Malignant canine mammary epithelial cells shed exosomes containing differentially expressed microRNA that regulate oncogenic networks

Background: 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. Methods: Three normal canine mammary epithelial cell (CMEC) cultures and 5 CMT cell lines were grown in serum-free media. Exosomes were isolated from culture media by ultracentrifugation then profiled by transmission electron microscopy, dynamic light scattering, and Western blot. Exosomal small RNA was deep-sequenced on an Illumina HiSeq2500 sequencer and validated by qRT-PCR. In silico bioinformatic analysis was carried out to determine microRNA gene and pathway targets. Results: CMEC and CMT cell lines shed round, "cup-shaped" exosomes approximately 150-200 nm, and were immunopositive for exosomal marker CD9. Deep-sequencing averaged similar to 15 million reads/sample. Three hundred thirty-eight unique miRs were detected, with 145 having > +/- 1.5-fold difference between one or more CMT and CMEC samples. Gene ontology analysis revealed that the upregulated miRs in this exosomal population regulate a number of relevant oncogenic networks. Several miRNAs including miR-18a, miR-19a and miR-181a were predicted in silico to target the canine estrogen receptor (ESR1a). Conclusions: CMEC and CMT cells shed exosomes in vitro that contain differentially expressed miRs. CMT exosomal RNA expresses a limited number of miRs that are up-regulated relative to CMEC, and these are predicted to target biologically relevant hormone receptors and oncogenic pathways. These results may inform future studies of circulating exosomes and the utility of miRs as biomarkers of breast cancer in women and dogs.