Cultures Of Mammary Epithelial Cells Research Articles

Malignant canine mammary epithelial cells shed exosomes containing differentially expressed microRNA that regulate oncogenic networks

BackgroundBreast (mammary) cancers in human (BC) and canine (CMT) patients share clinical, pathological, and molecular similarities that suggest dogs may be a useful translational model. Many cancers, including BC, shed exosomes that contain microRNAs (miRs) into the microenvironment and circulation, and these may represent biomarkers of metastasis and tumor phenotype.MethodsThree normal canine mammary epithelial cell (CMEC) cultures and 5 CMT cell lines were grown in serum-free media. Exosomes were isolated from culture media by ultracentrifugation then profiled by transmission electron microscopy, dynamic light scattering, and Western blot. Exosomal small RNA was deep-sequenced on an Illumina HiSeq2500 sequencer and validated by qRT-PCR. In silico bioinformatic analysis was carried out to determine microRNA gene and pathway targets.ResultsCMEC and CMT cell lines shed round, “cup-shaped” exosomes approximately 150–200 nm, and were immunopositive for exosomal marker CD9. Deep-sequencing averaged ~ 15 million reads/sample. Three hundred thirty-eight unique miRs were detected, with 145 having > ±1.5-fold difference between one or more CMT and CMEC samples. Gene ontology analysis revealed that the upregulated miRs in this exosomal population regulate a number of relevant oncogenic networks. Several miRNAs including miR-18a, miR-19a and miR-181a were predicted in silico to target the canine estrogen receptor (ESR1α).ConclusionsCMEC and CMT cells shed exosomes in vitro that contain differentially expressed miRs. CMT exosomal RNA expresses a limited number of miRs that are up-regulated relative to CMEC, and these are predicted to target biologically relevant hormone receptors and oncogenic pathways. These results may inform future studies of circulating exosomes and the utility of miRs as biomarkers of breast cancer in women and dogs.

IL-1β directly inhibits milk lipid production in lactating mammary epithelial cells concurrently with enlargement of cytoplasmic lipid droplets

Mammary epithelial cells (MECs) in lactating mammary glands produce milk lipid, which provides a large percentage of calories and bioactive lipids for appropriate infant growth. However, secreted milk lipid is often reduced concurrently with increases in IL-1β, IL-6, and TNF-α in mammary glands with mastitis. In this study, we investigated whether those cytokines directly influenced lipid production and secretion. A lactating MEC culture model with high lipid production ability was prepared by culture with oleic acid. TNF-α, IL-1β, and IL-6 differentially affected lipid production and secretion in lactating MECs. In particular, IL-1β treatment significantly reduced amounts of secreted triglycerides by 97% compared with the control concurrently with enlargement of cytoplasmic lipid droplets in MECs. IL-1β also decreased mRNA expression of Fabp3 and Srebp1 and the amount of aquaporin 3, GLUT-1 and adipophilin in the milk lipid production pathway. Furthermore, IL-1β inactivated STAT5 and glucocorticoid signaling to induce milk production in MECs, whereas STAT3 and NFκB signaling was activated. IL-1β induced mRNA expression of IL-6 and TNF-α in MECs. Therefore, we suggest that IL-1β is a key inhibitor of lipid production and secretion in lactating MECs.

Characterization of MCF-12A cell phenotype, response to estrogens, and growth in 3D

BackgroundThree-dimensional cultures of mammary epithelial cells allow for biologically-relevant studies of the development of the mammary gland in rodents and humans under normal and pathological conditions, like carcinogenesis. Under these conditions, mammotropic hormones play significant roles in tissue morphogenesis. Therefore, a system that recreates the normal, hormonally responsive epithelium would be a valuable tool to study the normal state and its transition to carcinogenesis. MCF-12A cells have been claimed to be non-tumorigenic mammary epithelial cells with reported sensitivity to estrogens. In this study, we aimed at characterizing MCF-12A cells for use in a hormone-responsive 3D culture system to determine their usefulness as a tool to identify normal and abnormal microenvironmental cues.MethodsMCF-12A cells were single-cell cloned in order to investigate their heterogeneous makeup. The parental cells were then treated with estradiol to investigate proliferative and transcriptional responses through the estrogen receptor alpha. Finally, parental cells and epithelial-like cell-derived clones were seeded in rat-tail collagen I to profile the morphogenesis of multicellular 3D structures. The resultant structures were then analyzed using unsupervised morphometric analysis.ResultsMCF-12A cells consist of epithelial-like colonies which shed elongated, freely growing cells on the colony’s edges. The cells express E-cadherin as well as mesenchymal vimentin but do not express markers associated with myoepithelial cells or fibroblasts. Treatment with estradiol does not affect either the proliferation rate or the induction of gene expression in MCF-12A cells. Parental MCF-12A cells form acini, solid spheres and elongated branching ducts when grown in rat-tail collagen type I matrix, the geometries and distribution of which are altered following the removal of fibroblast-like cells.ConclusionsMCF-12A cells are a heterogeneous pseudo-epithelial cell line capable of forming a variety of multicellular structures in 3D culture. We found no indication that the cells display estrogen-responsive characteristics, thus refuting previous studies which reported estrogen responsiveness. We report that MCF-12A cells are not suited for use in studies in which differential behaviors of “normal” and “cancerous” estrogen-responsive cells are to be compared.

