Lymph node metastasis (LNM) is a critical determinant of breast cancer prognosis. While oral contraceptives (OC) are associated with breast cancer risk, their relationship with LNM—particularly in understudied populations like Morocco—remains unclear. This study investigates whether tumor size modifies OC-LNM associations in Moroccan patients. We analyzed 356 breast cancer cases from Morocco’s National Institute of Oncology(2009). Adjusted odds ratios (ORs) and 95
Recent multicenter studies aim to define the effects of mothers’ blood glucose levels during pregnancy on mammary gland function and breast milk composition. Gene expression measured in cells in milk can serve as a liquid biopsy to evaluate the molecular biology of the mammary gland. Critical to this aim is reproducible and high-quality extraction of RNA from milk and harmonized collection protocols from across centers. To address this, we performed a study to optimize milk RNA quality metrics where samples are collected at multiple centers and shipped to a central laboratory for processing and analysis. Lactating mothers provided breast milk following informed consent. The treatments of the samples were as follows: (1) 200 µL of fresh, never frozen milk used as control (FRESH); (2) 1.7 or 5 mL of milk frozen and thawed on ice before adding TRIzol (FRZ); (3) 200 µL of milk frozen and thawed after adding TRIzol (FRZ 200); and (4) 200 µL of milk with 20 µL of RNA preservative added, frozen and thawed after adding TRIzol (FRZ + INH). In all scenarios, RNA was extracted using TRIzol followed by purification with a Qiagen RNeasy Mini kit. For the FRZ 200 and FRZ 200 + INH samples, TRIzol was directly added at the start of thawing, before extraction. Outcomes included RNA concentration, RNA purity (260/280 ratio), RNA fragmentation (DV 200), RNA integrity number (RIN), quantification via Qubit fluorescence-based assays, and visualization of RNA size on an Agilent TapeStation. A RIN cut-off value ≥ 7 indicated acceptable quality for transcriptomics studies. RNA quality metrics were modeled as continuous outcomes using linear mixed-effects regression models. For FRESH milk (n = 15) the estimated marginal mean (EMM) RIN was 7.48 (SE 0.29). For FRZ 200, (n = 22), the EMM RIN was 6.94 (SE 0.26). Samples of FRZ 200 + INH (n = 21) had an EMM RIN of 7.81 (SE 0.26). However, FRZ (n = 19) samples demonstrated markedly reduced RNA integrity with an EMM RIN of 1.92 (SE 0.26). RIN was not significantly different in FRESH versus FRZ 200 or FRZ 200 + INH milk. FRZ 200 + INH samples showed a statistically significant improvement in RIN compared to FRZ 200 (p = 0.0038). RNA quantities were sufficient for sequencing across all treatments. The addition of TRIzol directly to a 200 µL aliquot of milk at the start of thawing provided the highest integrity of extracted milk RNA, as measured by RIN. Adding RNase inhibitor at the time of sample collection, prior to freezing, also enhanced RNA integrity. We have developed a method to optimize the integrity of RNA from frozen human milk samples. This is a crucial methods improvement for multi-center studies where freezing of milk samples is often required prior to RNA extraction and analysis. These results can inform reproducible research protocols for evaluating the use of breastmilk as a liquid biopsy for mammary gland function.
