Canine Mammary Epithelial Cells

Malignant Canine Mammary Epithelial Cells Shed Exosomes Containing Differentially Expressed microRNA

Breast (mammary) cancers in human (BC) and canine (CMT) patients share clinical, pathological, and molecular similarities that suggest dogs may be a useful translational model. Many cancers, including BC, shed exosomes that contain microRNAs (miRs) into the microenvironment and circulation, and these may represent biomarkers of metastasis and tumor phenotype.

Fish et al. employed in vitro culture of normal and malignant canine mammary epithelial cells to isolate exosomes via ultracentrifugation. These exosomes were examined using electron microscopy, light scattering, and protein detection methods. The RNA bioanalyzer profiles showed a typical exosomal pattern, favoring small RNAs (~ 20–200 nucleotides) with minimal rRNA (Fig. 1a). A total of 338 unique miRs were identified in the cell-free RNA from CMEC and CMT samples. Principal component analysis divided the eight samples into two distinct CMEC and CMT clusters, although two CMT samples were outliers (Fig. 1b). Volcano plot analysis demonstrated that many miRs were significantly over- or under-expressed in CMT exosomes compared to CMEC (Fig. 1c). With criteria of p < 0.05 and a fold-change ≥ ±1.5, 170 miRs differed between CMT and CMEC. After excluding miR isoforms from varying chromosomal locations, 145 unique differentially expressed miRs were found, with 118 upregulated and 27 downregulated in CMT compared to CMEC.

Fig. 1. RNAseq profiling exosomal RNA (Fish E J, Irizarry K J, et al., 2018).

Targeting Canine Mammary Neoplastic Epithelial Cells with A Reengineered Anthrax Toxin

Mammary tumors, common in female dogs, resemble human breast cancer. da Fonseca et al. explored a reengineered anthrax toxin to treat these tumors without harming healthy cells. Given the role of metalloproteinases and urokinase in cancer metastasis, the toxin was modified to target these enzymes.

They employed five canine neoplastic epithelial cell lines and one normal cell line, treated with varying concentrations of the toxin. Cell cycle analysis, conducted via propidium iodide staining and flow cytometry, showed that the normal canine mammary epithelial cells (LOEC-NMG) and the LOEC-MCA1 mixed adenocarcinoma cell line had the lowest S phase proportions (5% and 5.12%). This suggests that less proliferative cells resist treatment better and are less affected by anthrax toxin. In their established cell lines, around 12% of adenoma (LOEC-MAd), complex adenocarcinoma (LOEC-MCA2), and simple adenocarcinoma (LOEC-MCA3) cells were in S phase. The adenocarcinoma cell line CF41.mg was the most proliferative, with 15.80% in S phase and 1.05 DNA index (Fig. 2). The G1 phase predominated across cell lines, with LOEC-MCA2 at 83.35% and LOEC-NMG at 89.4%. Cells had a DNA index between 1.00 and 1.05, denoting them as diploids, indicating low proliferation and favorable treatment response.

Fig. 2. Cell cycle analysis of canine mammary cell lines (da Fonseca I I M, Nagamine M K, et al., 2024).