- Open Access
Identification and transcript analysis of a novel wallaby (Macropus eugenii) basal-like breast cancer cell line
Molecular Cancervolume 7, Article number: 1 (2008)
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.
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.
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.
Breast cancer is the leading cause of mortality and morbidity of women in many countries and is truly a multidisiplinary problem. New ways are currently being sort to develop a new focus and new perspectives. One of these methods involves the study of alternative models of breast cancer for use in comparative oncology. Comparative oncology uses breast cancer models proffered by other species in an attempt to gather more information about breast cancer which may give rise to new human therapeutic targets and interventions.
Murine, feline and canine breast cancer models have been studied extensively showing numerous commonalities with human breast cancer [1, 2], . Carcinomas of the mammary gland are common among carnivorous animals and although rare in herbivores, have been documented in such animals as the horse . Mammary neoplasms in marsupials have not been reported to date, and interestingly, marsupials spend a relatively long period in a state of lactation.
The morphology of the wallaby mammary gland is similar to other mammals but the lactation strategy adapted by marsupials is different to eutherians. The gland undergoes lobular alveolar development during a short period of pregnancy (26.5 days) and the mother gives birth to an altricial young . During a relatively long lactation the mother progressively changes it's milk composition to regulate the considerable growth and development of the young [5–8]. The mammary gland grows considerably during lactation  and, at the end of lactation the mammary gland undergoes involution and returns to a virgin-like state .
The ability to establish primary cultures of tumor cells is an important prerequisite in cancer research, allowing the study of carcinogenesis, prognostic factors and therapeutic agents . In this report, a mammary carcinoma in a tammar wallaby was examined by histopathological techniques and a breast cancer cell line was established (WalBC). Transcriptional profiling using a custom tammar wallaby microarray was performed on the WalBC cell line identifying a gene expression profile consistent with human basal-like breast cancer. Moreover, in addition to the known markers for basal-like breast cancer, the WalBC cell line expressed a number of genes with homologues in the human genome, but have not previously been associated with breast cancer. Further study of these genes within human models of breast cancer may provide new clues in the development and progression of breast cancer which may in turn lead to new treatments and therapies.
Materials and methods
Animals, tumor detection and surgery
Tammar wallabies used in this study were part of a captive breeding colony maintained in an open enclosure at The University of Melbourne Macropod Research Facility (Melbourne, Victoria), with stock originally from Kangaroo Island, South Australia. Care and treatment of animals conformed to the National Health and Medical Research Council of Australia and were approved by the Victorian Department of Sustainability and Environment Ethics Committee. The animal presenting with a mammary lesion was part of the breeding colony for two years (February 2002 – February 2004) and was checked monthly during this time for signs of mating and presence of pouch young. The animal did not reproduce in this time but presented with a mammary lesion. The animal was subsequently monitored weekly for three weeks then euthanized. The lesion was measured, excised and either immediately frozen for future RNA extraction, placed in Hanks' Balanced Salt Solution (HBSS) (Sigma Aldridge, Sydney, Australia) at 4°C in preparation for isolation of mammary cells or fixed in formalin for histology. The tissue from the mammary gland was divided into two portions, one comprising the main body of the mammary lesion and the other comprising normal mammary tissue. Mammary gland tissue was also collected from a wallaby of comparable age and reproductive status for preparation of mammary epithelial cells.
Preparation of WalBC cell line and wallaby primary mammary epithelial cells
Tissue was immediately transferred to 1× Hanks' Balanced Salt Solution (HBSS) (Gibco, USA) with 20 μL/mL penicillin/streptomycin (Gibco, USA) and 5 μg/mL Fungizone (Gibco, USA) on ice and transported back to the laboratory for enzymatic digestion to harvest mammary epithelial cells.
Tissue was dissected free from fat, weighed, sliced finely and digested with 1 μg/mL Collagenase Class2 (Worthington UK), 10 mL/L penicillin/streptomycin (Gibco, USA), 10 mL/L fungizone (Gibco, USA), 0.35% Bovine Serum Albumin (Sigma) and a final concentration of 0.5 mM glucose. 10 g of tissue per 100 mL of media was digested shaking at 200 rpm at 37°C for 4 h. Cells were harvested by filtration through 150 μm nylon mesh using a Nalgene filter unit. The suspension was centrifuged at 80 g for 5 min and pellets were washed twice with HBSS containing 0.02 mg/mL DNase 1 (Invitrogen) and 1 mg/mL Trypsin Inhibitor (Sigma). Cells were again suspended in wash media, then filtered through 53 μM nylon mesh, re-centrifuged and finally resuspended in FCS/10%DMSO (DMSO-Sigma, Sydney, Australia), and frozen at a density of ~2 × 107 cells/mL.
WalBC cells and wallaby primary mammary cells were cultured in either 25 or 80 cm2 culture flasks in 10 mL or 20 mL respectively of M199/Hams/Hepes media with 1 μg/mL cortisol, 10 ng/mL EGF, 1 μg/mL insulin supplemented with 10% fetal bovine serum. WalBC cells were passaged (more than 20 times) when confluent by scraping and use of versine solution in phosphate buffered saline (PBS) (Sigma-Aldrich, Sydney, Australia). Passaging of cells with 0.1% trypsin-versine in phosphate buffered saline (PBS) (Sigma-Aldrich, Sydney, Australia) for 2 minutes at 37°C disrupted cellular aggregates but cells failed to retain viability following this treatment.
