ImmunoPET reveals tumor-associated macrophages in humanized MDA-MB-231 models

Background: Tumor-associated macrophages (TAMs) represent a major component of the tumor immune microenvironment and are associated with tumor progression and therapeutic resistance. Noninvasive detection of TAMs can provide valuable insight into immune infiltration and guide immunotherapy development. However, current macrophage imaging agents lack specificity for human pan-macrophages. In this study, we aim to image human macrophages in a humanized mouse model using our specific macrophage-targeted probe. Methods: We developed a human-specific CD68-Fab fragment (Fab14) and radiolabeled it with zirconium-89 to obtain [ 89 Zr]Zr-DFO-Fab14. Humanized NSG-SGM3 and control NSG-SGM3 mice were implanted with MDA-MB-231 triple-negative breast cancer cells into the mammary fat pad. When tumors were palpable, mice were injected intravenously with 6.25 µg of human-specific, macrophage-selective [ 89 Zr]Zr-DFO-Fab14 (~30 µCi per mouse). PET imaging was performed at 2-, 24- and 48-hours post-injection (p.i.), and radiotracer uptake was quantified using SUVmean. Ex vivo biodistribution at 48 hours was assessed by gamma counting to evaluate radiotracer specificity and retention. Tumors were harvested for immunofluorescence (IF) analysis of human CD68 expression. Results: At 2 hours p.i., quantitative analysis of [ 89 Zr]Zr-DFO-Fab14 uptake was similar in both humanized and control tumors, indicating comparable early distribution. By 24 hours, uptake of the immunoPET tracer was significantly higher and more sustained in humanized NSG-SGM3 mice (SUVmean = 0.29 ± 0.04) compared to control mice (SUVmean = 0.20 ± 0.03; ***p =0.0002). Biodistribution studies at 48 hours further validated the imaging specificity, showing markedly higher tumor retention in humanized NSG-SGM3 (4.66± 1.61 %ID/g) than in control NSG-SGM3 mice (1.71 ± 0.63 %ID/g). IF analysis confirmed abundant human CD68⁺ macrophage infiltration in humanized NSG-SGM3 tumors but not in NSG-SGM3 tumors, confirming the specificity of [ 89 Zr]Zr-DFO-Fab14 for human macrophages. Conclusions: The human-specific [ 89 Zr]Zr-DFO-Fab14 demonstrates macrophage-specific retention in humanized tumors, enabling noninvasive visualization of human TAMs in vivo. This radiotracer represents a promising tool for studying macrophage dynamics in human immune-reconstituted systems and evaluating efficacy of macrophage-targeted therapies in cancer.