Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins alpha-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, alpha-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background
Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.


Methods
We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.


Results
We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.


Conclusion
We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus)

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10–12 month lactation period, female fur seals suckle pups on shore for 2–3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 μm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (αS2-casein, β-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell–matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

Comparative genomics and transcriptomics of lactation

Lactation is an important characteristic of mammalian reproduction sometimes referred to as the quintessence of mammals. Comparative genomics and transcriptomics experiments are allowing a more in-depth molecular analysis of the evolution of lactation throughout the mammalian kingdom and these recent results are reviewed here. Milk cell and mammary gland gene expression analysis with sequencing methodology have started to reveal conserved or specific milk protein and components of the lactation system of monotreme, marsupial and eutherian lineages. These experiments have confirmed the ancient origin of the complex lactation system and provided useful insight into the function of specific milk proteins in the control of the lactation programme or the role of milk in the regulation of growth and development of the young beyond simple nutritive aspects.

Evolution of lactation : ancient origin and extreme adaptations of the lactation system

Lactation, an important characteristic of mammalian reproduction, has evolved by exploiting a diversity of strategies across mammals. Comparative genomics and transcriptomics experiments have now allowed a more in-depth analysis of the molecular evolution of lactation. Milk cell and mammary gland genomic studies have started to reveal conserved milk proteins and other components of the lactation system of monotreme, marsupial, and eutherian lineages. These analyses confirm the ancient origin of the lactation system and provide useful insight into the function of specific milk proteins in the control of lactation. These studies also illuminate the role of milk in the regulation of growth and development of the young beyond simple nutritive aspects.

Insulin, a key regulator of hormone responsive milk protein synthesis during lactogenesis in murine mammary explants

Murine milk protein gene expression requires insulin, hydrocortisone, and prolactin; however, the role of insulin is not well understood. This study, therefore, examined the requirement of insulin for milk protein synthesis. Mammary explants were cultured in various combinations of the lactogenic hormones and global changes in gene expression analysed using Affymetrix microarray. The expression of 164 genes was responsive to insulin, and 18 were involved in protein synthesis at the level of transcription and posttranscription, as well as amino acid uptake and metabolism. The folate receptor gene was increased by fivefold, highlighting a potentially important role for the hormone in folate metabolism, a process that is emerging to be central for protein synthesis. Interestingly, gene expression of two milk protein transcription factors, Stat5a and Elf5, previously identified as key components of prolactin signalling, both showed an essential requirement for insulin. Subsequent experiments in HCll cells confirmed that Stat5a and Elf5 gene expression could be induced in the absence of prolactin but in the presence of insulin. Whereas prolactin plays an essential role in phosphorylating and activating Stat5a, gene expression is only induced when insulin is present. This indicates insulin plays a crucial role in the transcription of the milk protein genes.

Identification and functional characterization of a novel monotreme - specific antibacterial protein expressed during lactation

Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother’s body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby (Macropus eugenii)

Lactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby.

