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

As an important cancer-associated fibroblast-specificbiomarker,fibroblast activation protein (FAP) has become an attractive targetfor tumor diagnosis and treatment. However, most FAP-based radiotracersshowed inadequate uptake and short retention in tumors. In this study,we designed and synthesized a novel FAP ligand (DOTA-GPFAPI-04) throughassembling three functional moieties: a quinoline-based FAP inhibitorfor specifically targeting FAP, a FAP substrate Gly-Pro asa linker for increasing the FAP protein interaction, and a 2,2 & PRIME;,2 & DPRIME;,2"'-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraaceticacid (DOTA) chelator for radiolabeling with different radionuclides.The FAP targeting ability of DOTA-GPFAPI-04 was investigated by moleculardocking studies. DOTA-GPFAPI-04 was then radiolabeled with Ga-68 to give [Ga-68]Ga-DOTA-GPFAPI-04 for positron emissiontomography (PET) imaging of glioblastoma. [Ga-68]Ga-DOTA-GPFAPI-04exhibited a purity of >98% and high stability analyzed by radio-HPLCin saline and mouse serum. Cell uptake studies demonstrated the targetingspecificity of the probe. Further in vivo pharmacokineticstudies in normal mice demonstrated the quick clearance of the probe.Moreover, compared with the widely studied [Ga-68]Ga-FAPI-04,[Ga-68]Ga-DOTA-GPFAPI-04 showed much higher U87MG tumoruptake values (4.467 & PLUSMN; 0.379 for [Ga-68]Ga-DOTA-GPFAPI-04and 1.267 & PLUSMN; 0.208% ID/g for [Ga-68]Ga-FAPI-04 at 0.5h post-injection, respectively). The area under the curve based ontime-activity curve (TAC) analysis for tumor radioactivityin small animal models was 422.5 for [Ga-68]Ga-DOTA-GPFAPI-04and 98.14 for [Ga-68]Ga-FAPI-04, respectively, demonstratingthat the former had longer tumor retention time. The tumor-to-muscle(T/M) ratio for [Ga-68]Ga-DOTA-GPFAPI-04 reached 9.15 ina U87MG xenograft animal model. PET imaging and blocking assays showedthat [Ga-68]Ga-DOTA-GPFAPI-04 had specific tumor uptake.In summary, this study demonstrates the successful synthesis and evaluationof a novel FAPI targeting probe, [Ga-68]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 fordeveloping FAP targeting tumor theranostic agents.