Background:

Canine mammary tumors (CMTs) have a poor prognosis, along with tumor recurrence and metastasis. Cell lines and organoids are vital in vitro models for CMT research. Although anti-HER2 therapy improves the patients’ outcome, anti-HER2 drug resistance is still a big challenge and yet no canine HER2-enriched cell line has been established. This study aimed at establishing such a novel canine mammary cell line. CMT-1 cell line was obtained from clinical tumor CMT-1 and characterized molecularly through qPCR, western blotting, immunochemistry, immunofluorescence. Its doubling time, migration rate and stemness were evaluated using growth study, wound healing assay and 3D cultures respectively. To determine its tumorigenesis, xenograft transplantation was performed. Results CMT-1 is a mixed canine mammary carcinoma that stains negative for estrogen receptors (ER) and progesterone receptors (PR), but positive for human epidermal growth receptor-2 (HER2), defined as HER2-enriched subtype. In this study, a CMT-1 cell line obtained from CMT-1 tumor was confirmed as myoepithelial cells and was successfully cultured for more than 50 passages showing the same immunoreactivity to ER, PR, and HER2. The doubling time of CMT-1 cell line was 26.67 h. A spontaneous epithelial to mesenchymal transition (EMT) was noticed during cell cultures. EMT-induced CMT-1 cells showed a faster migration rate compared to the original CMT-1 cells, but no significance was detected. CMT-1 cells grew on a 3D culture and formed grape-like, solid, and cystic mammosphere at different stages, indicating CMT-1 cell line has stem cell property. Inoculation of CMT-1 cells induced a mixed HER2-enriched mammary tumor with metastasis in mice. Conclusions A canine cancerous HER2-enriched myoepithelial cell line was successfully established and the first canine mammosphere was documented in this study. We are expecting this novel cell line and mammosphere could be used as a model for CMT tumorigenesis and new anti-HER2 drug screening in the future.