Innate defense capability of challenged primary bovine mammary epithelial cells after an induced negative energy balance in vivo.

Negative energy balance (NEB), if followed by metabolic imbalance, is a common problem in high-yielding dairy cows frequently associated with inflammation of the mammary gland. After entering the teat canal, mammary epithelium is the first line of defense against a pathogen invasion. To investigate the effect of NEB on the innate host defense of the mammary epithelium, primary bovine mammary epithelial cell (pbMEC) cultures were generated by cell extraction of milk derived from energy restricted and control feeding cows. pbMEC were obtained from 8 high-yielding dairy cows affected by induced NEB in mid-lactation due to a reduction to 51 ± 2% of total energy requirement (restriction group) and from 7 control cows (control group). They were exposed to heat-inactivated Escherichia coli and Staphylococcus aureus for 24 and 72 h to investigate the influence of NEB on gene expression profiles of cytokines, chemokines, genes associated with apoptosis and antimicrobial peptides plus their receptors (AMPR) of the innate immune response. The immune challenge of pbMEC demonstrated an effect of immune capacity and NEB in 15 differential expressed genes. NEB induced a substantial up-regulation in restriction compared to control cells by trend in E. coli and a down-regulation in S. aureus exposed cells. Our investigations showed that the dietary-induced NEB in vivo influenced the immune response of pbMEC in vitro and altered the expression of immunological relevant genes due to a difference in energy supply. These results demonstrate that pbMEC are a suitable model for mastitis research, in which even effects of feeding regimes can be displayed.

Blimp-1/PRDM1 is a critical regulator of Type III Interferon responses in mammary epithelial cells

The transcriptional repressor Blimp-1 originally cloned as a silencer of type I interferon (IFN)-β gene expression controls cell fate decisions in multiple tissue contexts. Conditional inactivation in the mammary gland was recently shown to disrupt epithelial cell architecture. Here we report that Blimp-1 regulates expression of viral defense, IFN signaling and MHC class I pathways, and directly targets the transcriptional activator Stat1. Blimp-1 functional loss in 3D cultures of mammary epithelial cells (MECs) results in accumulation of dsRNA and expression of type III IFN-λ. Cultures treated with IFN lambda similarly display defective lumen formation. These results demonstrate that type III IFN-λ profoundly influences the behavior of MECs and identify Blimp-1 as a critical regulator of IFN signaling cascades.

Lipid Metabolism in Mammary Epithelial Cells-A Comparison of Common In vitro Models

One of the main cellular functions of mammary epithelial cells (MEC) is the vast production of lipids to provide the new born with the energy and bioactive molecules that are essential for its development and survival. Milk lipids are secreted in a structure termed milk fat globule (MFG) which buds from the MEC, enveloped with the cellular bilayer membrane. MFG membrane composition changes as a function of lactation stage, nutrition and MFG size. In addition, MFG size differs at different stages of lactation and in response to nutritional treatments. Therefore, it is hypothesized that the MEC membrane changes according to metabolic and hormonal signals in the animal's body, and that these changes are associated with milk lipid production and secretion and MFG size. The aim of this work was to find a suitable model for studying lipogenic activity and its relation to membrane composition in the mammary gland. Two in vitro models were compared: mammary gland tissue culture ('explants') and primary culture of MEC. Prolactin treatment increased fat concentration in the explants. In addition, conjugated linoleic acid decreased fat content, but only in the presence of prolactin. Despite these interesting results, the model was not reproducible. Cells of the primary MEC culture showed cytokeratin-18 mRNA and protein expression, which validated the culture's epithelial content. In addition, this model was prolactin-sensitive, as reflected by induction of α-lactalbumin expression in response to prolactin administration. Oleic acid induced the formation of large lipid droplets, and methionine addition to the oleic acid treatment further increased lipid droplet size. In conclusion, primary culture of bovine MEC was found as an appropriate model for studying lipogenic activity with a focus on membrane composition and lipid droplet size in the mammary gland.