Tissue was fixed in 10% formalin for 24 h. Samples were processed (Citadel; Shandon Scientific Ltd., Cheshire, England), and embedded in paraffin using routine procedures. Paraffin-embedded sections of 5 μm thickness were cut, mounted on 3-aminopropyltriethoxysilane-coated slides and submerged in histolene to remove the paraffin. After rehydration, sections were stained with haematoxylin and eosin. Finally, sections were coverslipped and examined using an Olympus BX40 microscope and Coolscope digital microscope (Nikon) for light microscopy.
Paraffin-embedded sections of 5 μm thickness were cut and mounted on 3-aminopropyltriethoxysilane-coated slides and submerged in histolene to remove the paraffin. After rehydration, tissue peroxidases were blocked for 30 min with a 1% hydrogen peroxide solution and washed with 1× PBS. For detection of Vimentin, sections underwent treatment with sodium citrate buffer. Sections were then blocked for 60 minutes with 10% goat serum (Sigma)/1% BSA/PBS prior to addition of Vimentin antibody (V9, 1:800; Dako, Australia) at 4°C overnight. An HRP-conjugated goat anti-mouse secondary antibody (1:250 dilution; Dako, Australia) was then applied for 1 hour to allow a brown precipitate to develop using AEC (Dako, Australia). Finally, sections were counterstained with eosin, dehydrated, coverslipped and examined using a Coolscope digital microscope (Nikon) for light microscopy.
Proliferation assay and cell morphology
WalBC or wallaby MEC's cells were plated (2000 cells/well) in 96 well plate formats with growth media containing either insulin (I; 1 μg/ml), cortisol (F; 1 μg/ml) and prolactin (P; 1 μg/ml) or I, F, Epidermal growth factor (EGF; 10 ng/ml). Cells were grown for a further 10 days before being fixed and stained with Sulforhadamine B as previously described . Each time point was performed in quadruplicate. Cells were visualized by phase contrast microscopy using an Olympus BX40 microscope and photographed using a DigitalSight DSL1 (Nikon) camera.
Matrigel outgrowth assay
Matrigel outgrowth assays were performed in 48 well plates as previously described (Price and Thompson, 1999). Cells (2 × 104) were dispersed in 75 μL of undiluted Matrigel (approx.10 mg/mL) and then overlaid onto 100 μL of polymerized undiluted Matrigel. Once the top layer had polymerized the cultures were incubated in MEM/10%FBS media for up to 10 days and photographed at 20 × magnification by phase contrast microscopy using an Olympus BX40 microscope and photographed using a DigitalSight DSL1 (Nikon) camera.
In vivo studies
Mice (3–4 week old intact female Balb/C nu/nu) were purchased from Australian Resource Center (Perth, Australia), housed in individually ventilated cages under filtered air (Techniplast, Milan, Italy) and acclimatized for one week prior to manipulation. Anesthesia was achieved by i.p injection of ketamine/xylazine (Provet; Australia; 40 μg/g mouse and 16 μg/g mouse, respectively). The mice were allowed to recover from the anesthesia before being returned to their cages and monitored daily. Animal studies were conducted with ethical approval of the St. Vincent's Hospital Animal Ethics Committee (Melbourne, Australia), and in accordance with the Australian National Health and Medical Research Council's Guidelines for the Care and Use of Laboratory Animals.
Mammary fat pad inoculation of WalBC cells
WalBC cells were harvested from near confluent conditions, aspirated into cell suspension (cell aggregates and single cells were present), washed thrice and resuspended in PBS before inoculation. Two groups of 8 mice received mammary fat pad inoculation of WalBC cells (5 × 105 cells/15 μL) as described by Price et al . Tumor growth was assessed by monitoring the fat pad for palpable tumors at weekly intervals. Mice were sacrificed after 6 months.
WalBC cells grown on plastic were fixed in 4% paraformaldehyde (30 min) and washed three times in PBS. Cells were permeabilized with 0.1% Triton X/PBS (5–10 min) and washed thrice with PBS before blocking in 1% BSA for 30 min. Either Vimentin9 (Dako; 1/800) or Rabbit anti-cytokeratin (Bectin Dickinson1/400) antibody was added in blocking buffer and incubated overnight at 4°C. Cells were then washed five times in PBS to remove non-specific binding of primary antibody. Cells were then incubated in goat anti-mouse (FITC) (DAKO; 1/400) or goat anti-rabbit (FITC) (DAKO; 1/400) in blocking buffer for 1 hour and washed thrice with PBS. Nuclei were visualized using Propidium Iodide (Invitrogen). Images were visualized for fluorescence using an Olympus BX40 microscope and photographed using a DigitalSight DSL1 (Nikon) camera.
RNA preparation for gene expression by microarray analysis
WalBC cells, primary mammary cells cultured from the same animal and primary wallaby mammary cells cultured from a virgin animal of similar age were scraped from tissue culture dishes using Tripure (Roche). Total RNA was isolated from the aqueous phase and further purified using the Qiagen RNeasy miniprep kit (Sydney Australia) following the manufacturer's instructions.
