A Tissue Proteolysis Activity Mapping and Substrate Discovery Platform for Identifying Novel Tumor-Activated Biosensors

Dysregulated extracellular proteolytic activity is a prominent hallmark of cancer and can thus be exploited for tumor detection and therapeutic development. However, the discovery of tumor-responsive probes has been hindered by the lack of methods capable of capturing proteolytic events directly in tissue samples. Here, we report PSurf, a platform that enables the identification of tissue-specific protease sensors with tissue specimens. Through differential selection of tumor-specific sequences over healthy tissue, PSurf identified context-specific tumor-activated probes that precisely distinguish metastatic lesions in lung tissue slices. Using these substrates, we engineered nanobody-targeted biosensors that release urinary reporters upon tumor-specific cleavage in vivo , enabling precise noninvasive tumor detection in a murine lung metastasis model. PSurf provides a foundation for developing conditionally activated agents through tissue-specific activity mapping and probe discovery.