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Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth. (Cancer Res 2006; 66(23): 11271-8)

Supplementary Table 1 from Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2

Supplementary Table 2 from Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2

Supplementary Table 3 from Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2

Oral cavity cancer (OCC) is the predominant subtype of head and neck cancer (HNC) and has up to 50% mortality. Genome-wide microRNA (miR) sequencing data indicates overexpression of miR-9-5p in HNC tumours, however, the biological role of miR-9-5p in OCC is complex; it can either act as a tumour suppressor or an oncomir, regulating many target genes at the post-transcriptional level. We have investigated the overexpression of miR-9-5p in three OCC cell lines. We have evaluated its expression levels and Galectin-3 as potential biomarkers in saliva samples collected from controls and OCC patients. We found that over expression of miR-9-5p in OCC cell lines resulted in a significant reduction in cell proliferation and migration, and an increase in apoptosis, which was paralleled by an increase in Galectin-3 secretion and export of Galectin-3 protein. Our data are consistent with miR-9-5p being a modulator of Galectin-3 via the AKT/γ-catenin pathway. In addition, the positive correlation between the levels of miR-9-5p expression and secreted Galectin-3 in saliva reflects a similar relationship in vivo, and supports the utility of their integrative evaluation in OCC. Our findings indicate that both miR-9-5p and Galectin-3 are critical biomolecules in the progression of OCC.

BackgroundBreast cancers acquire aggressive capabilities via epithelial to mesenchymal transition (EMT), in which various integrins/integrin-linked kinase signalling are upregulated.MethodsWe investigated this in two patient-derived xenografts (PDXs) developed from breast-to-bone metastases, and its functional significance in a breast cancer cell line system. ED03 and EDW01 PDXs were grown subcutaneously in immunocompromised SCID mice through 11 passages and 7 passages, respectively. Tumour tissue was assessed using immunohistochemistry (IHC) for oestrogen receptor (ER)-alpha, E-cadherin, vimentin, Twist1, beta-catenin, P120-RasGAP, CD44, CD24 and Ki67, and RT-qPCR of EMT-related factors (CDH1,VIM,CD44,CD24), integrins beta 1 (ITGB1), alpha 2 (ITGA2) andILK. Integrin andILKexpression in epidermal growth factor (EGF)-induced EMT of the PMC42-ET breast cancer cell line was assessed by RT-qPCR and Western blotting, as were the effects of their transient knockdown via small interfering RNA +/− EGF. Cell migration, changes in cell morphology and adhesion of siRNA-transfected PMC42-ET cells to various extracellular matrix (ECM) substrates was assessed.ResultsThe ED03 (ER+/PR−/HER2−/lobular) and EDW01 (ER+/PR−/HER2−/ductal) PDXs were both classified as molecular subtype luminal A. ED03 xenografts exhibited mutated E-cadherin with minimal expression, but remained vimentin-negative across all passages. In EDW01, the hypoxic indicator gene CAIX and Twist1 were co-ordinately upregulated at passages 4–5, corresponding with a decrease in E-cadherin. At passages 6–7,VIMwas upregulated along withITGB1andITGA2, consistent with an increasing EMT. The ED03 PDX displayed minimal change over passages in mice, for all genes examined.ILK,ITGB1andITGA2mRNAs were also increased in the EGF-induced EMT of PMC42-ET cells (in whichCDH1was downregulated) although siRNA against these targets revealed that this induction was not necessary for the observed EMT. However, their knockdown significantly reduced EMT-associated adhesion and Transwell migration.ConclusionOur data suggest that despite an increase inITGA2andITGB1gene expression in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.

Mammographic Density (MD) is the degree of radio-opacity of the breast in an X-ray mammogram. It is determined by the Fibroglandular: Adipose tissue ratio. MD has major implications in breast cancer risk and breast cancer chemoprevention. This study aimed to investigate the feasibility of accurate, low-cost quantification of MD in vivo without ionising radiation. We used single-sided portable nuclear magnetic resonance ("Portable NMR") due to its low cost and the absence of radiation-related safety concerns. Fifteen (N = 15) healthy female volunteers were selected for the study and underwent an imaging routine consisting of 2D X-ray mammography, quantitative breast 3T MRI (Dixon and T

Blood samples, bone marrow, tumours and metastases where possible were collected from SCID mice bearing orthotopic xenografts of the triple-negative MDA-MB-468 cell line or a transplantable ER-positive patient derived xenograft (ED-03), and assessed using human-specific, tandem-nested RT-qPCR for markers relating to detection of circulating (CTCs) and disseminated tumour cells (DTCs), breast cancer clinicopathology, the 'cancer stem cell' phenotype, metabolism, hypoxia and epithelial-mesenchymal plasticity (EMP). Increased levels of SNAI1, ILK, NOTCH1, CK20, and PGR, and a decrease/loss of EPCAM in CTCs/DTCs were observed relative to the primary xenograft across both models. Decreased CD24 and EGFR was restricted to the MDA-MB-468 model, while increased TFF1 was seen in the ED-03 model. The major metabolic regulator PPARGC1A, and several hypoxia-related markers (HIF1A, APLN and BNIP3) were significantly elevated in both models. Increased expression of mesenchymal markers including SNAI1 was seen across both models, however CDH1 did not decrease concordantly, and several other epithelial markers were increased, suggesting an uncoupling of EMP to produce an EMP hybrid or partial-EMT. Single cell analysis of ED-03 CTCs, although limited, indicated uncoupling of the EMP axis in single hybrid cells, rather than distinct pools of epithelial or mesenchymal-enriched cells, however dynamic heterogeneity between CTCs/DTCs cannot be ruled out. Reduced CD24 expression was observed in the MDA-MB-468 CTCs, consistent with the 'breast cancer stem cell' phenotype, and metastatic deposits in this model mostly resembled the primary xenografts, consistent with the mesenchymal-epithelial transition paradigm.

