Optimal Sarcoma Exosome Isolation Techniques from Cells and Blood

Grant Winners

Deans

Abstract

Sarcomas cause significant morbidity and mortality, particularly among children, with about 13,000 new cases diagnosed each year in the U.S. The nvariety of sarcoma subtypes affect tissue of mesenchymal origin, such as bone, cartilage and fibrous tissue. Surgery is the first type of treatment; however, it carries high recurrence rates. While one half of patients respond to currently available treatment options, the other half of patients are completely resistant to all forms of treatment, which results in a significant unmet need. Despite much advancement in molecular diagnostics, no significant improvement has been made which enables the identification or characterization of a sarcoma early in the process, before invasive methods are required. Understanding the defining molecular characteristics of these tumors would have a profound effect on subsequent treatment decisions, potentially enabling the development of affordable early-stage treatments. Thus, there is a critical need for a reliable and accurate, non-invasive detection method to identify sarcomas in the early stage when non-surgical treatment options may still have the potential for success, improving patient outcomes. In an effort to address this unmet need, researchers have most recently been evaluating biomarkers of disease activity using exosomes, which are small extracellular membrane vesicles that are released by all cells, including cancer cells. In particular, tumor-derived exosomes have proven to play a role in tumor progression and metastasis in a variety of cancers, including sarcomas. Current research efforts have demonstrated advances in understanding the role of exosomes in sarcoma, but study results are varied and comparison across studies have been limited by the discrepancies in approaches taken. Therefore, in this pilot study, our team of a renowned sarcoma surgeon, experts in sarcoma tumors, primary tumor culture and exosome isolation will compare five common exosome isolation methods (ultracentrifugation, magnetic bead-based separation, precipitation, sequential microfiltration and fast protein liquid chromatography (FPLC) in the first six months to achieve our goal of proving that FPLC with a size exclusion column (SEC) will isolate intact and functional exosomes from both tumor cultures and patient plasma within the year. The resulting isolates will be evaluated for cost of isolation, efficiency of isolation and usability of the isolate for important assays in biomarker discovery. By 1) delivering this comparison and 2) proving the efficacy of our approach, we aim to 3) provide the foundation for future research efforts supported by extramural funding, including comprehensive biomarker discovery and eventual development of targeted sarcoma therapies.