RNA Amplification was done in 3 parts similarly to the Eberwine protocol . First strand synthesis utilized MMLV RNase H- (Promega M3681) and second strand synthesis was done with DNA Polymerase 1 (Promega, M2501). Lastly in vitro transcription was performed with the T7 Megascript Kit (Ambion 1334). The resulting amplified RNA was then further purified using the QIAGEN RNeasy miniprep kit.
The amplified RNA from each treatment group was labeled using amino allyl reverse transcription followed by Cy3 and Cy5 coupling. Samples of amplified RNA (10 mg) were reverse transcribed using 5 μg random hexamers (Geneworks), MMLV reverse transcriptase (Promega), RNAse H (Invitrogen) and 1× buffer at 42°C for 2.5 hours. The reaction mix was hydrolyzed by incubation at 65°C for 15 minutes in the presence of 55 mM NaOH, 55 mM EDTA followed by a subsequent addition of acetic acid to 50 mM. The cDNA was then adsorbed to a Qiagen QIAquick PCR Purification column. Coupling of either Cy3 or Cy5 dye was performed on the column by incubating the adsorbed cDNA with the appropriate dye in 0.1 M sodium bicarbonate pH 9.0 for 1 hour at room temperature in darkness. Each labeled cDNA was eluted in 80 μl of water and was then combined with its comparing sample during further purification on a second Qiagen QIAquick PCR Purification column. The joint Cy3 and Cy5 labeled probe in a final concentration of 0.4 mg/ml yeast tRNA, 1 mg/ml human Cot 1 DNA, 0.2 mg/mL Poly dA50, 1.25 × Denharts, 3.2 × SSC and 50% formamide was heated to 100°C for 3 minutes. SDS, to 0.1%, was added immediately after heating and just prior to application. Probes were hybridized to custom made tammar wallaby EST microarray slides overnight at 42°C in a HyPro20 (Integrated Science) humidified chamber. The slides were printed with 10,000 EST's from tammar mammary gland cDNA libraries generated from tissue collected across the lactation cycle (Lefevre, manuscript in preparation).
The tammar EST database was derived from several cDNA libraries comprising day 23 pregnant (n = 4), lactating at day 130 (n = 4), lactating at day 260 (n = 1), lactating at day 130 subtracted for all the major milk protein genes (n = 2), non-lactating (n = 2) and a normalized library (combined RNA from day 26 pregnant, lactating at day 55, day 87, day 130, day 180, day 220, day 260 and involuting at day 5).
Microarray's were washed in 0.5× SSC, 0.01% SDS for 1 minute, 0.5× SSC for 3 minutes then 0.006× SSC for 3 minutes at room temperature in the dark. Slides were centrifuged dry at 130 g for 5 minutes then scanned with a VersArray Scanner (BioRad). Images were analyzed using Versarray Software (Biorad).
Analysis of gene expression data
Gene expression data was normalized using the single channel normalization method in the Limma package of Bioconductor . These normalized expression values were analyzed using a two-stage process where all the expression values are considered simultaneously. The first stage involves fitting a linear mixed model of the formMadj = μ + Treatment + Probe + Treatment.Probe + ε
where Madj is the adjusted (loess-normalized) log intensity ratio for a probe on the cDNA array, Treatment is the fixed effect of the treatment (tumor cells, non-tumor cells, or virgin), Probe is the random effect of the probe on expression levels, regardless of the treatment, and Treatment.Probe is the random effect of a particular probe within a particular treatment, the effect of interest. Typically, the distribution of these random Treatment.Probe effects show a mixture of two distributions, one with small variance (non-differentially expressed genes, non-DE) and one with large variance (differentially expressed, DE). So the second stage involved fitting a two-component mixture model (DE vs non-DE) to these effects , and this will return the (posterior) probability that a particular gene is DE, given its Treatment.Probe effect, with a probability in excess of 0.5 indicating a gene is more likely DE, for that particular treatment. However, a stringent threshold has been used requiring a posterior probability in excess of 0.999 before a gene was classified as being DE, in order to reduce the false positive rate. The statistical package R was use for this two-stage process.
To determine the significance of EGF response within each cell type a paired students t-test was performed on quadruplicate samples at day 10. For Matrigel outgrowth, 20 outgrowths were measured for each cell type at day 4 in a given area and averaged. Unpaired t-test was used to determine significance differences in the length of outgrowths between the two cell types.
Pathology and immunoreactivity of a wallaby primary tumor
A 1.5 cm3 mammary lesion was detected in a female, non-pregnant, non-lactating tammar wallaby (Fig. 1). The female was not less than two years of age, although her true age was not determined as she was captured from the wild and held in a captive colony for two years prior to tumor presentation. Examination by histological techniques showed primary tumor cells were present within the gland arranged in solid masses which appeared to display a pushing margin that invaded the stroma (Fig. 2A). Anti-vimentin immunostaining revealed positive vimentin immunoreactivity of the stroma and negative immunoreactivity of the tumor cells (Fig. 2B).