Epithelial Mesenchymal Transition (EMT) refers to the transition of cells from a more differentiated epithelial phenotype to a less differentiated mesenchymal phenotype, a process that may be triggered by a range of therapeutic interventions including cytotoxic treatment, and which we have previously linked to poor breast cancer (BrCa) outcome after neoadjuvant chemotherapy (NAC)1. Mammographic breast density (MBD)represents the white radiographic appearance of epithelial and stromal breast tissue on a mammogram. High MBD in patients being treated for BrCa also associates with chemoresistance, correlating with lower pathological complete response rates (pCR)2. Linking these two stimuli, EMT can also be induced by artificial high-density stroma, where it also leads to chemoresistance in vitro3. Here we set out to validate the link between poor outcome after NAC and EMT in a larger validatory patient cohort, and to ascertain the molecular drivers through which EMT is triggered in this setting. Further we look to confirm the association of high MBD with poor chemoresponse in the same cohort, and to assess whether this chemoresistance is mediated through EMT with the same drivers. In a pilot cohort of 50 NAC-treated locally advanced BrCas with a pCR rate of 20%, pre-NAC biopsies and post-NAC surgical specimens were analysed for expression changes in a panel of EMT-related markers across treatment using 230 Nanostring assays. This included the EMT-driving transcription factors TWIST 1 and 2, SNAIL 1, 2 and 3 and ZEB 1 and 2, which were correlated with risk of relapse. Snail-3 showed significantly greater induction in relapsers compared to non-relapsers (OR=1.8, p=0.04) with a borderline significantly greater induction of TWIST-1 (OR=2.4, p=0.08) in relapsers in addition. In a subsequent 240-patient validation cohort with a pCR rate of 18%, contralateral cranio-caudal view mammograms from the time of diagnosis have been collated and digitized with MBD assessment employing Cumulus software ongoing. Percent breast density will be assessed both as a continuous variable and by quartiles. Immunohistochemistry on pre- and post-operative tissue sections with pan-cytokeratin-vimentin co-staining to identify EMT and staining for SNAIL-3 and TWIST-1 is also in progress. Associations between MBD, EMT before and after chemotherapy, pCR and relapse-free survival will be presented. The role of Snail-3 and TWIST-1 in the interplay between MBD, EMT and outcome is being explored and will be reported. Citation Format: Agarwal V, Spalding LJ, Blick T, Dobrovic A, Thompson EW, Redfern A. The interplay between stromal density, epithelial mesenchymal transition and chemoresistance in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-05.

High mammographic density (MD) increases breast cancer (BC) risk and creates a stiff tissue environment. BC risk is also increased in BRCA1/2 gene mutation carriers, which may be in part due to genetic disruption of the tumour suppressor gene Ras association domain family member 1 (RASSF1A), a gene that is also directly regulated by tissue stiffness. High MD combined with BRCA1/2 mutations further increase breast cancer risk, yet BRCA1/2 mutations alone or in combination do not increase MD. The molecular basis for this additive effect therefore remains unclear. We studied the interplay between MD, stiffness, and BRCA1/2 mutation status in human mammary tissue obtained after prophylactic mastectomy from women at risk of developing BC. Our results demonstrate that RASSF1A expression increased in MCF10DCIS.com cell cultures with matrix stiffness up until ranges corresponding with BiRADs 4 stiffnesses (~16 kPa), but decreased in higher stiffnesses approaching malignancy levels (&gt, 50 kPa). Similarly, higher RASSF1A protein was seen in these cells when co-cultivated with high MD tissue in murine biochambers. Conversely, local stiffness, as measured by collagen I versus III abundance, repressed RASSF1A protein expression in BRCA1, but not BRCA2 gene mutated tissues, regional density as measured radiographically repressed RASSF1A in both BRCA1/2 mutated tissues. The combinatory effect of high MD and BRCA mutations on breast cancer risk may be due to RASSF1A gene repression in regions of increased tissue stiffness.

Oral cavity cancer (OCC) is the predominant subtype of head and neck cancer (HNC) and has up to 50% mortality. Genome-wide microRNA (miR) sequencing data indicates overexpression of miR-9-5p in HNC tumours, however, the biological role of miR-9-5p in OCC is complex; it can either act as a tumour suppressor or an oncomir, regulating many target genes at the post-transcriptional level. We have investigated the overexpression of miR-9-5p in three OCC cell lines. We have evaluated its expression levels and Galectin-3 as potential biomarkers in saliva samples collected from controls and OCC patients. We found that over expression of miR-9-5p in OCC cell lines resulted in a significant reduction in cell proliferation and migration, and an increase in apoptosis, which was paralleled by an increase in Galectin-3 secretion and export of Galectin-3 protein. Our data are consistent with miR-9-5p being a modulator of Galectin-3 via the AKT/γ-catenin pathway. In addition, the positive correlation between the levels of miR-9-5p expression and secreted Galectin-3 in saliva reflects a similar relationship in vivo, and supports the utility of their integrative evaluation in OCC. Our findings indicate that both miR-9-5p and Galectin-3 are critical biomolecules in the progression of OCC.

