Cultures Of Mammary Cells Research Articles

Interplay of the transcription factor MRTF-A and matrix stiffness controls mammary acinar structure and protrusion formation

BackgroundOngoing differentiation processes characterize the mammary gland during sexual development and reproduction. In contrast, defective remodelling is assumed to be causal for breast tumorigenesis. We have shown recently that the myocardin-related transcription factor A (MRTF-A) is essential for forming regular hollow acinar structures. Moreover, MRTF-A activity is known to depend on the biochemical and physical properties of the surrounding extracellular matrix. In this study we analysed the mutual interaction of different matrix stiffnesses and MRTF-A activities on formation and maintenance of mammary acini.MethodsHuman MCF10A acini and primary mature organoids isolated from murine mammary glands were cultivated in 3D on soft and stiff matrices (200–4000 Pa) in conjunction with the Rho/MRTF/SRF pathway inhibitor CCG-203971 and genetic activation of MRTF-A.ResultsThree-dimensional growth on stiff collagen matrices (> 3000 Pa) was accompanied by increased MRTF-A activity and formation of invasive protrusions in acini cultures of human mammary MCF10A cells. Differential coating and synthetic hydrogels indicated that protrusion formation was attributable to stiffness but not the biochemical constitution of the matrix. Stiffness-induced protrusion formation was also observed in preformed acini isolated from murine mammary glands. Acinar outgrowth in both the MCF10A acini and the primary organoids was partially reverted by treatment with the Rho/MRTF/SRF pathway inhibitor CCG-203971. However, genetic activation of MRTF-A in the mature primary acini also reduced protrusion formation on stiff matrices, whilst it strongly promoted luminal filling matrix-independently.ConclusionOur results suggest an intricate crosstalk between matrix stiffness and MRTF-A, whose activity is required for protrusion formation and sufficient for luminal filling of mammary acini.Dfb3mXyQrGPoxja4C3DNjFVideo

Pathways Involved in the Development of Vasculogenic Mimicry in Canine Mammary Carcinoma Cell Cultures

Vasculogenic mimicry (VM) is the ability of highly aggressive cancer cells to form fluid-conducting channels that facilitate the nutrition and metastasis of cancer cells. Considering the importance of VM in the prognosis of canine mammary gland tumours, this study aimed to investigate global gene expression in two canine mammary carcinoma cell cultures associated with the capacity for VM invitro. The cell lines were subjected to an in-vitro assay to form VM channels (3D culture). Each cell line was then used in 2D conditions as controls and we compared the global gene expression with that of the 3D cultures. A total of 1,217 differentially expressed genes (DEGs) (P<0.05, fold change >2.0 or <2.0) were observed in 3D conditions compared with 2D culture in the UNESP-CM9 cell line, of which 677 were upregulated genes and 540 were downregulated. In contrast, the UNESP-CM60cell line had only one upregulated and two downregulated genes. Overall, we identified several genes and pathways involved in the development of VM and these molecular data will be useful for future studies aimed at identifying diagnostic and therapeutic targets for VM in canine mammary carcinoma.

HER2/p53 ratios among canine mammary tumours and respective derived cell culture

In this study, the expression of p53 and human epidermal growth factor receptor 2 (HER2) proteins were investigated in different canine mammary tumors (CMT) and cell cultures derived from them, for the purpose of determining neoplastic behavior in vitro. For this purpose, immunohistochemistry and immunocytochemistry were performed and results were compared according to histological diagnose. CMT were collected and divided into two halves, one for histological and immunohistochemistry analyses and another for cell cultures. Cell markers p53 and HER2 proteins were detected by immunohistochemistry in the original tumors and immunocytochemistry in CMT-derived cell lines. A total of CMT (n = 17) were classified and divided into benign mixed tumor (BMT), complex carcinoma (CC), simple carcinoma (SC), and squamous cell carcinoma (SCC). Low expression of p53 was verified in BMT and CC, averagely in SC and undetectable in SCC. In vitro, there was an increase in p53 expression by cultured cells. However, HER2 protein expression was not significant in BMT, CC and SCC. In contrast, MCT-derived cells were considered HER2-positive in all MCT types. In conclusion, the in vitro model is essential for the study of neoplastic behavior and understanding of the carcinogenic mechanisms that occur in vivo.