HER2-positive breast cancer (BC) is an aggressive subtype with poor long-term outcomes in advanced disease, largely due to resistance to HER2-targeted therapies. Within the tumor-immune microenvironment (TIME), tumor and immune cells face diverse stressors, including endoplasmic reticulum stress (ERS). ERS activates the Unfolded Protein Response (UPR), which aims to restore homeostasis or trigger apoptosis. In BC cells, chronic UPR activation promotes malignant progression, while in immune cells ERS impairs function and weakens antitumor immunity. Given the relatively high immune infiltration in HER2-positive BC, preserving immune competence is crucial for therapeutic efficacy. This study aims to elucidate TIME-associated molecular mechanisms in HER2-positive BC using in-vitro model and patient tumor samples analysis from 21 patients and to develop predictive models of treatment response using liquid biopsy-based mass spectrometry (MS) approach (21 patients, 15 healthy donors). ERS modulation was shown to significantly affect cancer–immune cell interactions in co-culture models. Key ERS-related genes were characterized at the transcriptional, translational, and spatial levels. These findings were used to model the effects of immunomodulatory therapies in vitro. Analysis of patient samples revealed distinct TIME patterns associated with treatment response. Serum-based MALDI-TOF MS enabled the development of a predictive model capturing systemic alterations associated with ERS driven tumor immune interactions, therefore discriminated responders from non-responders. Modulating ERS within the TIME represents a promising strategy to enhance immune-mediated antitumor activity in HER2-positive BC. This study provides a detailed molecular characterization of the HER2-positive BC TIME and presents an MS- and machine learning-based predictive model for patient stratification.
Understanding the cell hierarchy of the mammary gland has been a major focus of research for several decades. This review retraces the key advances enabled by lineage tracing approaches to study stem cells and cell hierarchy in the mammary gland. Although not exhaustive, this review highlights the paradigm shifts brought about by lineage tracing studies, which have, in the author’s view, profoundly reshaped our understanding of mammary epithelial organization. In particular, these studies have challenged traditional models of cell hierarchy and the concept of terminally differentiated cells in the mammary gland.
The isogenic, tumor-bearing Dark Agouti Mammary Adenocarcinoma (DAMA) model is commonly utilized in breast cancer research to evaluate mechanisms of, and interventions for chemotherapy-induced toxicity and tumor response. However, the biological subtype, immune landscape, and genomic profile of DAMA have remained uncharacterized. In this study, we comprehensively evaluated DAMA tumors naïve and exposed to methotrexate, assessing their histopathological, molecular, and genomic features. Our findings reveal that the DAMA model presents a platform to model the human triple-negative breast cancer (TNBC), as it exhibits substantial macrophage infiltration, the tumors display dysregulation of oncogenes Bcl2, Egfr, and potentially Myc, suggesting apoptotic resistance as a key mechanism driving growth. These characteristics position the DAMA model as a potential preclinical model for investigating TNBC biology, therapeutic responses, and drug toxicity.
Psychological stress during lactation decreases β-casein concentration in milk. Smoking-induced increases in miR-210 expression also decrease β-casein concentration in milk and inhibits its synthesis in mammary epithelial cells. However, the relationship between stress and miR-210 expression in the mammary epithelium has remained uninvestigated. Here, we investigated the association between stress and miR-210-5p (miR-210) expression in the mammary epithelium and evaluated whether miR-210 is involved in stress-induced suppression of β-casein content in breast milk. miR-210 expression in the milk and mammary epithelium of restraint-stressed mice was significantly higher than that in control mice. Additionally, β-casein mRNA expression and the protein levels of signal transducer and activator of transcription 5 (STAT5) and phosphorylated STAT5 were significantly lower than those in control mice. In clinical investigation, a significant positive correlation was observed between miR-210 levels in breast milk and the total mood disturbance (TMD) score determined using the Profile of Mood State 2nd Edition–Adult Short questionnaire. In contrast, the positive mood scale vigor–activity on the TMD subscale and the positive mood scale friendliness exhibited a significant negative correlation with miR-210 levels in breast milk. These results indicate that miR-210 levels in breast milk may be increased in nursing mothers with a strong negative mood. In conclusion, psychological stress during lactation may induce miR-210 expression in the mammary epithelium, resulting in increased miR-210 levels in breast milk. Additionally, stress-induced suppression of β-casein concentration in breast milk might be related to the induction of miR-210 expression in the mammary epithelium.
