Key words: xenograft; matrix metalloproteinases; MMP-13; collagenase-3; breast tumour; stroma; bone metastasis; microenvironment Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes with the ability to degrade extracellular matrix (ECM) components as well as numerous secreted and membrane-bound cell modulators and thus are considered key to the tissue rearrangements associated with malignancy. There are 24 soluble and membrane-anchored members of the MMP family in humans that demonstrate extensive sequence homology and overlapping but distinct substrate specificities. 1 Proteolytic activity of the MMPs is regulated at several levels, most notably via gene transcription, activation via proteolysis of a propeptide, cell compartmentalisation and inhibition by the endogenous tissue inhibitors of metalloproteinases (TIMPs). 2 Although endowed with pro-invasive properties, the functions of MMPs have been shown to be much more widespread than simply facilitating migration/invasion. They also are involved in tumour initiation and progression, activation of chemokines and growth factors, angiogenesis and apoptosis induction to name a few. 1,3 It is thus not surprising that many MMPs have been documented in cancer tissue. 2,4,5 Although MMPs have been implicated in several key steps of tumour progression, clinical trials using synthetic MMP inhibitors have not led to significant therapeutic benefit. This is in contrast to marked inhibition of tumour growth and metastasis in animal models. 6,7 A number of reasons have been proposed for these shortcomings, including the high tumour burden of selected patients for clinical trials, a lack of surrogate markers for dose efficacy and the revelation that some MMPs can inhibit tumour growth. 8,9 It is thus clear that our understanding of the source and specific function of each MMP in the various stages of tumour progression is not complete and that this knowledge is crucial to effectively apply MMP inhibitors in the treatment of cancer. 1,2,6,7 A tumour is defined as a local uncontrolled growth of abnormal tissue consisting of transformed cells, as well as other cell types including fibroblasts, macrophages, endothelial cells and connective tissue components known as the stroma. 10 Tumour cells are known to influence and manipulate the stroma such that the latter produces a permissive and supportive environment, which helps facilitate the growth of the carcinoma. Breast cancer (BrCa) tissue has been shown to contain most MMPs including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-13, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, [11][12][13][14][15][16][17][18][19][20] BrCa cells frequently and selectively metastasise to the bone, resulting in osteolysis, pathologic fracture, pain and significant clinical morbidity. 21 Numerous MMPs have been implicated in bone turnover, 22,23 and more recently bone metastasis in experimental breast and prostate cancer models were found to be sensitive to MMP inhibition. 24 -26 Determining which MMPs are more influential i...

Over 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 cell's 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. Citation 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