Fibroblast Activation Protein Targeting Probe with Gly–Pro Sequence for PET of Glioblastoma

As an important cancer-associated fibroblast-specific biomarker, fibroblast activation protein (FAP) has become an attractive target for tumor diagnosis and treatment. However, most FAP-based radiotracers showed inadequate uptake and short retention in tumors. In this study, we designed and synthesized a novel FAP ligand (DOTA-GPFAPI-04) through assembling three functional moieties: a quinoline-based FAP inhibitor for specifically targeting FAP, a FAP substrate Gly–Pro as a linker for increasing the FAP protein interaction, and a 2,2′,2″,2?-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) chelator for radiolabeling with different radionuclides. The FAP targeting ability of DOTA-GPFAPI-04 was investigated by molecular docking studies. DOTA-GPFAPI-04 was then radiolabeled with ⁶⁸Ga to give [⁶⁸Ga]Ga-DOTA-GPFAPI-04 for positron emission tomography (PET) imaging of glioblastoma. [⁶⁸Ga]Ga-DOTA-GPFAPI-04 exhibited a purity of >98% and high stability analyzed by radio-HPLC in saline and mouse serum. Cell uptake studies demonstrated the targeting specificity of the probe. Further in vivo pharmacokinetic studies in normal mice demonstrated the quick clearance of the probe. Moreover, compared with the widely studied [⁶⁸Ga]Ga-FAPI-04, [⁶⁸Ga]Ga-DOTA-GPFAPI-04 showed much higher U87MG tumor uptake values (4.467 ± 0.379 for [⁶⁸Ga]Ga-DOTA-GPFAPI-04 and 1.267 ± 0.208% ID/g for [⁶⁸Ga]Ga-FAPI-04 at 0.5 h post-injection, respectively). The area under the curve based on time–activity curve (TAC) analysis for tumor radioactivity in small animal models was 422.5 for [⁶⁸Ga]Ga-DOTA-GPFAPI-04 and 98.14 for [⁶⁸Ga]Ga-FAPI-04, respectively, demonstrating that the former had longer tumor retention time. The tumor-to-muscle (T/M) ratio for [⁶⁸Ga]Ga-DOTA-GPFAPI-04 reached 9.15 in a U87MG xenograft animal model. PET imaging and blocking assays showed that [⁶⁸Ga]Ga-DOTA-GPFAPI-04 had specific tumor uptake. In summary, this study demonstrates the successful synthesis and evaluation of a novel FAPI targeting probe, [⁶⁸Ga]Ga-DOTA-GPFAPI-04, with a Gly–Pro sequence. It shows favorable in vivo glioblastoma imaging properties and relatively long tumor retention, highlighting DOTA-GPFAPI-04 as a promising molecular scaffold for developing FAP targeting tumor theranostic agents.