Effects of Stryphnodendron adstringens extracts on murine 4T1 tumor line

Breast cancer appears as the main gynecological cancer and presents high morbidity and mortality. Because most diagnoses are made belatedly, it is necessary to seek therapeutic options that aim for advanced stages of the disease. This study aims to evaluate the antitumoral action of Stryphnodendron adstringens fruit extracts on 4T1 murine mammary carcinoma cell culture. The inhibitory potential of S. adstringens fruit extract on the metalloproteinases (MMPs) 2 and 9 was evaluated through zymography. From these results, MTT assays were performed to evaluate the extracts’ effects on the murine mammary carcinoma 4T1 line cell viability. From the crude extract, the following extracts were obtained: hydroalcoholic (SAFCEA), hexane (SAFCEB), chloroform (SAFCEC), and ethyl acetate (SAFCED). Lastly, the migration of the cells treated with extracts SAFCEA and SAFCED was verified. The hydroalcoholic extract (SAFCEA) was the most efficient in inhibiting gelatinases. During the phytochemical study, it was noted that alkaloids were present in all partitions. The 50 % growth inhibition (IC50) concentrations found were: 40.1 μg/mL (SAFCEA) and 70.14 μg/mL (SAFCED). After the cellular cytotoxicity assay, cell morphology was altered by treatment with the selected partitions (SAFCEA and SAFCED), obtaining morphology consistent with apoptosis. The results demonstrate that S. adstringens extracts exhibit the inhibitory activity of MMP-2 and MMP-9 as well as cytotoxicity toward 4T1 tumor cells. These findings indicate that follow-up studies of the partitions from S. adstringens may lead to the development of novel chemotherapeutics for oncological treatments.

Effects of Lapatinib on HER2-Positive and HER2-Negative Canine Mammary Carcinoma Cells Cultured In Vitro.

HER2 is a prognostic and predictive marker widely used in breast cancer. Lapatinib is a tyrosine kinase inhibitor that works by blocking the phosphorylation of the receptor HER2. Its use is related to relatively good results in the treatment of women with HER2+ breast cancer. Thus, this study aimed to verify the effects of lapatinib on four canine primary mammary gland carcinoma cell cultures and two paired metastatic cell cultures. Cultures were treated with lapatinib at concentrations of 100, 500, 1000 and 3000 nM for 24 h and the 50% inhibitory concentration (IC50) for each cell culture was determined. In addition, a transwell assay was performed to assess the ability of lapatinib to inhibit cell migration. Furthermore, we verified HER2 expression by RT-qPCR analysis of cell cultures and formalin-fixed paraffin-embedded tissues from samples corresponding to those used in cell culture. Lapatinib was able to inhibit cell proliferation in all cell cultures, but it was not able to inhibit migration in all cell cultures. The higher the expression of HER2 in a culture, the more sensitive the culture was to treatment. This relationship may be an indication that the expression of HER2 may be a predictive factor and opens a new perspective for the treatment of primary and metastatic mammary gland cancer.

Mammary Organoids and 3D Cell Cultures: Old Dogs with New Tricks.

3D cell culture methods have been an integral part of and an essential tool for mammary gland and breast cancer research for half a century. In fact, mammary gland researchers, who discovered and deciphered the instructive role of extracellular matrix (ECM) in mammary epithelial cell functional differentiation and morphogenesis, were the pioneers of the 3D cell culture techniques, including organoid cultures. The last decade has brought a tremendous increase in the 3D cell culture techniques, including modifications and innovations of the existing techniques, novel biomaterials and matrices, new technological approaches, and increase in 3D culture complexity, accompanied by several redefinitions of the terms "3D cell culture" and "organoid". In this review, we provide an overview of the 3D cell culture and organoid techniques used in mammary gland biology and breast cancer research. We discuss their advantages, shortcomings and current challenges, highlight the recent progress in reconstructing the complex mammary gland microenvironment in vitro and ex vivo, and identify the missing 3D cell cultures, urgently needed to aid our understanding of mammary gland development, function, physiology, and disease, including breast cancer.

