A murine model of bone marrow micrometastasis in breast cancer

Bone marrow (BM) is one of the most common sites and often the first clinical indication of metastatic progression of breast cancer. Multivariate analyses have shown that the presence of cytokeratin positive tumor cells in the marrow of women with newly diagnosed stage I, II or III breast cancer is an independent predictor of survival. The objective of this study was to develop an orthotopic model of spontaneous BM metastasis to facilitate studies of this process. A murine mammary adenocarcinoma cell line, Clone 66, was transduced with the neomycin resistance gene (Cl66neo) and injected orthotopically into female Balb/c mice. Polymerase chain reaction (PCR) for the neo gene performed on BM cells harvested from tumor bearing mice demonstrated as few as 10(2) injected tumor cells produced BM micrometastases at 4 weeks postinjection. Small foci of tumor cells were identified in the mammary fatpad (mfp) without gross evidence of primary tumors. Higher doses of tumor cells produced BM micrometastases, detectable by PCR, at one week post-injection. Constructs containing green fluorescent protein (GFP) and the neomycin resistance gene (neo) were also transduced into Clone 66 cells (Cl66-GFPneo) and injected into the mfp. GFP transduced tumor cells were identified in multiple tissues in addition to BM by flow cytometric analysis (FACS) but less 13% of the animals developed gross metastases. This model is a clinically relevant tool for the analysis of organ specificity of metastasis.