SPECT Imaging with Tc-99m-labeled HYNIC-FAPI-04 to Extend the Differential Time Window in Evaluating Tumorous Fibrosis

Introduction: Fibroblast activating protein (FAP) is highly expressed in a variety of malignant tumors and is an important receptor in designing molecular agents that served for tumor diagnosis and treatment in the field of nuclear medicine. However, the so-far used Ga-68- or F-18-labelled tracers were of a relative short time window in differentiating tumor fibrosis.

Methods: In this study, we synthesized a SPECT-adapted imaging probe, ^99mTc-HYNIC-FAPI-04, and evaluated it in animal models of FAP-positive glioma and FAP-negative hepatoma. Using SnCl₂ as reducing agent, ^99mTc was radiolabeled to small molecules HYNIC-FAPI-04 under the coordination of Tricine/EDDA. To investigate the specificity of ^99mTc-HYNIC-FAPI-04 to FAP, in vitro cellular uptake of ^99mTc-HYNIC-FAPI-04 was studied using U87 cells with high FAP expression, and competitive binding to FAP was studied using unlabeled DOTA-FAPI-04 as a blocker. ^99mTc-HYNIC-FAPI-04 SPECT/CT imaging was performed in severely immunodeficient ICR mice with U87 MG or HUH-7 xenografts, and then compared with ¹⁸F-FDG or ⁶⁸Ga-FAPI-04 PET/CT.

Results: The radio-labeling rate of ^99mTc-HYNIC-FAPI-04 was greater than 90%, and the radiochemical purity was > 99% after purification with sep-pak C18 column. The tracer was of good stability in PBS and 5% FBS. In vitro cell uptake experiments of ^99mTc-HYNIC-FAPI-04 showed good FAP binding specificity, and the significantly decreased cellular uptake was observed when blocked by DOTA-FAPI-04, reflecting the similar targeted uptake mechanism of HYNIC-FAPI-04 and DOTA-FAPI-04. As shown in Figure 1, SPECT/CT imaging showed that U87 MG tumor was clearly visible and of a high uptake of ^99mTc-HYNIC-FAPI-04 (2.26 ± 0.40 %ID/mL at 1.5 h post injection), while tumor signal of FAP-negative HUH-7 was very low (0.29 ± 0.08 %ID/mL). At 5 h post injection, U87 MG tumor was still distinguishable (1.04 ± 0.20 % ID/mL). Additionally, ^99mTc-HYNIC-FAPI-04 was mainly excreted by kidney and liver. In comparison with ¹⁸F-FDG imaging, U87 MG and HUH-7 tumors showed no statistical or visual difference in radioactive uptake. Although ⁶⁸Ga-FAPI-04 imaging results of U87 MG tumor showed that the tumor was of obvious uptake and was clearly visible at 1 h post injection, the tumorous radioactive signals were fuzzy.

Conclusions: A new ^99mTc-labeled HYNIC-FAPI-04 probe was synthesized. The probe specifically bound to FAP-positive tumors and qualified with the ability of evaluating tumor status in a longer time windows.

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