A new mechanism for a familiar mutation \u2013 bovine DGAT1 K232A modulates gene expression through multi-junction exon splice enhancement

BackgroundThe DGAT1 gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes. Since first described over 15 years ago, a protein-coding variant K232A has been assumed as the causative variant underlying these effects, following in-vitro studies that demonstrated differing levels of triglyceride synthesis between the two protein isoforms.ResultsWe used a large RNAseq dataset to re-examine the underlying mechanisms of this large milk production QTL, and hereby report novel expression-based functions of the chr14 g.1802265AA > GC variant that encodes the DGAT1 K232A substitution. Using expression QTL (eQTL) mapping, we demonstrate a highly-significant mammary eQTL for DGAT1, where the K232A mutation appears as one of the top associated variants for this effect. By conducting in vitro expression and splicing experiments in bovine mammary cell culture, we further show modulation of splicing efficiency by this mutation, likely through disruption of an exon splice enhancer as a consequence of the allele encoding the 232A variant.ConclusionsThe relative contributions of the enzymatic and transcription-based mechanisms now attributed to K232A remain unclear; however, these results suggest that transcriptional impacts contribute to the diversity of lactation effects observed at the DGAT1 locus.

Abstract 2785: Metabolic autofluorescence microscopy of 3D microscale macrophage-tumor co-cultures

Abstract Macrophages are ideal treatment targets due to their tumor-regulatory functions and high infiltration in the tumor microenvironment (TME). However, macrophage heterogeneity prevents effective therapeutic reprogramming. Macrophages have high plasticity and adopt phenotypes with diverse behavior and metabolism. The complex TME can drive this plasticity but is not well understood. Standard functional assays (e.g. flow cytometry, ELISA) lack sensitivity to heterogeneous cell populations. Also, their destructive sample processing limits spatial and temporal assessment. Thus, nondestructive, single cell imaging and analysis tools are needed to study macrophage heterogeneity within the TME. Optical metabolic imaging (OMI) measures two-photon excited fluorescence from the coenzymes NAD(P)H and FAD to resolve cellular metabolism within intact, 3D samples. The optical redox ratio (NAD(P)H intensity/FAD intensity) and fluorescence lifetimes reflect cell redox state and intracellular protein binding, respectively. Previous studies have shown that OMI detects spatial and temporal changes in stromal cells across in vivo and 3D in vitro models. Microscale models closely mimic the TME, are high throughput, and enable precise environmental control. Thus, microenvironmental stimulation of macrophage polarization and migration was mimicked in 3D microscale cultures of mouse macrophages (RAW264.7) and mammary carcinoma cells (PyVMT) in a collagen matrix. OMI captured redox ratio, NAD(P)H and FAD mean lifetime (τm) changes in mono-cultured and co-cultured macrophages 24-72 hours post-seeding. Intensity and lifetime volumes of macrophage layers further assessed metabolic changes in macrophages during migration. Co-cultured macrophages exhibited significantly increased (p&amp;lt;0.05) redox ratio and NAD(P)H τm compared to mono-cultured macrophages by 24 hours. Decreasing redox ratio in co-cultures over time suggested an oxidative metabolic shift, characteristic of M2-like phenotype. Distribution curves of redox ratio revealed macrophage subpopulations after 24 hours co-culture that disappear by 72 hours. Immunofluorescence markers of M1- or M2-like macrophages indicated heterogeneous polarization at 24 hours then transition to a homogeneous M2-like phenotype by 72 hours, consistent with metabolic imaging. Migration increased in co-cultured macrophages over time. Decreased redox ratio and increased NAD(P)H and FAD τm in migratory macrophages suggested proximity to the tumor affects macrophage metabolism. These results establish OMI for noninvasive monitoring of cell-level macrophage dynamics in 3D, in vitro TME models. Microscale co-culture and OMI were also demonstrated with primary mouse macrophage/Panc02 co-culture and human THP-1/MDA-MB-231 co-culture. Overall, these tools could better characterize heterogeneous macrophage behavior to improve regulation of anti-tumor activity. Citation Format: Tiffany M. Heaster, Jiaquan Yu, Margaret Edman, David J. Beebe, Melissa C. Skala. Metabolic autofluorescence microscopy of 3D microscale macrophage-tumor co-cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2785.

