Establishment and characterization of primary canine mammary epithelial cells as a normal-like epithelial reference model.

The lack of well-characterized normal canine mammary epithelial cell models limits mechanistic studies of mammary tumorigenesis and drug response. Here, we established CM-VZ, a primary canine mammary epithelial cell population derived from histologically normal-appearing epithelium sampled away from gross hyperplastic lesions in a dog with mammary hyperplasia. CM-VZ showed stable growth for 10-15 passages with predominantly glandular epithelial-like morphology and an estimated doubling time of 49.56 h. Conventional karyotyping indicated predominantly diploid chromosome counts (mode 78). Immunophenotyping demonstrated strong expression of epithelial markers (E-cadherin and CK8), low-level expression of ER, PR and Ki-67, and absence of HER-2, p63, and α-SMA. DAPI staining did not reveal obvious extranuclear DNA signals suggestive of mycoplasma contamination. In vivo tumorigenicity assays in nude mice showed no tumor formation up to three months post-inoculation, and histology of major organs revealed no abnormalities. Multiplex qPCR confirmed canine origin and excluded cross-species contamination. A 24-h CCK-8 assay showed dose-dependent reductions in metabolic viability, providing a baseline short-term tolerance profile under the tested conditions. CM-VZ represents a characterized, normal-like canine mammary epithelial model with finite lifespan. It provides a practical normal-like control for comparative studies of mammary tumorigenesis and drug response when healthy canine mammary tissue is unavailable. This model may facilitate comparisons with tumor-derived canine mammary cell lines in mechanistic and pharmacological research.