A dioxin-like compound induces hyperplasia and branching morphogenesis in mouse mammary gland, through alterations in TGF-β1 and aryl hydrocarbon receptor signaling

Hexachlorobenzene (HCB) is a widespread environmental pollutant and a dioxin-like compound that binds weakly to the aryl hydrocarbon receptor (AhR). Because AhR and transforming growth factor β1 (TGF-β1) converge to regulate common signaling pathways, alterations in this crosstalk might contribute to developing preneoplastic lesions. The aim of this study was to evaluate HCB action on TGF-β1 and AhR signaling in mouse mammary gland, through AhR+/+ and AhR−/− models. Results showed a differential effect in mouse mammary epithelial cells (NMuMG), depending on the dose: 0.05μM HCB induced cell migration and TGF-β1 signaling, whereas 5μM HCB reduced cell migration, promoted cell cycle arrest and stimulated the dioxin response element (DRE) -dependent pathway. HCB (5μM) enhanced α-smooth muscle actin expression and decreased TGF-β receptor II mRNA levels in immortalized mouse mammary fibroblasts AhR+/+, resembling the phenotype of transformed cells. Accordingly, their conditioned medium was able to enhance NMuMG cell migration. Assays in C57/Bl6 mice showed HCB (3mg/kg body weight) to enhance ductal hyperplasia, cell proliferation, estrogen receptor α nuclear localization, branch density, and the number of terminal end buds in mammary gland from AhR+/+ mice. Primary culture of mammary epithelial cells from AhR+/+ mice showed reduced AhR mRNA levels after HCB exposure (0.05 and 5μM). Interestingly, AhR−/− mice exhibited an increase in ductal hyperplasia and mammary growth in the absence of HCB treatment, thus revealing the importance of AhR in mammary development. Our findings show that environmental HCB concentrations modulate AhR and TGF-β1 signaling, which could contribute to altered mammary branching morphogenesis, likely leading to preneoplastic lesions and retaining terminal end buds.

Abstract 1539: Investigating the mechanisms of tumor suppression mediated by PTEN PDZ-binding domain in a murine breast cancer model

Abstract PTEN (Phosphatase and tensin homolog) functions as a key negative regulator of PI3K-Akt pathway through its lipid phosphatase activity. Mutations or loss of PTEN is frequently observed in brain, breast, prostate and endometrial cancer. PTEN possesses a PDZ-binding domain (PDZ-BD) at the end of its carboxyl terminus. Functionally, PDZ-BD mediates PTEN’s interactions with other PDZ domain-containing proteins, including ZO-1, PSD-95, DLG, etc, which are involved in the formation of cell junctions, post-synaptic densities, and serves as scaffolding proteins for multiple signaling complexes. However, whether the PTEN PDZ-binding domain is required to suppress tumorigenesis still remains obscure. To study the role of PTEN PDZ-BD in breast epithelial tumorigenesis, a PTEN-ΔPDZ mice lacking this domain was generated. PTEN expression level and activity of PI3K-Akt pathway are not changed in the mammary epithelial cells, indicating that PDZ-BD is dispensable for PTEN stability and negative regulation of the PI3K-Akt pathway. Further, we crossed the PTEN-ΔPDZ mice with the MMTV-PyMT breast cancer model and observed increased total tumor burden as well as higher tendency of lung metastasis in PTEN-ΔPDZ mice. This data indicates that PTEN PDZ-BD is indeed important for tumor suppression during breast cancer progression. Previously, PTEN has been reported to govern the integrity of tight junctions and apical-basal polarity of epithelial cells, which requires PTEN’s interactions with other proteins through the PDZ-BD. However, through in vitro three-dimensional culture of primary mouse mammary epithelial cells, we failed to observe any disruption of either apical-basal polarity or tight junction. Meanwhile, we investigated the alteration of the transcriptome of breast cancer tissue in PTEN-ΔPDZ mice by performing RNA-sequencing. Several enriched signaling pathways were identified by Ingenuity Pathway Analysis (IPA), which seems to be mainly associated with cell skeleton signaling as well as signaling interaction between extracellular matrix and the cancer cell. A list of genes were validated by RT-PCR, and functional validation of these genes during the tumorigenesis in our animal model is to be carried out in the near future. This work was supported by a General Research Fund grant (#460713) from the Hong Kong University Grants Committee to AC. YM was supported by a Hong Kong PhD Fellowship (PF12-13584), and a Lo Kwee-Seong Biomedical Research Seed Fund to AC. Citation Format: Mingfei Yan, Alexander Many, Hong Guan, Penelope M. Or, Andrew M. Chan. Investigating the mechanisms of tumor suppression mediated by PTEN PDZ-binding domain in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1539. doi:10.1158/1538-7445.AM2017-1539