Background: Breast cancers acquire aggressive capabilities via epithelial to mesenchymal transition (EMT), in which various integrins/integrin linked kinase signalling are upregulated. Methods: We investigated this in two patient-derived xenografts (PDXs) developed from breast-to-bone metastases, and it’s functional significance in a breast cancer cell line system. ED03 and EDW01 PDXs were grown subcutaneously in immunocompromised SCID mice through 11 passages and 7 passages, respectively. Tumour tissue was assessed using immunohistochemistry (IHC) for estrogen receptor (ER)-alpha, E-cadherin, vimentin, Twist1, beta-catenin, P120-RasGAP, CD44, CD24 and Ki67, and RT-qPCR of EMT-related factors (CDH1, VIM, CD44, CD24), integrins beta-1 (ITGB1), alpha-2 (ITGA2) and ILK. Integrin and ILK expression in epidermal growth factor (EGF) induced EMT of the PMC42-ET breast cancer cell line was assessed by RT-qPCR and Western blotting, as were the effects of their transient knockdown via small interfering RNA +/- EGF. Cell migration, changes in cell morphology and adhesion of siRNA-transfected PMC42-ET cells to various extracellular matrix (ECM) substrates was assessed.Results: The ED03 (ER+/PR-/HER2-/lobular) and EDW01 (ER+/PR-/HER2-/ductal) PDXs were both classified as molecular subtype luminal A. ED03 xenografts exhibited mutated E-cadherin with minimal expression, but remained vimentin-negative across all passages. In EDW01, the hypoxic indicator gene CAIX and Twist1 were co-ordinately upregulated at passage 4-5, corresponding with a decrease in E-cadherin. At passages 6-7, vimentin was upregulated along with ITGB1 and ITGA2, consistent with an increasing EMT. The ED03 PDX displayed minimal change over passages in mice, for all genes examined. ILK, ITGB1 and ITGA2 were also increased in the EGF-induced EMT of PMC42-ET cells (in which E-cadherin was downregulated) although siRNA against these targets revealed that this induction was not necessary for the observed EMT. However, their knockdown significantly reduced EMT-associated adhesion and Transwell migration.Conclusion: Our data suggest that despite an increase in integrins alpha-2 and beta-1 gene expression in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.

Background: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. Methods: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute&rsquo, s Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. Results: In total, 259 shRNAs were depleted with doxorubicin treatment (at p &lt, 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. Conclusions: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.

Background: Breast cancers acquire aggressive capabilities via epithelial to mesenchymal transition (EMT), in which various integrins/integrin linked kinase signalling are upregulated. Methods: We investigated this in two patient-derived xenografts (PDXs) developed from breast-to-bone metastases, and it’s functional significance in a breast cancer cell line system. ED03 and EDW01 PDXs were grown subcutaneously in immunocompromised SCID mice through 11 passages and 7 passages, respectively. Tumour tissue was assessed using immunohistochemistry (IHC) for estrogen receptor (ER)-alpha, E-cadherin, vimentin, Twist1, beta-catenin, P120-RasGAP, CD44, CD24 and Ki67, and RT-qPCR of EMT-related factors (CDH1, VIM, CD44, CD24), integrins beta-1 (ITGB1), alpha-2 (ITGA2) and ILK. Integrin and ILK expression in epidermal growth factor (EGF) induced EMT of the PMC42-ET breast cancer cell line was assessed by RT-qPCR and Western blotting, as were the effects of their transient knockdown via small interfering RNA +/- EGF. Cell migration, changes in cell morphology and adhesion of siRNA-transfected PMC42-ET cells to various extracellular matrix (ECM) substrates was assessed. Results: The ED03 (ER+/PR-/HER2-/lobular) and EDW01 (ER+/PR-/HER2-/ductal) PDXs were both classified as molecular subtype luminal A. ED03 xenografts exhibited mutated E-cadherin with minimal expression, but remained vimentin-negative across all passages. In EDW01, the hypoxic indicator gene CAIX and Twist1 were co-ordinately upregulated at passage 4-5, corresponding with a decrease in E-cadherin. At passages 6-7, vimentin was upregulated along with ITGB1 and ITGA2, consistent with an increasing EMT. The ED03 PDX displayed minimal change over passages in mice, for all genes examined. ILK, ITGB1 and ITGA2 were also increased in the EGF-induced EMT of PMC42-ET cells (in which E-cadherin was downregulated) although siRNA against these targets revealed that this induction was not necessary for the observed EMT. However, their knockdown significantly reduced EMT-associated adhesion and Transwell migration. Conclusion: Our data suggest that despite an increase in integrins alpha-2 and beta-1 in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.

