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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

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