Multimodal activity-affinity assay of ADAM-10 extracellular vesicles in untreated plasma reveals metastatic stage of colorectal cancer

Metalloproteinases (MPs) such as a-disintegrin and metalloproteinase-10 (ADAM-10) are key drivers of extracellular matrix remodeling during tumor progression, yet MP-based liquid biopsy tests have not reached clinical utility. Here, we show that active ADAM-10 is selectively enriched on the surface of circulating extracellular vesicles (EVs) in the plasma of colorectal cancer patients. Our findings further suggest ADAM-10+ EVs are locally enriched in dense pre-metastatic tumor extracellular matrices and subsequently accumulate in blood post-metastasis. To capture these unique signatures of disease progression, an ADAM-10 activity assay is integrated with a novel size-sensitive Immuno-Janus Particle affinity assay for characterizing ADAM-10+ EVs in untreated plasma. In a 43-patient colorectal cancer cohort, this multimodal platform distinguishes healthy, pre-metastatic, and metastatic states with 95% overall accuracy. When combined with lipidomics as a third modality, the platform correctly determines 97.4% cancer stage accuracy, with only one misclassification. This study establishes a multimodal EV-based activity/affinity assay as a robust framework for liquid biopsy, providing accurate cancer staging, improved prognostics, and offering a potential platform for pan-disease diagnostics.