Background Epithelial-to-mesenchymal transition (EMT) is associated with downregulated E-cadherin and frequently with decreased proliferation. Proliferation may be restored in secondary metastases by mesenchymal-to-epithelial transition (MET). We tested whether E-cadherin maintains epithelial proliferation in MDA-MB-468 breast cancer cells, facilitating metastatic colonization in severe combined immunodeficiency (SCID) mice. Methods EMT/MET markers were assessed in xenograft tumors by immunohistochemistry. Stable E-cadherin manipulation was effected by transfection and verified by Western blotting, immunocytochemistry, and quantitative polymerase chain reaction (qPCR). Effects of E-cadherin manipulation on proliferation and chemomigration were assessed in vitro by performing sulforhodamine B assays and Transwell assays, respectively. Invasion was assessed by Matrigel outgrowth; growth in vivo was assessed in SCID mice; and EMT status was assessed by qPCR. Hypoxic response of E-cadherin knockdown cell lines was assessed by qPCR after hypoxic culture. Repeated measures analysis of variance (ANOVA), one- and two-way ANOVA with posttests, and paired Student’s t tests were performed to determine significance (p

Circulating tumour cells (CTCs) are shed into the bloodstream from both primary and secondary tumours and provide a non-invasive means to study tumor progression and response to treatment. Assessment of ribonucleic acid (RNA) and monitoring dynamic changes in gene expression profiles of CTCs extends their clinical and prognostic power and establish their role in guiding treatment. Among these methods, droplet digital (RT-ddPCR) technique provides a high sensitivity and detectibility of CTCs. RNA-sequencing (RNAseq) is the most comprehensive method, that would allow the simultaneous measurement of a large number of genes and theoretically the whole transcriptome. Since CTCs are heterogeneous in nature, single cell RNAseq methods are very valuable in assessing population dynamics and functional states of CTCs. While RNA in situ hybridization (RNA-ISH) is used relatively less frequently, it also allows for the assessment of expression of multiple genes within individual CTCs. Epithelial to Mesenchymal Transition (EMT) is a major contributor to metastasis, providing a mechanism to allow cells to become migratory and invasive, and to survive in the bloodstream. Monitoring CTCs undergoing EMT may lead to improvement in their prognostic and predictive power. Here, we review various RNA analysis of CTCs and those that undergo EMT and their application in diagnosis, prognosis and treatment of cancers.

Dynamic interconversions between transitional epithelial and mesenchymal states underpin the epithelial mesenchymal plasticity (EMP) seen in some carcinoma cell systems. We have delineated epithelial and mesenchymal subpopulations existing within the PMC42-LA breast cancer cell line by their EpCAM expression. These purified but phenotypically plastic states, EpCAMHigh (epithelial) and EpCAMLow (mesenchymal), have the ability to regain the phenotypic equilibrium of the parental population (i.e., 80% epithelial and 20% mesenchymal) over time, although the rate of reversion in the mesenchymal direction (epithelial-mesenchymal transition; EMT) is higher than that in the epithelial direction (mesenchymal-epithelial transition; MET). Single-cell clonal propagation was implemented to delineate the molecular and cellular features of this intrinsic heterogeneity with respect to EMP flux. The dynamics of the phenotypic proportions of epithelial and mesenchymal states in single-cell generated clones revealed clonal diversity and intrinsic plasticity. Single cell-derived clonal progenies displayed differences in their functional attributes of proliferation, stemness marker (CD44/CD24), migration, invasion and chemo-sensitivity. Interrogation of genomic copy number variations (CNV) with whole exome sequencing (WES) in the context of chromosome count from metaphase spread indicated that chromosomal instability was not influential in driving intrinsic phenotypic plasticity. Overall, these findings reveal the stochastic nature of both the epithelial and mesenchymal subpopulations, and the single cell-derived clones for differential functional attributes.

Mammographic density (MD) is a strong independent risk factor for breast cancer. Traditional screening for MD using X-ray mammography involves ionising radiation, which is not suitable for young women, those with previous radiation exposure, or those having undergone a partial mastectomy. Therefore, alternative approaches for MD screening that do not involve ionising radiation will be important as the clinical use of MD increases, and as more frequent MD testing becomes desirable for research purposes. We have previously demonstrated the potential utility of spin relaxation-based, single-sided portable-NMR measurements for the purpose of MD quantification. We present here a more refined analysis by quantifying breast tissue density in excised samples on a continuous scale (0% to 100% fibroglandular tissue content) using micro-CT (μCT), and comparing the results to spin-relaxation and diffusion portable-NMR measurements of the same samples. μCT analysis of mammary tissues containing high- and low-MD (HMD and LMD, respectively) regions had Hounsfield Unit (HU) histograms with a bimodal pattern, with HMD regions exhibiting significantly higher HU values than LMD regions. Quantitative MD (%HMD) values obtained using μCT exhibited an excellent correlation with portable-NMR results, namely longitudinal spin-relaxation time constants (T1) and the relative tissue water content obtained from portable-NMR diffusion measurements (R2 = 0.92, p < 0.0001 and R2 = 0.96, p < 0.0001, respectively). These findings are consistent with our previous results demonstrating relatively high water content in HMD breast tissue, consistent with the high proportion of fibroglandular tissue, FGT, which in turn contains more abundant water-carrying HSPG proteins. We observed an excellent correlation between the T1 values and diffusion NMR-measured relative tissue water content (R2 = 0.94, p < 0.0001). These findings demonstrate, for the first time, the ability of single-sided portable NMR to accurately quantify MD in vitro on a continuous scale. The results also indicate that portable-NMR analysis can assist in the identification of features underpinning MD, namely FGT and adipose tissue content. Future work will involve application of portable NMR to quantifying MD in vivo.

