Background Canine mammary tumors (CMTs) have a poor prognosis, along with tumor recurrence and metastasis. Cell lines are vital in vitro models for CMT research. Many CMT epithelial cell lines were reported. However, canine mammary myoepithelial cells, the contractile component of the canine mammary tissue were overlooked. This study aimed at establishing such a cell line. CMT-1 cell line was obtained from a canine mammary tumor CMT-1 and characterized molecularly through qPCR, western blotting, immunochemistry and immunofluorescence. Its doubling time, cytogenetic analysis and migration rate were evaluated using growth study, karyotype analysis and wound healing assay respectively. To determine its tumorigenesis, xenograft transplantation was performed. Results CMT-1 tumor was a complex canine mammary carcinoma that stained negative to estrogen receptors (ER) and progesterone receptors (PR), but positive to 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 immune-positive to vimentin, α-SMA, p63 and negative to E-cadherin (E-cad), indicating CMT-1 cells were myoepithelial cells. It was successfully cultured for more than 50 passages showing the same immunoreactivity to ER, PR, and HER2 as the primary canine tumor. The doubling time of CMT-1 cell line was 26.67 h. The chromosome number of CMT-1 cells ranged from 31 to 64. A potential spontaneous epithelial to mesenchymal transition (EMT) was noticed during cell cultures. Potential EMT-induced CMT-1 cells showed no significance in migration rate compared to the original CMT-1 cells. CMT-1 cells was able to grow on a 3D culture and formed grape-like, solid, and cystic mammospheres at different time period. Inoculation of CMT-1 cells induced a complex HER2-enriched mammary tumor with metastasis in mice. Conclusions A canine cancerous HER2-enriched myoepithelial cell line was successfully established and a canine mammosphere developed from myoepithelial cells was documented in this study. We are expecting this novel cell line and its associated mammospheres could be used as a model to elucidate the role of myoepithelial cells in CMT carcinogensis in the future.
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.
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