Cultures Of Mammary Epithelial Cells Research Articles

Entourage effect for phenolic compounds on production and metabolism of mammary epithelial cells

Primary culture of mammary epithelial cells (MEC) was exposed to ethyl-acetate, chloroform and hexane extracts of Pistacia lentiscus (lentisk). The hexane extract contained mainly ethyl gallate whereas the chloroform extract contained mainly ethyl-gallate with smaller amount of gallic acid, and the ethyl-acetate extract contained mainly rutin, gallic acid and myricetin. Ethyl acetate extract increased secretion of protein and fat and improved mitochondrial activity. The enhancing effect on protein production was attributed to myricetin, one of the polyphenols in the ethyl-acetate extract whereas gallic acid did not affect protein production or secretion. Interestingly, exposure to the isolated polyphenols did not improve mitochondrial productivity and activity as effectively as exposure to the complete plant extract. The results indicated that polyphenols improve production of milk constituents by MEC, through different modes of action for different polyphenols suggesting an additive or even synergistic effect on production traits of mammary cells.

Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-\u03baB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures

Emergence of bovine mastitis caused by Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1β and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1β, and IL-1β proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.

Human Breast Extracellular Matrix Microstructures and Protein Hydrogel 3D Cultures of Mammary Epithelial Cells.

Simple SummaryHuman breast tissue extracellular matrix (ECM) is a microenvironment essential for the survival and biological activities of mammary epithelial cells. The ECM structural features of human breast tissues remain poorly defined. In this study, we identified the structural and mechanical properties of human normal breast and invasive ductal carcinoma tissue ECM using histological methods and atomic force microscopy. Additionally, a protein hydrogel was generated using human breast tissue ECM and defined for its microstructural features using immunofluorescence imaging and machine learning. Furthermore, we examined the three-dimensional growth of normal mammary epithelial cells or breast cancer cells cultured on the ECM protein hydrogel, where the cells exhibited biological phenotypes like those seen in native breast tissues. Our data provide novel insights into cancer cell biology, tissue microenvironment mimicry and engineering, and native tissue ECM-based biomedical and pharmaceutical applications.Tissue extracellular matrix (ECM) is a structurally and compositionally unique microenvironment within which native cells can perform their natural biological activities. Cells grown on artificial substrata differ biologically and phenotypically from those grown within their native tissue microenvironment. Studies examining human tissue ECM structures and the biology of human tissue cells in their corresponding tissue ECM are lacking. Such investigations will improve our understanding about human pathophysiological conditions for better clinical care. We report here human normal breast tissue and invasive ductal carcinoma tissue ECM structural features. For the first time, a hydrogel was successfully fabricated using whole protein extracts of human normal breast ECM. Using immunofluorescence staining of type I collagen (Col I) and machine learning of its fibrous patterns in the polymerized human breast ECM hydrogel, we have defined the microstructural characteristics of the hydrogel and compared the microstructures with those of other native ECM hydrogels. Importantly, the ECM hydrogel supported 3D growth and cell-ECM interaction of both normal and cancerous mammary epithelial cells. This work represents further advancement toward full reconstitution of the human breast tissue microenvironment, an accomplishment that will accelerate the use of human pathophysiological tissue-derived matrices for individualized biomedical research and therapeutic development.

IL-4 and IL-13 Promote Proliferation of Mammary Epithelial Cells through STAT6 and IRS-1.

T helper (Th)2 cytokines such as interleukin (IL)-4 and IL-13 control immune function by acting on leukocytes. They also regulate multiple responses in non-hematopoietic cells. During pregnancy, IL-4 and IL-13 facilitate alveologenesis of mammary glands. This particular morphogenesis generates alveoli from existing ducts and requires substantial cell proliferation. Using 3D cultures of primary mouse mammary epithelial cells, we demonstrate that IL-4 and IL-13 promote cell proliferation, leading to enlargement of mammary acini with partially filled lumens. The mitogenic effects of IL-4 and IL-13 are mediated by STAT6 as inhibition of STAT6 suppresses cell proliferation and improves lumen formation. In addition, IL-4 and IL-13 stimulate tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Prolonged treatment with these cytokines leads to increased IRS-1 abundance, which, in turn, amplifies IL-4- and IL-13-stimulated IRS-1 tyrosine phosphorylation. Through signaling crosstalk between IL-4/IL-13 and insulin, a hormone routinely included in mammary cultures, IRS-1 tyrosine phosphorylation is further enhanced. Lowering IRS-1 expression reduces cell proliferation, suggesting that IRS-1 is involved in IL-4- and IL-13-stimulated cell proliferation. Thus, a Th2-dominant cytokine milieu during pregnancy confers mammary gland development by promoting cell proliferation.