Epithelial−mesenchymal plasticity (EMP), encompassing epithelial−mesenchymal transition (EMT) and mesenchymal−epithelial transition (MET), are considered critical events for cancer metastasis. We investigated chromosomal heterogeneity and chromosomal instability (CIN) profiles of two sister PMC42 breast cancer (BC) cell lines to assess the relationship between their karyotypes and EMP phenotypic plasticity. Karyotyping by GTG banding and exome sequencing were aligned with SWATH quantitative proteomics and existing RNA-sequencing data from the two PMC42 cell lines; the mesenchymal, parental PMC42-ET cell line and the spontaneously epithelially shifted PMC42-LA daughter cell line. These morphologically distinct PMC42 cell lines were also compared with five other BC cell lines (MDA-MB-231, SUM-159, T47D, MCF-7 and MDA-MB-468) for their expression of EMP and cell surface markers, and stemness and metabolic profiles. The findings suggest that the epithelially shifted cell line has a significantly altered ploidy of chromosomes 3 and 13, which is reflected in their transcriptomic and proteomic expression profiles. Loss of the TGFβR2 gene from chromosome 3 in the epithelial daughter cell line inhibits its EMT induction by TGF-β stimulus. Thus, integrative ‘omics’ characterization established that the PMC42 system is a relevant MET model and provides insights into the regulation of phenotypic plasticity in breast cancer.

// Arutha Kulasinghe 1, 7 , Chris Perry 2 , Majid E. Warkiani 3 , Tony Blick 1, 7 , Anthony Davies 4, 7 , Ken O'Byrne 4, 7 , Erik W. Thompson 1, 7 , Colleen C. Nelson 5, 7 , Ian Vela 5, 6, 7 , Chamindie Punyadeera 1, 7 1 The School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia 2 Department of Otolaryngology, Princess Alexandra Hospital, Woolloongabba, QLD, Australia 3 School of Mechanical and Manufacturing Engineering, Australian Centre for NanoMedicine, University of New South Wales, Sydney, Australia 4 Translational Cell Imaging Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, QLD, Australia 5 Australian Prostate Cancer Research Centre-Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Princess Alexandra Hospital, QLD, Australia 6 Department of Urology, Princess Alexandra Hospital, Wolloongabba, QLD, Australia 7 Translational Research Institute, Woolloongabba, QLD, Australia Correspondence to: Chamindie Punyadeera, email: chamindie.punyadeera@qut.edu.au Keywords: circulating tumour cells, head and neck cancer, ex-vivo culture, metastasis, HPV Received: June 03, 2016 Accepted: July 20, 2016 Published: August 09, 2016 ABSTRACT Minimally invasive techniques are required for the identification of head and neck cancer (HNC) patients who are at an increased risk of metastasis, or are not responding to therapy. An approach utilised in other solid cancers is the identification and enumeration of circulating tumour cells (CTCs) in the peripheral blood of patients. Low numbers of CTCs has been a limiting factor in the HNC field to date. Here we present a methodology to expand HNC patient derived CTCs ex-vivo . As a proof of principle study, 25 advanced stage HNC patient bloods were enriched for circulating tumour cells through negative selection and cultured in 2D and 3D culture environments under hypoxic conditions (2% O 2 , 5% CO 2 ). CTCs were detected in 14/25 (56%) of patients (ranging from 1–15 CTCs/5 mL blood). Short term CTC cultures were successfully generated in 7/25 advanced stage HNC patients (5/7 of these cultures were from HPV+ patients). Blood samples from which CTC culture was successful had higher CTC counts ( p = 0.0002), and were predominantly from HPV+ patients ( p = 0.007). This is, to our knowledge, the first pilot study to culture HNC CTCs ex-vivo . Further studies are warranted to determine the use of short term expansion in HNC and the role of HPV in promoting culture success.

There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.

Whilst locoregional control of head and neck cancers (HNCs) has improved over the last four decades, long-term survival has remained largely unchanged. A possible reason for this is that the rate of distant metastasis has not changed. Such disseminated disease is reflected in measurable levels of cancer cells in the blood of HNC patients, referred to as circulating tumour cells (CTCs). Numerous marker-independent techniques have been developed for CTC isolation and detection. Recently, microfluidics-based platforms have come to the fore to avoid molecular bias. In this pilot, proof of concept study, we evaluated the use of the spiral microfluidic chip for CTC enrichment and subsequent detection in HNC patients. CTCs were detected in 13/24 (54%) HNC patients, representing both early to late stages of disease. Importantly, in 7/13 CTC-positive patients, CTC clusters were observed. This is the first study to use spiral microfluidics technology for CTC enrichment in HNC.

