Proteomic Profiles of Circulating Exosomes as Biomarkers for Disease Detection in Canine Osteosarcoma

Graduation date: 2018Osteosarcoma (OSA) is the most common primary skeletal tumor in dogs accounting for 85% of bone tumors and nearly 6% of all canine neoplasms. They are highly aggressive tumors that carry a poor prognosis despite intensive treatment, highlighting the need for more effective diagnostic and therapeutic tools. Recent research in human medicine has underlined the importance of vesicular transport via exosomes, particularly related to their role in the progression of cancer and drug resistance. Exosomes are small vesicles with a phospholipid bilayer membrane that are secreted by many cells in the body, and have been shown to transfer oncogenic proteins and nucleic acids that play influential roles in tumorigenesis, metastasis, and response to treatment. More recently, exosome-associated proteins in biofluids have been studied for their potential as minimally invasive biomarkers for a variety of human neoplasms, including OSA. The objectives of our study were twofold. First, to determine if serum-derived exosomes contain a unique protein signature that can be ascribed to canine OSA patients, which could serve as a biomarker for disease and predict response to therapy. Second, we sought to evaluate if exosomes are capable of transferring chemotherapeutic resistance to susceptible osteoblasts, with the hypothesis that acquisition of resistance will correspond with a distinct proteomic profile.\ud \ud Exosomes that were isolated from sera of canine patients were measured using light scatter technology and determined to be of appropriate size. Proteomic analysis via mass spectrometry exhibited unique protein profiles for dogs with OSA, traumatic bone fracture, and normal animals. Additionally, 10 proteins were identified that could differentiate OSA from control patients (normal or traumatic fracture) with 85% accuracy. Select discriminating proteins were validated with western blot. Furthermore, unique exosomal protein signatures were evident for dogs with OSA at different disease stages (diagnosis, 2-weeks post-amputation, and detection of metastatic disease), with 2 proteins identified that could distinguish between the disease stages with 75% accuracy.\ud \ud A carboplatin-resistant cell line (HMPOS-R) was created by repetitive incubations of the cell line with increased concentrations of carboplatin and expansion of the survived cells. A viability assay demonstrated that HMPOS-R had considerably improved viability compared with its naïve equivalent (HMPOS-S). Exosomes isolated from HMPOS-R were incubated with HMPOS-S and subsequent viability assay showed increased viability of resistant exosome-treated cells (HMPOS-EX), suggesting exosomal transfer of resistance. Proteins contained in exosomal preparations had a distinctive profile in comparison to cell lysates. Additionally, proteomic profiling of exosomes and cell lysates at various stages in acquiring resistance demonstrated a unique change in protein expression that may contribute to chemotherapeutic resistance. \ud \ud In summary, our findings demonstrate the potential for circulating exosomes as powerful tools for discovery of novel biomarkers and potential therapeutic targets as a unique protein signature is ascribed to exosomal cargo from serum of dogs with OSA compared with control animals, and throughout various stages of disease. Our research also demonstrates the capability of exosomes to confer chemotherapeutic resistance on susceptible cells, similar to what has been demonstrated in many human malignancies. Further investigation into the biomarker potential and functional significances of exosomal cargo in veterinary medicine is warranted