Tissue specific stem cells are critical in maintaining organ function throughout life. Understanding the dynamics and heterogeneity of stem cells and progenitors is essential to understanding how diseased cell states such as cancer evolve. Studies in the mammary gland have revealed significant heterogeneity in stem cell identity during embryonic development, puberty, adulthood and aging. In this review, we discuss the dynamics of lineage commitment in the mammary gland and the current understanding of mammary stem cells versus lineage committed progenitors at different stages. As breast cancer risk increases with age, we also review recent studies on the aged mammary gland in relation to lineage identity and provide perspectives for future research in understanding the niche of mammary epithelial cells.
Postnatal mammary gland development involves the formation of a highly branched epithelial ductal tree, primarily through the elongation and branching morphogenesis of mammary epithelial ducts. Multiple factors are involved in this process, in which both estrogen and progesterone receptors (ER/PR) play a crucial role. In this study, we identified a role of DCAF8 in promoting mammary ductal elongation and branching through its impacts on ER/PR signaling. Homozygous Dcaf8 knockout mice exhibited significant delay in mammary ductal elongation during puberty, which was characterized by a reduction in mammary ductal elongation area and distance, and terminal end buds (TEBs); abnormal branching morphogenesis of mammary ducts was also observed in adult Dcaf8 null mice, which was characterized by a reduction of lateral branches and an increase of ductal bifurcation. To further elucidate the mechanism underlying DCAF8’s role in mouse mammary development, we performed transcriptomic sequencing and biochemical experiments. The results revealed that downstream key effectors of the PR signaling pathway were significantly downregulated, while the expression of ERβ, a potential inhibitor of ERα/PR signaling, was significantly elevated in the mammary gland of Dcaf8 null mice. Collectively, this study suggests that DCAF8 may play an important role in mammary development by promoting ductal elongation and branching morphogenesis, through ERβ-mediated inhibition of ERα/PR signaling pathway.
Clonal hematopoiesis of indeterminate potential (CHIP) is associated with systemic inflammation and solid tumor progression, but its impact on breast cancer therapy remains unclear. This study investigated whether CHIP influences neoadjuvant chemotherapy (NAC) response and shapes the tumor microenvironment (TME) in breast cancer. We analyzed tumor sequencing data from The Cancer Genome Atlas (TCGA), focusing on mutations in CHIP-driver genes within a variant allele frequency range of 2–33.3
Human breast milk contains RNA in various fractions, including milk cells and milk fat globules (MFG), making it a valuable resource for studying lactation physiology. However, preserving RNA integrity, especially in low-resource or at-home collection settings, is challenging due to rapid RNA degradation. This study aimed to evaluate RNA preservation methods for milk cells and MFG, using RNAlater for stabilization before freezing. Human milk samples (n = 26) were collected from lactating participants and either frozen (standard practice) or mixed with RNAlater (1:1, v/v) before freezing. RNA was extracted from separated cellular and MFG fractions and assessed for concentration, quality (RNA quality number-RQN and 28 S/18S ratio), and gene expression (ACTB, LALBA, PRLR, PTPRC) using RT-qPCR. Samples preserved with RNAlater showed significantly improved RNA integrity, particularly in the MFG fraction, compared to those frozen without RNAlater. Gene expression was largely stable across preservation methods. Delays in mixing with RNAlater led to declining RQN values in milk cell fractions, underscoring the need for prompt stabilization. Lastly, we show that this method can be used in low-resource countries by extracting RNA from samples collected in a randomized clinical trial in Guinea-Bissau and shipped to the US for analysis. This procedure led to improved yield and integrity of these samples. These findings demonstrate that RNAlater pre-freezing stabilization enhances RNA quality and yield, and supports its use for milk gene expression analysis. This approach provides a practical, scalable solution for RNA preservation in clinical and field research, including remote and low-resource settings, as it requires minimal experience and equipment.