Palmitic acid increases the abundance of mRNA of genes involved in de novo synthesis of fat in mammary explants from lactating ewes

Culture of mammary cells with palmitic acid (PA) has shown contradictory results for gene expression of enzymes involved in de novo synthesis pathway. The objective of this study was to evaluate the effects of PA on the mRNA abundance of enzymes involved in de novo fatty acid (FA) synthesis pathway, acetyl-CoA carboxylase alpha from promoters II and III (ACACA-α PII and ACACA-α PIII), fatty acid synthase (FASN), fatty acid internalization (CD36 molecule), intracellular transport (fatty acid binding proteins; FABP3 and FABP4) and desaturation (stearoyl-CoA desaturase 1; SCD1) of FA during milk fat synthesis. Mammary explants obtained through biopsy from three Lacaune ewes with 40 days in milk (DIM) were used. The explants were cultured for 24 h using the following treatments: Control (culture medium + bovine serum albumin (BSA, 98%) and Palmitic Acid (culture medium + 75 μM of C16:0 - 99%). Immediately after incubation, total RNA was extracted, complementary DNA was synthesized, and quantitative real time polymerase chain reaction was performed. The PA treatment upregulated the mRNA expression levels of ACACA-α PIII, ACACA-α PII, and FASN by 1.5, 1.3, and 1.3 fold, respectively, compared to Control. The gene expression levels of CD36, FABP3, FABP4, and SCD1 were not affected by the PA treatment. Thus, PA treatment upregulated the mRNA expression of genes codifying proteins involved in the de novo synthesis pathway.

A multi-centre study on the role of the thioredoxin system in breast cancer cell proliferation

Redox proteins (thioredoxin, glutaredoxin) are key macromolecules capable of modulating intracellular processes. This determines research choices in the field of redox-dependent cell proliferation management. The study of the molecular mechanisms of the onset, development and progression of malignant neoplasms underlies the search for tumor-associated markers and potential targets for personalized antitumor therapy.Purpose.To establish the role of the “thioredoxin – thioredoxin-reductase” system in the impaired proliferation of mammary adenocarcinoma cells under the action of the cyclin-dependent protein kinase roskovitin blocker.Materials and methods.The study was carried out using the culture of mammary adenocarcinoma cells of the MCF-7 line incubated in the presence and absence of roskovitin at a final concentration of 20 μM for 18 h. The intracellular content of thioredoxin and protein regulators of proliferation (cyclin E and cyclin-dependent protein kinase 2) were determined by Western blotting technique, the expression level of thioredoxin mRNA was determined by real-time polymerase chain reaction and the activity of thioredoxin-reductase was measured by a spectrophotometric method.Results.It was established that the decrease in proliferative activity of MCF-7 tumor cells incubated in the presence of roskovitin was accompanied by a decrease in the content of cyclin E and cyclin-dependent kinase on the background of a decrease in the expression level of thioredoxin mRNA and an increase in the activity of thioredoxin-reductase.Conclusion.The involvement of the components of the thioredoxin system (thioredoxin, thioredoxinreductase) in disrupting the proliferation of MCF-7 tumor cells was detected under the action of the cyclindependent protein kinases of roskovitin.

Programming Niche Accessibility and In Vitro Stemness with Intercellular DNA Reactions.

