Molecular analysis of tammar (Macropus eugenii) mammary epithelial cells stimulated with lipopolysaccharide and lipoteichoic acid.

The immunological function of the metatherian mammary gland plays a crucial part in neonatal survival of the marsupial young. Marsupial pouch young do not develop adult like immune responses until just prior to leaving the pouch. The immune components of the maternal milk secretions are important during this vulnerable early post-partum period. In addition, infection of the mammary gland has not been recognized in metatherians, despite the ready availability of pathogens in the pouch. Regardless of which, little is known about the immunobiology of the mammary gland and the immune responses of mammary epithelial cells in metatherians. In this study, a molecular approach was utilized to examine the response of tammar (Macropus eugenii) mammary epithelial cells to Escherichia coli derived lipopolysaccharide (LPS) and Staphylococcus aureus derived lipoteichoic acid (LTA). Using custom-made cDNA microarrays, candidate genes were identified in the transciptome, which were involved in antigen presentation, inflammation, cell growth and proliferation, cellular damage and apoptosis. Quantification of mRNA expression of several of these candidate genes, along with seven other genes (TLR4, CD14, TNF-alpha, cathelicidin, PRDX1, IL-5 and ABCG2) associated with innate immunity in LPS and LTA challenged mammary epithelial cells and leukocytes, was assessed for up to 24 h. Differences in genes associated with cellular damage and pro-inflammatory cytokine production were seen between stimulated mammary epithelial cells and leukocytes. LTA challenge tended to result in lower level induction of pro-inflammatory cytokines, increased PRDX1 mRNA levels, suggesting increased oxidative stress, and increased CD14 expression, but in a non-TLR4-dependent manner. The use of functional genomic tools in the tammar identified differences in the response of tammar mammary epithelial cells (MEC) and leukocytes to challenge with LPS and LTA, and validates the utility of the approach. The results of this study are consistent with a model in which tammar mammary epithelial cells have the capacity to elicit a complex and robust immune response to pathogens.

Identification and transcript analysis of a novel wallaby (Macropus eugenii) basal-like breast cancer cell line.

BACKGROUND: A wide variety of animal models have been used to study human breast cancer. Murine, feline and canine mammary tumor cell lines have been studied for several decades and have been shown to have numerous aspects in common with human breast cancer. It is clear that new comparative approaches to study cancer etiology are likely to be productive. RESULTS: A continuous line of breast carcinoma cells (WalBC) was established from a primary breast cancer that spontaneously arose in a female tammar wallaby (Macropus eugenii). The primary tumor was 1.5 cm3 and although large, did not appear to invade the stroma and lacked vimentin expression. The WalBC cell line was cultured from the primary tumor and passaged for 22 months. WalBC cells displayed an epithelial morphology when grown on plastic, were not EGF responsive, stained strongly for cyto-keratin and negatively for vimentin. WalBC cells were shown to be non-invasive within a Matrigel invasion assay and failed to produce tumors following transplantation into nude mice. Gene expression profiling of WalBC cells was performed using a cDNA microarray of nearly 10,000 mammary gland cDNA clones and compared to normal primary mammary cells and profiles of human breast cancer. Seventy-six genes were down-regulated and sixty-six genes were up-regulated in WalBC cells when compared to primary mammary cells. WalBC cells exhibited expression of known markers of basal invasive human breast cancers as well as increased KRT17, KRT 14 and KRT 19, DSP, s100A4, NDRG-1, ANXA1, TK1 and AQP3 gene expression and decreased gene expression of TIMP3, VIM and TAGLN. New targets for breast cancer treatment were identified such as ZONAB, PACSIN3, MRP8 and SUMO1 which have human homologues. CONCLUSION: This study demonstrates how novel models of breast cancer can provide new fundamental clues regarding cancer etiology which may lead to new human treatments and therapies.

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 microm 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 (alphaS2-casein, beta-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.

A population of mammary epithelial cells do not require hormones or growth factors to survive.

Hormonal stimulation of mammary explants mimics many of the biochemical changes observed during lactogenesis. Previous studies using eutherian species conclude that mammary explants require addition of exogenous macromolecules to remain hormone responsive in culture. The present study examines the survival of mammary explants from the wallaby and mouse using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants from pregnant tammars and mice showed that milk protein gene expression was significantly elevated after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. Surprisingly, mammary explants remained hormone responsive and expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. Furthermore, the alveolar architecture was maintained. Global functional microarray analysis showed that classic involution markers were not differentially expressed, although two stress-induced survival genes were significantly up-regulated. We report that a population of mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. We propose that the mammary explant culture model uncouples the first phase of involution, as milk accumulation that normally provides involution stimuli is absent in this culture model allowing a population of cells to survive.