Systematic analysis of different degrees of haemolysis on miRNA levels in serum and serum-derived extracellular vesicles from dogs.

BACKGROUND: Circulating microRNAs (miRNAs) are described as promising non-invasive biomarkers for diagnostics and therapeutics. Human studies have shown that haemolysis occurring during blood collection or due to improper sample processing/storage significantly alters the miRNA content in plasma and serum. Nevertheless, no similar research has been performed in dogs so far. We therefore investigated the effects of different degrees of haemolysis on the levels of selected miRNAs in serum and serum-derived extracellular vesicles (EVs) from dogs, by inducing a controlled in vitro haemolysis experiment. RESULTS: The abundance of miR-16, miR-92a, miR-191, miR-451 and miR-486 was significantly sensitive to haemolysis in serum and serum-derived EVs, while other selected miRNAs were not influenced by haemolysis. Furthermore, we found that the abundance of some canine miRNAs differs from data reported in the human system. CONCLUSIONS: Our results describe for the first time the impact of haemolysis on circulating miRNAs not only in whole serum, but also in serum-derived EVs from dogs. Hence, we provide novel data for further analyses in the discovery of canine circulating biomarkers. Our findings suggest that haemolysis should be carefully assessed to assure accuracy when investigating circulating miRNA in serum or plasma-based tests.

Deregulation of miR-27a may contribute to canine fibroblast activation after coculture with a mast cell tumour cell line.

The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of significant interest as these cells are reprogrammed during tumorigenesis to become cancer-associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2-derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha-smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR-27a and let-7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR-27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR-27a in canine fibroblasts and involved in their activation. Importantly, miR-27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin-6 (IL-6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR-27a may play an important role in the tumour-microenvironment crosstalk.

Exploration of serum- and cell culture-derived exosomes from dogs.

BACKGROUND: Exosomes are defined as extracellular membrane vesicles, 30-150 nm in diameter, derived from all types of cells. They originate via endocytosis and then they are released through exocytosis to the extracellular space, being found in various biological fluids as well as in cell culture medium. In the last few years, exosomes have gained considerable scientific interest due to their potential use as biomarkers, especially in the field of cancer research. This report describes a method to isolate, quantify and identify serum- and cell culture-derived exosomes from dog samples, using small volumes (100 µL and 1 mL, respectively). RESULTS: Quantification and sizing of exosomes contained in serum and cell culture samples were assessed by utilizing nanoparticle tracking analysis, transmission electron microscopy and immunoelectron microscopy. Detected particles showed the normal size (30-150 nm) and morphology described for exosomes, as well as presence of the transmembrane protein CD63 known as exosomal marker. CONCLUSIONS: Based on a validated rapid isolation procedure of nanoparticles from small volumes of different types of dog samples, a characterization and exploration of intact exosomes, as well as facilitation for their analysis in downstream applications was introduced.