Stem cells generally exist in low abundance and tend to lose stemness in the absence of self-renewal signals. While extracellular-matrix-mimicking techniques have been developed to support stem cell proliferation, the lack of niche cells in these synthetic systems often hampers continuous stem cell expansion and maintenance of pluripotency, which are indispensable for regenerative medicine. Here, an intercellular DNA-reaction-programmed ESPN (expansion of stem cells with pairing niches) strategy is developed for 3D culture of mammary stem cells (MaSCs). Boolean logic operations are implemented to confer DNA-programmed mechanical signaling and genetically engineered morphogen signaling by niche cells, resulting in sustained expansion of MaSCs in vitro. The creation of stem cell niches improves the proliferation of pluripotent cells by four times during one-week culture. This method thus provides a novel approach for logical regulation of stemness and proliferation of stem cells for biomedicine.

Abstract A59: Assessment of conditional reprogramming to generate 2D and 3D primary human mammary cell culture models

Abstract Human mammary gland development and differentiation are tightly regulated by hormones, growth factors, and microenvironmental cues. Rodent models have been used to help gain knowledge about mammary gland biology, but there are significant structural and hormonal response differences between the human and rodent mammary glands. Moreover, cultured immortalized human mammary cell lines have been widely used for in vitro experiments to study epithelial cell biology, but it has been questioned whether they faithfully recapitulate normal breast cells. Conditional reprogramming has recently emerged as an efficient method to induce rapid and inexhaustible in vitro proliferation of primary epithelial cells from normal and malignant tissues in two-dimensional (2D) culture conditions. However, studies using this method have not shown whether conditionally reprogrammed mammary epithelial cells can form defined structures in three-dimensional (3D) culture conditions. Therefore, our goal is to develop an appropriate in vitro model using conditional reprogramming to study human mammary cell and tissue function under 2D and 3D culture conditions. We cultured primary human mammary cells from normal prophylactic tissues or breast tumors using this method. Cell type heterogeneity, cellular marker expression, and structural arrangement were examined using immunofluorescence staining. We found that normal breast cells grown under this culture condition exhibited morphologic features of luminal cells (CK18, desmoglein 3, and CK19) and myoepithelial cells (vimentin, p63, and CK14), indicating maintenance of in vivo heterogeneity. CD49f and EpCAM double staining is commonly used to separate luminal, basal, and progenitor populations. Immunofluorescence and FACS analysis further revealed subpopulations with varying CD49f and EpCAM expression profiles in the normal primary cultures, as well as detectable expression of ERα in earlier passages. Treatment with estradiol also stimulated cellular proliferation as detected by positive EdU staining. When grown in Matrigel/Collagen I gel, normal primary cells self-organize into two distinct 3D structures that are composed of densely packed cells or a spherical structure containing a lumen, which express either luminal or myoepithelial cell markers, respectively. CK8-positive luminal cells that form the lumen can differentiate into milk-producing cells in the presence of a prolactogenic growth condition. Tumor cells extracted from breast cancer patients showed expression for either basal (CK18 and FOXC1) or luminal (CK14 and ER-positive) markers in 2D cultures. Our ongoing work entails delineating the long-term culture effect on primary mammary cell fate and function and the tumorigenic property of primary breast tumor cells. The current findings uncover an in vitro model that may be a valuable tool to study mammary cell function and can potentially be used to elucidate mechanisms involved in mammary tumorigenesis. Citation Format: Stacey Chung, Liting Jin, Ying Qu, Liliana J. Gomez, Bingchen Han, Bowen Gao, Xuefeng Liu, Farin Amersi, Catherine Dang, Armando E. Giuliano, Xiaojiang Cui. Assessment of conditional reprogramming to generate 2D and 3D primary human mammary cell culture models [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A59.