A FACS-Free Purification Method to Study Estrogen Signaling, Organoid Formation, and Metabolic Reprogramming in Mammary Epithelial Cells.

Few in vitro models are used to study mammary epithelial cells (MECs), and most of these do not express the estrogen receptor α (ERα). Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs. These MECs remain differentiated for up to six days with >85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland’s morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ERα signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favoring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed mass spectrometry (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ERα reprograms cell metabolism in mammary organoids. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis.

Direct effects of phenolic compounds on the mammary gland: In vivo and ex vivo evidence

We assessed the potential of Pistacia lentiscus (lentisk) phenolic compounds to enhance production of milk composition in lactating goats and caprine primary mammary epithelial cells (MEC). Damascus goats were given a lentisk infusion (LI) or fresh water (FW) to drink, in a crossover design. Milk from LI vs. FW goats was 0.43% richer in fat and 30% in omega 3 fatty acids. Lentisk infusion enhanced antioxidant capacity of plasma and milk by 37% and 30% respectively, and induced transcriptional activation of antioxidant genes. To assess the direct effect of polyphenols on milk quality in terms of composition and antioxidant capacity, we used plasma collected from goats fed hay (HP) or browsed on phenolic compounds-rich pasture (primarily lentisk; PP) as a conditioning medium for primary culture of MEC. PP increased 2-fold cellular triglyceride content and 2.4-fold intracellular casein, and increased ATP production and non-mitochondrial oxygen consumption. Taken together, the results imply that lentisk phenolic compounds affect blood, MEC and milk oxidative status, which increase fat production by the mammary gland.

Development of a Pig Mammary Epithelial Cell Culture Model as a Non-Clinical Tool for Studying Epithelial Barrier-A Contribution from the IMI-ConcePTION Project.

Simple SummaryThe information about the risks related to the use of medication during breastfeeding is lacking for most commonly used drugs. The ConcePTION project aims to fill this gap using multiple approaches. Within the project, the pig has been selected as the most appropriate in vivo animal model. In agreement with the application of the “3Rs” principle (Replacement, Reduction and Refinement) and international legislations, the present paper reports the establishment of cellular lines of porcine mammary epithelial cells as a valid tool to study the mammary epithelial barrier function in vitro.The ConcePTION project aims at generating further knowledge about the risks related to the use of medication during breastfeeding, as this information is lacking for most commonly used drugs. Taking into consideration multiple aspects, the pig model has been considered by the consortium as the most appropriate choice. The present research was planned to develop an efficient method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study the mammary epithelial barrier in vitro. Mammary gland tissues were collected at a local slaughterhouse, dissociated and the selected cellular population was cultured, expanded and characterized by morphology, cell cycle analysis and immunophenotyping. Their ability to create a barrier was tested by TEER measurement and sodium fluorescein transport activity. Expression of 84 genes related to drug transporters was evaluated by a PCR array. Our results show that primary cells express epithelial cell markers: CKs, CK18, E-Cad and tight junctions molecules ZO-1 and OCL. All the three pMEC cellular lines were able to create a tight barrier, although with different strengths and kinetics, and express the main ABC and SLC drug transporters. In conclusion, in the present paper we have reported an efficient method to obtain primary pMEC lines to study epithelial barrier function in the pig model.

Identifying Enthalpic Barriers to Entropically-Driven Structural Disruption in Breast Cancers

