Activity-Based Nanoprobes for Multiscale Functional Analysis of Proteases in Medulloblastomas

Enzymes mediate diverse cancer processes by exerting their functional activities. Precise profiling of enzyme activity in the tumor microenvironment (TME) is therefore critical to understanding and targeting the pathological roles of enzymes in cancer. Here, we report biomodified nanoprobes for the highly sensitive detection of specific protease activities in medulloblastoma (MB) across scales. The surface-enhanced Raman scattering (SERS)-based nanoprobes use peptide-functionalized three-dimensional (3D) surfaces rich in electromagnetic hotspots to facilitate both enzymatic hydrolysis and plasmonic enhancement of Raman signals. We apply the nanoprobes to analyze multiple protease activities in 2D in vitro culture and 3D tumor cell spheroids, uncovering heterogeneous activity maps among different cell types, therefore allowing for recognition of distinct MB cellular subtypes. Through spatially resolved in situ localization of protease activity, we observe the suppressed protease function in the core region of tumor spheroids. Furthermore, in a pilot clinical assay (<i>n</i> = 27), the nanoprobe-based SERS assay reveals elevated levels of protease activity in sera of MB patients, and achieves great accuracy in discriminating MB from noncancer controls with the area under the receiver operating characteristic curve of 1.0. Together, this study offers a framework for functional, multiscale measurement of protease dysregulation in cancer.