A Peptide Heterodimer Tracer Targeting CXCR4 and Integrin α_vβ₃ for Pancreatic Cancer Imaging

Objectives: Nowadays, pancreatic cancer is a formidable disease to diagnose. The CXC chemokine receptor 4 (CXCR4) and integrin α_vβ₃ play an important role in tumor development, progression, invasion and metastasis, which are overexpressed in many types of human cancers. Studies have indicated that probes which can recognize multiple targets could achieve higher targeting efficiency than their corresponding monospecific counterparts. Therefore, in this study, we synthesized a heterodimeric tracer ⁶⁸Ga-yG5-RGD targeting both CXCR4 and integrin α_vβ₃ and evaluated its feasibility and utility in PET imaging of pancreatic cancer.

Methods: yG5 peptide, an analogy of peptide FC131, was first modified with bifunctional chelator NO2A_tBu-N₃ and then conjugated to BCN-PEG₄-c(RGDyK) via copper free click chemistry. The resulting precursor was identified and then radiolabeled with ⁶⁸Ga. The in vitro and in vivo stability was confirmed by radio-HPLC. The expression levels of CXCR4 and integrin α_vβ₃ of BXPC3 (human pancreatic cancer cell) and MX-1 (human breast cancer cell) cells and their corresponding tumor tissues were characterized by western blot and immunohistochemical analysis. The in vitro cell studies were performed. PET/CT scan and biodistrbution studies of ⁶⁸Ga-yG5-RGD, ⁶⁸Ga-yG5 and ⁶⁸Ga-RGD were performed using BXPC3 xenograft tumor mice. As negative control, the in vivo performance of ⁶⁸Ga-yG5-RGD was also evaluated in MX-1 tumor-bearing mice.

Results: yG5 and yG5-RGD were successfully synthesized and labeled with ⁶⁸Ga with high yield (> 99%) and purity (> 99%) at molar activity of 74~92 MBq/nmol (n = 6). Less than 1% disassociation was observed after incubation in PBS and fresh human serum at 37 ℃ for 2 h in vitro. ⁶⁸Ga-yG5-RGD also showed high in vivo metabolic stability. BXPC3 showed moderate expression level of CXCR4 as well as integrin α_vβ₃, while MX-1 low or not. The uptake of ⁶⁸Ga-yG5-RGD was higher than ⁶⁸Ga-yG5 (P < 0.001) and ⁶⁸Ga-RGD (P < 0.001) and could be blocked by excess amounts of AMD3100 (FDA-approved CXCR4 antagonist) or unlabeled RGD (P < 0.001). On small animal PET/CT images, ⁶⁸Ga-yG5-RGD was mainly excreted via liver and kidney. BXPC3 tumor was visualized more clearly post injection of ⁶⁸Ga-yG5-RGD than that of ⁶⁸Ga-yG5 and ⁶⁸Ga-RGD within observation time. What’s more, BXPC3 tumors were still clearly visible at 2 h post injection of ⁶⁸Ga-yG5-RGD. In addition, in vivo blocking studies showed that the tumor visualization could be blocked at 30 min post co-injection of ⁶⁸Ga-yG5-RGD and block agents. As control, no elevated uptake of ⁶⁸Ga-yG5-RGD was found in MX-1 tumors. The biodistribution results were consistent with imaging.

Conclusions: In this study, a peptide-based heterodimeric tracer ⁶⁸Ga-yG5-RGD was successfully developed with excellent in vitro and in vivo performance. The dual-receptor targeting strategy achieves improved tumor-targeting efficiency and prolonged tumor retention in BXPC3 tumor, making ⁶⁸Ga-yG5-RGD a promising tracer for noninvasive detection of tumors which express either CXCR4 or integrin α_vβ₃ or both, showing promising clinical translation prospects. Acknowledgements: This work was supported by the National Natural Science Foundation of China (No. 81801738, 81630049 and 81771863).

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