Cancer-associated fibroblasts (CAFs) play a key role in breast cancer progression and exhibit a procoagulant phenotype within the tumour microenvironment (TME). We hypothesised that this procoagulant phenotype correlates with a CAF-like phenotype and that fibroblasts distant from the immediate TME are less procoagulant. We also proposed that the procoagulant phenotype contributes functionally to breast cancer progression. Primary fibroblasts were cultured from human breast tumour tissue and matched normal breast tissue from regions distant to the tumour. Conditioned media (CM) from these cells were collected for analysis. We conducted immunocytochemistry, western blotting, transforming growth factor beta 1 (TGFβ1) ELISA, tissue factor (TF) activity and procoagulant activity assays. A positive correlation was found between the expression of TF and alpha-smooth muscle actin (α-SMA), a CAF marker, and between fibroblast procoagulant activity and secretion of the CAF inducer, TGFβ1. Interestingly, fibroblasts from distant breast tissue exhibited CAF-like and procoagulant phenotypes similar to tumour-associated fibroblasts. To assess functional relevance, scratch wound migration assays were performed using MCF-7 breast cancer cells. Inhibition of TF derived from both tumour and distant fibroblasts significantly reduced MCF-7 cell migration. Combined inhibition of TF and TGFβ1 in distant fibroblast CM further suppressed migration. These findings suggest that tumour-derived influences may extend beyond the immediate TME, inducing a CAF-like, procoagulant phenotype in fibroblasts from histologically normal breast tissue. Furthermore, fibroblast-derived TF promotes breast cancer cell migration which is important for the processes of invasion and metastasis. This further highlights TF as a promising therapeutic target in breast cancer.
The study of breast cancer is complicated by the heterogeneity inherent within the disease. Numerous models have been developed to study the initiation, progression, and treatment of breast cancer. These include carcinogen induced mouse models, genetically engineered mouse models, and patient derived xenografts. The relevance of these mouse models to humans must be precisely defined for appropriate understanding of disease mechanisms to derive intervening treatments. Sequencing projects such as The Cancer Genome Atlas Project (TCGA) and Catalogue Of Somatic Mutations In Cancer (COSMIC) were pivotal developments in understanding driving events in human cancers. These studies have revealed that in addition to activation of strong oncogenes, or loss of tumor suppressors, that secondary events are necessary for tumor development and progression. These techniques should also be applied to mouse models of human breast cancer. For all the available models studied and reviewed here, whole genome sequencing (WGS) in conjunction with gene expression analysis has revealed conserved events between human and mouse model systems. This identification of conserved, critical events driving breast cancer has led to novel targets based on breast cancer subtype, ultimately resulting in new therapeutic opportunities. The combination of sequencing and choice of the appropriate mouse model can provide a powerful tool in developing appropriate pre-clinical models of breast cancer.
Studies in breast cancer have demonstrated that apatinib exhibits both antiangiogenic and antitumor effects, while PD-L1 inhibitors have similarly shown meaningful clinical benefit. Building upon these observations, this study evaluated the potential synergistic antitumor effects of combining apatinib with a PD-L1 inhibitor and examined the mechanistic basis for their interaction in breast cancer. Notably, we found that this regimen could significantly suppress the proliferation, migration, and invasion of MCF-7 and MDA-MB-231 cells, and promote cell apoptosis. In addition, the levels of p-ERK, NF-κB, and Slug were markedly reduced in vitro. Collectively, these findings support the potential clinical utility of combining apatinib and PD-L1 inhibition, as evidenced by consistent in vitro and in vivo synergy.