Tissues can span a large structural phase space, but only occupy a small set of configurations as interfacial tension-driven self-organization counters entropically-favored disorganization. Historical self-organization models often do not address structural variability and transitions commonly observed during morphogenesis and disease. In breast cancer, structural breakdown of the bilayered mammary epithelium, comprised of inner luminal (LEP) layer surrounded by an outer myoepithelial (MEP) layer, is directly linked increased patient risk upon invasion. Organotypic cultures of patient-derived human mammary epithelial cells self-organize in vitro, largely driven by differences in favorability of LEP and MEP interface with the extracellular matrix. The observed distribution of organoid structures closely aligns with Boltzmann statistics - a function of the underlying interfacial energies (enthalpy), geometric constraints (entropy), and mechanical fluctuations (activity) of the tissue. We predict that transformations which increase the probability of LEP occupancy in the basal compartment can destabilize tissue structure and promote invasion, consistent with observations in murine organoid models.To test these predictions experimentally, we examined the ability of 15 cancer-associated genetic changes to alter interfacial tensions of LEP and disrupt self-organization in reconstituted human mammary organoids. While most perturbations only minimally impacted self-organization, PIK3CA activation in LEP uniquely reduced their ECM interfacial energy and disrupted tissue structure. Modeling predicts that normalization of PIK3CA-LEP interfacial energy or decreasing overall tissue activity can correct tissue structure, which we confirm experimentally. Consistently, upregulation of basal adhesion (enthalpy) is observed during progression from in situ to invasive human cancers. Additionally changes in tissue composition (entropy) and remodeling (activity) are linked to changes in cancer risk post-pregnancy. Collectively, this statistical mechanical framework presents a new strategy for understanding and targeting cancer progression, emphasizing the importance of structural probability distributions rather than average structures.

In vitro culture and evaluation of bovine mammary epithelial cells from Ukraine dairy cows.

Mammary epithelial cells (MECs) have been widely-used over the years as models to understand the physiological function of mammary disease. This study aimed to establish a culture system and elucidate the unique characteristics of bovine mammary epithelial cells (BMECs) from the milk of Ukraine Holstein dairy cows in order to develop a general in vitro model. The milk from a three-year-old lactating dairy cow was used as a source of the epithelial cell, characteristics of BMECs were examined using real time cell assay (RTCA), immunocytochemistry (ICC), reverse transcription-polymerase chain reaction (RT-PCR), and Western blot (WB). The results showed that BMECs can be recovered from milk, grown in culture, and exhibit the characteristic cobblestone morphology of epithelial cells. The established BMECs retained MEC characteristics and secreted β-caseins even when grew on plastic substratum. Thus, the established cell line had normal morphology, growth characteristics, as well as secretory characteristics, and it could be considered as a model system and useful tool for understanding the biology of dairy cow mammary glands.

Menthol from Mentha piperita Suppresses the Milk Production of Lactating Mammary Epithelial Cells In Vivo and In Vitro.

Peppermint is traditionally used as an antigalactagogue in breastfeeding women. However, the suppressive mechanism remains unclear. The authors investigate whether and how peppermint influences milk production at the cellular and molecular levels. A lactating mammary epithelial cell (MEC) culture model that produces major milk components is prepared. Peppermint oil (PMO) suppresses β-casein production in conjunction with the induced enlargement of cytoplasmic lipid droplets (CLDs). PMO also significantly inactivates STAT5 and mTOR in the lactogenic signaling pathway. Menthol treatment, which is a primary PMO component, leads to decreases in β-casein production, enlarged CLDs, the inactivated STAT5 and mTOR. Eucalyptol and menthyl acetate, other components of peppermint, does not show suppressive effects on lactating MECs. The inactivation of STAT5 and mTOR upon menthol administration is also evident in alveolar MECs of lactating mice. Furthermore, lactating MECs expresses TRPM8 and TRPA1, which are menthol receptors known as cold receptors. Agonists of TRPM8 and TRPA1 suppresses β-casein production and inactivation of STAT5 and mTOR in the lactating MECs. These findings indicate that peppermint has potential as an antigalactagogue. Menthol is suggested to be an active compound in peppermint. TRPM8 and TRPA1 may function as receptors for menthol.

Suppressive effects of curcumin on milk production without inflammatory responses in lactating mammary epithelial cells