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

Hyperactive inflammatory responses following cancer initiation have led to cancer being described as a 'wound that never heals'. These inflammatory responses elicit signals via NFκB leading to IL-6 production, and IL-6 in turn has been shown to induce epithelial to mesenchymal transition in breast cancer cells in vitro, implicating a role for this cytokine in cancer cell invasion. We previously have shown that conditioned medium derived from cancer-associated fibroblasts induced an Epithelial to Mesenchymal transition (EMT) in PMC42-LA breast cancer cells and we have now identify IL-6 as present in this medium. We further show that IL-6 is expressed approximately 100 fold higher in a cancer-associated fibroblast line compared to normal fibroblasts. Comparison of mouse-specific (stroma) and human-specific (tumor) IL-6 mRNA expression from MCF-7, MDA MB 468 and MDA MB 231 xenografts also indicated the stroma rather than tumor as a significantly higher source of IL-6 expression. Mast cells (MCs) feature in inflammatory cancer-associated stroma, and activated MCs secrete IL-6. We observed a higher MC index (average number of mast cells per xenograft section/average tumor size) in MDA MB 468 compared to MDA MB 231 xenografts, where all MC were observed to be active (degranulating). This higher MC index correlated with greater mouse-specific IL-6 expression in the MDA MB 468 xenografts, implicating MC as an important source of stromal IL-6. Furthermore, immunohistochemistry on these xenografts for pSTAT3, which lies downstream of the IL-6 receptor indicated frequent correlations between pSTAT3 and mast cell positive cells. Analysis of publically available databases for IL-6 expression in patient tissue revealed higher IL-6 in laser capture microdissected stroma compared to adjacent tissue epithelium from patients with inflammatory breast cancer (IBC) and invasive non-inflammatory breast cancer (non-IBC) and we show that IL-6 expression was significantly higher in Basal versus Luminal molecular/phenotypic groupings of breast cancer cell lines. Finally, we discuss how afferent and efferent IL-6 pathways may participate in a positive feedback cycle to dictate tumor progression.

Epithelial-mesenchymal transition (EMT) is a feature of migratory cellular processes in all stages of life, including embryonic development and wound healing. Importantly, EMT features cluster with disease states such as chronic fibrosis and cancer. The dissolution of the E-cadherin-mediated adherens junction (AJ) is a key preliminary step in EMT and may occur early or late in the growing epithelial tumour. This is a first step for tumour cells towards stromal invasion, intravasation, extravasation and distant metastasis. The AJ may be inactivated in EMT by directed E-cadherin cleavage; however, it is increasingly evident that the majority of AJ changes are transcriptional and mediated by an expanding group of transcription factors acting directly or indirectly to repress E-cadherin expression. A review of the current literature has revealed that these factors may regulate each other in a hierarchical pattern where Snail1 (formerly Snail) and Snail2 (formerly Slug) are initially induced, leading to the activation of Zeb family members, TCF3, TCF4, Twist, Goosecoid and FOXC2. Within this general pathway, many inter-regulatory relationships have been defined which may be important in maintaining the EMT phenotype. This may be important given the short half-life of Snail1 protein. We have investigated these inter-regulatory relationships in the mesenchymal breast carcinoma cell line PMC42 (also known as PMC42ET) and its epithelial derivative, PMC42LA. This review also discusses several newly described regulators of E-cadherin repressors including oestrogen receptor-α and new discoveries in hypoxia- and growth factor-induced EMT. Finally, we evaluated how these findings may influence approaches to current cancer treatment.

Background: Metastasis in HNC patients is reflected by measurable levels of circulating tumor cells (CTCs) in the peripheral blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. For patients without CTCs at presentation, the detection of CTCs in the blood and analysis of biomarkers within them provide an opportunity to identify patients "at risk" of developing overt metastasis, accelerating targeted treatment in addition to routing care with the clear aim of improving cure. Methods: Our study aimed to assess whether CTCs from the blood of HNC patients attending the Princess Alexandra Hospital and Royal Brisbane and Women's Hospital provided early cues of distant metastases (n=250). Results: With significant advances in CTC isolation technologies, we could demonstrate a higher CTC capture efficiency using epitope-independent platforms. By assessment of single and clustered CTCs, our data showed that HNC patients can be identified 4-6 months prior to developing clinical/radiographically evident metastasis. In these patients, a window for treatment escalation could become a possibility. In a proof-of-principle study, using novel culture formulations and hypoxic conditions (1-2% O2), we were able to demonstrate, for the first time, short-term patient-derived CTC cultures ex vivo from 7/18 HNC samples (4/7 HPV-positive, oropharyngeal) in a clinically relevant time period. Recent advancements have shown that PD-1 immune checkpoint therapies have durable responses in metastatic HNC patients that fail 1st- and 2nd-line therapy. Our preliminary data suggest PD-L1 is frequently expressed on HNC CTCs, and an immunoscore may be able to stratify patients likely to respond to immunotherapy. Conclusion: Expanding CTCs outside the patient's body allows for the recapitulation of the molecular diversity present within the tumor, understanding the disease progression and testing of therapies. Patients with a high percentage of PD-L1+ CTCs could be potential candidates for anti-PD-L1 therapy, a promising new immunotherapy. Citation Format: Arutha Kulasinghe, Chris Perry, Liz Kenny, Tony Blick, Majid Warkiani, Ian Vela, Ken O'Byrne, Jean-Paul Thiery, Erik Thompson, Colleen Nelson, Chamindie Punyadeera. Circulating tumor cells: The tumor trail left in the blood [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5572.