Human milk is rich in bioactive components beyond nutrition, including RNAs—especially miRNAs. It is the most RNA-rich biofluid. While storage effects on other milk components are well studied, RNA preservation has been minimally explored, primarily focusing on miRNAs in exosomes under refrigeration. The goal of this clinical study was to determine optimal collection and storage methods to preserve RNA quantity and quality in human milk, predominantly focusing on RNA stability in research laboratory settings when isolation of milk fat globules is not feasible. The study recruited 30 lactating women in central Arkansas. Milk samples were collected in-clinic and processed immediately for baseline RNA data. Remaining aliquots were stored at 4 °C, -20 °C, or -80 °C with or without RNAlater. RNA was extracted at 4 h, 24 h, 1 week, 4 weeks, 12 weeks, and 24 weeks using Qiagen miRNeasy and MinElute kits. RNA quality (RINe) and concentration (ng/µL) were assessed with Agilent TapeStation with additional quality analysis through qPCR. RNA quality declined over time at all storage temperatures in samples without RNAlater. Addition of RNAlater prevented decreases in RINe scores at most time points, maintaining RINe > 7 for up to 6 months, though it reduced total RNA concentration. qPCR also showed improved mRNA stability with RNAlater. miRNAs were analyzed using Agilent Bioanalyzer Small RNA kits with qPCR for quality analysis. RNAlater was not beneficial for miRNA preservation, and miRNAs remained stable when stored frozen without RNAlater. qPCR verified miRNA stability when frozen. In conclusion, RNA in human milk degrades over time. For mRNA analysis, storage with RNAlater at -80 °C is recommended. For miRNA analysis, storage at -20 °C or -80 °C without preservatives is optimal. These findings offer preliminary guidance for HM RNA storage, and researchers are advised to conduct method testing before full study implementation.
High mammographic breast density relates to the abundance of fibroglandular tissue in comparison to fatty tissue in the breast and is associated with increased breast cancer risk. Chronic low-level inflammation has been implicated as a driver of high density and cancer risk, however little is understood of the underlying cause of inflammation. This research aimed to investigate the role of the innate immune recognition receptor toll-like receptor-4 (TLR4) in inflammation associated with high fibroglandular density. Immunohistochemical analysis was performed on paired breast tissue samples of high and low fibroglandular density tissue (n = 22 pairs) to investigate the expression of TLR4, TLR4 agonists lipopolysaccharide (LPS) and damage response protein high-mobility group protein 1 (HMGB1), as well as activation of downstream mediators myeloid differentiation primary response 88 (MYD88) and nuclear factor kappa B (NFKB). Mammary epithelial cell organoids (n = 5) were cultured in vitro with LPS to investigate the expression of genes associated with inflammation. TLR4 was primarily expressed in basal epithelial cells, stromal macrophages, and some expression was detected in luminal epithelial cells. Increased expression of TLR4, MYD88, NFKB, and HMGB1 was observed in epithelial cells in high fibroglandular density tissue. There was increased expression of the genes encoding inflammatory cytokines tumour necrosis factor alpha (TNFA) and C-C motif ligand 2 (CCL2) in mammary epithelial cell organoids treated with LPS. The TLR4 signalling pathway may be a mediator of local breast inflammation associated with regions of high breast density and the damage response protein HMGB1 may be a trigger for TLR4 activation.
Cellular plasticity in mammary epithelial cells enables dynamic cell state changes essential for normal development but can be hijacked by breast cancer cells to drive tumor progression and metastasis. However, the molecular factors that maintain cellular plasticity through the regulation of a hybrid cell state (epithelial/mesenchymal) are not fully defined. As LMO2 has been previously shown to regulate metastasis in breast cancer, here we determine the role of LMO2 in normal mammary epithelial cells. Using lineage tracing and knockout mouse models, we find that Lmo2 lineage-traced cells are present in the luminal and basal layer of the mammary gland but have limited proliferative potential. Lmo2 loss does not impact mammary gland development, but acute deletion decreases in vivo reconstitution. Moreover, LMO2 knockdown in mouse and human mammary epithelial cells (MECs) reduces organoid formation. We find that LMO2 regulates the epithelial cell state in MECs and LMO2 knockdown promotes mesenchymal differentiation. Transcriptional profiling of LMO2 knockdown cells reveals significant enrichment in the epithelial-mesenchymal transition (EMT) pathway and upregulation of MCAM, a mesenchymal marker and negative regulator of regenerative capacity in the mammary gland. Altogether, we show that LMO2 plays a role in maintaining cellular plasticity in MECs, adding insight into the normal differentiation programs hijacked by cancer cells to drive tumor progression.
