Deborah Sultan

BACKGROUND: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte–associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages. METHODS: We used an established murine ICI myocarditis model ( Ctla4 +/– Pdcd1 –/– mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies. RESULTS: We observed marked increases in CCR2 (C-C chemokine receptor type 2) + monocyte-derived macrophages and CD8 + T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2 + subpopulation highly expressing Cxcl9 (chemokine [C-X-C motif] ligand 9), Cxcl10 (chemokine [C-X-C motif] ligand 10), Gbp2b (interferon-induced guanylate-binding protein 2b), and Fcgr4 (Fc receptor, IgG, low affinity IV) that originated from CCR2 + monocytes. It is important that a similar macrophage population expressing CXCL9 , CXCL10 , and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9 + Cxcl10 + macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8 + T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of Cxcl9 + Cxcl10 + macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis. CONCLUSIONS: These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ–induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.

Gold nanoparticles, especially positron-emitter- labeled gold nanostructures, have gained steadily increasing attention in biomedical applications. Of the radionuclides used for nanoparticle positron emission tomography imaging, radiometals such as (64) Cu have been widely employed. Currently, radiolabeling through macrocyclic chelators is the most commonly used strategy. However, the radiolabel stability may be a limiting factor for further translational research. We report the integration of (64) Cu into the structures of gold nanoparticles. With this approach, the specific radioactivity of the alloyed gold nanoparticles could be freely and precisely controlled by the addition of the precursor (64) CuCl2 to afford sensitive detection. The direct incorporation of (64) Cu into the lattice of the gold nanoparticle structure ensured the radiolabel stability for accurate localization in vivo. The superior pharmacokinetic and positron emission tomography imaging capabilities demonstrate high passive tumor targeting and contrast ratios in a mouse breast cancer model, as well as the great potential of this unique alloyed nanostructure for preclinical and translational imaging.

Rationale: Paradigm shifting studies have revealed that the heart contains functionally diverse populations of macrophages derived from distinct embryonic and adult hematopoietic progenitors. Under steady-state conditions, the heart is largely populated by CCR2− (C-C chemokine receptor type 2) macrophages of embryonic descent. After tissue injury, a dramatic shift in macrophage composition occurs whereby CCR2+ monocytes are recruited to the heart and differentiate into inflammatory CCR2+ macrophages that contribute to heart failure progression. Currently, there are no techniques to noninvasively detect CCR2+ monocyte recruitment into the heart and thus identify patients who may be candidates for immunomodulatory therapy. Objective: To develop a noninvasive molecular imaging strategy with high sensitivity and specificity to visualize inflammatory monocyte and macrophage accumulation in the heart. Methods and Results: We synthesized and tested the performance of a positron emission tomography radiotracer ( 68 Ga-DOTA [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ECL1i [extracellular loop 1 inverso]) that allosterically binds to CCR2. In naive mice, the radiotracer was quickly cleared from the blood and displayed minimal retention in major organs. In contrast, biodistribution and positron emission tomography demonstrated strong myocardial tracer uptake in 2 models of cardiac injury (diphtheria toxin induced cardiomyocyte ablation and reperfused myocardial infarction). 68 Ga-DOTA-ECL1i signal localized to sites of tissue injury and was independent of blood pool activity as assessed by quantitative positron emission tomography and ex vivo autoradiography. 68 Ga-DOTA-ECL1i uptake was associated with CCR2+ monocyte and CCR2+ macrophage infiltration into the heart and was abrogated in CCR2 −/ − mice, demonstrating target specificity. Autoradiography demonstrated that 68 Ga-DOTA-ECL1i specifically binds human heart failure specimens and with signal intensity associated with CCR2+ macrophage abundance. Conclusions: These findings demonstrate the sensitivity and specificity of 68 Ga-DOTA-ECL1i in the mouse heart and highlight the translational potential of this agent to noninvasively visualize CCR2+ monocyte recruitment and inflammatory macrophage accumulation in patients.

Abstract Gold nanoparticles, especially positron‐emitter‐ labeled gold nanostructures, have gained steadily increasing attention in biomedical applications. Of the radionuclides used for nanoparticle positron emission tomography imaging, radiometals such as 64 Cu have been widely employed. Currently, radiolabeling through macrocyclic chelators is the most commonly used strategy. However, the radiolabel stability may be a limiting factor for further translational research. We report the integration of 64 Cu into the structures of gold nanoparticles. With this approach, the specific radioactivity of the alloyed gold nanoparticles could be freely and precisely controlled by the addition of the precursor 64 CuCl 2 to afford sensitive detection. The direct incorporation of 64 Cu into the lattice of the gold nanoparticle structure ensured the radiolabel stability for accurate localization in vivo. The superior pharmacokinetic and positron emission tomography imaging capabilities demonstrate high passive tumor targeting and contrast ratios in a mouse breast cancer model, as well as the great potential of this unique alloyed nanostructure for preclinical and translational imaging.

As an emerging class of nanomaterial, nanoclusters hold great potential for biomedical applications due to their unique sizes and related properties. Herein, we prepared a (64)Cu doped gold nanocluster ((64)CuAuNC, hydrodynamic size: 4.2 ± 0.5 nm) functionalized with AMD3100 (or Plerixafor) for targeted positron emission tomography (PET) imaging of CXCR4, an up-regulated receptor on primary tumor and lung metastasis in a mouse 4T1 orthotopic breast cancer model. The preparation of targeted (64)CuAuNCs-AMD3100 (4.5 ± 0.4 nm) was done via one-step reaction with controlled conjugation of AMD3100 and specific activity, as well as improved colloid stability. In vivo pharmacokinetic evaluation showed favorable organ distribution and significant renal and fecal clearance within 48 h post injection. The expression of CXCR4 in tumors and metastasis was characterized by immunohistochemistry, Western blot, and reverse transcription polymerase chain reaction analysis. PET imaging with (64)CuAuNCs-AMD3100 demonstrated sensitive and accurate detection of CXCR4 in engineered tumors expressing various levels of the receptor, while competitive receptor blocking studies confirmed targeting specificity of the nanoclusters. In contrast to nontargeted (64)CuAuNCs and (64)Cu-AMD3100 alone, the targeted (64)CuAuNCs-AMD3100 detected up-regulated CXCR4 in early stage tumors and premetastatic niche of lung earlier and with greater sensitivity. Taken together, we believe that (64)CuAuNCs-AMD3100 could serve as a useful platform for early and accurate detection of breast cancer and metastasis providing an essential tool to guide the treatment.