Murine Mammary Tumor Cell Lines for Cancer Research

The EO771 cell line was derived from a spontaneous mammary tumour in a C57BL/6 mouse (Casey et al., 1951 (link)) and was stored in liquid nitrogen vapour phase. Early-passage parental EO771 cells were transduced with the pMSCV (murine stem cell virus) retroviral vector expressing the mCherry fluorescent protein (Denoyer et al., 2011 (link)). The lungs from a mouse orthotopically implanted with EO771_mCherry cells in our laboratory were excised and sorted by flow cytometry for mCherry-positive cells that were expanded in culture. This sequence of orthotopic growth in vivo followed by recovery of mCherry-positive cells from the lung was repeated. Upon the second round of mammary fat-pad injections of these mCherry-positive cells, visible lung nodules were detected. One of these nodules was designated Lung Metastasis nodule B and, after being returned to culture, became the EO771.LMB cell line. 67NR and 4T1 cell lines were derived from a subpopulation of a single mammary tumour that arose in a BALB/c/C3H mouse (Aslakson and Miller, 1992 (link)), with the 4T1.2 cell line being derived from a single-cell clone of the 4T1 population (Lelekakis et al., 1999 (link)). EMT6.5 (Ellis et al., 2000 (link)) is a single-cell clone derived from the EMT6 mammary tumour (Rockwell and Kallman, 1973 (link)). NMuMG immortal murine mammary epithelial cells were obtained from ATCC. The Pik3ca-mutant murine mammary tumour line MH248 was a kind gift from Dr Wayne Phillips (Tikoo et al., 2012 (link)). The murine mammary tumour line AT3, derived from polyoma-middle T antigen transgenic mice (Stewart and Abrams, 2007 (link)), was a kind gift from Dr Trina Stewart (Griffith University, Queensland, Australia). 67NR and 4T1.2 mammary adenocarcinoma cells were maintained in Eagle’s minimum essential medium (alpha modification) supplemented with 5% (v/v) fetal bovine serum (FBS) (SAFC Biosciences, Brooklyn, Victoria, Australia) and 1% (v/v) penicillin-streptomycin, whereas EO771, EO771.LMB, EMT6.5, AT3, MH248 and NMuMG cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing HEPES (20 mM) supplemented with 10% (v/v) FBS, penicillin (100 IU/ml) and streptomycin (100 μg/ml). All cells were cultured at 37°C in 5% CO₂ (v/v) in air and were maintained in culture for a maximum of 4-5 weeks.