Characterization of the WalBC cell line: Morphology, immunoreactivity and growth/invasive potential
The WalBC cell line was grown from a mixture of cells containing both normal mammary stromal/epithelial cells and primary cancer cells (Fig. 3A). The WalBC cell line formed a monolayer comprising a homogeneous cell population which exhibited epithelial morphology and strong cell-cell interactions. Once established, the WalBC cell line was passaged for 2 years, cells maintained a cuboidal morphology and proliferated to islands of confluent cells. Single cells did not survive in the absence of cell contact and passaging of cells required the presence of large cellular aggregates which attached to the tissue culture treated plastic before initiating proliferation. Use of trypsin for passaging was unsuccessful due to the break down of cellular aggregates. The morphology of wallaby primary cells and WalBC cells grown in culture was markedly different with WalBC cells appearing smaller and exhibiting strong cell-cell contact (Fig. 3B, C).
Immunostaining with anti-cytokeratin of WalBC cells grown in culture showed 90% of cells exhibited strong cytokeratin immunoreactivity which localized to the cytoplamic region of positive cells (Fig. 3D). A small population of cells showed weaker cytokeratin immunoreactivity, however all cells appeared to show some degree of immunoreactivity.
The proliferation rate of the WalBC cell line was compared to primary wallaby epithelial cells (MEC's) in the presence of insulin (I), cortisol (F) and prolactin (P) or I, F and epidermal growth factor (EGF). Wallaby MEC's exhibited a higher proliferation rate compared to WalBC (Fig. 4). Wallaby MEC's also showed an increase in proliferation in the presence of EGF compared to growth in the presence of prolactin (P < 0.01) while the WalBC cell line failed to respond to EGF treatment.
The invasive potential of the WalBC cell line was examined by Matrigel invasion assay. This assay has previously associated stellate Matrigel morphology with invasiveness in human breast cancer cell lines [11, 17]. The WalBC cell line failed to exhibit stellate growth but grew mammary-like structures that resemble ducts and lobules (Fig. 5). These structures also mimicked the morphology exhibited by the in situ primary lesion which showed masses of tumor cells surrounded by stromal cells. Matrigel morphology analysis was also performed on a known human invasive breast cell line, MDA-MB-231 which demonstrated stellate outgrowth as expected. Comparative quantitative analysis was performed to determine the average length of outgrowth between WalBC and MDA-MB-312 cells. The average outgrowth for WalBC cells in Matrigel was found to be 145 μm (standard deviation of the mean = 72.147) and MDA-MB-231 cells showed an average outgrowth of 50.75 μm (standard deviation of the mean = 20.601). The difference between the length of outgrowths between the two cell types was determined to be highly significant (P < 0.0001). An in vivo model of tumor growth in nude mice was used to study the degree of tumorgenicity of the WalBC cell line. WalBC cells were tested on two separate occasions using two groups of eight mice and a different WalBC passage numbers and on both occasions WalBC cells failed to generate palpable tumors (data not shown).
WalBC gene expression profile
The gene expression profile of the WalBC cells was compared to primary mammary epithelial cells (MEC's) from the same animal and virgin mammary epithelial cells grown from a different animal using a microarray with 10,000 tammar mammary ESTs . Genes were considered differentially expressed if there was a 2-fold or more increase or decrease of intensity between WalBC and both normal MEC's from the same animal and virgin MEC's from a different animal, with a posterior probability of >0.999. Gene expression profiles revealed a large number of differentially expressed genes were associated with carcinogenesis (Table 1 and 2). The highest up-regulated gene in the WalBC cell line was cytokeratin 17 while vimentin was down regulated. Notably WalBC cells showed up regulated expression of DSP, s100A4, ANXA1, NDRG-1, TK1 and down regulation of genes such as tissue inhibitor of TIMP-3 and TAGLN. The overall expression profile revealed a pattern of gene expression consistent with basal-type breast cancer. A number of EST's were also identified that have not been previously associated with breast cancer. These included CAP43, SALL1, ZONAB, SUMO-1 and MRP8 and a number of hypothetical proteins with homologues within the human genome.
A wide variety of animal models have been used to study human breast cancer . For example, murine, feline and canine mammary tumor cell lines have been studied for several decades and have shown to have numerous aspects in common with human breast cancer [1, 2, 20]. We present here the first reported discovery of a primary breast lesion in a marsupial and the subsequent establishment and characterization of the first wallaby breast cancer cell line for comparative analysis of breast cancer. The primary lesion lacked vimentin expression and the cell line was shown to be cytokeratin positive which is consistent with basal type invasive carcinoma.
