific imaging and bioactive agents. Herein, we explore the feasibility of delivering a bioactive agent through cancer-relevant fructose-specific transporter GLUT5. We employed specific targeting of GLUT5 by 2,5-anhydro-D-mannitol and investigated several drug conjugates for their ability to induce cancer-specific cytotoxicity. The proof-of-concept analysis was carried out for conjugates of chlorambucil (CLB) in GLUT5-positive breast cancer cells and normal breast cells. The cytotoxicity of conjugates was assessed over 24 h and 48 h, and significant dependence between cancer-selectivity and conjugate size was observed. The differences were found to relate to the loss of GLUT5-mediated uptake upon increased conjugate size and hydrophobicity. The findings provide information on the substrate tolerance of GLUT5 and highlight the importance of maintaining appropriate hydrophilicity for GLUT-mediated delivery.

High-throughput surface marker screen on primary human breast tissues reveals further cellular heterogeneity.

BACKGROUND: Normal human breast tissues are a heterogeneous mix of epithelial and stromal subtypes in different cell states. Delineating the spectrum of cellular heterogeneity will provide new insights into normal cellular properties within the breast tissue that might become dysregulated in the initial stages of cancer. Investigation of surface marker expression provides a valuable approach to resolve complex cell populations. However, the majority of cell surface maker expression of primary breast cells have not been investigated. METHODS: To determine the differences in expression of a range of uninvestigated cell surface markers between the normal breast cell subpopulations, primary human breast cells were analysed using high-throughput flow cytometry for the expression of 242 cell surface proteins in conjunction with EpCAM/CD49f staining. RESULTS: We identified 35 surface marker proteins expressed on normal breast epithelial and/or stromal subpopulations that were previously unreported. We also show multiple markers were equally expressed in all cell populations (e.g. CD9, CD59, CD164) while other surface markers were confirmed to be enriched in different cell lineages: CD24, CD227 and CD340 in the luminal compartment, CD10 and CD90 in the basal population, and CD34 and CD140b on stromal cells. CONCLUSIONS: Our dataset of CD marker expression in the normal breast provides better definition for breast cellular heterogeneity.

Tumor-Associated Stromal Cellular Density as a Predictor of Recurrence and Mortality in Breast Cancer: Results from Ethnically Diverse Study Populations.

PURPOSE: Tumor-associated stroma is comprised of fibroblasts, tumor-infiltrating lymphocytes (TIL), macrophages, endothelial cells, and other cells that interactively influence tumor progression through inflammation and wound repair. Although gene-expression signatures reflecting wound repair predict breast cancer survival, it is unclear whether combined density of tumor-associated stromal cells, a morphologic proxy for inflammation and wound repair signatures on routine hematoxylin and eosin (H&E)-stained sections, is of prognostic relevance. METHODS: By applying machine learning to digitized H&E-stained sections for 2,084 breast cancer patients from China (n = 596; 24-55 years), Poland (n = 810; 31-75 years), and the United States (n = 678; 55-78 years), we characterized tumor-associated stromal cellular density (SCD) as the percentage of tumor-stroma that is occupied by nucleated cells. Hazard ratios (HR) and 95% confidence intervals (CI) for associations between SCD and clinical outcomes [recurrence (China) and mortality (Poland and the United States)] were estimated using Cox proportional hazard regression, adjusted for clinical variables. RESULTS: SCD was independently predictive of poor clinical outcomes in hormone receptor-positive (luminal) tumors from China [multivariable HR (95% CI)fourth(Q4) vs. first(Q1) quartile = 1.86 (1.06-3.26); P trend = 0.03], Poland [HR (95% CI)Q4 vs. Q1 = 1.80 (1.12-2.89); P trend = 0.01], and the United States [HR (95% CI)Q4 vs. Q1 = 2.42 (1.33-4.42); P trend = 0.002]. In general, SCD provided more prognostic information than most classic clinicopathologic factors, including grade, size, PR, HER2, IHC4, and TILs, predicting clinical outcomes irrespective of menopausal or lymph nodal status. SCD was not predictive of outcomes in hormone receptor-negative tumors. CONCLUSIONS: Our findings support the independent prognostic value of tumor-associated SCD among ethnically diverse luminal breast cancer patients. IMPACT: Assessment of tumor-associated SCD on standard H&E could help refine prognostic assessment and therapeutic decision making in luminal breast cancer.

CC chemokine ligand 2 (CCL2) stimulates aromatase gene expression in mammary adipose tissue.

Obesity is a risk factor for postmenopausal breast cancer. Obesity-related inflammation upregulates aromatase expression, the rate-limiting enzyme for estrogen synthesis, in breast adipose tissue (BAT), increasing estrogen production and cancer risk. The regulation of aromatase gene (CYP19A1) in BAT is complex, and the mechanisms linking obesity and aromatase dysregulation are not fully understood. An obesity-associated factor that could regulate aromatase is the CC chemokine ligand (CCL) 2, a pro-inflammatory factor that also activates signaling pathways implicated in CYP19A1 transcription. By using human primary breast adipose stromal cells (ASCs) and aromatase reporter (hARO-Luc) mouse mammary adipose explants, we demonstrated that CCL2 enhances the glucocorticoid-mediated CYP19A1 transcription. The potential mechanism involves the activation of PI.4 via ERK1/2 pathway. We also showed that CCL2 contributes to the pro-inflammatory milieu and aromatase expression in obesity, evidenced by increased expression of CCL2 and CYP19A1 in mammary tissues from obese hARO-Luc mice, and subcutaneous adipose tissue from obese women. In summary, our results indicate that postmenopausal obesity may promote CCL2 production in BAT, leading to exacerbation of the menopause-related inflammatory state and further stimulation of local aromatase and estrogens. These results provide new insights into the regulation of aromatase and may aid in finding approaches to prevent breast cancer.