The importance of bone marrow micrometastasis to outcome of breast cancer has recently been confirmed {lcub}357,360{rcub}. The paucity of animal models for the study of marrow micrometastasis has limited the investigation of micrometastatic cells. Therefore, a murine model of marrow micrometastasis was developed and characterized. Following injection of Cl66neo tumor cells (Cl66 tumor cells transduced with the neomycin marker) into the mammary fatpad (mfp) and development of a primary tumor, the marrow was analyzed for the presence of Cl66neo cells. As few as 100 cells were shown to produce marrow micrometastases detectable by polymerase chain reaction (PCR). Injection of 1 × 105 Cl66neo cells produced marrow metastasis detectable after 1–2 weeks. To simulate micrometastatic breast cancer following removal of a primary tumor (clinical complete remission), Cl66neo cells were injected into the mfp of recipient mice to produce a primary tumor. Marrow was then harvested and transplanted into lethally irradiated recipients that subsequently received cytokine. PCR/ neo and immunocytochemical analysis for cytokeratin (CK) expression was performed on various tissues. Tumor cells were found in the marrow and spleen by immunocytochemistry, and in the blood, marrow and spleen by PCR. Although neo cells were detected in blood, suggesting mobilization of tumor cells from the marrow by cytokine, no gross metastases were observed. C166 cells were also transduced with a construct carrying green fluorescent protein (GFP). The GFP/neo marked CL66 cells were injected into the mfp and their emigration tracked. Many more tumor cells migrated to various organs than actually formed metastases. Finally, C166 cells were serially passaged through mice to obtain a highly marrow metastatic variant. Preliminary analysis of the marrow derived clone showed increased metastatic characteristics when compared to the parental cell line. In this model, migration of breast cancer cells was an early and seemingly frequent event. However, metastasis is an inherently inefficient process that is enhanced by selection of a cell population that has developed mechanisms which provides a survival advantage, a process enhanced by the marrow microenvironment.
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