Synthesis and Evaluation of [64Cu]Cu-NOTA-HFn for PET Imaging of Transferrin Receptor 1 Expression in Nasopharyngeal Carcinoma

Abstract

As recurrent and metastatic nasopharyngeal carcinoma (NPC) is the most common cause of death among patients with NPC, there is an urgent clinical need for the development of precision diagnosis to guide personalized treatment. Recent emerging evidence substantiates the increased expression of transferrin receptor 1 (also known as cluster of differentiation 71, CD71) within tumor tissues and the inherent targeting capability of natural heavy-chain ferritin (HFn) toward CD71. This study aimed to synthesize and assess a radiotracer ([⁶⁴Cu]Cu-NOTA-HFn) designed to target CD71 for positron emission tomography (PET) imaging in an NPC tumor-bearing mouse model. The entire radiolabeling process of [⁶⁴Cu]Cu-NOTA-HFn was completed within 15 min with high yield (>98.5%) and high molar activity (72.96 ± 21.33 GBq/μmol). The in vitro solubility and stability experiments indicated that [⁶⁴Cu]Cu-NOTA-HFn had a high water solubility (log P = -2.42 ± 0.52, n = 6) and good stability in phosphate-buffered saline (PBS) for up to 48 h. The cell saturation binding assay indicated that [⁶⁴Cu]Cu-NOTA-HFn had a nanomolar affinity (K_d = 10.9 ± 6.1 nM) for CD71-overexpressing C666-1 cells. To test the target engagement in vivo, prolonged-time PET imaging was performed at 1, 6, 12, 24, and 36 h postinjection (p.i.) of [⁶⁴Cu]Cu-NOTA-HFn to C666-1 NPC tumor-bearing mice. The C666-1 tumors could be visualized by [⁶⁴Cu]Cu-NOTA-HFn and blocked by nonradiolabeled HFn. PET imaging quantitative analysis demonstrated that the uptake of [⁶⁴Cu]Cu-NOTA-HFn in C666-1 tumors peaked at 6 h p.i. and the best radioactive tumor-to-muscle ratio was 10.53 ± 3.11 (n = 3). Ex vivo biodistribution assay at 6 h p.i. showed that the tumor uptakes were 1.43 ± 0.23%ID/g in the nonblock group and 0.92 ± 0.2%ID/g in the block group (n = 3, p < 0.05). Immunohistochemistry and immunofluorescence staining confirmed positive expression of CD71 and the uptake of HFn in C666-1 tumor tissues. In conclusion, our experiments demonstrated that [⁶⁴Cu]Cu-NOTA-HFn possesses a very high target engagement for CD71-positive NPC tumors and provided a fundamental basis for further clinical translation.

Figure 1

CD71 expression in NPC cell lines and cell uptake of HFn-FITC. (a,b) Western blotting of CD71 expressions in C666-1 and HK-1 (a), and quantification of CD71 expressions presented as the ratio of CD71 vs GAPDH (b). (c) Representative fluorescence images of C666-1 incubated with IgG, anti-CD71 + HFn-FITC, respectively, with a scale bar of 40 μm. The colocalization coefficient in the shown image is 0.77. (d) Flow cytometry analysis of the cellular uptake of IgG, anti-CD71, HFn-FITC, and anti-CD71 + HFn-FITC, respectively. The data of (b) are expressed as mean ± SD and the Mann–Whitney test was used to calculate the p value. “ns” not significant.

Figure 2

Synthesis of [⁶⁴Cu]Cu-NOTA-HFn and cell saturation binding of [⁶⁴Cu]Cu-NOTA-HFn. (a) Labeling scheme of [⁶⁴Cu]Cu-NOTA-HFn. (b) Radio-TLC of copper-64 and [⁶⁴Cu]Cu-NOTA-HFn. (c) In vitro stability of [⁶⁴Cu]Cu-NOTA-HFn in PBS within 48 h. (d) [⁶⁴Cu]Cu-NOTA-HFn specific binding to C666-1 cells, which could be blocked by excess nonradiolabeled HFn. (e) One-site saturation binding curve and Scatchard plot showed the binding affinity of [⁶⁴Cu]Cu-NOTA-HFn to C666-1 cells (K_d = 10.9 ± 6.1 nM, n = 3).

Figure 3

Tumor-bearing mice PET/CT imaging, ex vivo imaging, and biodistribution. (a) Flowchart for PET/CT imaging of [⁶⁴Cu]Cu-NOTA-HFn in C666-1 xenograft models. (b) Representative whole-body Maximum Intensity Projection (MIP) PET/CT images of [⁶⁴Cu]Cu-NOTA-HFn in C666-1 tumor-bearing mice at 1, 6, 12, 24, and 36 h p.i. The upper row is the nonblock group, and the bottom row is the block group. The dotted circles and white arrows refer to the tumor. (c,e) Quantitative analysis of PET/CT imaging of [⁶⁴Cu]Cu-NOTA-HFn (n = 4). (c) Time–activity curve of tumor uptake from 1 to 36 h. (d) Tumor-to-muscle (T/M) ratios in mice postinjection. (e) Tumor-to-blood (T/B) ratios in mice postinjection. (f) Ex vivo photograph of interesting organs, the black arrow refers to the tumor. Photograph courtesy of Yanfang Shen. Copyright 2024. (g) PET imaging of ex vivo organs, the white arrow refers to the tumor. (h) Biodistribution of the [⁶⁴Cu]Cu-NOTA-HFn after 6 h p.i. uptake (n = 3). The data are expressed as mean ± SD. Mann–Whitney test in (c–e) and unpaired, two-tailed Student’s t-test in (f) were used to calculate p values. *p < 0.05.

Figure 4

Histopathological confirmation of tumor tissues of C666-1 tumor-bearing mice and NPC patients: HE (a), IHC (b), and IF (c) staining of C666-1 cells of xenograft tumors; IF (d) staining of NPC tissues of patients. (a) HE staining of tumors and adjacent muscle tissues, with a scale bar of 2 mm for the overall image and 40 μm for the enlarged view. (b) IHC staining of tumors and adjacent muscle tissues for the expression of CD71, with a scale bar of 2 mm for the overall image and 40 μm for the enlarged view. “T” denotes tumor, and “M” represents muscle. (c) IF staining of mice tumor tissues with IgG, anti-CD71, and anti-CD71 + HFn-FITC, respectively. (d) IF staining of NPC tissues of patients with anti-CD71 + HFn-Cy5.5 (c,d) with a scale bar of 40 μm.