PO-300 CIP2A-mediated regulation of senescence in basal-like breast cancer

IntroductionBasal-like breast cancer (BLBC) is an aggressive breast cancer molecular subtype, and it lacks efficient therapy options. Expression levels of oncoprotein CIP2A are increased in BLBC as compared to other breast cancer subtypes. Our results demonstrate that high CIP2A expression significantly predicts poor patient survival in triple negative breast cancer, that constitutes a major subset of BLBC. CIP2A inhibition also significantly impairs tumour growth of human BLBC xenografts. Previously, we have shown that CIP2A promotes breast cancer by inhibiting senescent growth arrest (Laine et al. Cancer Discov 2013). Induction of senescence and senescence associated secretome (SASP) has been shown to alter function of tumour infiltrating immune cells. Based on this we hypothesise that in addition to effects of CIP2A-mediated senescence inhibition to tumour cell-intrinsic mechanisms, it may impact infiltrating immune cells and thus therapy response.Material and methodsTo investigate the role of CIP2A in BLBC a carcinogen DMBA-induced tumour formation approach in Cip2a-/- mouse model was applied. In order to test our hypothesis about the effect of CIP2A-targeted senescence in immune cells we have set up mouse mammary tumour cell and organoid cultures from a spontaneous BLBC genetically engineered mouse model K14Cre;Brca1fl/fl;Tp53fl/fl. These cultures are genetically manipulated by CRISPR/Cas9 system to knock out CIP2A both prior to transplantation into recipient mice and in established tumours. To validate the in vivo findings we are using several different human breast cancer patient cohorts.Results and discussionsOur results demonstrate that CIP2A-deficient mice are severely impaired in a formation of DMBA-induced basal-like mammary tumours. Together with the clinical data, these results suggest that CIP2A is a novel driver oncoprotein, and a potential therapeutic target, in human BLBC. In addition, we have validated the tumour cell-intrinsic role of CIP2A in K14Cre;Brca1fl/fl;Tp53fl/fl BLBC mouse model as loss of CIP2A induces senescent growth arrest in mammary gland tumour cells in vitro. Currently, we are studying the interplay between the immune system and CIP2A-inhibited tumours in vitro and in vivo.ConclusionOverall, this study will address feasibility of CIP2A targeting as a novel approach to combat basal-like breast cancer. Results of this project will also enhance our general understanding of cross-talk between senescent cancer cells and the tumour environment in this clinically challenging human cancer type.

Technical Note: Culturing sheep lactating mammary explants for gene expression studies

Mammary cell cultures have been used to evaluate gene expression under many types of treatments. Additionally, culturing explants of mammary gland of lactating animals could allow the evaluation of the effects of bioactive molecules, considering that the tissue maintains its normal cellular activity as it does in the living animal. The objectives of this study were to evaluate mammary gland explants of lactating ewes as an in vitro model to test the gene expression of transcription factors and lipogenic genes involved in milk synthesis. Explants were cultured for 3 or 24 h and subjected or not to a challenge dose of conjugated linoleic acid (CLA = 290 μM of trans-10, cis-12) and; No CLA = culture medium. The Comet Assay was performed to evaluate DNA fragmentation. RNA was extracted, cDNA was synthesized, and RT-qPCR was carried out. The Comet Assay showed absence of DNA damage in culture at 24 h. Collectively, our results showed gene expression of lipogenic genes and alpha lactalbumin, supporting that mammary gland explants of lactating ewes can be cultured for 24 h for molecular studies.

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.

In vitro culture of ovine mammary gland cells expressing beta-lactoglobulin and beta-casein

