Establishment and molecular characterization of novel luminal A and luminal B canine mammary cancer cell lines for comparative oncology.

Abstract

Background and aim: Canine mammary cancer (CMC) is the most frequently diagnosed malignancy in female dogs, sharing significant pathological and molecular similarities with human breast cancer (HBC). Despite the availability of various CMC cell lines, most represent triple-negative orepidermal growth factor receptor 2 (ErbB2)-enriched subtypes, which limit research on hormone receptor-positive cancers. This study aimed to establish and characterize novel CMC cell lines representing luminal A and B subtypes.

Materials and methods: Between 2020 and 2021, 31 canine mammary tumors (CMTs) were collected from clinical cases. Tumor tissues were processed for primary culture, and two cell lines - CMGT_071020 and CMGT_180321 - were successfully established. Immunohistochemistry (IHC) was used to assess expression of estrogen receptor alpha (ERα), progesterone receptor (PR), ErbB2, Ki-67, vimentin, and multi-cytokeratin. Functional assays (wound-healing and transwell migration) assessed metastatic behavior. Gene expression (EGFR, TP53, Bcl-2, PTEN, SNAIL, N-cadherin, and E-cadherin) was analyzed using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Cell line authentication was confirmed through short tandem repeat (STR) profiling and mycoplasma testing.

Results: The CMGT_071020 (luminal B) and CMGT_180321 (luminal A) cell lines were derived from malignant epithelial tumors and maintained stable growth over 30 passages. IHC confirmed molecular subtype classifications. CMGT_071020 exhibited a fibroblast-like morphology, a high Ki-67 index (67%), and superior migratory capacity compared to CMGT_180321 and the commercial ErbB2-enriched REM134 cell line. E-cadherin expression was significantly elevated in CMGT_071020 (p < 0.05), whereas the expression levels of other genes were comparable. STR analysis verified their genetic uniqueness, and both lines were free from mycoplasma contamination.

Conclusion: This study successfully established and characterized two novel hormone receptor-positive CMC cell lines, representing luminal A and luminal B subtypes. The CMGT_071020 line exhibited higher metastatic potential, offering a promising model for aggressive hormone-responsive CMC. These cell lines provide valuable tools for comparative oncology and may facilitate subtype-specific therapeutic research.