Reproduction studies are important for the conservation of cetaceans (whales, dolphins, and porpoises) because they provide essential information for assessing populations and species dynamics, particularly in relation to the management of the diverse cetacean species in human care. A vast majority of literature on the female cetacean reproductive anatomy and physiology has focused on the ovaries, which can be used to infer reproductive history, or genital diseases and anomalies. However, literature regarding the morphology, physiology, and developmental pattern of cetacean mammary glands is scarce, despite their fundamental role in providing vital nutrients for the growth of offspring. This review describes current diagnostic tools applied in human and veterinary medicine to assess mammary glands and how marine mammal medicine could benefit from incorporating these tools into standard evaluation of the mammary glands in free-ranging and captive cetaceans. By evaluating the strengths and weaknesses of the current tools used to assess the mammary glands in humans and domestic animals —such as mammography, CT, MRI and ultrasonography— we frame a collection of diagnostic approaches that might be adapted to the particular challenges faced by marine mammal veterinarians, to enhance the evaluation of cetacean mammary gland morphology, physiology and development.
Fibroblast growth factor receptors (FGFRs) are critical mediators of cellular signaling involved in development, tissue repair, and metabolic homeostasis. Dysregulated FGFR signaling is also a common feature in multiple cancer types, including breast cancer. In breast cancer, aberrant FGFR signaling can occur by amplification, mutation, isoform switching, or gene fusion and has emerged as a driver of tumor progression, metastasis, and therapeutic resistance. Beyond its canonical roles in proliferation and survival, recent evidence highlights FGFRs as key regulators of cancer cell metabolism. This review summarizes current findings on how FGFR signaling reprograms metabolic pathways in breast cancer, specifically glycolytic and lipid metabolism. We explore the interplay between FGFR activity and metabolic enzymes, transcription factors, and nutrient-sensing pathways, emphasizing subtype-specific metabolic vulnerabilities. Furthermore, we discuss how FGFR-mediated metabolic plasticity contributes to tumor heterogeneity and resistance to targeted therapies. Understanding the metabolic functions of FGFR signaling offers new opportunities for therapeutic intervention and biomarker development in breast cancer.
Among breast cancer subtypes, basal-like breast cancer (BLBC) is a highly aggressive form characterized by a lack of estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor (HER2) expression and is associated with poor prognosis, leaving chemotherapy as the sole treatment option available. Loss-of-function mutations in BRCA1 are strongly associated with the development of BLBC. Patients with this subtype are more likely to have grade III tumors and larger average tumor sizes than those with other subtypes of breast cancer. It is not known whether BRCA1 loss of function affects all cell types equally within breast tissue or if it has a preferential malignant impact on specific cell types, leading to the progression of lineage-specific tumorigenesis in the breast epithelium of women carrying BRCA1 mutations. Lineage tracing experiments using genetically engineered mouse models have provided critical insights into how BRCA1 loss alters cellular hierarchy within the mammary gland. These studies have demonstrated that BRCA1-deficient luminal progenitors can aberrantly differentiate into basal-like cells, suggesting that BLBC may arise from a misregulated luminal compartment rather than pre-existing basal stem cells. Understanding the mechanisms underlying BRCA1-mediated lineage plasticity offers novel therapeutic avenues to target early-stage tumor initiation and progression in BRCA1-mutated breast cancer. This review perspective sheds light on the role of BRCA1 in lineage plasticity and highlights probable mechanisms by which BRCA1 could promote this lineage plasticity.
The neglect of research into women’s health and female biology has had major impacts for the fields of mammary biology and cancer. A quarter of the way through the twenty-first century, we still lack basic knowledge regarding the formation and function of the organ that gives its name to all mammals, and which provides important health benefits for children and their breastfeeding parent through the creation and delivery of breast milk. In this review, we highlight key similarities and differences in mouse and human mammary glands, and discuss how both systems of investigation are important and necessary to fill outstanding knowledge gaps. We discuss important discoveries that have arisen through mouse models as well as methodological advances that have enabled more widespread investigations in human samples. Finally, we contend that the translatability of mammary gland research requires thoughtful design, careful evaluation and continued review, irrespective of the system of investigation.