Surgical treatment of gynaecomastia in a Saanen goat

Background: Gynaecomastia in male goats is characterized by abnormal development of the mammary gland. Enlarged udder may be observed cranially to the scrotum, which can occasionally reach the size of the testicles. The udder may carry functional glands and impair the animal's reproductive performance and welfare. The case of a successful surgical treatment of gynaecomastia in a high reproductive performance Saanen buck-goat is reported in the present study.Material, Methods & Results: The animal was admitted presenting significant augmentation of the mammary glands, which was clinically diagnosed as gynaecomastia. The male goat owned optimal phenotypic characteristics for the Saanen breed, which had been producing high performance descendents. The mammary glands had been impairing the goat's locomotion and sexual performance. Manual milking resulted in great amount of milk secretion. The animal presented anorexia and impaired sexual performance. After clinical and laboratorial evaluation, the animal was submitted to radical mastectomy. An elliptic skin incision was performed around each mammary gland. Subcuticular blunt dissection was accomplished to isolate the mammarian tissue from the abdominal muscular layer and the spermatic chord. The excised mass was sampled for histological assessment. Subcuticular layer and skin closure was carried in a routine fashion. Hygienization of the surgical wound was performed with 2,5% PVP-I solution for ten days. Additionally, an association of penicillin G benzathine and streptomycin, and fluxinin meglumine were also given. The surgical procedure was successfully accomplished without any peroperative complication. The excised mass was sampled for anatomic/histological assessment. Macroscopically, the left mammary gland presented 22 cm in length, 12 cm wide and 26 cm in diameter. The right gland presented 16 cm in length, 7 cm wide and 13,5 cm in diameter. The microscopic assessment revealed hyperplasia of the glandular ducts. No abnormalities resembling malignant mammary neoplasms or degeneration were observed. At the end of the treatment, the animal was completely recovered. The animal convalesced satisfactorily and surgical wound healed completely within the first 10 days post-op. The goat was not culled and returned to normal reproductive activity. Within 12 months of follow-up, the animal was able to produce high milk yield performance progenies.Discussion: This case report presented relevant aspects of the surgical management of gynaecomastia, especially to veterinary practitioners dealing with milk goats. Gynaecomastia is not as common as other reproductive disorders in domestic animals. In opposition to the findings of the present study, other trials revealed that gynaecomastia usually does not affect fertility, libido, ejaculate parameters and sexual performance of goats. However, it is important to consider that neoplasic disorders such as mammary adenocarcinoma may be present, even though these are rare complications. Last but not least, the decision making on mastectomy in the present study was crucial in order to reestablish the animal's welfare and its functionality in the farms reproduction program.

Lymphatic drainage on healthy and neoplasic mammary glands in female dogs: Can it really be altered?

The purpose of this research was to study the mammary lymphatic drainage under a macroscopic and mesoscopic view, comparing the vascular pattern of healthy and neoplasic mammary glands injected with drawing ink alcoholic and fluorescein solutions, in 46 mongrel female dogs. The results pointed out that the thoracic gland is drained by the axillary lymph centre, but in mammary neoplasia either superficial cervical or ventral thoracic lymph centres can be involved. Cranial and caudal abdominal glands may be drained by the axillary, inguinofemoral and popliteal lymph centres. However, the popliteal drainage is specific for the healthy caudal abdominal mammary gland. The inguinal gland can be drained by both inguinofemoral and popliteal lymph centres in both neoplasic and healthy conditions. Regarding the mammary lymphatic communications, this research demonstrated that neoplasic glands present more types of anastomosis (40.9%), than healthy glands (33.33%), and an increase in contralateral anastomosis (50%) compared with healthy ones (33%). Given the data, the mammary neoplasia can change the lymphatic drainage pattern in terms of lymph centres and vascular arborization, thus forming new drainage channels and recruiting a larger number of lymph nodes. Lastly, some comments were made about the severity of a specific neoplasic mammary gland and conditions to be considered before making a decision in terms of the most adequate operative procedure, and suggestions for further investigations.

Ocular melanoma and mammary mucinous carcinoma in an African lion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Modifying Effects of Lemongrass Essential Oil on Specific Tissue Response to the Carcinogen N-Methyl-N-Nitrosurea in Female BALB/c Mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Potential effects of the herbicide Diuron on mammary and urinary bladder two-stage carcinogenesis in a female Swiss mouse model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud: Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.Case presentation: Hematopoietic stem cells were found in a benign mixed mammary tumor of a 7-year-old female mongrel dog that presents a nodule in the left inguinal mammary gland. The patient did not have any hematological abnormalities. Cytological evaluation demonstrated two distinct cell populations, composed of either epithelial or mesenchymal cells, sometimes associated with a fibrillar acidophilic matrix, apart from megakaryocytes, osteoclasts, metarubricytes, prorubricytes, rubricytes, rubriblasts, promyelocytes, myeloblasts. Histological examination confirmed the presence of an active hematopoietic bone marrow within the bone tissue of a benign mammary mixed tumor.Conclusions: EMH is a rare condition described in veterinary medicine that can be associated with mammary mixed tumors. It's detection can be associated with several neoplastic and non-neoplastic mammary lesions, i.e. osteosarcomas, mixed tumors and bone metaplasia.