B2R-Targeting Radiotracer for PET/MR Imaging of Hepatocellular Carcinoma and Guiding Anti-B2R Therapy

Abstract

The bradykinin B2 receptor (B2R) is overexpressed in a wide variety of tumors and is a well-defined target for tumor imaging and therapy. The hybrid positron emission tomography/magnetic resonance imaging (PET/MRI) scanner is considered a noninvasive and advanced instrument for precise tumor imaging. In this work, we developed a novel B2R-targeting radiotracer, ⁶⁸Ga-DOTA-icatibant, for quantifying B2R expression. ⁶⁸Ga-DOTA-icatibant showed high stability, fast clearance and specific binding to B2R. PET/MR imaging revealed excellent tumor accumulation, and the uptake in tumors could be blocked by DOTA-icatibant. Icatibant-mediated anti-B2R therapy downregulated B2R expression in tumor cells and inhibited the growth of HepG2 tumors, and the decrease in tumor uptake was monitored by timely PET/MR imaging. Hematoxylin and eosin (H&E) and immunohistochemical staining results further demonstrated that the efficacy of anti-B2R could be accurately monitored with the developed PET/MR imaging radiotracer. ⁶⁸Ga-DOTA-icatibant can be utilized to noninvasively determine B2R expression and dynamically and sensitively monitor the efficacy of anti-B2R therapy.

Figure 1

(a) Schematic diagram of ⁶⁸Ga-DOTA-icatibant synthesis and PET/MRI scan. (b) Experimental protocol for monitoring B2R expression in the tumor microenvironment with ⁶⁸Ga-DOTA-icatibant.

Figure 2

(a) Radio-TLC chromatogram of ⁶⁸Ga-DOTA-icatibant. (b) Radio-HPLC chromatograms of ⁶⁸GaCl₃, ⁶⁸Ga-DOTA, and ⁶⁸Ga-DOTA-icatibant. Stability of ⁶⁸Ga-DOTA-icatibant in (c) PBS, (d) FBS, (e) mouse plasma, and (f) human serum.

Figure 3

(a) CCK-8 assay of HepG2 cells treated with 6.25 μM, 12.5 μM, 25 μM, 50 μM, or 100 μM DOTA-icatibant. (b) Cell uptake curve of ⁶⁸Ga-DOTA-icatibant in HepG2 and HEK-293T cells. (c) Cell uptake and block study of ⁶⁸Ga-DOTA-icatibant on HepG2 cells. (d) Cell binding assay of ⁶⁸Ga-DOTA-icatibant to B2R in HepG2 cells. Each result is presented as the mean ± standard deviation (SD); n = 3 independent measurements; ***P < 0.001.

Figure 4

(a) Pharmacokinetic study of ⁶⁸Ga-DOTA-icatibant in HepG2 tumor-bearing mice (n = 5 for each group). Blood samples at different time points (2, 5, 10, 15, 30, 45, 60, 90, and 120 min) were collected, weighed, and counted using a γ-counter. (b) Biodistribution study of ⁶⁸Ga-DOTA-icatibant in HepG2 tumor-bearing mice model at 15, 30, and 60 min postinjection (n = 3 for each group). (c) T/M, (d) T/L, and (e) T/H ratios in part b. Each result is presented as mean ± SD; ***P < 0.001.

Figure 5

(a) Representative whole-body coronal PET/MR slice images of HepG2 tumor-bearing mice at different time points after injection of 3.7 MBq ⁶⁸Ga-DOTA-icatibant or 3.7 MBq ⁶⁸Ga-DOTA-icatibant + Block via the tail vein (n = 3 for each group). White circles indicate tumors. (b) Uptake in the tumor, muscle, liver, and blood in ⁶⁸Ga-DOTA-icatibant and ⁶⁸Ga-DOTA-icatibant + Block groups at different time points. Data were derived from PET quantification. (c) T/M, (d) T/L, and (e) T/B ratios in part b. Each result is presented as mean ± SD; **P < 0.01; ***P < 0.001.

Figure 6

(a) Representative whole-body coronal PET/MR slice images of HepG2 tumor-bearing mice at 30 min postinjection of 3.7 MBq ⁶⁸Ga-DOTA-icatibant before and after treatment with PBS (group A: 50 μL of PBS via subcutaneous injection daily, n = 6) or icatibant (group B: 50 μL of icatibant (2 mg/kg) via subcutaneous injection daily, n = 6). White circles indicate tumors. (b) Tumor uptake, (c) T/M and (d) T/L ratios in HepG2 tumor-bearing mice before and after treatment with PBS or icatibant. (e) T/M and T/L ratios 14 days after treatment with PBS or icatibant. (f) Changes in tumor volume and (g) mouse body weight after different treatments. Each result is presented as mean ± SD; “NS” indicates no statistically significant difference between groups. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

(a) H&E and immunohistochemical staining of HepG2 tumor sections treated with PBS or icatibant (scale bar, 50 μm). (b) Expression levels of B2R in groups A and B 14 days after treatment. (c) Correlation analysis between the expression levels of B2R, tumor uptake, and T/M and T/L ratios. Each result is presented as mean ± SD; ***P < 0.001.