In all species studied extensively so far, the ability of invasive tumor cells to interact specifically with, and invade, the extracellular matrix (ECM) has been linked to breast cancer progression [21–23]. From these studies it can be concluded that malignant progression is a stepwise process and tumor growth occurs after a series of molecular events that parallel morphological changes indicative of cell transformation. It has been well established that the invasive capability of a breast cancer cell line can be accurately predicted by performance in an in vitro three dimensional Matrigel outgrowth assay [17, 24–29]. When placed on reconstituted basement membrane (Matrigel) breast cancer cell lines with the potential to metastasize have been shown to demonstrate stellate branching morphology and invade the matrix, while non-invasive cell lines fail to grow or develop into spherical bunches of cells without invading the matrix . This in vitro phenomenon is thought to mimic the in vivo interaction of tumor cells with the surrounding matrix and demonstrates the ability of the tumor cell to degrade the basement membrane, which encapsulates the primary tumor, and allows individual cells to migrate from the initial tumor mass into the breast stroma and eventually establish within the lymph gland which aids dispersal to other organs of the body. In stark contrast, normal mammary epithelial cells form polar acini, similar to alveoli breast structures when plated on a layer of Matrigel [30–32]. The WalBC cells appeared to exhibit a non-invasive phenotype, however the growth pattern in Matrigel did not resemble the spherical bunches of cells exhibited by other non-invasive cell lines  but appeared to exhibit a normal branching and lobular development. This difference in morphology could be due to difference in cellular signaling via the components within Matrigel. It has been shown that mammary epithelial cells exhibit species-specific cell interaction with their surrounding extracellular matrix. Fur seal (Arctocephalus pusillus pusillus) mammary epithelial cells only form acinar structures capable of expressing milk protein genes when grown on their own matrix and display invasive morphology when grown on Matrigel . A similar effect is also seen with primary wallaby mammary cells, which exhibit stellate outgrowth on Matrigel and only form acinar structures capable of producing milk when grown on their own matrix (Mailer and Nicholas unpublished data). These observations suggest Matrigel may not be an adequate substrate to test the invasive potential of wallaby breast cancer cell lines and a wallaby derived ECM would be better suited to use with these cells as it has the potential to more closely mimic the in vivo environment. Similarly, the failure of WalBC cell to establish tumorgenicity using the nude mice xenograph model may also be subject to the same species specific restraints.
Epidermal growth factor (EGF), a polypeptide found in human and animal blood and secretions, is an important mitogen in breast epithelial cells. EGF has been found to stimulate a variety of tissues including normal rodent breast tissue and rodent breast cancer  and human breast epithelial cells in culture  and fibridomas . In MCF-7 cells as little as 0.01 ng/ml of EGF stimulates cell growth and 10 ng/ml was maximal, however, EGF shows no effect on another human breast cancer cell line, MDA-MB-231. . Similarly, primary wallaby epithelial cells shows a growth response to EGF while the WalBC cell line was not EGF responsive as these maximal doses. It is suggested that some breast cancers retain EGF sensitivity observed with nonmalignant mammary cells, while it is lost in others. The slow growth rate observed for the WalBC cell line compared with the primary cells may indicate that this cell line requires other hormones/factors for maximal growth.
Newly emerging data about the genomes of other species such as the marsupial and the availability of 15,000 breast specific wallaby cDNA's expressed at all stages during the lactation cycle, and a 10, 000 ESTs array offers the rare opportunity to profile the WalBC cell line for gene expression patterns. Variations in transcriptional programs account for much of the biological diversity of human cells and tumors. Despite this molecular diversity, analyses of invasive breast carcinomas using microarrays have identified gene expression signatures that characterize many of the essential qualities important for biological and clinical classification . DNA microarray profiling studies on breast tumors show distinct and reproducible subtypes of breast carcinoma associated with different outcomes. Expression profiles have characterized invasive breast carcinomas into five groups: luminal A, luminal B, HER2+/estrogen receptor (ER)-, basal-like, and normal breast-like. The basal-like is typically ER- and HER2- and shows some characteristics of breast myoepithelial cells. The basal-like subtype has been shown to have the highest proliferation rates and poorest outcomes [39, 40], and has been described in association with BRCA-1-associated carcinomas . Myoepithelial cells typically express cytokeratin 17, while luminal cells typically express cytokeratins 8 and 18. The prevalence and poor prognosis of basal-like breast carcinomas have been validated immunohistochemically; in a 564-case tissue microarray, it was demonstrated that 16% of tumors stained positive for cytokeratin5/6 or cytokeratin 17 and that basal cytokeratin expression was associated with a poor prognosis .
The WalBC transcript profile was compared to normal wallaby mammary cells obtained from the same animal and to mammary cells obtained from a virgin animal. Microarray analysis revealed that KRT17 (30 fold up regulated) was the highest up-regulated gene in the WalBC cell line when grown as a mono-layer culture. Expression of this gene is characteristic of basal carcinomas . A 4.7 fold down regulation of VIM expression and absence of vimentin immunoreactivity in the in situ tumor compared to normal mammary cells suggests both the tumor and the derived cell line have not undergone an epithelial-to-mesenchymal transition associated with increased invasive/migratory properties of epithelial cells [43, 44]. WalBC cells showed regulated expression of DSP, s100A14 and ANXA1 which are genes associated with well-differentiated, epithelioid breast cancer cell lines with weak invasive potential and poorly invasive tumors [45–47]. WalBC cells also over expressed the tumor metastasis suppressor NDRG1, which has been shown to be negatively correlated with tumor metastasis. In vitro and in vivo studies have also demonstrated a significant reduction in the metastatic ability of cells over-expressing NDRG1. The morphology of the in situ tumor also resembled a basal-like carcinoma  which exhibited solid architecture and a pushing margin.
