Surgery (St Vincent's) - Research Publications
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ItemHuman glandular organoid formation in murine engineering chambers after collagenase digestion and flow cytometry isolation of normal human breast tissue single cells(WILEY, 2016-11)Women with high mammographic density (MD) are at increased risk of breast cancer (BC) after adjustment for age and body mass index. We have developed a murine biochamber model in which both high MD (HMD) and low MD (LMD) tissue can be propagated. Here, we tested whether cells isolated by collagenase digestion and fluorescence-activated cell sorting (FACS) from normal breast can be reconstituted in our biochamber model, which would allow cell-specific manipulations to be tested. Fresh breast tissue was collected from women (n = 7) undergoing prophylactic mastectomy. The tissue underwent collagenase digestion overnight and, in some cases, additional FACS enrichment to obtain mature epithelial, luminal progenitor, mammary stem, and stromal cells. Cells were then transferred bilaterally into biochambers in SCID mice (n = 5-7) and incubated for 6 weeks, before harvesting for histological analyses, and immunohistochemical staining for cytokeratins (CK), vimentin, Ki-67, murine macrophages, and Cleaved Caspase-3. Biochambers inoculated with single cells after collagenase digestion or with flow cytometry contained glandular structures of human origin (human vimentin-positive), which expressed CK-14 and pan-CK, and were proliferating (Ki-67-positive). Glandular structures from the digested tissues were smaller than those in chambers seeded with finely chopped intact mammary tissue. Mouse macrophage infiltration was higher in the chambers arising from digested tissues. Pooled single cells and FACS fractionated cells were viable in the murine biochambers and formed proliferating glandular organoids of human origin. This is among the first report to demonstrate the success of formed human glandular organoids from isolated primary mammary cells in the murine biochamber model.
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ItemTransverse relaxation-based assessment of mammographic density and breast tissue composition by single-sided portable NMR(WILEY, 2019-09)PURPOSE: Elevated mammographic density (MD) is an independent risk factor for breast cancer (BC) as well as a source of masking in X-ray mammography. High-frequency longitudinal monitoring of MD could also be beneficial in hormonal BC prevention, where early MD changes herald the treatment's success. We present a novel approach to quantification of MD in breast tissue using single-sided portable NMR. Its development was motivated by the low cost of portable-NMR instrumentation, the suitability for measurements in vivo, and the absence of ionizing radiation. METHODS: Five breast slices were obtained from three patients undergoing prophylactic mastectomy or breast reduction surgery. Carr-Purcell-Meiboom-Gill (CPMG) relaxation curves were measured from (1) regions of high and low MD (HMD and LMD, respectively) in the full breast slices; (2) the same regions excised from the full slices; and (3) excised samples after H2 O-D2 O replacement. T2 distributions were reconstructed from the CPMG decays using inverse Laplace transform. RESULTS: Two major peaks, identified as fat and water, were consistently observed in the T2 distributions of HMD regions. The LMD T2 distributions were dominated by the fat peak. The relative areas of the two peaks exhibited statistically significant (P < .005) differences between HMD and LMD regions, enabling their classification as HMD or LMD. The relative-area distributions exhibited no statistically significant differences between full slices and excised samples. CONCLUSION: T2 -based portable-NMR analysis is a novel approach to MD quantification. The ability to quantify tissue composition, combined with the low cost of instrumentation, make this approach promising for clinical applications.
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ItemT1-based sensing of mammographic density using single-sided portable NMR(WILEY, 2018-09)Purpose A single‐sided NMR instrument was used to investigate the ability of the T1 relaxation constant to distinguish between regions of low and high mammographic density in human breast tissue. Methods Measurements were performed on 5 breast slices obtained from 3 women undergoing breast reduction surgery or prophylactic mastectomy. Results T1 values measured in regions of high mammographic density in both the full breast slices (T1 = 170 ± 30 ms) and excised regions (T1 = 160 ± 30 ms) were found to be significantly different (P < .001) from those measured in regions of low mammographic density, in which T1 = 120 ± 10 ms was observed both in full slices and excised regions. There was no statistically significant difference between the T1 values measured in the full breast slices and those measured in the excised regions. Conclusion The findings suggest that portable NMR may provide a low‐cost means of assessing mammographic density in vivo.
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ItemIntegrin alpha-2 and beta-1 expression increases through multiple generations of the EDW01 patient-derived xenograft model of breast cancer-insight into their role in epithelial mesenchymal transition in vivo gained from an in vitro model system(BMC, 2020-12-04)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 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) 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 passages 4-5, corresponding with a decrease in E-cadherin. At passages 6-7, VIM 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 mRNAs were also increased in the EGF-induced EMT of PMC42-ET cells (in which CDH1 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 ITGA2 and ITGB1 gene expression in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.
