Preliminary Observations of a Substrate-Based Radiotracer Biosensor for <i>In Vivo</i> Positron Emission Tomography Imaging of Tumor Transmembrane Protease ST14

Imaging protease proteolysis with positron emission tomography (PET) has not been well documented in the literature, primarily due to the absence of suitable radiotracers. This study aims to develop a substrate-based radiotracer biosensor for ST14 protease to facilitate direct <i>in vivo</i> PET imaging of proteolysis. The design of the substrate-based radiotracer RQARK-DOTA-⁶⁸Ga is characterized by the inclusion of an ST14 substrate RQAR moiety and a Lys-DOTA-⁶⁸Ga moiety, linked via an ST14 cleavage site. The enzymatic cleavage of this radiotracer by ST14 protease was characterized <i>in vitro</i>, and the proteolysis of ST14 was further confirmed through <i>in vivo</i> PET imaging in tumors expressing ST14. RQARK-DOTA-⁶⁸Ga was specifically cleaved by ST14 protease to yield Lys-DOTA-⁶⁸Ga and RQAR moieties, whereas the d-isomer, rqark-DOTA-68Ga, was not susceptible to cleavage by ST14 protease. <i>In vivo</i> PET imaging demonstrated high tumor uptake of radioactive signal postinjection RQARK-DOTA-⁶⁸Ga in ST14-expressing AsPC-1 xenografts, with optimal accumulation observed 1 h postinjection. In contrast, the d-isomer radiotracer, rqark-DOTA-⁶⁸Ga, exhibited negligible tumor uptake, indicating a distinct preference for the substrate-based radiotracer in regions of ST14-mediated proteolysis. Radio-HPLC analysis following extraction from AsPC-1 tumors injected with RQARK-DOTA-⁶⁸Ga identified a radioactive peak corresponding to Lys-DOTA-⁶⁸Ga, confirming enzymatic cleavage and the generation of the anticipated radioactive product in the tumor tissue. Preliminary results indicate that a novel strategy for noninvasive in vivo positron emission tomography imaging of the transmembrane protease ST14 in tumors has been introduced through the development and application of a substrate-based radiotracer biosensor. The radiotracer RQARK-DOTA-⁶⁸Ga, capable of producing imaging signals through structural changes triggered by substrate cleavage, has proven its ST14-targeting potency in both in vitro enzymatic assays and in vivo PET imaging.