Abstract 1798: Mortalin precursor as potential marker for chemoprevention with SHetA2

Abstract Introduction: The flexible heteroarotinoid (Flex-Het), SHetA2, is a novel anticancer drug that induces both intrinsic and extrinsic apoptosis and autophagy in cancer cells, but not in normal cells. Protein isolation / mass spectrometry analysis using SHetA2-coupled polystyrene magnetic beads yielded three SHetA2-binding proteins all belonging to HSPA family namely, HSPA9/mortalin, HSPA8/Hsc70 and HSPA5/BiP. Mortalin, in addition to its vital chaperoning roles in other organelles of the cell, is essential for import and folding of nuclear-encoded mitochondrial proteins. The precursor form of mortalin has a 46-amino acid N-terminal region that functions as a mitochondrial localization sequence (MLS) and is cleaved by proteases after import into the mitochondrial matrix. Hypothesis: SHetA2 binding to mortalin causes alterations that can be measured to study mechanism and monitor drug effects in animal models and clinical trials. Methods: Western blots of whole cell or subcellular protein extracts made from cultures of normal human fallopian tube secretory or mammary epithelial cells, rat mammary tumors or human ovarian cancer cell lines treated with SHetA2 or solvent were probed with an antibody that recognizes total mortalin, a custom-made rabbit polyclonal antibody specific for the mortalin MLS (PA8238) or antibodies to loading control proteins. Immunocytochemistry using these antibodies was performed with an automated (Leica Bond III) IHC work station on cells treated with SHetA2 or solvent. Results: Using a commercial antibody against total mortalin, we observed a lower-mobility band closely moving above the specific (mature) mortalin band in Western blots. Our mortalin MLS-specific antibody recognized only the lower-mobility band confirming it as the precursor form of mortalin in the SHetA2-treated cell extracts. Subcellular fractionation of the drug-treated cells revealed that the precursor protein accumulated only in the cytoplasm and not in the mitochondria. Staining of whole cells with an antibody to total mortalin showed no effect of SHetA2 treatment on the punctate pattern of expression consistent with mitochondrial localization. In contrast, staining with the mortalin MLS-specific antibody demonstrated that SHetA2 increased the intensity of the diffuse cytoplasmic staining consistent with localization of mortalin throughout the cytoplasm. These results were consistently observed among the various cell types. Conclusions: SHetA2 is the only known drug that blocks the translocation of the precursor mortalin to mitochondria and its processing to mature protein. Such cytoplasmic accumulation of the precursor form of mortalin can potentially serve as pharmacodynamic endpoint to study SHetA2’s effect in laboratory experimental tumor models as well as in human clinical trials. Funding: NCI PREVENT Task Order HHSN26100002 Citation Format: Elangovan Thavathiru, Vishal Chandra, Rajani Rai, Doris Benbrook. Mortalin precursor as potential marker for chemoprevention with SHetA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1798. doi:10.1158/1538-7445.AM2017-1798

Tightly controlled MRTF-A activity regulates epithelial differentiation during formation of mammary acini

BackgroundMyocardin-related transcription factors (MRTF) A and B link actin dynamics and mechanotransduction to gene expression. In mice, MRTF-A is involved in mammary gland differentiation, but its role in human mammary epithelial cells remains unclear.MethodsThree-dimensional cultures of human mammary epithelial MCF10A cells were used to model acinar morphogenesis. Stable MRTF-A knockdown, MRTF-A/B rescue and MRTF-A/B overexpression was established to characterize the functional role during morphogenesis using confocal microscopy and expression analysis. Breast cancer patient databases were analyzed for MRTF-A expression.ResultsWe showed that a precise temporal control of MRTFs is required for normal morphogenesis of MCF10A mammary acini. MRTF transcriptional activity, but not their protein amounts, is transiently induced during 3D acini formation. MRTF-A knockdown dramatically reduces acini size and prevents lumen formation. These effects are rescued by re-expression of MRTF-A, and partially by MRTF-B. Conversely, overexpression of MRTF-A and MRTF-B increases acini size, resulting in irregular spheroids without lumen and defective apico-basal polarity. These phenotypes correlate with deregulated expression of cell cycle inhibitors p21/Waf1, p27/Kip1 and altered phosphorylation of retinoblastoma protein. In MRTF overexpressing spheroids, proliferation and apoptosis are simultaneously increased at late stages, whilst neither occurs in control acini. MRTFs interfere with anoikis of the inner cells and cause an integrin switch from α6 to α5, repression of E-cadherin and induction of mesenchymal markers vimentin, Snai2 and Zeb1. Moreover, MRTF-overexpressing spheroids are insensitive to alteration in matrix stiffness. In two breast cancer cohorts, high expression of MRTF-A and known target genes was associated with decreased patient survival.ConclusionMRTF-A is required for proliferation and formation of mammary acini from luminal epithelial cells. Conversely, elevated MRTF activity results in pre-malignant spheroid formation due to defective proliferation, polarity loss and epithelial-mesenchymal transition.