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ItemAberrant pregnancy-associated plasma protein-A expression in breast cancers prognosticates clinical outcomes(NATURE PUBLISHING GROUP, 2020-08-13)Elevated levels of pregnancy-associated plasma protein-A (PAPP-A) have been implicated in the pathogenesis of various malignancies, including breast cancers. Breast cancer is one of the most frequent carcinomas and is the second most common cancer type detected in women of child-bearing age. Throughout pregnancy PAPP-A is produced and secreted by the placental syncytiotrophoblast cells; co-incidentally pregnancy-associated breast cancers often have an aggressive clinical course. The components of the PAPP-A/IGF axis was assessed in a panel of breast cancer cell lines. Using neutralising antibodies the impact of PAPP-A/IGF axis on cell motility was evaluated. PAPP-A was expressed in four of the twelve breast cancer cell lines tested. Blocking PAPP-A and IGFBP4 with neutralising antibodies significantly decreased motiliy of MDA-MB-231 cells. Upregulation of PAPP-A expression in breast tumours resulted in a trend towards worse overall survival. Notably, PAPP-A expression also positively correlated with epithelial-to-mesenchymal transition markers. In conclusion, these results indicate that PAPP-A plays an important role in breast cancer progression and it may be a promising therapeutic target in breast cancer.
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ItemDNA Methylation Profiling of Breast Cancer Cell Lines along the Epithelial Mesenchymal Spectrum-Implications for the Choice of Circulating Tumour DNA Methylation Markers(MDPI, 2018-09)(1) Background: Epithelial⁻mesenchymal plasticity (EMP) is a dynamic process whereby epithelial carcinoma cells reversibly acquire morphological and invasive characteristics typical of mesenchymal cells. Identifying the methylation differences between epithelial and mesenchymal states may assist in the identification of optimal DNA methylation biomarkers for the blood-based monitoring of cancer. (2) Methods: Methylation-sensitive high-resolution melting (MS-HRM) was used to examine the promoter methylation status of a panel of established and novel markers in a range of breast cancer cell lines spanning the epithelial⁻mesenchymal spectrum. Pyrosequencing was used to validate the MS-HRM results. (3) Results: VIM, DKK3, and CRABP1 were methylated in the majority of epithelial breast cancer cell lines, while methylation of GRHL2, MIR200C, and CDH1 was restricted to mesenchymal cell lines. Some markers that have been used to assess minimal residual disease such as AKR1B1 and APC methylation proved to be specific for epithelial breast cell lines. However, RASSF1A, RARβ, TWIST1, and SFRP2 methylation was seen in both epithelial and mesenchymal cell lines, supporting their suitability for a multimarker panel. (4) Conclusions: Profiling DNA methylation shows a distinction between epithelial and mesenchymal phenotypes. Understanding how DNA methylation varies between epithelial and mesenchymal phenotypes may lead to more rational selection of methylation-based biomarkers for circulating tumour DNA analysis.
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ItemHypoxia-induced reactive oxygen species mediate N-cadherin and SERPINE1 expression, EGFR signalling and motility in MDA-MB-468 breast cancer cells(NATURE PORTFOLIO, 2017-11-09)One of the hallmarks of the tumour microenvironment is hypoxia resulting from increased oxygen consumption by proliferative cancer cells and altered vasculature. Hypoxic tension initiates various cellular signals and can drive epithelial to mesenchymal transition (EMT), a process important in cancer progression. In this study, using the antioxidant N-acetylcysteine (NAC), we show that hypoxia-induced reactive oxygen species (ROS) in MDA-MB-468 breast cancer cells, selectively regulate hypoxia-induced increases in N-cadherin and SERPINE1, two proteins involved in cell adhesion. Treatment of cells with NAC also attenuated hypoxia-mediated activation of EGFR, but did not have any effect on hypoxia-mediated induction of HIF1α. Exogenous hydrogen peroxide phenocopied the effects of hypoxia on N-cadherin and SERPINE1 expression and EGFR activation, suggesting its possible involvement in these hypoxia-mediated events. Reflective of their effect on cell adhesion proteins and EGFR (associated with migratory phenotypes), NAC also reduced cell migration under hypoxic conditions, a crucial event in metastasis. Our findings suggest a selective role for redox signalling in the regulation of specific components of the responses to hypoxia and induction of EMT in breast cancer cells. This study provides new evidence supporting the potential of targeting ROS as a therapeutic strategy for the control of breast cancer metastasis.
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ItemNo Preview AvailableCOMPLEXO: identifying the missing heritability of breast cancer via next generation collaboration(BMC, 2013)Linkage analysis, positional cloning, candidate gene mutation scanning and genome-wide association study approaches have all contributed significantly to our understanding of the underlying genetic architecture of breast cancer. Taken together, these approaches have identified genetic variation that explains approximately 30% of the overall familial risk of breast cancer, implying that more, and likely rarer, genetic susceptibility alleles remain to be discovered.