Abstract: Background – Many cancers are diagnosed when advanced, highlighting the need for early detection. As part of developing a canine osteosarcoma liquid biopsy test, we examined physical parameters of extracellular vesicles (EVs) in canine cancers and benign musculoskeletal diseases (MD), important comparisons for osteosarcoma due to overlapping clinical signs. Hypothesis/Objectives – We aimed to characterize plasma EV size and concentration from dogs with cancer, dogs with MD, and healthy dogs. We hypothesized that physical EV parameters would be distinct between groups. Animals – Two sets of client-owned dogs were studied. Set-1: Healthy dogs (n=26), dogs with cancer (n=29; osteosarcoma n=8). Set-2: Healthy dogs (n=10), dogs with MD (n=50). Methods – Plasma EVs were enriched by precipitation (Set-1) or size exclusion chromatography (Set-2), and characterized by nanoparticle tracking analysis. Medians/IQRs are reported and Wilcoxon tests compared characteristics between groups. Results – Set-1: osteosarcoma dogs had a smaller median EV size than healthy dogs (osteosarcoma median/IQR: 78.7/24.1nm; healthy: 113.0/32.8nm; p=0.0065) and dogs with other cancers (median/IQR: 104.5/36.6nm, p=0.04). Set-2: the median EV size in dogs with MD was smaller than healthy dogs (MD median/IQR: 79.9/12.0nm; healthy: 96.4/18.6nm; p=0.00025). Median EV concentrations were higher in dogs with osteosarcoma and MD than in their respective healthy dog groups. Importantly, the size and concentration distributions overlapped between groups. Conclusions and Clinical Importance – Physical parameters of canine plasma EVs alone cannot differentiate between benign and malignant conditions. Molecular analysis of EV contents will robustly improve differentiation, creating opportunities to develop clinically useful diagnostic tools.