CCN5/WISP-2 restores ER-\u221d in normal and neoplastic breast cells and sensitizes triple negative breast cancer cells to tamoxifen

CCN5/WISP-2 is an anti-invasive molecule and prevents breast cancer (BC) progression. However, it is not well understood how CCN5 prevents invasive phenotypes of BC cells. CCN5 protein expression is detected in estrogen receptor-α (ER-α) -positive normal breast epithelial cells as well as BC cells, which are weakly invasive and rarely metastasize depending on the functional status of ER-α. A unique molecular relation between CCN5 and ER-α has been established as the components of the same signaling pathway that coordinate some essential signals associated with the proliferation as well as delaying the disease progression from a non-invasive to invasive phenotypes. Given the importance of this connection, we determined the role of CCN5 in regulation of ER-α in different cellular settings and their functional relationship. In a genetically engineered mouse model, induced expression of CCN5 in the mammary ductal epithelial cells by doxycycline promotes ER-α expression. Similarly, CCN5 regulates ER-α expression and activity in normal and neoplastic breast cells, as documented in various in vitro settings such as mouse mammary gland culture, human mammary epithelial cell and different BC cell cultures in the presence or absence of human recombinant CCN5 (hrCCN5) protein. Mechanistically, at least in the BC cells, CCN5 is sufficient to induce ER-α expression at the transcription level via interacting with integrins-α6β1 and suppressing Akt followed by activation of FOXO3a. Moreover, in vitro and in vivo functional assays indicate that CCN5 treatment promotes response to tamoxifen in triple-negative BC (TNBC) cells possibly via restoring ER-α. Collectively, these studies implicates that the combination treatments of CCN5 (via activation of CCN5 or hrCCN5 treatment) and tamoxifen as potential therapies for TNBC.

Abstract P3-05-05: PTHrP affects breast cancer initiation and progression through Stat5 signaling pathway

Abstract PTHrP affects mammary development and breast cancer. PTHrP is expressed in basal myoepithelial cells until late pregnancy, when it also becomes expressed in alveolar epithelial cells. During lactation, PTHrP is secreted into milk and into the circulation, and regulates systemic calcium and bone metabolism. Secretion of PTHrP by breast cancers stimulates bone resorption, promoting the skeletal metastases and/or development of hypercalcemia. Less is known about the contribution of PTHrP to development and/or progression of primary breast cancers. Recent GWAS reports have identified the PTHrP gene as a breast cancer susceptibility locus. Using data from the Cancer Genome Atlas (TCGA) Project and a Yale tissue microarray (YTMA49) we have found that increased breast tumor PTHrP expression predicts a more aggressive phenotype and increased mortality, signifying the clinical relevance of high tumor PTHrP levels. In an attempt to define how PTHrP affects breast cancer, we assessed the effects of PTHrP overexpression on the development of mammary tumors in mice. We developed a tetracyline-regulated, MMTV-driven transgenic model of PTHrP overexpression targeted to mammary epithelial cells (MMTV-rtTA;tetO-hPTHrP). Surprisingly, we found that PTHrP overexpression in luminal epithelial cells causes alveolar hyperplasia, secretory differentiation and milk production in virgin mice, associated with a lower number of luminal progenitor cells and basal stem cells upon FACS analysis. Additionally, Signal Transducer and Activator of Transcription 5 (Stat5) is highly activated in the setting of PTHrP-induced alveolar hyperplasisa. This activation is functionally relevant as evidenced by higher levels of β-Casein mRNA and protein, the transcription of which is regulated by activated Stat5 (pStat5). This effect of PTHrP was reversible upon withdrawal of doxycycline (Dox). Addition of Dox to primary mammary epithelial cell cultures from these mice recapitulated the findings. Knocking out PTHrP receptor (PTH1R) in these mice did not affect this phenotype, suggesting that PTHrP acts in a receptor-independent manner. Overexpression of PTHrP in MMTV-PyMT mice dramatically accelerated formation of mammary tumors reducing both latency of tumor formation and survival of the mice. It caused a higher rate of proliferation and a lower rate of apoptosis in vivo. All MMTV-rtTA;tetO-hPTHrP;MMTV-PyMT mice developed palpable tumors in all mammary glands within 3 weeks of age, became hypercalcemic and died before 4.5 weeks of age. These mice had higher levels of pStat5 and βCasein. Tumor cells cultured ex vivo and cell lines established from these tumors overexpressed PTHrP when stimulated with Dox. PTHrP overexpression, and not exogenous PTHrP, caused higher levels of proliferation, pStat5, and β-Casein. This PTHrP-induced activity of Stat5 was blocked by treatment of these cells with a pharmacological inhibitor of Jak2, a tyrosine kinase upstream of Stat5. Additionally, T47D cells transfected with PTHrP showed higher levels of pStat5, suggesting that PTHrP could activate Stat5 signaling regardless of the cell type. We show that PTHrP overexpression results in Stat5 activation and increased proliferation in breast cancer cells in a cell-autonomous fashion and independent of its receptor. Citation Format: Takyar FM, Boras-Granic K, Kim W, Dann P, Wysolmerski JJ. PTHrP affects breast cancer initiation and progression through Stat5 signaling pathway [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-05.