A expressão in vitro de proteínas do leite e essencial para explorar as células da glândula mamaria como um modelo biológico. A desagregação tecidual via enzimática e amplamente utilizada para o estabelecimento cultivo de células mamarias. No entanto, seu efeito a longo prazo no cultivo de células da glândula mamaria ovina ainda não e bem elucidado. Este estudo tem como objetivo comparar dois métodos de dissociação tecidual, mecânico/enzimático e mecânico, para estabelecer cultivo celular de glândula mamaria ovina. A indução da diferenciação celular, por adição de soro de ovelha lactante ou soro fetal bovino, foi avaliada pelos níveis de expressão de proteínas do leite (beta-lactoglobulina, alpha s1-caseina e beta-caseína). Células mecanicamente dissociadas foram positivamente marcadas para a presença de citoqueratina 8.13, marcador para células epiteliais mamarias. Essas células são as responsáveis pela produção das proteínas do leite e são pouco marcadas para a presença de vimentina, marcador para células de origem mesenquimal. Já as células obtidas da dissociação mecânica/ enzimática foram positivamente marcadas para presença de vimentina. A maior velocidade de crescimento (células/hora) foi observado para o grupo com dissociação mecânica cultivado em meio com 10% de soro fetal bovino. As células obtidas tanto da dissociação mecânica quanto mecânica/enzimática foram capazes de expressar beta-lactoglobulina e beta-caseína, mas não alfa s1-caseina. A expressão relativa de beta-lactoglobulina não foi afetada pelo método de dissociação ou meio de cultivo. A expressão relativa da beta-caseína foi negativamente regulada para células mecanicamente dissociadas e cultivadas na presença de soro de ovelha lactante (P = 0,019). A expressão relativa da beta-caseína também foi negativamente regulada para células dissociadas de forma mecânica/enzimática e cultivadas com soro fetal bovino (P = 0,021). Nas condições do presente estudo, concluímos que o método de dissociação mecânica seguido pelo cultivo em meio com 10% de soro fetal bovino foi o método mais eficiente para induzir a expressão mRNA de proteínas do leite por células epiteliais mamarias ovinas in vitro.

Mammary Gland Cell Culture of Macaca fascicularis as a Reservoir for Stem Cells

The mammary gland contains adult stem cells that are capable of self-renewal and are likely target for neoplastic transformation leading to breast cancer. In this study, we developed a cell culture derived from the mammary glands of cynomolgus monkeys (Macaca fascicularis) (MfMC) and furthermore identified the expression of markers for stemness and estrogen receptor-associated activities. We found that the primary culture can be successfully subcultured to at least 3 passages, primarily epithelial-like in morphology, the cultured cells remained heterogenous in phenotype as they expressed epithelial cell markers CD24, CK18, and marker for fibroblast S1004A. Importantly, the cell population also consistently expressed the markers of mammary stem cells (ITGB1 or CD29 and ITGA6 or CD49f), mesenchymal stem cells (CD73 and CD105) and pluripotency (NANOG, OCT4, SOX2). In addition to this, the cells were also positive for Estrogen Receptor (ER), and ER-activated marker Trefoil Factor 1, suggesting an estrogen responsiveness of the culture model. These results indicate that our cell culture model is a reliable model for acquiring a population of cells with mammary stem cell properties and that these cultures may also serve as a reservoir from which more purified populations of stem cell populations can be isolated in the future.

Antiproliferative effect of berberine on canine mammary gland cancer cell culture.

Canine mammary gland tumors are the most frequent cause of cancer in female dogs. Numerous studies using cancer cell lines and clinical trials have indicated that various natural products and antioxidants reduce or possibly prevent the development of cancer. Berberine (BBR), the most important alkaloid in the Berberidaceae, which exerts a wide range of pharmacological and biochemical effects, has drawn much attention due to its particularly high antitumor activity in vitro and in animal studies. The aim of the present study was to investigate the antiproliferative effect of BBR against a canine mammary gland carcinoma cell line (CF41.Mg) in vitro. CF41.Mg cells were cultured in RPMI-1640 medium containing 10% heat inactived fetal bovine serum (FBS) and 100 mg/ml peniciline-streptomycin. Subsequently the cells were treated with different concentrations of BBR chloride (10, 25, 50, 100 and 200 µM) at a density of 12,000 cells/well in 96-well plates. Following treatment, the MTT assay was used to detect cell viability after 24-, 48- and 72-h incubations at 37°C with 5% CO2. The results indicated that BBR inhibited proliferation of canine mammary gland carcinoma cells, as treatment with 100 µM BBR for 24 h resulted in a significant decrease in cell viability (P<0.005). As the present study demonstrated that BBR (10-200 µM) induced cancer cell death, it is proposed that BBR may serve as a candidate agent against canine mammary tumor cells via its antiproliferative activity.

