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Evaluation of 64Cu Labeled GX1: A Phage Display Peptide Probe for PET Imaging of Tumor Vasculature

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Abstract

Purpose

Molecular imaging using positron emission tomography (PET) radiotracers targeted to tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The previous in vitro results demonstrated that the GX1 peptide, identified by phage display technology, is a tumor vasculature endothelium-specific ligand. In this study, we evaluated a 64Cu-labeled GX1 peptide as a potential radiotracer for microPET imaging of tumor vasculature in a U87MG tumor xenografted mouse model.

Methods

Macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid (DOTA)-conjugated GX1 peptide was synthesized and radiolabeled with 64Cu (t 1/2 = 12.7 h) in ammonium acetate buffer. The 64Cu-labeled GX1 peptide was then subjected to in vitro tumor cell uptake study, small animal PET and direct tissue sampling biodistribution studies in a U87MG tumor xenografted mouse model.

Results

The in vitro experiment demonstrated that 64Cu-DOTA-GX1 is stable in PBS with more than 91% of 64Cu-DOTA-GX1 peptide remaining intact after 24 h of incubation. Cellular uptake and retention studies revealed 64Cu-DOTA-GX1 binds to U87MG glioma cells and has good tumor cell retention. For small animal PET imaging studies, the U87MG tumors were all clearly visible with high contrast to contralateral background at all measured time points after injection of 64Cu-DOTA-GX1 while high accumulation in liver and kidneys were also observed at early time points. The U87MG tumor uptake was determined to be the highest (7.97 ± 0.75%ID/g) at 24 h pi. The blocking experiment was achieved by co-injection of 64Cu-DOTA-GX1 with non-radiolabeled GX1 peptide (20 mg/kg) at 24 h pi, suggesting 64Cu-DOTA-GX1 is a target-specific tracer. Furthermore, the biodistribution results were consistent with the quantification of microPET imaging, demonstrating the highest ratio (16.09 ± 1.21) of tumor/muscle uptake of 64Cu-DOTA-GX1 at 24 h pi for non-blocking group and significant decreased ratio (6.57 ± 0.58) for blocking group. Finally, metabolic studies suggested that 64Cu-DOTA-GX1 is stable in mouse blood and urine in vivo at early time point while the metal transchelation may also occur in mouse liver and kidneys.

Conclusion

Our studies demonstrate that 64Cu-DOTA-GX1 is a promising radiotracer for imaging tumor vasculature.

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Abbreviations

PET:

positron emission tomography

HPLC:

high-performance liquid chromatography

TLC:

thin-layer chromatography

%ID/g:

percentage injected dose per gram of tissue

pi:

postinjection

GX1:

cyclo(CGNSNPKSC) peptide

PBS:

phosphate-buffered saline

DOTA:

1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid

Boc:

t-butoxycarbonyl

NHS:

N-hydroxysuccinimide

TFA:

trifluoroacetic acid

EDTA:

ethylenediaminetetraacetic acid

DMF:

N,N-dimethylformamide

DIPEA:

diisopropylethylamine

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Acknowledgments

This study was supported in part by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health, the USC Department of Radiology, and the Provost’s Biomedical Imaging Science Initiative.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of Southern California, 2250 Alcazar Street, CSC 103, Los Angeles, CA, 90033-9061, USA

    Kai Chen, Li-Peng Yap & Peter S. Conti

  2. Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), 31 Center Drive, Suite 1C14, Bethesda, MD, 20892-2281, USA

    Kai Chen, Xilin Sun, Gang Niu, Ying Ma & Xiaoyuan Chen

  3. State Key Laboratory of Cancer Biology & Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, 710032, Xi’an, Shanxi, China

    Xiaoli Hui, Kaichun Wu & Daiming Fan

Authors
  1. Kai Chen
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  2. Xilin Sun
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  3. Gang Niu
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  10. Xiaoyuan Chen
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Correspondence to Kai Chen or Xiaoyuan Chen.

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Chen, K., Sun, X., Niu, G. et al. Evaluation of 64Cu Labeled GX1: A Phage Display Peptide Probe for PET Imaging of Tumor Vasculature. Mol Imaging Biol 14, 96–105 (2012). https://doi.org/10.1007/s11307-011-0479-1

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