Abstract C24: Interplay between ECM stiffness, miR-203 and mammographic density

Abstract High mammographic density (MD) is associated with a greater risk of breast cancer and is characterized by high extracellular matrix (ECM) collagen. Elevated collagen increases ECM stiffness and can promote the malignant transformation of oncogenically-primed mammary epithelial cells in culture and in vivo. Whether or not high MD reflects a breast stroma that is stiff and if ECM stiffness accounts for the elevated breast cancer risk in high MD tissue and how has yet to be resolved. To address this question we are conducting a comprehensive biophysical and molecular analysis of BIRADS status in prophylactic mastectomies in women with low (BIRADS 1) versus high MD (BIRADS 4). Data revealed that the intra-lobular ECM associated with the terminal end-buds contains anisotropic compliant and relaxed collagen fibrils. By contrast, the inter-lobular ECM contains stiff, oriented collagen fibrils. Preliminary data suggest that the ECM associated with the terminal end-buds in the upper outer quadrant may be stiffer in women with high MD as compared to low MD. These same tissues also expressed lower levels of the microRNA miR-203, which has been implicated in epithelial-to-mesenchymal transition (EMT) inhibition. Consistently, in vitro studies revealed that miR-203 was repressed in MECs cultured on a stiff ECM. We also found that a stiffened mammary ECM decreases miR-203 expression and promotes an EMT. Additionally, inhibiting ECM stiffening restored miR-203 levels and reduced an EMT. Moreover and importantly, we showed that triple negative breast tumors that often express EMT markers have reduced miR-203 levels. Studies are underway to elaborate these preliminary findings and to assess their clinical relevance. (This work is supported by the: NIH NSRA T32 CA 108462-11 to Ivory Dean, Susan G Komen PDF12230246 to Irene Acerbi, W81XWH-13-1-0216, USMRAA DOD-BCRP BC122990, NIH R01 CA138818-01A1 and NIH R01 CA192914-01 to Valerie Weaver.) Citation Format: Ivory Dean, Irene Acerbi, Janna Mouw, Quanming Shi, Yunn-Yi Chen, Jan Liphardt, Shelley Hwang, Valerie Weaver. Interplay between ECM stiffness, miR-203 and mammographic density. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C24.

Abstract A58: Cell motility in a basement membrane gel concentrates ECM around breast epithelial cells, a feature lost in malignant cells

