Isolation and characterization of a canine mammary cell line prepared for proteomics analysis

Mammary cancer is the most common tumor in female dogs. Canine mammary tumor serves as an excellent model for human breast cancer biology. Cancer cell lines are routinely used as the source of protein for proteomics studies because antigen homogeneity is essential for protein profiling of tumors. In this study, we sought to isolate and characterize a canine mammary cell line that was subject to protein profiling analysis through 2-dimensional electrophoresis (2-DE) method. Mammary tumor was collected from a 6-year-old terrier dog. Tumor fragments were treated with collagenase, and dissociated cells were cultured. The cell line was subcultured over 50 times. Characterization profile included population doubling time, colony forming assay, spheroid formation/migration potency, immunocytochemistry for steroid receptors and intermediate filaments, karyotyping, RT-PCR for cytokeratins 8, 14, and 18, and 2-DE pattern. The cell line revealed three growth phases including normal, dormant, and immortal phase. Immunocytochemistry showed that the cell line was positive for estrogen receptor, pancytokeratin, cytokeratin-low and vimentin, and negative for progesterone receptor, cytokeratin-high. RT-PCR supported the immunocytochemistry results. 2-DE pattern and proteome analysis of the cell line revealed that protein composition was stable, indicating the cell line as an appropriate source of protein for canine mammary proteomics studies.