Tissue-resident macrophages are complementary to proinflammatory macrophages to promote the progression of atherosclerosis. The noninvasive detection of their presence and dynamic variation will be important to the understanding of their role in the pathogenesis of atherosclerosis. The goal of this study was to develop a targeted PET radiotracer for imaging CD163-positive (CD163+) macrophages in multiple mouse atherosclerosis models and assess the potential of CD163 as a biomarker for atherosclerosis in humans. Methods: CD163-binding peptide was identified using phage display and conjugated with a NODAGA chelator for ⁶⁴Cu radiolabeling ([⁶⁴Cu]Cu-ICT-01). CD163-overexpressing U87 cells were used to measure the binding affinity of [⁶⁴Cu]Cu-ICT-01. Biodistribution studies were performed on wild-type C57BL/6 mice at multiple time points after tail vein injection. The sensitivity and specificity of [⁶⁴Cu]Cu-ICT-01 in imaging CD163+ macrophages upregulated on the surface of atherosclerotic plaques were assessed in multiple mouse atherosclerosis models. Immunostaining, flow cytometry, and single-cell RNA sequencing were performed to characterize the expression of CD163 on tissue-resident macrophages. Human carotid atherosclerotic plaques were used to measure the expression of CD163+ resident macrophages and test the binding specificity of [⁶⁴Cu]Cu-ICT-01. Results: [⁶⁴Cu]Cu-ICT-01 showed high binding affinity to U87 cells. The biodistribution study showed rapid blood and renal clearance with low retention in all major organs at 1, 2, and 4 h after injection. In an ApoE^–/– mouse model, [⁶⁴Cu]Cu-ICT-01 demonstrated sensitive and specific detection of CD163+ macrophages and capability for tracking the progression of atherosclerotic lesions; these findings were further confirmed in Ldlr^–/– and PCSK9 mouse models. Immunostaining showed elevated expression of CD163+ macrophages across the plaques. Flow cytometry and single-cell RNA sequencing confirmed the specific expression of CD163 on tissue-resident macrophages. Human tissue characterization demonstrated high expression of CD163+ macrophages on atherosclerotic lesions, and ex vivo autoradiography revealed specific binding of [⁶⁴Cu]Cu-ICT-01 to human CD163. Conclusion: This work reported the development of a PET radiotracer binding CD163+ macrophages. The elevated expression of CD163+ resident macrophages on human plaques indicated the potential of CD163 as a biomarker for vulnerable plaques. The sensitivity and specificity of [⁶⁴Cu]Cu-ICT-01 in imaging CD163+ macrophages warrant further investigation in translational settings.

组织驻留巨噬细胞与促炎巨噬细胞互补，促进动脉粥样硬化的进展。无创检测它们的存在和动态变化对于理解它们在动脉粥样硬化发病机制中的作用非常重要。本研究的目的是开发一种靶向 PET 放射性示踪剂，用于对多种小鼠动脉粥样硬化模型中的 CD163 阳性 (CD163+) 巨噬细胞进行成像，并评估 CD163 作为人类动脉粥样硬化生物标志物的潜力。方法：使用噬菌体展示技术鉴定 CD163 结合肽，并与 NODAGA 螯合剂缀合进行⁶⁴ Cu 放射性标记 ([ ⁶⁴ Cu]Cu-ICT-01)。 CD163过表达的U87细胞用于测量[ ⁶⁴ Cu]Cu-ICT-01的结合亲和力。在尾静脉注射后的多个时间点对野生型 C57BL/6 小鼠进行生物分布研究。在多种小鼠动脉粥样硬化模型中评估了[ ⁶⁴ Cu]Cu-ICT-01 在动脉粥样硬化斑块表面上调的 CD163+ 巨噬细胞成像中的敏感性和特异性。通过免疫染色、流式细胞术和单细胞 RNA 测序来表征组织驻留巨噬细胞上 CD163 的表达。使用人颈动脉粥样硬化斑块测量CD163+常驻巨噬细胞的表达并测试[ ⁶⁴ Cu]Cu-ICT-01的结合特异性。结果： [ ⁶⁴ Cu]Cu-ICT-01 对 U87 细胞表现出高结合亲和力。生物分布研究显示，注射后 1、2 和 4 小时，血液和肾脏清除迅速，所有主要器官的滞留率较低。在 ApoE ^–/–小鼠模型中，[ ⁶⁴ Cu]Cu-ICT-01 表现出对 CD163+ 巨噬细胞的灵敏且特异的检测能力以及跟踪动脉粥样硬化病变进展的能力；这些发现在 Ldlr ^–/–和 PCSK9 小鼠模型中得到了进一步证实。免疫染色显示斑块上 CD163+ 巨噬细胞的表达升高。流式细胞术和单细胞 RNA 测序证实了 CD163 在组织驻留巨噬细胞上的特异性表达。人体组织表征表明，动脉粥样硬化病变上 CD163+ 巨噬细胞高表达，离体放射自显影显示 [ ⁶⁴ Cu]Cu-ICT-01 与人 CD163 特异性结合。结论：这项工作报告了结合 CD163+ 巨噬细胞的 PET 放射性示踪剂的开发。人类斑块上 CD163+ 常驻巨噬细胞的表达升高表明 CD163 作为易损斑块生物标志物的潜力。 [ ⁶⁴ Cu]Cu-ICT-01 在 CD163+ 巨噬细胞成像中的敏感性和特异性值得在转化环境中进行进一步研究。