Abstract Introduction: The basement membrane (BM) underlying epithelial cells plays a key role in regulating cell function and suppressing malignancy; loss of this layer of ECM proteins is a hallmark of invasive cancer. Using 3D extracellular matrix (ECM) gels, we sought to understand how this structure develops by comparing nonmalignant breast epithelial cells (BEC), which develop into polarized, growth arrested structures to malignant breast cancer cells (BCC), which form disorganized masses under the same conditions. These two cell types show different movement patterns in the ECM gels: BEC rotate around their center of mass in a process termed coherent angular motion, whereas BCC migrate randomly, with unknown consequences for assembly of ECM into a basement membrane-like layer. We investigated whether coherent angular motion could concentrate exogenous ECM into a BM like structure, and whether these changes were due to the physical forces applied by the cells on their microenvironment. Methods: To determine if cells undergoing coherent angular motion were better able to concentrate exogenous ECM at their surface compared to malignant cells, single human BEC and BCC from the HMT3522 breast cancer progression series were cultured in fluorescent laminin rich ECM gels (LrECM; using BD Matrigel), and ECM density at the cell surface was measured during growth with confocal microscopy. To determine how motility translated into forces on the microenvironment, cells were embedded in lrECM labeled with a dilute suspension of fluorescent microbeads. Confocal microscopy was performed over the first several days of culture, and traction force microscopy was used to determine the magnitude and spatial pattern of forces applied by the cells. After 5-6 days of culture, cells stained to determine whether cells had developed into polarized structures. Results: Over the first several days of culture, non-malignant BEC rotated as a coherent mass. At 24 hours, labeled ECM proteins were observed to concentrate in a spherical shell on surface of rotating clusters of BEC, whereas malignant, non-rotating cell masses had lower levels of ECM at the surface of the mass and higher levels of ECM proteins between the cells. Traction patterns between rotating and non-rotating cell masses were observed to differ- cells undergoing random motility applied strains to the ECM along the line of invasion, whereas cells undergoing coherent angular motion showed shear strains at the cell surface and even inward tractions. Conclusions: Coherent angular motion appears to affect concentration of exogenous ECM by physically pulling ECM proteins into an organized shell at the cell surface as an early aspect of formation of a basement membrane-like ECM structure in mammary epithelial cells in culture whereas this process is defective in malignant cells undergoing random motility. Further investigation will determine whether this process of motility, physically altering ECM density, is seen during normal development. Citation Format: Claire Robertson, Mina J. Bissell. Cell motility in a basement membrane gel concentrates ECM around breast epithelial cells, a feature lost in malignant cells. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A58.

Optimized protocol for establishment of primary goat mammary fibroblast and epithelial cell cultures by selective trypsinization

Optimized protocol for establishment of primary goat mammary fibroblast and epithelial cell cultures by selective trypsinization

Role of Prolactin in Promotion of Immune Cell Migration into the Mammary Gland.

Immune cells in the mammary gland play a number of important roles, including protection against infection during lactation and, after passing into milk, modulation of offspring immunity. However, little is known about the mechanism of recruitment of immune cells to the lactating gland in the absence of infection. Given the importance of prolactin to other aspects of lactation, we hypothesized it would also play a role in immune cell recruitment. Prolactin treatment of adult female mice for a period equivalent to pregnancy and the first week of lactation increased immune cell flux through the mammary gland, as reflected in the number of immune cells in mammary gland-draining, but not other lymph nodes. Conditioned medium from luminal mammary epithelial HC11 cell cultures was chemo-attractive to CD4+ and CD8+ T cells, CD4+ and CD8+ memory T cells, B cells, macrophages, monocytes, eosinophils, and neutrophils. Prolactin did not act as a direct chemo-attractant, but through effects on luminal mammary epithelial cells, increased the chemo-attractant properties of conditioned medium. Macrophages and neutrophils constitute the largest proportion of cells in milk from healthy glands. Depletion of CCL2 and CXCL1 from conditioned medium reduced chemo-attraction of monocytes and neutrophils, and prolactin increased expression of these two chemokines in mammary epithelial cells. We conclude that prolactin is an important player in the recruitment of immune cells to the mammary gland both through its activities to increase epithelial cell number as well as production of chemo-attractants on a per cell basis.

Using 3D Culture of Primary Mammary Epithelial Cells to Define Molecular Entities Required for Acinus Formation: Analyzing MAP Kinase Phosphatases.

Three-dimensional (3D) cell cultures on reconstituted basement membrane (rBM) enable the study of complex interactions between extracellular matrix (ECM) components and epithelial cells, which are crucial for the establishment of cell polarity and functional development of epithelia. 3D cultures of mammary epithelial cells (MECs) on Matrigel (a laminin-rich ECM derived from the Engelbreth-Holm-Swarm (EHS) murine tumor) promote interactions of MECs with the matrix via integrins, leading to formation of spherical monolayers of polarized cells surrounding a hollow lumen (acini). Acini closely resemble mammary alveoli found in the mammary gland. Thus, it is possible to study ECM-cell interactions and signalling pathways that regulate formation and maintenance of tissue-specific shape and functional differentiation of MECs in 3D under in vitro conditions. Here we present experimental protocols used to investigate the role of mitogen-activated protein kinase phosphatases (MKPs) during development of the alveoli-like structures by primary mouse mammary epithelial cells (PMMEC) cultured on Matrigel. We present detailed protocols for PMMEC isolation, and establishment of 3D cultures using an "on top" method, use of specific kinase and phosphatases inhibitors (PD98059 and pervanadate, respectively) administered at different stages of acinus development, and give examples of analyses carried out post-culture (Western blot, immunofluorescence staining, and confocal imaging).

