Dexing Huang

An MMP13-Selective Inhibitor Delays Primary Tumor Growth and the Onset of Tumor-Associated Osteolytic Lesions in Experimental Models of Breast Cancer

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.

High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers

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 6xa0weeks 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 (pxa0=xa00.0002) and lower adipose (pxa0=xa00.0006) 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 (pxa0=xa00.4) or count (pxa0=xa00.4) 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.

An optimised direct lysis method for gene expression studies on low cell numbers.

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.

Abstract 2977: Epithelial mesenchymal plasticity in xenograft models of circulating and disseminated tumour cells from human breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnOver the past several decades, there have been numerous breast cancer patient studies aimed at detecting disseminated (DTC) and circulating tumor cells (CTC) within bone marrow and blood respectively. Although they offer prognostic and predictive value they are not yet used clinically, and qualitative analysis of these cells may provide additional valuable information. In view of this, we have set about to establish reproducible and robust mouse models for breast cancer DTC/CTC research. We have developed a species-specific tandem nested RT-qPCR approach which enables us to detect and measure a panel of human markers associated with epithelial-mesenchymal plasticity (EMP; CDH1, ILK, CD24 and VIM, normalised to RPL32), which are hypothesised to be involved in the generation and function of DTC/CTC. Mock experiments have demonstrated the ability to detect high abundance transcripts from a single cells worth of RNA amongst a very large amount of mouse background using our assays. Blood, bone marrow and tumor tissue were collected from xenografts generated utilizing the MDA-MB-231 (mesenchymal) and MDA-MB-468 (epithelial) cell lines, and a transplantable breast cancer xenograft (ED03). MDA-MB-468 xenografts exhibit two zones of VIM expression, one at the stromal interface and another at the necrotic interface, which may correspond to the EGF- and hypoxia-inducible EMP seen with these cells in vitro. Large secondary deposits in lymph node or lungs are intensely epithelial, while small lymphovascular deposits appear mesenchymal. No evidence of EMP is seen in the ED-03 xenografts despite these xenografts producing the most CTC. The MDA-MB-231 xenografts appear mesenchymal with widespread VIM staining and lack of CDH1. Preliminary analysis of the blood of mice with MDA-MB-231 xenografts revealed human RPL32 levels significantly higher than the background levels measured in RNA collected from the blood of control mice (p = <0.01), however the blood burden was too low to allow measurement of other transcripts. Very low levels of human RNA were detected in the blood of MDA-MB-468 mice, necessitating the use of various transfected vector markers for RT-qPCR analysis. A reduction in CD24 expression relative to the primary tumour was seen, suggestive of reduced epithelial nature, however no changes were seen in VIM compared to the primary site. Cells in the blood of ED-03 xenograft-bearing mice showed higher CHD1 levels than seen in the tumour. The CDH1 levels in the ED-03 CTCs decreased with increased blood burden, which may reflect altered intravasation or intracellular interactions in the blood. IHC analysis on cytospin slides of bone marrow from the MDA-MB-468 and ED-03 xenografts supported the presence of low numbers of DTC in these models. These data provide evidence for altered expression of some EMP markers in these CTC models, and provide a test system for further analyses.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2977. doi:1538-7445.AM2012-2977

Abstract 3428: Coordinated regulation of mesenchymal epithelial transition in the PMC42-LA breast cancer cell line variant

PMC42 cells were derived from a breast cancer pleural effusion and exhibit stem cell-like features when first characterized (Whitehead, Bertoncello et al. 1983; Whitehead, Monaghan et al. 1983). The PMC42-LA subline (Ackland, Michalczyk et al. 2001; Ackland, Newgreen et al. 2003) is distinctly less mesenchymal than parental PMC42-ET cells, but both exhibit EGF- and TGFβ-inducible mesenchymal-like change. Together they provide an ideal model in which to study the regulation of epithelial-mesenchymal plasticity in a breast cancer context. We have assessed in vitro parameters including migration, 3D colony formation and EMT marker expression, and undertook both miRNA profiling (mirVana probe set V1, Ambion) and alternative splice usage (Affymetrix human 1.0 ST whole genome exon arrays) analysis. Several miRNAs were expressed differently in the two sublines and/or up- or down-regulated in response to EMT-inducing treatments. Relative to the mesenchymal ET subline, the, miR-200 family members were elevated in the epithelial LA subline, where a corresponding loss of Zeb1 expression was seen. The LA subline also showed reduced E-Cadherin promoter methylation, and increased methylation of the Zeb1 promoter. Manipulation of the miR 200 family affected the EMT marker expression in these cells, as did Zeb1 suppression with shRNA. Matrigel invasion was also inhibited with Zeb1 knockdown. Additional gene expression changes and alternative splice usage, promoter methylation changes, and miR expression changes are under study. These studies may identify molecules and pathways that are important in cell specification across the epithelial mesenchymal axis, and represent diagnostic and therapeutic targets in breast cancer. Ackland, M. L., A. Michalczyk, et al. (2001). “PMC42, A novel model for the differentiated human breast.” Exp Cell Res 263(1): 14-22. Ackland, M. L., D. F. Newgreen, et al. (2003). “Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells.” Lab Invest 83(3): 435-48. Whitehead, R. H., I. Bertoncello, et al. (1983). “A new human breast carcinoma cell line (PMC42) with stem cell characteristics. I. Morphologic characterization.” J Natl Cancer Inst 70(4): 649-61. Whitehead, R. H., P. Monaghan, et al. (1983). “A new human breast carcinoma cell line (PMC42) with stem cell characteristics. II. Characterization of cells growing as organoids.” J Natl Cancer Inst 71(6): 1193-203. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2011-3428