BackgroundCurcumin is a naturally occurring polyphenol found in Curcuma longa with multiple therapeutic properties, such as anti-inflammatory, wound healing and anti-cancer effects. Curcuma longa is also used as a galactagogue to improve milk production during lactation. PurposeTo assess curcumin could have therapeutic potential for breastfeeding mothers, we investigated whether and how curcumin influences milk production in lactating mammary epithelial cells (MECs) at the cellular and molecular levels. MethodsWe prepared a lactating MEC culture model that produced milk components and formed less-permeable tight junctions (TJs) to investigate the molecular mechanism of curcumin on milk production, TJs, and inflammation in vitro. ResultsCurcumin downregulated milk production in lactation MECs concurrently with inactivation of lactogenesis-relating signaling (STAT5 and glucocorticoid receptor). The maintenance of a less-permeable TJ barrier was also confirmed, although the TJ protein claudin-4 increased. Curcumin inactivated NFκB and STAT3 signaling, which are closely involved in inflammatory responses in weaning and mastitis mammary glands. The expression levels of IL-1β and TNF-α were also decreased by curcumin treatment. Furthermore, curcumin blocked activation of inflammatory signaling by lipopolysaccharide treatment in MECs, similar to those in MECs that were treated with diclofenac sodium. The drastic phosphorylation of ERK was induced by curcumin treatment in the absence of EGF. U0126, an inhibitor of ERK phosphorylation, attenuated the adverse effects of curcumin on lactating MECs. ConclusionThe results of the present study suggests that curcumin downregulates milk production via inactivation of STAT5 and GR signaling with concurrent suppression of inflammatory responses via STAT3 and NFκB signaling in MECs. These findings provide new insights into the role of curcumin as a mild suppressor of milk production without inflammatory damages in breastfeeding mothers.

Mycoplasma bovis-generated reactive oxygen species and induced apoptosis in bovine mammary epithelial cell cultures

Mycoplasma bovis is an important cause of bovinemastitis in China and worldwide. We hypothesized that M. bovis damages bovine mammary epithelial cells (bMEC), with the degree of damage varying among field isolates. Our objective was to evaluate 2 novel sequence type (ST) field strains of M.bovis (ST172 and ST173) for their ability to induce oxidative stress, cytotoxicity, pathomorphological changes, and apoptosis in bMEC, as a model for pathogenesis of M. bovis-induced bovine mastitis. Cytotoxicity (as indicated by release of lactate dehydrogenase, LDH) from bMEC depended on multiplicity of infection (MOI), with a high MOI (1:1,000) being required to induce cytotoxicity. Morphological changes in bMEC, including shrinkage, loss of cell integrity, and heavy staining (hematoxylin and eosin) of cytoplasm were apparent 24 h after infection with ST172 or ST173 M.bovis, with more severe changes being induced by the latter strain. Adhesion and invasion assays both had curvilinear patterns, peaking 12 h after infection with MOI of 1:1,000. Both production of reactive oxygen species (ROS) and proportion of apoptotic cells increased with time after infection. Increased Bax/Bcl-2 ratios and activation of caspase-3 implied involvement of mitochondria-dependent pathways ofapoptosis. Furthermore, intracellular ROS generation, apoptosis, and cleaved caspase-3 were mitigated by N-acetyl-l-cysteine, a ROS scavenger. Both interleukin (IL)-1β and IL-6 were significantly upregulated by ST172 and ST173 M. bovis, with little change in expression of tumor necrosis factor-α. One ST173 M. bovis isolate had the greatest cytotoxicity of all of our field isolates, with the highest LDH release, adhesion, invasion, ROS production, and apoptosis. In conclusion, our hypothesis was supported: M. bovis damaged bMEC by generating ROS and initiating a mitochondria-dependent pathway ofapoptosis, with the degree of damage varying among field isolates. This study provided new knowledge regarding pathogenesis of M. bovis-induced bovine mastitis.

