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Breast cancer metastasis to brain in a new in vivo chick embryo model system

Breast cancer metastases to brain are often rapidly lethal. This project has established the chick embryo as a new model for studying extravasation of breast cancer to brain. Human MDA‐MB‐231 and MDA‐MB‐435 breast cancer cell lines were infected with retroviral vectors expressing the lacZ marker gene and/or the green fluorescent protein (GFP) gene for identification of cells in vivo and in vitro. Approximately 50,000 cells were injected into the extraembryonic vasculature of 5 day embryos, followed by dissection of the brain, fixation and beta‐galactosidase staining, and preparation of routine H&E paraffin sections less than 2 weeks later. Cryosections were also prepared and subsequently immunostained for marker genes. Numerous lacZ(+) tumor cells were present on the outside of the brain after 10 days in the embryo, and cells were clearly located in brain tissue outside blood vessels, demonstrating their extravasation. GFP expressing tumor cells were recovered from dissociated brains and subjected to quantitation by flow cytometry and clonal formation analyses after puromycin drug selection. They were detectable by flow cytometry, but drug selection and clonal analysis was a more sensitive and accurate method of quantitation of tumor cell extravasation into brain. These experiments have validated the use of this new model for the study of breast cancer metastasis to brain, and now will allow molecular mechanisms of metastasis to be investigated. Furthermore, lacZ and GFP both were useful markers for localization and quantitation of tumor cells.