|Canine mammary tumors (CMT) are one of the most common forms of cancer in intact female dogs. CMTs share clinical, epidemiologic, and genetic features with women with breast cancer (BC). BC and CMT cells have altered profiles of microRNA (miRNA), small 18-22 nucleotide non-coding RNA molecules that down-regulate gene translation. Human BC cells secrete exosomes that contain microRNA (miRNA) cargo that get into peripheral blood, which may facilitate tumor progression and cancer aggressiveness. These circulating exosomal miRNA can serve as minimally invasive biomarkers of disease and provide prognostic information. The objectives of this research were (1) to isolate and characterize CMT exosomes and their miRNA profiles in vitro; (2) profile circulating serum miRNA from clinical CMT patients and determine their diagnostic utility, and; (3) evaluate the association between differentially expressed circulating miRNA and histopathologic tumor characteristics and overall survival.
Cell-free conditioned media was harvested from normal and malignant canine mammary epithelial cells. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) identified that this media contained numerous irregularly round, 150-200 nm diameter, “cup-shaped” vesicles. Western blot for CD9 confirmed these vesicles were exosomes. Exosomal miRNA was submitted for RNA deep-sequencing (RNAseq) and found hundreds of differentially expressed miRNA between CMT and normal mammary cells. Among the significantly up-regulated miRNA were miR-18a, miR-19a,
and miR-181a, which were validated by qRT-PCR. In silico bioinformatic analysis found that the CMT exosomal miRNA profile was predicted to regulate a number of cell proliferation, apoptosis, and hormone receptor pathways, including the estrogen receptor (ESR1) and tumor suppressor PTEN.
Next, serum was collected from 10 dogs with histologically-confirmed malignant CMT and 10 healthy female control dogs (5 intact and 5 spayed). RNAseq revealed 65 differentially expressed circulating miRNA. A subset of seven miRNA were validated by digital droplet PCR (dPCR). Serum miR-19a and miR-125a showed good diagnostic performance in discriminating CMT patients from controls. This population of serum miRNA showed overlap with the in vitro exosomal miRNA, and were predicted to regulate a number of important tumor suppressors, pathways relevant to metastasis, and chemoresistance.
Finally, circulating miRNA were analyzed for their relationship to clinical and pathological characteristics. Serum miR-18a by RNAseq was significantly higher in patients with evidence lymphatic invasion (metastasis), and nearly significant for CMT patients with high-grade (Grade III) versus low-grade (Grade I/II) tumors. No miRNA correlated with survival times. In situ hybridization (ISH) revealed neoplastic epithelial cells expressed pre-miR-18a, pre-miR-19b, and pre-miR-34c, indicating those specific tumor cells represent one likely source of the circulating serum miRNA (as opposed to stromal cells, inflammatory cells, adnexa, or other tissues). This research is the first to characterize CMT exosomes and circulating miRNA, identified miR-19b and miR-18a as candidate diagnostic and prognostic biomarkers (respectively), and informs future research on serum miRNA for CMT.