Abstract 5818: The immortalization process as a therapeutic target

Abstract Normal human somatic cells have a finite lifespan and intact tumor suppressor barriers, whereas most carcinoma cells have gained immortality and overcome multiple tumor suppressor barriers. We have developed a comprehensive human mammary epithelial cell (HMEC) culture system to examine the alterations involved in the transition from normal finite to malignant immortal; such data can suggest prevention strategies. Our studies have indicated that HMEC initially encounter a stress-associated senescence barrier (stasis) enforced by retinoblastoma (RB). Errors in the RB pathway allow stasis bypass; similar errors are seen early in progression in vivo. Replicative senescence due to telomere erosion is a second extremely stringent barrier, with critically short telomeres leading to genomic instability. Overcoming this barrier requires reactivation of endogenous telomerase, similar to what is seen in high-grade DCIS in vivo. Resulting immortal HMEC are resistant to oncogene-induced-senescence, and exposure to oncogenes that cause finite cells to senesce, can now give rise to malignancy, underscoring the critical importance of the immortalization step in progression. Also like DCIS, the molecular properties of our non/pre-malignant immortal HMEC lines are more similar to malignant immortal than normal finite cells, highlighting the abnormal, cancer-like qualities of immortalized cells. We suggest this similarity is due to a novel process that we have discovered to be involved in cancer-associated immortal transformation, that we have called conversion. HMEC that gain an error permissive for telomerase reactivation still need to undergo additional changes to assume the cancer-associated immortal phenotype: expression of sufficient telomerase activity to maintain short telomeres, mean TRF ~4 kb (which is shorter than what is present in normal finite cells). Conversion appears initiated by a mean TRF of <4 kb, and may entail changes in telomere structure. Possibly, the observed similar alterations in immortal cells ± malignancy results from changes induced by conversion. Importantly, cancer-associated immortalization is unique to cells progressing to cancer, and its inhibition may thus be a valuable therapeutic target. Surprisingly, little has been done to explore this possibility, we believe because: 1) small short-lived animals like mice do not stringently repress telomerase in adult cells; lacking a significant immortalization barrier, they cannot model this critical step in human carcinogenesis; 2) Immortally transformed cells are commonly referred to as normal or untransformed; 3) hTERT immortalization does not model cancer-associated immortalization; there is no conversion to short telomeres. Our preliminary data suggest potential approaches to inhibiting conversion. Citation Format: Martha R. Stampfer, Tara Fresques, Sun-Young Lee, Mark LaBarge, James Garbe. The immortalization process as a therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5818.

Uptake of copper from hemopexin (HPX) in the absence and presence of heme by hepatic and mammary epithelial cells in culture

Hemopexin (HPX) captures heme units in the blood in conditions such as hemolysis. The resulting heme‐HPX complex is transported mainly to hepatocytes, taken up by receptor mediated endocytosis, and degraded, the heme degradation being initiated by heme oxygenases that release the iron for recycling. This process also provides a safe way to dispose of heme, which would otherwise be toxic [1]. Uptake of the heme‐HPX complex also brings copper into cells, and that copper appears to mediate a coordinate response resulting in induction of heme oxygenase and metallothionein [1]. We recently isolated HPX from porcine blood plasma, and found copper bound to it very tightly (that could not be removed by extensive dialysis with 100 uM histidine (pH 7). EPR analysis showed that Cu(II) was bound to oxygen and nitrogen ligands in HPX. The UV‐Vis spectrum of the same sample showed a typical heme absorbance peak. Treatment with chelating Sepharose however removed not only the copper but also the heme. These findings led us to wonder whether perhaps HPX might normally have a role in the distribution and transport of copper to cells from the circulation. To examine this, we obtained human HPX purified from blood plasma by Athens Research and Technology (Georgia), dialyzed it at pH 5 to remove any heme, added traces of radioactive 67Cu (as the NTA complex), and applied it to size exclusion chromatography on Sephadex G100 equilibrated with MOPS buffer pH 7.4, containing electrolytes to make it isotonic for tissue culture studies. The 67Cu eluted with the HPX protein peak, and no low molecular weight 67Cu was detected, indicating it had all stuck to the HPX. The peak fractions were pooled and incubated with human hepatic (HepG2) or mammary epithelial cells (PMC42) for various lengths of time. 67Cu radioactivity in the cells (washed with medium containing EDTA) was then determined, and rates of uptake (percent dose/h/mg cell protein) were calculated. For both cell types, the uptake was linear over several hours, but were 2–3 times faster with the hepatic cells. In subsequent experiments, the effects of heme on copper uptake were also measured. When heme was added to the 67Cu‐prelabeled HPX at a molar ratio of 1.1:1.0 (heme:HPX), we expected the rate of copper uptake to increase stimulated, since adding heme to HPX would allow receptor‐mediated endocytosis of the complex. Instead, heme addition markedly reduced copper uptake. The potential effects of three different endocytosis inhibitors were also examined with mixed results. However, some significant inhibition of uptake was observed, including when HPX with 67Cu but no added heme was administered. These findings indicate that copper attached to HPX can be delivered to hepatic and non‐hepatic cells even in the absence of heme and suggest that copper may have more than one role in the function of this plasma protein.

Antimicrobial Effects of Conditioned Medium From Amniotic Progenitor Cells in vitro and in vivo: Toward Tissue Regenerative Therapies for Bovine Mastitis.

