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

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.

Melatonin Treatment Combined with TGF-β Silencing Inhibits Epithelial- Mesenchymal Transition in CF41 Canine Mammary Cancer Cell Line

Background: Mammary cancer is the most prevalent type of cancer in female dogs. The main cause of mortality is the occurrence of metastasis. The metastatic process is complex and involves the Epithelial- Mesenchymal Transition (EMT), which can be activated by Transforming Growth Factor beta (TGF-β) and involves changes in cellular phenotype, as well as, in the expression of proteins such as E-cadherin, N-cadherin, vimentin and claudin-7. Melatonin is a hormone with oncostatic and anti-metastatic properties and appears to participate in the TGF-β pathway. Thus, the present work aimed to evaluate the expression of EMT markers, E-cadherin, N-cadherin, vimentin and claudin-7, as well as, the cell migration of the canine mammary cancer cell line, CF41, after treatment with melatonin and TGF-β silencing. Methods: Canine mammary cancer cell line, CF41, was cultured and characterized in relation to markers ER, PR and HER2. Cell line CF41 with reducing expression level of TGF-βwas performed according to Leonel et al. (2017). Expression of the protein E-caderin, N-cadherin, vimentin and claudin-7 was evaluated by immunocytochemistry and quantified by optical densitometry. The analysis of cell migration was performed in transwell chambers with 8μM pore size membrane. Results: CF41 cells present a triple negative phenotype, which is an aggressive phenotype. Immunocytochemistry staining showed increased expression of E-caderin and claudin-7 (P˂0.05) and decreased expression of N-cadherin and vimentin (P˂0.05) in CF41 cells after treatment with 1mM melatonin and TGF-β silencing. Moreover, treatment with melatonin and TGF-β silencing was able to reduce migration in cell line CF41 (P˂0.05). Conclusion: Our data suggests that therapies combining TGF- β1 silencing and melatonin may be effective in suppressing the process of EMT, corroborating the hypothesis that melatonin acts on the TGF-β1 pathway and can reduce the metastatic potential of CF41 cells. This is so far the first study that reports melatonin treatment in CF41 cells with TGF-β1 silencing and its effect on EMT. Thus, further studies are needed to confirm this hypothesis.

The EO771 mammary cancer cell line displays a luminal B phenotype and it is sensitive to anti-estrogen treatments

ABSTRACTDespite decades of therapeutic trials, detection, many drugs available and numerous studies on breast cancer, it remains the most deadly cancer in women. In order to choose the most appropriate treatment and to know the prognosis of the patients, the breast cancer is divided into different subtypes using a molecular classification. Thus, the need to discover new effective therapy is necessary, requiring to have models to test them. The EO771 (also named E0771 or EO 771) murine mammary cancer cell line is originally isolated from a spontaneous tumor in C57BL/6 mouse. Although frequently used, this cell line remains poorly characterized. Therefore, the EO771 phenotype was investigated. Transcriptomic and protein analysis allowed to classify the EO771 as luminal B subtype and more precisely estrogen receptor α negative, estrogen receptor β positive, progesterone receptor positive and ErbB2 positive. This phenotype was associated to a sensitivity to anti-estrogen treatments such as tamoxifen, 4-hydroxy-tamoxifen, endoxifen and fulvestrant.