In addition to exhibiting a gene expression profile that correlates with poorly invasive breast cancers the WalBC cell line also displayed a gene profile that correlated with highly invasive cells. For example the up-regulation of TK1 seen in WalBC cells has previously been associated with high proliferation activity and invasiveness potential which is related to a more aggressive phenotype . Down regulation of genes such as TIMP-3 and TAGLN in the WalBC cell line, which have been previously shown to be associated with highly invasive breast cancer cell lines or tumors with poor prognosis [50, 51], suggests the WalBC cell line appears to exhibit a gene expression profile in common with both poorly invasive and highly invasive cell types. WalBC also exhibited overexpression of AQP3 which is associated with inflammatory breast cancer . Expression of these gene in the non-invasive WalBC cell line may indicate that although these may be markers for invasive potential expression of these genes alone cannot invoke the invasive process.
It is clear the specific targets responsible for tumor progression need to be identified. A number of new targets such as hypothetical proteins, CAP4, SALL1, ZONAB, and SUMO1 were identified. These genes encoding these proteins have been found to have human ortholgoues, and with further study, may also prove to be expressed in human cancers representing possible new molecular targets for the treatment of breast cancer. In addition, MRP8, a newly discovered member of the ATP-binding cassette transporter superfamily, previously identified by EST database mining and gene prediction programming was found to be highly expressed in human breast cancer  and thus was identified as a putative molecular target for the treatment of breast cancer. Expression of MRP8 was also detected in the WalBC cell line and further supports this prediction demonstrating the effectiveness the WalBC cell line in the search for new targets of breast cancer therapy and treatment. Further study of these newly identified targets within human models of breast cancer may provide fresh clues in the development and progression of breast cancer which may in turn lead to new treatments and therapies.
Observation of analogous gene expression profiles between human basal-like breast cancer and wallaby breast cancer has identified a common pattern of gene expression that appears to be characteristic for this type of cancer regardless of species. Therefore, the use of comparative oncology provides a useful tool to identify new potential molecular targets relevant to other species. The comparative study of breast cancer in species such as the wallaby may provide new fundamental clues to the etiology of breast cancer which may in turn lead to new treatments and therapies. In many fields unique approaches to drug discovery and design are being sort in order to unearth naturally occurring factors that may be used as potential drug therapies. One example is the use of disintegrins derived from snake venom as a potential therapeutic for treatment of breast cancer progression [54–56]. The discovery of new genes identified in wallaby breast cancer with human homologous by comparative biology may provide useful informative for further study of aspects of human breast cancer research, which may, in turn, lead to new interventions and treatment regimes.
Fetal Calf Serum.
Minke JM, Weijer K, Misdorp W: Allotransplantation of K248 feline mammary carcinoma cell line in cats. A model for monoclonal antibody guided detection and therapy of human breast cancer. Lab Invest. 1991, 65 (4): 421-432.
Heppner GH, Miller FR, Shekhar PM: Nontransgenic models of breast cancer. Breast Cancer Res. 2000, 2 (5): 331-334.
Hirayama K, Honda Y, Sako T, Okamoto M, Tsunoda N, Tagami M, Taniyama H: Invasive ductal carcinoma of the mammary gland in a mare. Vet Pathol. 2003, 40 (1): 86-91.
Lincoln DW, Renfree MB: Mammary gland growth and milk ejection in the agile wallaby, Macropus agilis, displaying concurrent asynchronous lactation. J Reprod Fertil. 1981, 63 (1): 193-203.
Trott JF, Simpson KJ, Moyle RL, Hearn CM, Shaw G, Nicholas KR, Renfree MB: Maternal regulation of milk composition, milk production, and pouch young development during lactation in the tammar wallaby (Macropus eugenii ). Biol Reprod. 2003, 68 (3): 929-936.
Nicholas K, Simpson K, Wilson M, Trott J, Shaw D: The tammar wallaby: a model to study putative autocrine-induced changes in milk composition. J Mammary Gland Biol Neoplasia. 1997, 2 (3): 299-310.
Ballard FJ, Grbovac S, Nicholas KR, Owens PC, Read LC: Differential changes in the milk concentrations of epidermal growth factor and insulin-like growth factor-I during lactation in the tammar wallaby, Macropus eugenii. Gen Comp Endocrinol. 1995, 98 (3): 262-268.
Nicholas KR: Asynchronous dual lactation in a marsupial, the tammar wallaby (Macropus eugenii). Biochem Biophys Res Commun. 1988, 154 (2): 529-536.
Hood CJ, Parham DM: A simple method of tumour culture. Pathol Res Pract. 1998, 194 (3): 177-181.
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR: New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990, 82 (13): 1107-1112.
Price JTT: Models for studying cellular invasion of basement membranes. Methods Mol Biol. 1999, 129: 231-249.
Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas JD, Eberwine JH: Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A. 1990, 87 (5): 1663-1667.
Thomson PC: Analysis of microarray data: a mixed-model finite-mixture approach. Paper presented at the XXIIIrd International Biometric Conference. (Montreal, Canada, July 2006). 2006
Wolfinger RD, Gibson G, Wolfinger ED, Bennett L, Hamadeh H, Bushel P, Afshari C, Paules RS: Assessing gene significance from cDNA microarray expression data via mixed models. J Comput Biol. 2001, 8 (6): 625-637.
