Pro-tumoral M2 tumor-associated macrophages (TAMs) play a critical role in the tumor immune microenvironment (TIME), making them an important therapeutic target for cancer treatment. Approaches for imaging and monitoring M2 TAMs, as well as tracking their changes in response to tumor progression or treatment are highly sought-after but remain underdeveloped. Here, we report an M2-targeted magnetic resonance imaging (MRI) probe based on sub-5 nm ultrafine iron oxide nanoparticles (uIONP), featuring an anti-biofouling coating to prevent non-specific macrophage uptake and an M2-specific peptide ligand (M2pep) for active targeting of M2 TAMs. The targeting specificity of M2pep-uIONP was validated in vitro, using M0, M1, and M2 macrophages, and in vivo, using an orthotopic patient-tissue-derived xenograft (PDX) mouse model of glioblastoma (GBM). MRI of the mice revealed hypointense contrast in T-weighted images of intracranial tumors 24 h after receiving intravenous (i.v.) injection of M2pep-uIONP. In contrast, no noticeable contrast change was observed in mice receiving scrambled-sequence M2pep-conjugated uIONP (scM2pep-uIONP) or the commercially available iron oxide nanoparticle formulation, Ferumoxytol. Measurement of nanoparticle-induced Tvalue changes in tumors showed 38 %, 9 %, and 2 % decrease for M2pep-uIONP, scM2pep-uIONP, and Ferumoxytol, respectively. Moreover, M2pep-uIONP exhibited 88.7-fold higher intra-tumoral accumulation compared to co-injected Ferumoxytol at 24 h post-injection. Immunofluorescence-stained tumor sections showed that CD68/CD163M2 TAMs were highly co-localized with Cy7-M2pep-uIONP, but not with Cy7-scM2pep-uIONP and Cy7-Ferumoxytol. Flow cytometry analysis revealed 26 ± 10 % of M2 TAMs were targeted by M2pep-uIONP, which was significantly higher than Ferumoxytol (16 ± 1 %) and scM2pep-uIONP (13 ± 4 %) with the same dosage (20 mg Fe/kg). These findings demonstrate that M2pep-uIONP functions as a ligand-mediated MRI probe for targeted imaging of M2 TAMs in GBM, with potential applications for imaging of M2 TAM in other cancer types. STATEMENT OF SIGNIFICANCE: Targeting the pro-tumoral M2 subtype of tumor-associated macrophages (TAMs) to modulate the tumor immune microenvironment (TIME) is an emerging strategy for developing novel cancer therapies and enhancing the efficacy of existing treatments. In this study, we have developed a magnetic resonance imaging (MRI) probe using sub-5 nm ultrafine iron oxide nanoparticles (uIONP), which are coated with an anti-biofouling polymer and conjugated to an M2-specific peptide ligand (M2pep). Our results demonstrate that M2pep-uIONP exhibits an 88.7-fold higher accumulation in intracranial tumors in an orthotopic patient-derived xenograft (PDX) model of glioblastoma compared to the commercial iron oxide nanoparticle, Ferumoxytol. This enhanced accumulation enables M2pep-uIONP to induce significant MRI contrast, providing a non-invasive imaging tool to visualize M2 TAMs and monitor changes in the TIME of brain tumors and potentially other cancers. 2 2 + +