To establish the suitability of multiplex tandem polymerase chain reaction (MT-PCR) for rapid identification of oestrogen receptor (ER) and Her-2 status using a single, formalin-fixed, paraffin-embedded (FFPE) breast tumour section.Tissue sections from 29 breast tumours were analysed by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). RNA extracted from 10 mum FFPE breast tumour sections from 24 of 29 tumours (14 ER positive and 5 Her-2 positive) was analysed by MT-PCR. After establishing a correlation between IHC and/or FISH and MT-PCR results, the ER/Her-2 status of a further 32 randomly selected, archival breast tumour specimens was established by MT-PCR in a blinded fashion, and compared to IHC/FISH results.MT-PCR levels of ER and Her-2 showed good concordance with IHC and FISH results. Furthermore, among the ER positive tumours, MT-PCR provided a quantitative score with a high dynamic range. Threshold values obtained from this data set applied to 32 archival tumour specimens showed that tumours strongly positive for ER and/or Her-2 expression were easily identified by MT-PCR.MT-PCR can provide rapid, sensitive and cost-effective analysis of FFPE material and may prove useful as triage to identify patients suited to endocrine or trastuzumab (Herceptin) treatment.

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed Luminal) or mixed basal/luminal (termed Basal A) features (Neve et al Cancer Cell 2006). Studies providing molecular and cellular analyses of EMT features in these cell lines are summarised, and the expression levels of EMT-associated factors in these cell lines are analysed. Recent clinical studies supporting the presence of EMT-like changes in vivo are summarised. Human breast cancer cell lines with mesenchymal properties continue to hold out the promise of directing us towards key mechanisms at play in the metastatic dissemination of breast cancer.

The influence of alphaVbeta3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing beta3 integrin status. Overexpression of beta3 integrin caused increased cell surface expression of alphaV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. beta3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, alphaVbeta3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of beta3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with beta3 integrin expression. Although our studies confirm important biological effects of alphaVbeta3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, beta3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by alphaVbeta3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.

Like a set of bookends, cellular, molecular, and genetic changes of the beginnings of life mirror those of one of the most common cause of death--metastatic cancer. Epithelial to mesenchymal transition (EMT) is an important change in cell phenotype which allows the escape of epithelial cells from the structural constraints imposed by tissue architecture, and was first recognized by Elizabeth Hay in the early to mid 1980's to be a central process in early embryonic morphogenesis. Reversals of these changes, termed mesenchymal to epithelial transitions (METs), also occur and are important in tissue construction in normal development. Over the last decade, evidence has mounted for EMT as the means through which solid tissue epithelial cancers invade and metastasize. However, demonstrating this potentially rapid and transient process in vivo has proven difficult and data connecting the relevance of this process to tumor progression is still somewhat limited and controversial. Evidence for an important role of MET in the development of clinically overt metastases is starting to accumulate, and model systems have been developed. This review details recent advances in the knowledge of EMT as it occurs in breast development and carcinoma and prostate cancer progression, and highlights the role that MET plays in cancer metastasis. Finally, perspectives from a clinical and translational viewpoint are discussed.

There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.

Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth. (Cancer Res 2006; 66(23): 11271-8)

Objective Human ovarian carcinoma samples were orthotopically implanted into SCID mice to investigate the contribution of matrix metalloproteases (MMPs) to the spread of ovarian tumors. Methods Mice were inoculated with patient tumor samples, and developed ovarian tumors over a 16-week period with metastasis occurring in some mice. Species-specific quantitative RT-PCR was used to identify the source of tumor-associated MMPs. Results Membrane-type (MT)1-MMP mRNA was significantly increased in high-grade tumors, tumors with evidence of serosal involvement, and tumors in which distant metastases were detected. The increase in MT1-MMP expression was predominantly from the human tumor cells, with a minor contribution from the mouse ovarian stroma. Neither human nor mouse MT2-MMP were correlated with tumor progression and MT3-MMP levels were negligible. While tumor cells did not produce significant amounts of MMP-2 or MMP-9, the presence of tumor was associated with increased levels of MMP-2 expression by mouse ovarian stroma. Stromal-derived MT1-MMP was greater in large tumors and was associated with stromal MMP-2 expression but neither was significantly linked with metastasis. Conclusions These studies indicate that tumor-derived MT1-MMP, more so than other gelatinolytic MMPs, is strongly linked to aggressive tumor behavior. This orthotopic model of human ovarian carcinoma is appropriate for studying ovarian tumor progression, and will be valuable in the further investigation of the metastatic process.

Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding β1 integrins in collagen-stimulated MMP-2 activation. Three β1 integrin siRNAs were used to elucidate the involvement of β1 integrins in the Col I-induced MMP-2 activation mechanism. were used to elucidate the involvement of1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of β1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography. All three β1 integrin siRNAs were efficient at β1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins α2 and α3, which are heterodimeric partners of β1, but not αV, which is not. All three β1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of β1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped β1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the β1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by β1 integrin siRNA knockdown. Together, the data reveals that strong abrogation of β1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of β1 integrin.

