Establishment and Partial Characterization of Canine Mammary Tumor Cell Lines

Abstract

Mammary tumors are the most common neoplasms diagnosed in female dogs and have been considered excellent models for studying human breast cancer. Establishing cell lines from primary cultures of canine mammary tumors provides an in vitro model to better understand the disease and develop new treatments. This study aimed to establish and characterize canine mammary tumor cell lines. Ten cell cultures were generated from tumor tissue obtained from affected dogs, including seven from primary mammary tumors and three from metastatic sites. Characterization included molecular marker expression (ER, PR, HER2, cytokeratin 5/6 (CK5/6), vimentin, and the marker of cell proliferation Ki67) and in vitro tumorigenic capacity assessment. Additionally, the susceptibility of five cell lines to DOX, 5-FU, paclitaxel, colchicine, and carboplatin was evaluated using the MTT assay. ICC analysis revealed negative expression of hormonal receptors (ER and PR) in five cell lines, while only one cell line was positive for both. Six cell lines were HER2-negative and positive for vimentin. Five cell lines exhibited in vitro tumorigenic capacity, forming colonies in soft agar. DOX showed the highest growth-inhibitory effect (DOX > Paclitaxel > Colchicine > 5-FU > Carboplatin). Two cell lines had a minimal concentration for 50% inhibition in vitro (IC₅₀) < 0.63 µM and 4.37 ± 0.40 µM for DOX, while one was sensitive to colchicine and paclitaxel (IC₅₀ 0.19 µM and 0.04 µM, respectively). All tested cell lines were resistant to carboplatin and 5-FU. These cell lines provide a valuable model for studying breast cancer in humans and dogs and evaluating new potential therapeutic strategies.

Keywords: breast cancer; cancer; canine mammary tumors; cell culture; cell lines.

Figure 1

Histopathological classification of primary canine mammary tumors. (A–H) Tumor paraffin sections, stained with hematoxylin and eosin. (Scale bar = 50 μm).

Figure 2

Workflow of cell line generation from different biological samples of canines with mammary tumors. (A) Obtention of biological sample, (B) mechanical dissociation and/or enzymatic digestion, (C) generation of primary cell culture at 48 h, visualizing different cell morphology, small pieces of tissue known as organoids, (D) followed by subcultures for 2–3 months until the generation of cell line.

Figure 3

In vitro tumorigenic capacity of the cell lines CMTF4, CMTG5, CMTN7, CMTP8, and CMTL9. (400×). Soft agar colony formation assay was used to evaluate the capability of the cell lines to grow and form colonies in suspension. MCF7 cell line was used as a positive control for colony formation. Images were obtained on day 30.

Figure 4

Immunocytochemistry results and morphological analysis of canine mammary cancer cell lines. In the figure, rows represent the cell markers used, and columns correspond to the analyzed cell lines (A1–F6). Some images include insets in the upper right corner: red-framed insets show close-ups of positive cells, while green-framed insets highlight cellular morphology. Isotype and positive controls: ER—MCF7, PR—T47D, HER2—SKBR-3, Ki67—MDA-MB-231, CK5/6—skin tissue, vimentin—appendix tissue. ND: Not determined.