Iron–quercetin complex reprograms tumor-associated macrophages toward M1 phenotype and enables MRI tracking in ex vivo tumor-mimicking spheroids

Abstract Background Tumor-associated macrophages (TAMs) frequently adopt an M2-like, immunosuppressive state that promotes tumor growth and therapy resistance. Reprogramming TAMs toward a pro-inflammatory M1 phenotype is a promising strategy to remodel the tumor microenvironment (TME) and enhance treatment responses. We evaluated an iron(III)–quercetin complex (IronQ) as a macrophage-directed, magnetic resonance imaging (MRI)-visible theranostic. Methods IronQ stability was tested in protein-containing media by dynamic light scattering (DLS). Biocompatibility was assessed in fibroblasts (MRC-5), THP-1–derived macrophages, and non-small-cell lung cancer (NSCLC) cell lines (A549, H460). Cellular uptake was examined by Prussian blue staining and in vitro MRI. Reactive oxygen species (ROS; DCFH-DA), ERK/NF-κB activation (Western blot), and polarization markers (flow cytometry, qRT-PCR, ELISA) were quantified after IronQ exposure. Functional anti-tumor effects were evaluated in non-contact Transwell co-cultures and in 3D tri-culture spheroids (tumor cells, fibroblasts, macrophages). For imaging, agar-embedded spheroids containing IronQ-labeled macrophages were scanned by T1-weighted MRI. Results IronQ was colloidally stable and well tolerated across cell types within a broad non-toxic window. It accumulated preferentially in macrophages. At low doses, IronQ increased intracellular ROS, activated ERK/NF-κB signaling, upregulated M1 markers ( CD86 , NOS2 , TNFA , IL1B ), and downregulated M2 markers ( CD206 , TGFB1 , IL10 , CCL18 ); these effects attenuated at higher doses, indicating a biphasic response. Functionally, IronQ repolarized IL-4–primed M2 macrophages and indirectly reduced tumor viability in Transwell assays. In 3D spheroids, IronQ limited growth and shifted the secretome toward higher TNF-α and lower IL-10, with IL-6 unchanged. As an imaging agent, IronQ produced dose-dependent T1 hyperintensity in labeled cells and yielded positive T1 signal in spheroids containing IronQ-labeled macrophages. Conclusions IronQ integrates macrophage-selective uptake, actionable M1 repolarization within a defined dose window, and positive-contrast MRI visibility. These properties support its development as a cost-efficient, multifunctional platform for TAM-focused cancer immunotherapy and image-guided response monitoring.