Mammographic density (MD) is a strong risk factor for breast cancer. It is altered by exogenous endocrine treatments, including hormone replacement therapy and Tamoxifen. Such agents also modify breast cancer (BC) risk. However, the biomolecular basis of how systemic endocrine therapy modifies MD and MD-associated BC risk is poorly understood. This study aims to determine whether our xenograft biochamber model can be used to study the effectiveness of therapies aimed at modulating MD, by examine the effects of Tamoxifen and oestrogen on histologic and radiographic changes in high and low MD tissues maintained within the biochamber model. High and low MD human tissues were precisely sampled under radiographic guidance from prophylactic mastectomy fresh specimens of high-risk women, then inserted into separate vascularized murine biochambers. The murine hosts were concurrently implanted with Tamoxifen, oestrogen or placebo pellets, and the high and low MD biochamber tissues maintained in the murine host environment for 3 months, before the high and low MD biochamber tissues were harvested for histologic and radiographic analyses. The radiographic density of high MD tissue maintained in murine biochambers was decreased in Tamoxifen-treated mice compared to oestrogen-treated mice (p = 0.02). Tamoxifen treatment of high MD tissue in SCID mice led to a decrease in stromal (p = 0.009), and an increase in adipose (p = 0.023) percent areas, compared to placebo-treated mice. No histologic or radiographic differences were observed in low MD biochamber tissue with any treatment. High MD biochamber tissues maintained in mice implanted with Tamoxifen, oestrogen or placebo pellets had dynamic and measurable histologic compositional and radiographic changes. This further validates the dynamic nature of the MD xenograft model, and suggests the biochamber model may be useful for assessing the underlying molecular pathways of Tamoxifen-reduced MD, and in testing of other pharmacologic interventions in a preclinical model of high MD.

Mammographic density (MD) is a strong heritable risk factor for breast cancer, and may decrease with increasing parity. However, the biomolecular basis for MD-associated breast cancer remains unclear, and systemic hormonal effects on MD-associated risk is poorly understood. This study assessed the effect of murine peripartum states on high and low MD tissue maintained in a xenograft model of human MD. Method High and low MD human breast tissues were precisely sampled under radiographic guidance from prophylactic mastectomy specimens of women. The high and low MD tissues were maintained in separate vascularised biochambers in nulliparous or pregnant SCID mice for 4 weeks, or mice undergoing postpartum involution or lactation for three additional weeks. High and low MD biochamber material was harvested for histologic and radiographic comparisons during various murine peripartum states. High and low MD biochamber tissues in nulliparous mice were harvested at different timepoints for histologic and radiographic comparisons. Results High MD biochamber tissues had decreased stromal (p = 0.0027), increased adipose (p = 0.0003) and a trend to increased glandular tissue areas (p = 0.076) after murine postpartum involution. Stromal areas decreased (p = 0.042), while glandular (p = 0.001) and adipose areas (p = 0.009) increased in high MD biochamber tissues during lactation. A difference in radiographic density was observed in high (p = 0.0021) or low MD biochamber tissues (p = 0.004) between nulliparous, pregnant and involution groups. No differences in tissue composition were observed in high or low MD biochamber tissues maintained for different durations, although radiographic density increased over time. Conclusion High MD biochamber tissues had measurable histologic changes after postpartum involution or lactation. Alterations in radiographic density occurred in biochamber tissues between different peripartum states and over time. These findings demonstrate the dynamic nature of the human MD xenograft model, providing a platform for studying the biomolecular basis of MD-associated cancer risk.

Metastasis is the major cause of cancer mortality. A strong link between metastatic behaviour and epithelial–mesenchymal plasticity (EMP) has been demonstrated in human breast cancer (BC). EMP can provide carcinoma cells with invasive ability to leave the primary tumour, enter into the circulation as circulating tumour cells (CTCs), arrive at a distant organ and ultimately form a metastasis. This project aims to investigate EMP in BC CTCs.

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.

We review here the recently emerging relationship between epithelial-mesenchymal transition (EMT) and breast cancer stem cells (BCSC), and provide analyses of published data on human breast cancer cell lines, supporting their utility as a model for the EMT/BCSC state. Genome-wide transcriptional profiling of these cell lines has confirmed the existence of a subgroup with mesenchymal tendencies and enhanced invasive properties ('Basal B'/Mesenchymal), distinct from subgroups with either predominantly luminal ('Luminal') or mixed basal/luminal ('Basal A') features (Neve et al. Cancer Cell, 2006). A literature-derived EMT gene signature has shown specific enrichment within the Basal B subgroup of cell lines, consistent with their over-expression of various EMT transcriptional drivers. Basal B cell lines are found to resemble BCSC, being CD44(high)CD24(low). Moreover, gene products that distinguish Basal B from Basal A and Luminal cell lines (Basal B Discriminators) showed close concordance with those that define BCSC isolated from clinical material, as reported by Shipitsin et al. (Cancer Cell, 2007). CD24 mRNA levels varied across Basal B cell lines, correlating with other Basal B Discriminators. Many gene products correlating with CD24 status in Basal B cell lines were also differentially expressed in isolated BCSC. These findings confirm and extend the importance of the cellular product of the EMT with Basal B cell lines, and illustrate the value of analysing these cell lines for new leads that may improve breast cancer outcomes. Gene products specific to Basal B cell lines may serve as tools for the detection, quantification, and analysis of BCSC/EMT attributes.