There is increasing evidence to suggest that, in addition to their regenerative effect, mesenchymal stromal cells (MSCs), and their secretome have an anti-inflammatory and antimicrobial role in the innate immune response in conditions such as sepsis. However, there is no published information on the effect of MSCs in bovine mastitis. Mastitis often results in extensive tissue damage due to multi-microorganism co-infection. This study investigated the ability of amniotic-derived conditioned medium (CM), in vitro and in vivo, to counteract microbial action and restore healthy tissue capable of milk production. Following determination of a dose–response curve, 10,000 colony-forming units (CFU) of Staphylococcus aureus (S. aureus) were inoculated into bovine mammary epithelial cell culture with and without 10% CM (supplemented either at the time of bacteria inoculation or after 4 h). Acridine orange staining was used to assess cell viability/apoptosis. Additionally, an in vivo study was performed using 48 dairy cows with acute and chronic mastitis, treated with CM (treated group) or antibiotics (control group). In vitro results showed that CM can attenuate bacterial growth, as evaluated by the number of CFU. After 24 h of culture with S. aureus, 89.67% of mammary epithelial cells treated with CM were still alive, whereas all cells cultured without CM were dead. Rates of epithelial cell survival (60.67%) were similar when CM was added 4 h after bacteria inoculation. There was no difference in somatic cell count between cases of acute mastitis in the CM-treated or control group in the in vivo study. However, relapses in chronic mastitis were less common in the group receiving CM. Our results show that CM is able to mitigate bacterial growth in vitro and may be particularly useful in the treatment of chronic mastitis, aiding restoration of milk production in cows that would otherwise be removed from the production cycle.

Translin restricts the growth of pubertal mammary epithelial cells estrogen-independently in mice

Translin, a ubiquitous RNA/DNA-binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses endoribonuclease activity and plays a physiological role in restricting the size and differentiation of mesenchymal precursor cells. However, the precise role of Translin in epithelial cells remains unclear. Here, we show evidence that Translin restricts the growth of pubertal mammary epithelial cells. The mammary epithelia of Translin-null females exhibited retarded growth before puberty, but highly enhanced growth and DNA synthesis with increased ramification after the onset of puberty. Primary cultures of Translin-null mammary epithelial cells showed augmented DNA synthesis in a ligand-independent and ligand-enhanced manner. Translin-null ovariectomized mice implanted with slow-release estrogen pellets showed enhanced length and ramification of the mammary glands. Mammary epithelial growth was also observed in ovariectomized Translin-null mice implanted with placebo pellets. Luciferase reporter assays using embryonic fibroblasts from Translin-null mice showed unaltered estrogen receptor α function. These results indicate that Translin plays a physiological role in restricting intrinsic growth, beyond mesenchymal cells, of pubertal mammary epithelial cells.

Functional analysis of recombinant buffalo lactoferrin and monoferric lobes and their cytotoxic effect on buffalo mammary epithelial cells.

Lactoferrin (Lf) has been involved in diverse type of cellular activities and its biochemical properties are species specific. Lf is a bilobal molecule in which each lobe binds with one Fe2+/Fe3+ ion. A lot of physiological effects of Lf are regulated by its iron binding and release properties; however these properties are species-specific. To understand the iron-binding, thermal stability and cytotoxic effect of buffalo Lf (buLf) and contribution of individual N- and C-terminal lobes therein, buLf and the truncated monoferric lobes were expressed in Kluyveromyces lactis or Pichia pastoris yeast expression systems. The iron-uptake/release behavior and thermal stability of recombinant buLf was observed similar to the Lf purified from buffalo milk. Supplementation of recombinant buLf to the buffalo mammary epithelial cells (BuMEC) culture decreased their proliferation and the cell viability in a dose dependent manner. The cell growth decreased by 37% at 1.0mg/ml Lf. C-lobe decreased the viability of BuMEC by 15% at 1mg/ml. The C-lobe showed greater cytotoxic effect against BuMEC in comparison to N-lobe. buLf caused a reduced expression of the casein in BuMEC. At 1.0mg/ml of buLf, CSN2 transcript level was reduced by 74% and 78% in the normal and hormone free media, respectively. The expression of IL-1β gene in BuMEC increased by 4-5 fold in the presence of 1.0mg/ml of Lf. The effect was similar to that observed in the involutory mammary gland, suggesting the role of elevated level of Lf in remodeling of buffalo mammary tissue during involution.

