Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role in cell-to-cell communication. They exert pleiotropic biological functions via the horizontal transfer of bioactive molecules (DNA, RNAs, proteins, and lipids) within the tumour microenvironment and throughout the body. In human cancer, EVs are known to interfere with pathways that lead to tumour progression and are used as novel cancer biomarkers. In veterinary medicine, very little is known on cancer-derived EVs. In this study, we preliminarily characterized EVs in mammary gland cancer of dogs and cats. EVs were isolated by ultracentrifugation from canine (CYPp), feline (FMCp) and human (MCF7) mammary tumour cell lines. EVs were visualized by transmission electron microscopy (TEM), counted using nanoparticle tracking analysis (NTA) and characterized by immunogold (CD63 and Alix) and western blot (Alix and TSG101). Additionally, EV production by "donor" cells (palmtdTomato ) and uptake by "recipient" cells (GFP ) were assessed. EVs were successfully isolated from all 3 cell lines by ultracentrifugation. Membrane-bound structures (50-400 nm) were identified by TEM and were positive for both CD63 and Alix at immunogold. Western blot showed positivity of EVs to Alix and TSG101. NTA analysis detected EVs from cell culture media ranging from 1.67 to 2.56 × 10 as number of EVs/cell and from 80 to 600 nm in size. Confocal microscopy identified the presence of palmtdTomato EVs into the cytoplasm of GFP cells. This preliminary study identified and characterized canine and feline mammary tumour cell-derived EVs, opening in veterinary medicine a new interesting unexplored field with several applications and limitless potential.

Feline mammary tumors are usually malignant and aggressive carcinomas. Most cases are simple monophasic carcinomas (1 epithelial population), and additional phenotyping is usually not needed. In this study, we describe 10 malignant mammary tumors from 9 female cats that had unusual histomorphology: they appeared biphasic, with 2 distinct cell populations. Initially, they were morphologically diagnosed as either carcinosarcoma (1/10) or malignant pleomorphic tumor (9/10) of the mammary gland, as the latter did not match any previously described histological subtype. Immunohistochemistry (IHC) was performed for pancytokeratin, cytokeratins 8 and 18, cytokeratin 14, cytokeratins 5 and 6, vimentin, p63, calponin, alpha-smooth muscle actin, Ki-67, ERBB2, estrogen receptor alpha, and progesterone receptor. In 7 of 10 cases, the biphasic nature was confirmed and, on the basis of the IHC results, they were classified as carcinoma and malignant myoepithelioma (4/10), ductal carcinoma (1/10), and carcinosarcoma (2/10). The other 3 of 10 cases were monophasic based on IHC. In the cases of carcinoma and malignant myoepithelioma, the malignant myoepithelial cells were 100% positive for vimentin (4/4) and variably positive for p63, calponin, and cytokeratins (4/4). These findings show that, although rare, biphasic mammary carcinomas do occur in cats. In dogs and humans, tumors composed of malignant epithelial and myoepithelial cells have a less aggressive behavior than certain simple carcinomas, and therefore, their identification might also be clinically significant in the cat.

Age-dependent differences in methylation at specific cytosine-guanosine sites (CpGs) have been used in “epigenetic clock” formulas to predict age. Deviations of epigenetic age from chronological age are informative of health status and are associated with adverse health outcomes, including mortality. In most cases, epigenetic clocks are performed on methylation from DNA extracted from circulating blood cells. However, the effect of neoplastic cells in the circulation on estimation and interpretation of epigenetic clocks is not well understood. Here, we explored this using Fischer 344 (F344) rats, a strain that often develops large granular lymphocyte leukemia (LGL). We found clear histological markers of LGL pathology in the spleens and livers of 27 out of 61 rats aged 17-27 months. We assessed DNA methylation by reduced representation bisulfite sequencing with coverage of 3 million cytosine residues. Although LGL broadly increased DNA methylation variability, it did not change epigenetic aging. Despite this, inclusion of rats with LGL in clock training sets significantly altered predictor selection probability at 83 of 121 commonly utilized CpGs. Furthermore, models trained on rat samples that included individuals with LGL had greater absolute age error than those trained exclusively on LGL-free rats (39% increase; p<0.0001). We conclude that the epigenetic signals for aging and LGL are distinct, such that LGL assessment is not necessary for valid measures of epigenetic age in F344 rats. The precision and architecture of constructed epigenetic clock formulas, however, can be influenced by the presence of neoplastic hematopoietic cells in training set populations.