In 2013, the parental MMTV-PyMT cells (PyMT-B6) (Kindly provided by Dr. DeNardo, Washington University in St. Louis) were isolated from a fully invasive mammary tumor that spontaneous arose at day 120 in a C57bL/6 background MMTV-PyMT mouse, a mouse model that represents an anti-estrogen sensitive, luminal B breast cancer. The tumor was collagenase treated, grown in single-cell suspension on a collagen-coated plate, and cloned to establish the parent PyMT-B6 cell line. Parent PyMT-B6 cells were injected into the mammary fat pad (MFP) tissue of a female C57BL/6J mouse, and after reaching a tumor size approaching 1cm, tumor cells were collagenase treated and cultured in a cell culture dish. The cultured tumor cells were intracardially injected into a 6-week-old female C57BL/6J mouse to establish bone metastases. 12 days post-intracardiac inoculation, the bone tumor was harvested and cultured in a cell culture dish with DMEM media plus 10% FBS to establish the PyMT-BO1 subline, which when compared to the parent PyMT-B6 cells, had a higher incidence of inducing bone metastases after either orthotopic MFP or intracardiac injection. The PyMT-BO1 cells were infected with lentivirus containing the GFP-firefly luciferase genes as described previously (28 (link)). GFP-expressing PyMT-BO1 cells were FACS sorted, cultured, and validated for luciferase expression; this cell line was named PyMT-BO1-GFP-Luc. PyMT-B6, PyMT-BO1, and PyMT-BO1-GFP-Luc cells were evaluated by qPCR, and all express the PyMT, Esr1, Esr2, and Itgb3 genes. These cell lines were tested as CD45 negative and pan-Keratin positive by FACS in 2013 and 2015.
The B16-F10 C57BL/6 murine melanoma cell line (ATCC) was modified to express firefly luciferase (B16F10-Luc) as described (29 (link)). In 2015, this cell line was tested as CD45 negative and integrin beta3 positive by FACS.