Quantitation of CXCR4 Expression in Myocardial Infarction Using 99mTc-Labeled SDF-1α

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Abstract
The chemokine stromal-derived factor-1α (SDF-1α, CXCL12) and its receptor CXCR4 are implicated as key mediators of hematopoietic stem cell retention, cancer metastasis, and HIV infection. Their role in myocardial infarction (MI) is not as well defined. The noninvasive in vivo quantitation of CXCR4 expression is central to understanding its importance in these diverse processes as well in the cardiac response to injury. Methods: Recombinant SDF-1α was radiolabeled under aprotic conditions and purified by gel-filtration chromatography (GFC) using high-specific-activity 99mTc-S-acetylmercaptoacetyltriserine-N-hydroxysuccinimide ([99mTc-MAS3]-NHS) prepared by solid-phase preloading. Radiotracer stability and transmetallation under harsh conditions were quantified by GFC. Affinity, specificity, and maximum number of binding sites (Bmax) were quantified, with adenoviral-expressed CXCR4 on nonexpressing cells and endogenous receptor on rat neonatal cardiomyocytes, using a high-throughput live-cell–binding assay. Blood half-life, biodistribution, and clearance of intravenously injected [99mTc-MAS3]-SDF-1α were quantified in Sprague–Dawley rats before and after experimentally induced MI. Results: [99mTc-MAS3]-SDF-1α could be prepared in 2 h total with a specific activity of 8.0 × 107 MBq/mmol (2,166 Ci/mmol) and a radiochemical purity greater than 98%. Degradation of the radiotracer after boiling for 5 min, with and without 1 mM dithiothreitol, and transmetallation in 100% serum at 37°C for 4 h were negligible. [99mTc-MAS3]-SDF-1α exhibits high specificity for CXCR4 on the surface of living rat neonatal cardiomyocytes, with an affinity of 2.7 ± 0.9 nM and a Bmax of 4.8 × 104 binding sites per cell. After intravenous injection, 99mTc-labeled SDF-1α displays a blood half-life of 25.8 ± 4.6 min, rapid renal clearance with only 26.2 ± 6.1 percentage injected dose remaining in the carcass at 2 h, consistently low uptake in most organs (99mTc-MAS3]-SDF-1α and confirmed using confocal immunofluorescence. Conclusion: We describe a 99mTc-labeled SDF-1α radiotracer that can be used as a sensitive and specific probe for CXCR4 expression in vivo and demonstrate that this radiotracer is able to quantify changes in CXCR4 expression under different physiologic and pathologic states. Taken together, CXCR4 levels should now be quantifiable in vivo in a variety of animal model systems of human diseases.