0853 Interactions among serotonin and circadian systems in the mammary gland

The circadian and serotonin systems are reciprocally regulated in the brain and function to maintain homeostasis and respond to internal and external stimuli. Both systems play regulatory functions throughout the lactation cycle in the mammary gland. We hypothesized that the serotoninergic and circadian systems are reciprocally regulated in the mammary gland to mediate mammary homeostasis and respond to metabolic demands of milk synthesis. The objective of this study was to determine whether there is reciprocal regulation among the systems using approaches that encompassed bioinformatics analysis, mammary cell and tissue culture, and temporal expression analysis of mammary gene expression using sheep milk. Bioinformatics analysis of the 2000-bp upstream region of the SLC6A4 (serotonin reuptake transporter, SERT) gene revealed the presence of a canonical E-box sequence (CACGTG) that circadian transcription factor CLOCK:BMAL1 binds. qPCR analysis of steady-state SLC6A4 mRNA in samples isolated from sheep milk fat globules showed that this gene exhibits a circadian rhythm of expression. SLC6A4 pattern of expression was similar to PER2, a core clock gene that is a transcriptional target of CLOCK:BMAL1. Moreover, comparison of PER2 and SLC6A4 temporal expression in wild-type HC11 cells also showed similar expression patterns in this mammary epithelial cell line. In HC11 cultures that carry shRNA that targeted CLOCK, SLC6A4 expression was decreased across all time points (P < 0.05), supporting that the mammary clock regulates this serotonergic factor. To study the effect of serotonin (5-HT) on the mammary clock, mammary explants were prepared from lactating mice and divided into two treatments: control (lactogenic culture media–prolactin, glucocorticoids, and insulin) and 5-HT (lactogenic culture media and 200 µM of 5-HT). Samples were collected every 4 h over a 24-h period. One-way ANOVA of temporal variation within treatments found time had a significant (P < 0.05) impact on BMAL1 expression in the control, but not the 5-HT treated samples, indicating that 5-HT attenuates BMAL1 expression rhythm. PER2 temporal variation was significant in both treatments (P < 0.001), but the differences were due to different time points, indicating that 5-HT shifted the phase of expression rhythm. Together, these data support that circadian and serotonergic systems interact in mammary gland. Further studies are needed to understand the significance of these interactions and how they may affect productivity.

Detailed analysis of the promoter activity of an attenuated lentivirus.

In spite of an eradication campaign that eliminated clinical cases of caprine arthritis encephalitis virus-induced arthritis in the Swiss goat population, seroconversions are still observed. In the affected flocks, viruses belonging mainly to the small ruminant lentivirus A4 subtype are regularly isolated. These viruses are considered attenuated, except in the mammary gland, where high viral loads and histopathological lesions have been observed. We previously characterized and sequenced such field isolates, detecting several potentially attenuating mutations in their LTR. Here we present a detailed analysis of the promoter activity of these genetic elements, which was comparable to those of virulent isolates. An AP-1 binding site was shown to be crucial for promoter activity in reporter gene assays and also in the context of a replicating molecular clone. Other sites, such as AML(vis) and a conserved E-box, appeared to be less crucial. Analysis of a unique AP-4 site showed a clear discrepancy between results obtained with reporter gene assays and those with mutated viruses. Within the limits of this in vitro study, we did not find evidence pointing to the LTR as the genetic correlate of attenuation for these viruses. Finally, the limited replication of SRLV A4 in mammary cell culture could not explain the suggested mammary tropism. In contrast, and in view of the abundance of macrophages in the mammary gland, it is the striking replication capacity of SRLV A4 in these cells, unaffected by all LTR mutations tested, which may explain the apparent mammary tropism of these viruses.