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Quantitation of CXCR4 expression in myocardial infarction using 99mTc-labeled SDF-1alpha
Preeti Misra1, Djamel Lebeche, Hung Ly
1Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
Insights
A novel technetium-99m-labeled stromal-derived factor-1alpha ([99mTc-MAS3]-SDF-1alpha) radiotracer allows for sensitive and specific in vivo quantification of CXCR4 expression. This tool revealed a five-fold increase in myocardial CXCR4 after myocardial infarction.
Area of Science:
- Biochemistry
- Molecular Biology
- Radiochemistry
Background:
- Stromal-derived factor-1alpha (SDF-1alpha, CXCL12) and its receptor CXCR4 are crucial in stem cell biology, cancer, and HIV.
- Their role in myocardial infarction (MI) and cardiac injury response requires further elucidation.
- Noninvasive in vivo quantitation of CXCR4 expression is essential for understanding its diverse functions.
Purpose of the Study:
- To develop and characterize a novel radiotracer for in vivo CXCR4 detection.
- To assess the utility of this radiotracer in quantifying CXCR4 expression changes in a myocardial infarction model.
Main Methods:
- Radiolabeling of SDF-1alpha with high-specific-activity 99mTc-S-acetylmercaptoacetyltriserine-N-hydroxysuccinimide ([99mTc-MAS3]-NHS).
- Assessment of radiotracer stability, affinity, and specificity using cell-binding assays.
- Evaluation of biodistribution, blood half-life, and clearance in Sprague-Dawley rats before and after experimentally induced MI.
Main Results:
- [99mTc-MAS3]-SDF-1alpha was prepared with high specific activity (>8.0 x 10(7) MBq/mmol) and radiochemical purity (>98%).
- The radiotracer demonstrated high specificity and affinity (2.7 +/- 0.9 nM) for CXCR4 on cardiomyocytes.
- A >5-fold increase in myocardial CXCR4 expression was observed post-MI, confirmed by immunofluorescence.
Conclusions:
- A stable and specific 99mTc-labeled SDF-1alpha radiotracer for in vivo CXCR4 quantification has been developed.
- This radiotracer effectively quantifies changes in CXCR4 expression in physiological and pathological states, including post-MI.
- CXCR4 levels are now quantifiable in vivo across various animal disease models.
Unlabelled:
The chemokine stromal-derived factor-1alpha (SDF-1alpha, 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-1alpha 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-1alpha were quantified in Sprague-Dawley rats before and after experimentally induced MI.
Results:
[99mTc-MAS3]-SDF-1alpha could be prepared in 2 h total with a specific activity of 8.0 x 10(7) 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 degrees C for 4 h were negligible. [99mTc-MAS3]-SDF-1alpha 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 x 10(4) binding sites per cell. After intravenous injection, 99mTc-labeled SDF-1alpha 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 (<0.1 percentage injected dose per gram), and no evidence of blood-brain barrier penetration. After MI was induced, CXCR4 expression levels in the myocardium increased more than 5-fold, as quantified using [99mTc-MAS3]-SDF-1alpha and confirmed using confocal immunofluorescence.
Conclusion:
We describe a 99mTc-labeled SDF-1alpha 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.
