A Novel Canine Mammary Cancer Cell Line: Preliminary Identification and Utilization for Drug Screening Studies

Abstract

Canine malignant mammary tumor is a dangerously fatal neoplastic disease with poor survival in female dogs. The aim of this study was to preliminary characterize a novel canine mammary cancer cell line, B-CMT, from canine primary mammary gland tumor, and to utilize it as a cell model for in vitro screening of possible therapeutic drugs. The successfully established cell line, B-CMT, was cultured over 50 passages. B-CMT has a fast proliferation rate, and a population doubling time (PDT) of 33.6 h. The B-CMT cell line lacked human epidermal growth factor receptor-2 (HER-2), estrogen receptors (ER) and progesterone receptors (PR) expression by qRT-PCR. Compared with MDCK cells, CDH1 expression of CMT cell line was significantly decreased or even absent, but GATA3 expression dramatically increased, while TGF-β expression was at a similar level. Interestingly, the B-CMT cell line from canine primary tumor also showed positive hypoxia inducible factor-1α (HIF-1α) results in immunofluorescence (IF), western blot, and qRT-PCR analysis. Ten days post inoculation with EGFP-B-CMT (B-CMT cells stably expressing EGFP), the experimental mice developed palpable soft tissue masses which histologically resembled the canine primary tumor, and was approved to be derived from B-CMT cell line through detection of EGFP by immunohistochemical (IHC) analysis. Moreover, we investigated the cytotoxicity of five drugs to B-CMT cells, and the results showed that rapamycin and imatinib significantly inhibited the proliferation of the cells in vitro within a certain range of concentration. They also induced cell cycle arrest of B-CMT cells at G1 and G2 phase, respectively. In summary, the results of this report showed that B-CMT cell line might serve as a tool for future studies on tumor microenvironment and drug resistance.

Anticancer effects of alpelisib on PIK3CA-mutated canine mammary tumor cell lines

“…However, there is currently no canine cell line that specifically focuses on the novel breast cancer-associated mutational gene, PIK3CA. While other studies have developed various canine cell lines originating from mammary tumors ( 3 – 5 ), PIK3CA mutation in these established cell lines are yet to be characterized. Therefore, we established novel CMT cell lines that we named VCL.…”

“…Further in vitro research is necessary to develop alternative CMT treatment methods. Despite the increasing interest in CMTs, CMT-derived cell lines have only recently been developed for laboratory research (3)(4)(5)(6)(7), and are not as diverse as human cell lines. Many studies have analyzed the specific mutations in various genes, such as BRCA1, BRCA2, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), using human breast cancer cell lines (8)(9)(10)(11).…”

Large Animal Models of Breast Cancer

“…There are several other canine BC cell lines that were characterized and used in development for BC in nude mice (44)(45)(46)(47). A newly developed canine TNBC cell line, B-CMT, also showed BC in nude mice after 14 days of orthotopic inoculation (48). Canine normal mammary tissue and BC tissue were used to develop organoids, which showed multi lineage potential and existence of different population of self-renewing stem cells (24).…”

Establishment of canine mammary gland tumor cell lines harboring PI3K/Akt activation as a therapeutic target

“…The B-CMT does not express the human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR). In vitro experiments using cytotoxic drugs, such as imatinib (a tyrosine kinase inhibitor) and rapamycin (a mTOR inhibitor), significantly reduced the proliferation and induced cell cycle arrest at the G1 and G2 phases in B-CMT cell lines [ 6 ].…”