Translational development of osteosarcoma (OS) extracellular vesicle (EV) matrix metalloproteinase (MMP) Activity Assay for assessing patient treatment responses

PROJECT SUMMARY Proteases, particularly matrix metalloproteinases (MMPs), are pivotal in the malignant progression of solid tumors, mediating invasion, migration, and metastasis via remodeling of extracellular matrix (ECM). Recent studies have shifted focus from the abundance of MMPs to their functional roles, especially in profiling proteolytic activities. However, detecting MMP activities within tumor tissue pose challenges, necessitating complicated procedures involving invasive acquisition of tumor tissues and labor-intensive purification of MMPs. This is especially more challenging for bone cancer tissues, as bone tissue biopsies typically undergo decalcification, which can severely damage the proteins in the tissue. Tumor extracellular vesicles (EVs) transport MMPs from tumor cells while preserving their functional activities, making them ideal surrogates for the parent tumor tissue for noninvasive profiling of MMP activities. Osteosarcoma (OS), the most common pediatric bone cancer is in pressing need of a quantitative liquid biopsy assay for assessing treatment responses for both localized OS and metastatic OS. Therefore, assessing OS EV MMP activities holds significant promise to serve as a liquid biopsy assay that can supplement radiographic imaging for assessment of treatment responses. Over the past decade, our UCLA team has pioneered click chemistry-mediated tumor EV enrichment technologies, i.e., Click Beads and Click Chips, enabling diverse downstream molecular analyses, such as mRNA profiling and protein quantification. Recently, we demonstrated the feasibility of coupling functional analysis, i.e., MMP activity assay using the enriched tumor EVs, offering a strong potential to deepen our understanding of MMPs’ pathological roles and to develop new cancer diagnostic solutions. The long-term goal of this TTNCI R01 proposal is to further refine, validate, and translate a streamlined OS EV MMP Activity Assay for monitoring treatment responses in OS patients who receive treatment interventions, e.g., a combination of neoadjuvant chemotherapy, surgery, and adjuvant chemotherapy. This two-step assay involves: i) click chemistry-mediated enrichment of subpopulations of OS EVs using EV Click MagBeads, in the presence of trans-cyclooctene-grafted antibodies targeting the respective OS EV surface markers, and ii) FRET peptide probes for assessing the activities of OS- associated MMPs in the enriched OS EVs. The innovation of this proposal lies in i) innovative use of nanotechnology-enabled OS EV MMP Activity Assay to address an unmet clinical need in noninvasive assessment of treatment responses in an aggressive pediatric tumor, and ii) a rigorously developed design of experiments (DOE) plan to identify optimal parameters for the assay. The successful development of the proposed OS EV MMP Activity Assay is expected to achieve translational readiness for eventual GLP manufacturing and a Phase-2 biomarker study.