In vitro characterization and endocrine regulation of cholesterol and phospholipid transport in the mammary gland

Cell-based studies previously showed that the ATP-binding cassette transporter A1 (ABCA1) transfers cholesterol across mammary epithelial cells (MEC). Data for phospholipid transport are lacking, and it is unclear from which cellular source the transported cholesterol stems, whether this transport activates signaling pathways, and how lactogenic hormones regulate it. To clarify these aspects, lipid transport and expressional analyses were performed in bovine primary (bMEC) and/or immortalized (MAC-T) MEC cultures. Lipid efflux and ABCA1, ABCG1 and liver X receptorα mRNA levels were higher in MAC-T than bMEC. In MAC-T, the transported cholesterol originated mainly from the plasma membrane. ABCA1 dependent cholesterol efflux was higher than phosphatidylcholine efflux, was suppressed by probucol (ABCA1 inhibitor), AG490 (janus kinase-2 inhibitor), PD98059 (mitogen activated protein kinase kinase inhibitor) and pretreatment with β-cyclodextrin (lowering membrane cholesterol). Insulin was the only hormone significantly increasing cholesterol efflux. In conclusion, this study gives novel mechanistic and regulatory insights into the transport of cholesterol and phospholipids in MEC.

Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro.

Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46(+)/CD3(-)) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis.

Abstract 656: Histological and 3D morphological evaluation of mammary cancers and primary cells from genetically engineered mice with only one copy of Brca1 disrupted in combination with Trp53 haploinsufficiency

Abstract Background: Previously established genetically engineered mouse (GEM) models with spontaneous mammary cancer development have both Brca1 alleles disrupted withTrp53 haploinsufficiency (Brca1f11/f11/MMTV-Cre/Trp53+/-). Cell culture plates with nanoimprinted scaffolds (SCIVAX Life Sciences, Inc) purportedly increase concordance between in vitro/in vivo results but reports are limited to human cancer cell lines. Here we characterized hyperplasia and cancer development in Brca1f11/WT11/MMTV-Cre/Trp53+/- mice and established conditions for 3D culture of primary mammary epithelial cells (MEC) using the imprinted plates. Methods: Time-course of mammary hyperplasia and cancer development was defined in female Brca1f11/WT11/MMTV-Cre/Trp53+/- (n = 23) and Brca1f11/f11/MMTV-Cre/Trp53+/- (n = 13) mice euthanized at age 6 and 12 months (m) or when largest tumor reached 1 cm3. Mammary tissue was taken at necropsy for histology (age 6/12m) and primary MEC culture (age 6m) using EpiCult-B (StemCell Technologies) on nanoimprinted scaffold plates. Primary normal (wild-type, n = 2) and precancerous MEC (Brca1f11/f11/MMTV-Cre/Trp53+/-, n = 2; Brca1f11/WT11/MMTV-Cre/Trp53+/-; n = 2) were cultured. Optimal conditions for spheroid growth were established by comparison of spheroid formation from thoracic vs. inguinal mammary glands with different numbers of plated cells and percentage FBS after 7 days culture. Endpoints included sphere number, size and presence/absence of concurrent monolayer growth. Spheres were harvested for histological examination. Hematoxylin and eosin (H&E) stained sections were read for histology and immunohistochemistry (IHC) for Pan-Cytokeratin (Pan-CK) and Ki67 performed for in vivo/in vitro comparisons. Results: Forty-two percent of Brca1f11/WT11/MMTV-Cre/Trp53+/- mice developed mammary cancer by age 12m compared to 100% of Brca1f11/f11/MMTV-Cre/Trp53+/- mice. Cancers appeared by age 6m only in Brca1f11/f11/MMTV-Cre/Trp53+/- mice. Mammary cancers in both models were carcinomas of either adeno, anaplastic or sarcomatoid types. All were Pan-CK positive and proliferation rates were similar between the two genetic models. Spheres developed from all primary MEC attempted. Optimal conditions for sphere formation were plating of 10,000 cells obtained from inguinal mammary gland per well in 5% FBS EpiCult-B. Conclusions: Here we report development of spontaneous mammary cancers in Brca1 insufficiency mice that are more representative of disease development in women who carry only one allele with a BRCA1-mutation and established conditions for use of cell culture plates with nanoimprinted scaffolds for 3D culture of normal and preneoplastic primary murine mammary epithelial cells. Supported by NCI, NIH 1RO1CA112176 (PAF). Citation Format: Sahar J. Alothman, Weisheng Wang, Bhaskar V. Kallakury, Priscilla Furth. Histological and 3D morphological evaluation of mammary cancers and primary cells from genetically engineered mice with only one copy of Brca1 disrupted in combination with Trp53 haploinsufficiency. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 656.