Noninvasive Assessment of Protease Activity in Osteosarcoma via Click Chemistry‐Mediated Enrichment of Extracellular Vesicles

Abstract Osteosarcoma (OS), the most common bone cancer in children, is characterized by aggressive tumors and subclinical metastasis. Metastasis significantly impacts OS patient survival rate, highlighting the need for frequent assessment of disease progression and treatment response. The study introduces OS extracellular vesicles (EV) matrix metalloproteinase (MMP) Activity Assay, noninvasively analyzing six combinations of OS EV surface markers and OS‐associated MMPs to generate a unique OS EV MMP profile for each patient. An OS EV MMP Activity Score is established from logistic regression of three top‐performing combinations to specifically distinguish metastatic OS from localized OS, achieving an area under the receiver operating characteristic (AUROC) curve of 0.97. The Scores from longitudinal monitoring of six OS patients strongly correlate with disease progression and treatment response, as confirmed by radiographic imaging. The OS EV MMP Activity Assay enables noninvasive and timely monitoring of disease progression and treatment response during the critical disease window of progression to metastatic OS, enhancing clinical management for OS patients.