Reverter A, Byrne KA, Brucet HL, Wang YH, Dalrymple BP, Lehnert SA: A mixture model-based cluster analysis of DNA microarray gene expression data on Brahman and Brahman composite steers fed high-, medium-, and low-quality diets. J Anim Sci. 2003, 81 (8): 1900-1910.
Thompson EW, Paik S, Brunner N, Sommers CL, Zugmaier G, Clarke R, Shima TB, Torri J, Donahue S, Lippman ME: Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines. J Cell Physiol. 1992, 150 (3): 534-544.
Lefèvre CM, Digby MR, Whitley JC, Strahm Y, Nicholas KR: Lactation transcriptomics in the Australian marsupial, Macropus eugenii: transcript sequencing and quantification. BMC Genomics. 2007, 8 (1): 417-425.
Vail DM, MacEwen EG: Spontaneously occurring tumors of companion animals as models for human cancer. Cancer Invest. 2000, 18 (8): 781-792.
Lee JL, Chang CJ, Chueh LL, Lin CT: Expression of secreted frizzled-related protein 2 in a primary canine mammary tumor cell line: a candidate tumor marker for mammary tumor cells. In Vitro Cell Dev Biol Anim. 2003, 39 (5-6): 221-227.
Tester AM, Ruangpanit N, Anderson RL, Thompson EW: MMP-9 secretion and MMP-2 activation distinguish invasive and metastatic sublines of a mouse mammary carcinoma system showing epithelial-mesenchymal transition traits. Clin Exp Metastasis. 2000, 18 (7): 553-560.
Hazan RB, Qiao R, Keren R, Badano I, Suyama K: Cadherin switch in tumor progression. Ann N Y Acad Sci. 2004, 1014: 155-163.
Lee JL, Chang CJ, Wu SY, Sargan DR, Lin CT: Secreted frizzled-related protein 2 (SFRP2) is highly expressed in canine mammary gland tumors but not in normal mammary glands. Breast Cancer Res Treat. 2004, 84 (2): 139-149.
Liang Z, Yoon Y, Votaw J, Goodman MM, Williams L, Shim H: Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res. 2005, 65 (3): 967-971.
Sommers CL, Thompson EW, Torri JA, Kemler R, Gelmann EP, Byers SW: Cell adhesion molecule uvomorulin expression in human breast cancer cell lines: relationship to morphology and invasive capacities. Cell Growth Differ. 1991, 2 (8): 365-372.
Tsai MS, Bogart DF, Castaneda JM, Li P, Lupu R: Cyr61 promotes breast tumorigenesis and cancer progression. Oncogene. 2002, 21 (53): 8178-8185.
Glondu M, Liaudet-Coopman E, Derocq D, Platet N, Rochefort H, Garcia M: Down-regulation of cathepsin-D expression by antisense gene transfer inhibits tumor growth and experimental lung metastasis of human breast cancer cells. Oncogene. 2002, 21 (33): 5127-5134.
Schiemann S, Schwirzke M, Brunner N, Weidle UH: Molecular analysis of two mammary carcinoma cell lines at the transcriptional level as a model system for progression of breast cancer. Clin Exp Metastasis. 1998, 16 (2): 129-139.
Lee SW: H-cadherin, a novel cadherin with growth inhibitory functions and diminished expression in human breast cancer. Nat Med. 1996, 2 (7): 776-782.
Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ: Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development. 1989, 105 (2): 223-235.
Petersen OW, Ronnov-Jessen L, Howlett AR, Bissell MJ: Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci U S A. 1992, 89 (19): 9064-9068.
Hall HG, Farson DA, Bissell MJ: Lumen formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture. Proc Natl Acad Sci U S A. 1982, 79 (15): 4672-4676.
Sharp JA, Cane KN, Mailer SL, Oosthuizen WH, Arnould JPY, Nicholas KR: 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). Matrix Biology. 2006, 25 (7): 430-442.
Turkington RW: Stimulation of mammary carcinoma cell proliferation by epithelial growth factor in vitro. Cancer Res. 1969, 29 (7): 1457-1458.
Taylor-Papadimitriou J, Shearer M, Stoker MG: Growth requirements of human mammary epithelial cells in culture. Int J Cancer. 1977, 20 (6): 903-908.
Stoker MG, Pigott D, Taylor-Papadimitriou J: Response to epidermal growth factors of cultured human mammary epithelial cells from benign tumours. Nature. 1976, 264 (5588): 764-767.
Osborne CK, Hamilton B, Titus G, Livingston RB: Epidermal growth factor stimulation of human breast cancer cells in culture. Cancer Res. 1980, 40 (7): 2361-2366.
Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D: Molecular portraits of human breast tumours. Nature. 2000, 406 (6797): 747-752.
Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL: Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001, 98 (19): 10869-10874.
Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, Demeter J, Perou CM, Lonning PE, Brown PO, Borresen-Dale AL, Botstein D: Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A. 2003, 100 (14): 8418-8423.
