[⁶⁸Ga]Ga-labeled FAPI Conjugated with Gly-Pro Sequence for PET Imaging of Malignant Tumors

To improve tumor uptake and prolong tumor retention, a novel fibroblast activation protein (FAP) ligand based on a quinoline-based FAP inhibitor (FAPI) conjugated with the Gly-Pro sequence and 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) was radiolabeled with [⁶⁸Ga]GaCl₃ ([⁶⁸Ga]Ga-DOTA-GPFAPI-04). Due to the tumor heterogeneity, this study aimed to further validate the preclinical value of [⁶⁸Ga]Ga-DOTA-GPFAPI-04 PET imaging in tumor mice models with different FAP expression levels.

[⁶⁸Ga]Ga-DOTA-GPFAPI-04 was synthesized and its partition coefficient was measured. The stability of [⁶⁸Ga]Ga-DOTA-GPFAPI-04 was tested in phosphate-buffered saline (PBS, pH 7.4) and fetal bovine serum (FBS). Small animal PET and semi-quantitative studies were conducted in Panc-1 and A549 xenograft tumor mice models compared with [⁶⁸Ga]Ga-DOTA-FAPI-04. Immunofluorescent and immunohistochemical staining and western blot assay were performed to confirm FAP expression in xenograft tumors.

[⁶⁸Ga]Ga-DOTA-GPFAPI-04 exhibited a radiochemical purity of > 99% and high stability in PBS and FBS. [⁶⁸Ga]Ga-DOTA-GPFAPI-04 had higher hydrophilic property than [⁶⁸Ga]Ga-DOTA-FAPI-04 (-4.09 ± 0.05 vs -3.45 ± 0.05). Small animal PET and semi-quantitative analysis revealed Panc-1 xenograft tumor displayed higher tumor uptake of [⁶⁸Ga]Ga-DOTA-GPFAPI-04 and tumor-to-background ratios compared to A549 xenograft tumor, consistent with the results of immunofluorescence, immunohistochemistry, and western blot. Moreover, [⁶⁸Ga]Ga-DOTA-GPFAPI-04 demonstrated higher tumor accumulation and longer tumor retention than [⁶⁸Ga]Ga-DOTA-FAPI-04 in both Panc-1 and A549 xenograft tumors. Furthermore, the FAP-binding specificity of [⁶⁸Ga]Ga-DOTA-GPFAPI-04 was confirmed in vivo by co-injection of unlabeled GPFAPI-04.

[⁶⁸Ga]Ga-DOTA-GPFAPI-04 showed more favorable in vivo tumor imaging and longer tumor retention compared to [⁶⁸Ga]Ga-DOTA-FAPI-04, which has high potential to be a promising PET probe for detecting FAP-positive tumors.