Abstract
Purpose
Single photon emission computed tomography (SPECT) radionuclide pairs having distinct decay rates and different energy maxima enable simultaneous detection of dual gamma signals and real-time assessment of dynamic functional and molecular processes in vivo. Here, we report image acquisition and quantification protocols for a single molecule labeled with two different radionuclides for functional SPECT imaging.
Procedures
LS370 and LS734 were prepared using modular solid phase peptide synthesis. Each agent has a caspase-3 cleavable reporting motif, flanked by a tyrosine residue and a chelator at the opposite end of molecule. Cell uptake and efflux were assessed in human MDA-MB-231 breast cancer cells. Biodistribution studies were conducted in tumor naive and orthotopic 4T1 metastatic breast cancer tumor mice. NanoSPECT dual-imaging validation and attenuation correction parameters were developed using phantom vials containing varying radionuclide concentrations. Proof-of-principle SPECT imaging was performed in MMTV-PyMT transgenic mice.
Results
LS370 and LS734 were singly or dually radiolabeled with 125I and 111In or 99mTc. Cell assays demonstrated 11-fold higher percent uptake (P < 0.001) of [125I]LS734 (3.6 ± 0.5) compared to [125I]LS370 (0.3 ± 0.3) at 2 h. Following chemotherapy, cellular retention of [125I]LS734 was 3-fold higher (P < 0.05) than untreated cells. Pharmacokinetics at 1 h postinjection demonstrated longer blood retention (%ID/g) for [125I]LS734 (3.2 ± 0.9) compared to [125I]LS370 (1.6 ± 0.1). In mice bearing bilateral orthotopic 4T1 tumors, the uptake (%ID/g) was 2.4 ± 0.3 for [125I]LS734 and 1.2 ± 0.03 for [125I]LS370. The iodinated tyrosine peptide residue label was stable under in vitro conditions for up to 24 h; rapid systemic deiodination (high thyroid uptake) was observed in vivo. Phantom studies using standards demonstrated deconvolution of radionuclide signals based on different gamma ray energies. In MMTV-PyMT mice imaged with dual-labeled [111In]–[125I]LS734, the gamma signals were separable and quantifiable.
Conclusions
Image processing protocols were developed for quantitative signal separation resulting from a caspase-3 responsive dual-radiolabeled SPECT probe. Crosstalk unmixing was obtained for multiradionuclide NanoSPECT imaging. In vitro and in vivo data demonstrated structure–activity relationships for developing functional agents for ratiometric SPECT imaging.
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References
Choi KY, Swierczewska M, Lee S, Chen X (2012) Protease-activated drug development. Theranostics 2:156–178
Boya P, Kroemer G (2008) Lysosomal membrane permeabilization in cell death. Oncogene 27:6434–6451
Johansson AC, Appelqvist H, Nilsson C et al (2010) Regulation of apoptosis-associated lysosomal membrane permeabilization. Apoptosis 15:527–540
Graham MM (2012) Clinical molecular imaging with radiotracers: current status. Med Princ Pract: Int J Kuwait University, Health Sci Centre 21:197–208
Benard F, Turcotte E (2005) Imaging in breast cancer: single-photon computed tomography and positron-emission tomography. Breast Cancer Res 7:153–162
Chen DL, Zhou D, Chu W et al (2012) Radiolabeled isatin binding to caspase-3 activation induced by anti-Fas antibody. Nucl Med Biol 39:137–144
Beekman F, van der Have F (2007) The pinhole: gateway to ultra-high-resolution three-dimensional radionuclide imaging. Eur J Nucl Med Mol Imaging 34:151–161
Edwards WB, Akers WJ, Ye Y et al (2009) Multimodal imaging of integrin receptor-positive tumors by bioluminescence, fluorescence, gamma scintigraphy, and single-photon emission computed tomography using a cyclic RGD peptide labeled with a near-infrared fluorescent dye and a radionuclide. Mol Imaging 8:101–110
Yang TJ, Haimovitz-Friedman A, Verheij M (2012) Anticancer therapy and apoptosis imaging. Exp Oncol 34:269–276
Wang J, Lenardo MJ (2000) Roles of caspases in apoptosis, development, and cytokine maturation revealed by homozygous gene deficiencies. J Cell Sci 113(Pt 5):753–757
Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305–1308
Zhang Z, Fan J, Cheney PP et al (2009) Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo. Mol Pharmaceutics 6:416–427
Ma CX, Cai S, Li S et al (2012) Targeting Chk1 in p53-deficient triple-negative breast cancer is therapeutically beneficial in human-in-mouse tumor models. J Clin Investig 122:1541–1552
Bullok KE, Maxwell D, Kesarwala AH et al (2007) Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis. Biochemistry 46:4055–4065
Eckelman WC, Bonardi M, Volkert WA (2008) True radiotracers: are we approaching theoretical specific activity with Tc-99m and I-123? Nucl Med Biol 35:523–527
Chau I, Rigg A, Cunningham D (2003) Matrix metalloproteinase inhibitors—an emphasis on gastrointestinal malignancies. Crit Rev Oncol Hematol 45:151–176
Luker GD, Luker KE (2008) Optical imaging: current applications and future directions. J Nucl Med 49:1–4
Ntziachristos V, Bremer C, Graves EE, Ripoll J, Weissleder R (2002) In vivo tomographic imaging of near-infrared fluorescent probes. Mol Imaging 1:82–88
Bremer C, Tung CH, Weissleder R (2001) In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat Med 7:743–748
Glazer DI, Brown RK, Wong KK, Savas H, Gross MD, Avram AM (2013) SPECT/CT evaluation of unusual physiologic radioiodine biodistributions: pearls and pitfalls in image interpretation. Radiographics 33:397–418
van Schaijk FG, Broekema M, Oosterwijk E et al (2005) Residualizing iodine markedly improved tumor targeting using bispecific antibody-based pretargeting. J Nucl Med 46:1016–1022
Acknowledgments
We thank Rui Tang, Kvar Black, and Mingzhou Zhou for helpful discussions.
Funding
This work was funded primarily by grant from the Department of Defense Breast Cancer Research Program (W81XWH-09-1-0333) and supported in part by grant from the National Institutes of Health NIBIB R01 EB021048 and resources from the Washington University Molecular Imaging Center (NCI P50 CA094056). WJA is supported in part by an award from the NIH Office of Research Infrastructure Programs (K01RR026095). MS is supported in part by an R01 award from NIH (R01CA176221).
Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Conflict of Interest
The authors declare that they have no conflict of interest.
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Xu, B., Shokeen, M., Sudlow, G.P. et al. Utilizing the Multiradionuclide Resolving Power of SPECT and Dual Radiolabeled Single Molecules to Assess Treatment Response of Tumors. Mol Imaging Biol 17, 671–679 (2015). https://doi.org/10.1007/s11307-015-0842-8
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DOI: https://doi.org/10.1007/s11307-015-0842-8