Foulkes WD, Stefansson IM, Chappuis PO, Begin LR, Goffin JR, Wong N, Trudel M, Akslen LA: Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. J Natl Cancer Inst. 2003, 95 (19): 1482-1485.
van de Rijn M, Perou CM, Tibshirani R, Haas P, Kallioniemi O, Kononen J, Torhorst J, Sauter G, Zuber M, Kochli OR, Mross F, Dieterich H, Seitz R, Ross D, Botstein D, Brown P: Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am J Pathol. 2002, 161 (6): 1991-1996.
Ackland ML, Newgreen DF, Fridman M, Waltham MC, Arvanitis A, Minichiello J, Price JT, Thompson EW: Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells. Lab Invest. 2003, 83 (3): 435-448.
Fuchs IB, Lichtenegger W, Buehler H, Henrich W, Stein H, Kleine-Tebbe A, Schaller G: The prognostic significance of epithelial-mesenchymal transition in breast cancer. Anticancer Res. 2002, 22 (6A): 3415-3419.
Sommers CL, Byers SW, Thompson EW, Torri JA, Gelmann EP: Differentiation state and invasiveness of human breast cancer cell lines. Breast Cancer Res Treat. 1994, 31 (2-3): 325-335.
de Silva Rudland S, Martin L, Roshanlall C, Winstanley J, Leinster S, Platt-Higgins A, Carroll J, West C, Barraclough R, Rudland P: Association of S100A4 and osteopontin with specific prognostic factors and survival of patients with minimally invasive breast cancer. Clin Cancer Res. 2006, 12 (4): 1192-1200.
Kreunin P, Urquidi V, Lubman DM, Goodison S: Identification of metastasis-associated proteins in a human tumor metastasis model using the mass-mapping technique. Proteomics. 2004, 4 (9): 2754-2765.
Kovacevic Z, Richardson DR: The metastasis suppressor, Ndrg-1: a new ally in the fight against cancer. Carcinogenesis. 2006
Livasy CA, Karaca G, Nanda R, Tretiakova MS, Olopade OI, Moore DT, Perou CM: Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol. 2006, 19 (2): 264-271.
Ami Y, Shimazui T, Akaza H, Uematsu N, Yano Y, Tsujimoto G, Uchida K: Gene expression profiles correlate with the morphology and metastasis characteristics of renal cell carcinoma cells. Oncol Rep. 2005, 13 (1): 75-80.
Kotzsch M, Farthmann J, Meye A, Fuessel S, Baretton G, Tjan-Heijnen VC, Schmitt M, Luther T, Sweep FC, Magdolen V, Span PN: Prognostic relevance of uPAR-del4/5 and TIMP-3 mRNA expression levels in breast cancer. Eur J Cancer. 2005, 41 (17): 2760-2768.
Dressman HK, Hans C, Bild A, Olson JA, Rosen E, Marcom PK, Liotcheva VB, Jones EL, Vujaskovic Z, Marks J, Dewhirst MW, West M, Nevins JR, Blackwell K: Gene expression profiles of multiple breast cancer phenotypes and response to neoadjuvant chemotherapy. Clin Cancer Res. 2006, 12 (3 Pt 1): 819-826.
Bera TK, Lee S, Salvatore G, Lee B, Pastan I: MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer. Mol Med. 2001, 7 (8): 509-516.
Swenson S, Costa F, Ernst W, Fujii G, Markland FS: Contortrostatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity. Pathophysiol Haemost Thromb. 2005, 34 (4-5): 169-176.
Jokhio R, Ansari AF: Cobra snake venom reduces significantly tissue nucleic acid levels in human breast cancer. J Pak Med Assoc. 2005, 55 (2): 71-73.
Yang RS, Tang CH, Chuang WJ, Huang TH, Peng HC, Huang TF, Fu WM: Inhibition of tumor formation by snake venom disintegrin. Toxicon. 2005, 45 (5): 661-669.
Lee JL, Chang CJ, Chueh LL, Lin CT: Expression of secreted frizzled-related protein 2 in a primary canine mammary tumor cell sentence: a candidate tumor marker for mammary tumor cells. In Vitro Cell Dev Biol Anim. 2003, 39: 221-227.
This work was supported by grants from the Geoffrey Gardiner Foundation, CRC for Co-operative Research of Innovative Dairy Products and Dairy Australia. We thank Dr. Matthew Digby (Department of Zoology, Melbourne University) for scanning of microarray slides, Dr. Kylie Cane (Department of Zoology, Melbourne University) for Vimentin immunohistochemical staining and in vivo tumor photography and Ms Emma Walker (University of Melbourne, Department of Medicine, St. Vincent's Hospital) for expert mammary fat pad injections.
The author(s) declare that they have no competing interests.
JS conceived of the study, carried out the immunoassays, histological and morphologic studies, interpretation of microarray results and participated in the isolation of the cell line and in vivo studies and drafted the manuscript. SM carried out the microarray analysis and participated in collection of material and development of the cell line, proliferation assay and in vivo studies. PT carried out the data statistical analysis of the data. CL carried out normalisation of microarray data. KN performed the examination and surgery, collected material and participated in the design and coordination of the study. All authors helped to draft the manuscript, read and approved the final manuscript.