Click chemistry-mediated enrichment of circulating tumor cells and tumor-derived extracellular vesicles for dual liquid biopsy in differentiated thyroid cancer

Circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tEVs) are two crucial methodologies of liquid biopsy. Given their distinct size differences and release dynamics, CTCs and tEVs potentially offer synergistic capabilities in the non-invasive detection of differentiated thyroid cancer (DTC), a typically indolent tumor. We present the Combined DTC CTC/tEV Assay, integrating dual liquid biopsy processes: i) DTC CTC enrichment by Click Chips, followed by analysis of seven DTC-specific genes, and ii) DTC tEV enrichment by Click Beads, succeeded by mRNA cargo quantification in DTC tEVs. This method utilizes click chemistry, leveraging a pair of biorthogonal , highly reactive functional motifs (tetrazine, Tz , trans-cyclooctene, TCO), to over- come the challenges encountered in the conventional immunoaffinity-based enrichment of CTCs and t EVs. The Combined DTC CTC/tEV t EV Assay synergistically combines the diagnostic precision of CTCs with the sensitivity of t EVs, demonstrating superior diagnostic accuracy in DTC detection and boasting an AUROC of 0.99. This out- performs the individual diagnostic performance of using either DTC CTC or DTC t EV alone. This integration enables full utilization of a patient's blood sample, and marks a significant evolution in the development of nanomaterial-based liquid biopsy technologies to address challenging unmet clinical needs in cancer care.