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. 2021 Jan 1;12(1):244-252.
doi: 10.7150/jca.49628. eCollection 2021.

Synthesis and evaluation of 68Ga-labeled dimeric cNGR peptide for PET imaging of CD13 expression with ovarian cancer xenograft

Yi Yang  1   2 , Jun Zhang  3 , Huifeng Zou  2 , Yang Shen  2 , Shengming Deng  1 , Yiwei Wu  1
Affiliations

Synthesis and evaluation of 68Ga-labeled dimeric cNGR peptide for PET imaging of CD13 expression with ovarian cancer xenograft

Yi Yang et al. J Cancer. .

Abstract

Introduction: Previous studies have shown that peptides containing the asparagine-glycine-arginine (NGR) sequence can specifically bind to CD13 (aminopeptidase N) receptor, a tumor neovascular biomarker that is over-expressed on the surface of angiogenic blood vessels and various tumor cells, and it plays an important role in angiogenesis and tumor progression. In the present study, we aimed to evaluate the efficacy of a gallium-68 (68Ga)-labeled dimeric cyclic NGR (cNGR) peptide as a new molecular probe that binds to CD13 in vitro and in vivo. Materials and Methods: A dimeric cNGR peptide conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) was synthesized and labeled with 68Ga. In vitro uptake and binding analyses of the 68Ga- DOTA-c(NGR)2 were performed in two ovarian tumor cell lines, ES2 and SKOV3, which had different CD13 expression patterns. An in vivo biodistribution study was performed in normal mice, and micro positron emission tomography (PET) imaging was conducted in nude mice bearing ES2 and SKOV3 tumors. Results: 68Ga-DOTA-c(NGR)2 was prepared with high radiochemical purity (>95%), and it was stable both in saline at room temperature and in bovine serum at 37°C for 3 h. In vitro studies showed that the uptake of 68Ga-DOTA-c(NGR)2 in ES2 cells was higher compared with SKOV3 cells, and such uptake could be blocked by the cold DOTA-c(NGR)2. Biodistribution studies demonstrated that 68Ga-DOTA-c(NGR)2 was rapidly cleared from blood and mainly excreted from the kidney. MicroPET imaging of ES2 tumor xenografts showed the focal uptake of 68Ga-DOTA-c(NGR)2 in tumors from 1 to 1.5 h post-injection. The high-contrast tumor visualization occurred at 1 h, corresponding to the highest tumor/background ratio of 10.30±0.26. The CD13-specific tumor targeting of the 68Ga-DOTA-c(NGR)2 was further supported by the reduced uptake of the probe in ES2 tumors by co-injection of the unlabeled cold peptide. In SKOV3 tumor models, the tumor was not obviously visible under the same imaging conditions. Conclusions: 68Ga-DOTA-c(NGR)2 was easily synthesized, and it showed favorable CD13-specific targeting ability by in vitro data and microPET imaging with ovarian cancer xenografts. Collectively, 68Ga-DOTA-c(NGR)2 might be a potential PET imaging probe for non-invasive evaluation of the CD13 receptor expression in tumors.

Keywords: 68Ga labeling; CD13; NGR peptide; Tumor angiogenesis; microPET imaging.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Structural formula of DOTA-c(NGR)2; chemical formula: C99H159N25O41S4; molecular weight: 2,483.74.
Figure 2
Figure 2
Radio-HPLC of 68Ga-DOTA-c(NGR)2 (retention time was 5.23 min).
Figure 3
Figure 3
In vitro stability of 68Ga-DOTA-c(NGR)2 in saline at room temperature and in bovine serum at 37 °C for 30 min, 1 h, 2 h and 3 h.
Figure 4
Figure 4
FACS of CD13 expression on ES2 (A) and SKOV3 (B) cells.
Figure 5
Figure 5
Immunohistochemical staining. (A) and (B) CD13 expression in SKOV3 tumor tissue sections; (C) and (D) CD13 expression in ES2 tumor cells (black arrows) and tumor vascular endothelia (white arrows). (A and C magnification × 200, B and D magnification ×400).
Figure 6
Figure 6
Uptake of 68Ga-DOTA-c(NGR)2 in ES2 and SKOV3 cells.
Figure 7
Figure 7
Competitive binding assay. The uptake in ES2 cells could be competitively inhibited by unlabeled DOTA-c(NGR)2 in a dose-dependent manner, and the IC50 value was 160.1 nM.
Figure 8
Figure 8
Biodistribution of 68Ga-DOTA-c(NGR)2 in normal mice (n=6).
Figure 9
Figure 9
Representative micro-PET images of 68Ga-DOTA-c(NGR)2 in xenograft models. Dotted circles indicate the tumor regions.
See this image and copyright information in PMC

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