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Preclinical evaluation of 68 Ga-labeled peptide CK2 for PET imaging of NRP-1 expression in vivo

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European Journal of Nuclear Medicine and Molecular Imaging Aims and scope Submit manuscript

Abstract

Purpose

Neuropilin-1 (NRP-1) is a multifunctional protein involved in a variety of biological processes such as angiogenesis, tumorigenesis and immunomodulation. It was usually overexpressed in many cancer cell lines and correlated with poor prognosis of breast cancer. Positron emission tomography (PET) is an advanced imaging technique for detecting the function and metabolism of tumor-associated molecules in real time, dynamically, quantitatively and noninvasively. To improve the level of early diagnosis and evaluate the prognosis of breast cancer, an NRP-1 targeting peptide-based tracer [68 Ga]Ga-NOTA-PEG4-CK2 was designed to sensitively and specifically detect the NRP-1 expression in vivo via PET imaging.

Methods

In silico modeling and microscale thermophoresis (MST) assay were carried out to design the NRP-1 targeting peptide NOTA-PEG4-CK2, and it was further radiolabeled with 68 Ga to prepare the tracer [68 Ga]Ga-NOTA-PEG4-CK2. The radiochemical yield (RCY), radiochemical purity (RCP), molar activity (Am), lipid-water partition coefficient (Log P) and stability of [68 Ga]Ga-NOTA-PEG4-CK2 were assessed. The targeting specificity of the tracer for NRP-1 was investigated by in vitro cellular uptake assay and in vivo PET imaging as well as blocking studies. The sensitivity of the tracer in monitoring the dynamic changes of NRP-1 expression induced by chemical drug was also investigated in vitro and in vivo. Ex vivo biodistribution, autoradiography, western blot, and immunofluorescence staining were also performed to study the specificity of [68 Ga]Ga-NOTA-PEG4-CK2 for NRP-1.

Results

[68 Ga]Ga-NOTA-PEG4-CK2 was designed and synthesized with high RCY (> 98%), high stability (RCP > 95%) and high affinity to NRP-1 (KD = 25.39 ± 1.65 nM). In vitro cellular uptake assay showed that the tracer [68 Ga]Ga-NOTA-PEG4-CK2 can specifically bind to NRP-1 positive cancer cells MDA-MB-231 (1.04 ± 0.04% at 2 h) rather than NRP-1 negative cancer cells NCI-H1299 (0.43 ± 0.05%). In vivo PET imaging showed the maximum tumor uptake of [68 Ga]Ga-NOTA-PEG4-CK2 in MDA-MB-231 xenografts (4.16 ± 0.67%ID/mL) was significantly higher than that in NCI-H1299 xenografts (1.03 ± 0.19%ID/mL) at 10 min post injection, and the former exhibited higher tumor-to-muscle uptake ratio (5.22 ± 0.18) than the latter (1.07 ± 0.27) at 60 min post injection. MDA-MB-231 xenografts pretreated with nonradioactive precursor NOTA-PEG4-CK2 showed little tumor uptake of [68 Ga]Ga-NOTA-PEG4-CK2 (1.67 ± 0.38%ID/mL at 10 min post injection). Both cellular uptake assay and PET imaging revealed that NRP-1 expression in breast cancer MDA-MB-231 could be effectively suppressed by SB-203580 treatment and can be sensitively detected by [68 Ga]Ga-NOTA-PEG4-CK2. Ex vivo analysis also proved the high specificity and sensitivity of [68 Ga]Ga-NOTA-PEG4-CK2 for NRP-1 expression in MDA-MB-231 xenografts.

Conclusion

A promising NRP-1 targeting PET tracer [68 Ga]Ga-NOTA-PEG4-CK2 was successfully prepared. It showed remarkable specificity and sensitivity in monitoring the dynamic changes of NRP-1 expression. Hence, it could provide valuable information for early diagnosis of NRP-1 relevant cancers and evaluating the prognosis of cancer patients.

Graphical Abstract

A novel promising NRP-1 targeting PET tracer [68 Ga]Ga-NOTA-PEG4-CK2 was developed based on a series of in vitro and in vivo investigations. The tracer showed remarkable specificity and sensitivity in detecting the expression of NRP-1. It could be applied for noninvasively and dynamically monitoring the NRP-1 expression in tumors and predicting the prognosis of breast cancer.

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Data availability

Data generated in the present study are available from the corresponding authors upon reasonable request.

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Funding

This work was supported by grants from the National Natural Science Foundation of China (22076069, 82202907, U23A20478 and 62171167), Science and Technology Program of Guangzhou (202102020138), Guangdong Basic and Applied Basic Research Foundation (2020A1515011374), Scientific Research Project of Jiangsu Commission of Health (ZD2022036 and M2020028), Development Project of Wuxi (Y20212013) and Wuxi Municipal Health Commission (Q202347). We also acknowledge the financial support received from Jiangsu Provincial Medical Key Laboratory (Key Laboratory of Nuclear Medicine).

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Authors and Affiliations

  1. NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, 214063, China

    Qingzhu Liu, Shuyue Cai, Quan Xie, Ling Qiu & Jianguo Lin

  2. Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China

    Jiacong Ye

  3. Department of Ultrasound, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China

    Rongbin Liu

  4. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China

    Rongbin Liu

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  1. Qingzhu Liu
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Contributions

J. Lin, L. Qiu and R. Liu. concepted and designed the project researches; Q. Liu and S. Cai performed theoretical modeling, radiolabeling, cell culture, animal model construction, PET scanning, pharmacokinetics and biodistribution study as well as corresponding data analysis; Q. Liu and Q. Xie performed the western blot, immunofluorescence staining and cellular uptake assay; J. Ye performed the microscale thermophoresis and corresponding data analysis; Q. Liu wrote the manuscript; J. Lin and L. Qiu reviewed and edited the manuscript.

Corresponding authors

Correspondence to Rongbin Liu, Ling Qiu or Jianguo Lin.

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This study only involved small animals. All the animal-based experiments were in compliance with the Laboratory Animal Guidelines for the Ethical Review of Animal Welfare of China (No. GB/T 35892–2018) and the Institutional Ethics Board of Jiangsu Institute of Nuclear Medicine.

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Liu, Q., Cai, S., Ye, J. et al. Preclinical evaluation of 68 Ga-labeled peptide CK2 for PET imaging of NRP-1 expression in vivo. Eur J Nucl Med Mol Imaging (2024). https://doi.org/10.1007/s00259-024-06632-x

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