Co-administering Melatonin With an Estradiol-Progesterone Menopausal Hormone Therapy Represses Mammary Cancer Development in a Mouse Model of HER2-Positive Breast Cancer.

Melatonin has numerous anti-cancer properties reported to influence cancer initiation, promotion, and metastasis. With the need for effective hormone therapies (HT) to treat menopausal symptoms without increasing breast cancer risk, co-administration of nocturnal melatonin with a natural, low-dose HT was evaluated in mice that develop primary and metastatic mammary cancer. Individually, melatonin (MEL) and estradiol-progesterone therapy (EPT) did not significantly affect mammary cancer development through age 14 months, but, when combined, the melatonin-estradiol-progesterone therapy (MEPT) significantly repressed tumor formation. This repression was due to effects on tumor incidence, but not latency. These results demonstrate that melatonin and the HT cooperate to decrease the mammary cancer risk. Melatonin and EPT also cooperate to alter the balance of the progesterone receptor (PR) isoforms by significantly increasing PRA protein expression only in MEPT mammary glands. Melatonin significantly suppressed amphiregulin transcripts in MEL and MEPT mammary glands, suggesting that amphiregulin together with the higher PRA:PRB balance and other factors may contribute to reducing cancer development in MEPT mice. Melatonin supplementation influenced mammary morphology by increasing tertiary branching in the mouse mammary glands and differentiation in human mammary epithelial cell cultures. Uterine weight in the luteal phase was elevated after long-term exposure to EPT, but not to MEPT, indicating that melatonin supplementation may reduce estrogen-induced uterine stimulation. Melatonin supplementation significantly decreased the incidence of grossly-detected lung metastases in MEL mice, suggesting that melatonin delays the formation of metastatic lesions and/or decreases aggressiveness in this model of HER2+ breast cancer. Mammary tumor development was similar in EPT and MEPT mice until age 8.6 months, but after 8.6 months, only MEPT continued to suppress cancer development. These data suggest that melatonin supplementation has a negligible effect in young MEPT mice, but is required in older mice to inhibit tumor formation. Since melatonin binding was significantly decreased in older mammary glands, irrespective of treatment, melatonin supplementation may overcome reduced melatonin responsiveness in the aged MEPT mice. Since melatonin levels are known to decline near menopause, nocturnal melatonin supplementation may also be needed in aging women to cooperate with HT to decrease breast cancer risk.

Quantitative Imaging of Morphometric and Metabolic Signatures Reveals Heterogeneity in Drug Response of Three-Dimensional Mammary Tumor Spheroids.

In order to monitor the drug responses of three-dimensional mammary tumor spheroids and to elucidate the role of inter- and intra-spheroid heterogeneity in determining drug sensitivity in the spheroids, an integrated image analysis framework was developed for morphometric and metabolic characterization of the three-dimensional tumor spheroids. Three-dimensional spheroid cultures of primary mammary tumor epithelial cells isolated from freshly excised tumors from a transgenic mouse model of adenocarcinoma (MMTV-PyMT) were imaged by using vital dyes and mitochondrial membrane potential markers. Custom-developed java and python program codes facilitated image processing, numerical computation, and graphical analysis of large datasets generated from the experiments. A panel of cancer drugs (rapamycin, BEZ235, MK2206, and flavopiridol) was tested to determine the degree of drug sensitivity as well as heterogeneity in drug response. A new quantitative metric (growth/toxicity) was developed based on morphometric parameters that were found to track the growth and apoptotic cell populations. Further, this study identified two parameters, namely, skew and kurtosis-which report the spatial heterogeneity in mitochondrial metabolism within the spheroids. The results of this study show that three-dimensional tumor spheroids selectively respond to cancer drugs depending on the specific metabolic pathways (AKT inhibition pathway in the present study), and there exists significant heterogeneity in the untreated tumor spheroids. Drug sensitivity of the spheroids was found to be associated with significant alterations in mitochondrial heterogeneity within the spheroids. In conclusion, the quantitative imaging of morphometric and metabolic analysis in large image datasets can serve as an excellent tool box for characterizing tumor heterogeneity in three-dimensional tumor spheroids and potentially, in intact tumors as well.

Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures.

The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection post-stasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serum-free medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ.