Dual-protease exosome sensing enables liquid-biopsy detection of chordoma

Chordoma is a rare and aggressive bone tumor arising from notochordal remnants. Its diagnosis is frequently delayed due to the absence of sensitive and specific circulating biomarkers, while current liquid biopsy modalities remain vastly underutilized in this pathology. Through EV proteomic profiling and perturbation analysis of the chordoma-defining factor TBXT (brachyury), we identified and biologically validated ADAM19 (a disintegrin and metalloproteinase 19) as a novel TBXT-regulated chordoma oncogene, which may function via the ADAM19/TNF-α/NF-κB axis. Paired with the validated pro-invasive marker MMP-2 (matrix metalloproteinase-2), we constructed an anti-interference dual-channel Au-Se fluorescent nanoprobe to achieve specific EV-associated ADAM19/MMP-2 activity readout. ADAM19 signals showed ~ 10.2-12.4-fold (in vitro cell supernatant) and ~ 9.7-10.6-fold (xenograft mouse serum) elevation vs. controls; MMP-2 signals reached ~ 8.5-10.5-fold and ~ 11.9-13.3-fold elevation respectively. Preliminary clinical plasma cohort data confirmed this dual-marker strategy separates chordoma patients from benign spinal disease cases and healthy controls, establishing preliminary proof-of-concept diagnostic performance. This exosome-based dual-channel fluorescent nanoprobe targeting ADAM19 and MMP-2 for chordoma demonstrates promising preliminary diagnostic discriminative potential across preliminary cohorts, and warrants further systematic clinical validation in larger independent patient cohorts.