Development of a CD163-Targeted PET Radiotracer That Images Resident Macrophages in Atherosclerosis.

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.

Development of a CCR2 targeted 18F-labeled radiotracer for atherosclerosis imaging with PET.

Atherosclerosis is a chronic inflammatory disease and the leading cause of morbidity and mortality worldwide. CC motif chemokine ligand 2 and its corresponding cognate receptor 2 (CCL2/CCR2) signaling has been implicated in regulating monocyte recruitment and macrophage polarization during inflammatory responses that plays a pivotal role in atherosclerosis initiation and progression. In this study, we report the design and synthesis of a novel 18F radiolabeled small molecule radiotracer for CCR2-targeted positron emission tomography (PET) imaging in atherosclerosis. The binding affinity of this radiotracer to CCR2 was evaluated via in vitro binding assay using CCR2+ membrane and cells. Ex vivo biodistribution was carried out in wild type mice to assess radiotracer pharmacokinetics. CCR2 targeted PET imaging of plaques was performed in two murine atherosclerotic models. The sensitive detection of atherosclerotic lesions highlighted the potential of this radiotracer for CCR2 targeted PET and warranted further optimization.

Chemokine Receptor 2 Targeted PET/CT Imaging Distant Metastases in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and treatment-refractory malignancies. The lack of an effective screening tool results in the majority of patients being diagnosed at late stages, which underscores the urgent need to develop more sensitive and specific imaging modalities, particularly in detecting occult metastases, to aid clinical decision-making. The tumor microenvironment of PDAC is heavily infiltrated with myeloid-derived suppressor cells (MDSCs) that express C-C chemokine receptor type 2 (CCR2). These CCR2-expressing MDSCs accumulate at a very early stage of metastasis and greatly outnumber PDAC cells, making CCR2 a promising target for detecting early, small metastatic lesions that have scant PDAC cells. Herein, we evaluated a CCR2 targeting PET tracer (68Ga-DOTA-ECL1i) for PET imaging on PDAC metastasis in two mouse models. Positron emission tomography/computed tomography (PET/CT) imaging of 68Ga-DOTA-ECL1i was performed in a hemisplenic injection metastasis model (KI) and a genetically engineered orthotopic PDAC model (KPC), which were compared with 18F-FDG PET concurrently. Autoradiography, hematoxylin and eosin (H&E), and CCR2 immunohistochemical staining were performed to characterize the metastatic lesions. PET/CT images visualized the PDAC metastases in the liver/lung of KI mice and in the liver of KPC mice. Quantitative uptake analysis revealed increased metastasis uptake during disease progression in both models. In comparison, 18F-FDG PET failed to detect any metastases during the time course studies. H&E staining showed metastases in the liver and lung of KI mice, within which immunostaining clearly demonstrated the overexpression of CCR2 as well as CCR2+ cell infiltration into the normal liver. H&E staining, CCR2 staining, and autoradiography also confirmed the expression of CCR2 and the uptake of 68Ga-DOTA-ECL1i in the metastatic foci in KPC mice. Using our novel CCR2 targeted radiotracer 68Ga-DOTA-ECL1i and PET/CT, we demonstrated the sensitive and specific detection of CCR2 in the early PDAC metastases in two mouse models, indicating its potential in future clinical translation.

Ketosis prevents abdominal aortic aneurysm rupture through C-C chemokine receptor type 2 downregulation and enhanced extracellular matrix balance.

Abdominal aortic aneurysms (AAAs) are prevalent with aging, and AAA rupture is associated with increased mortality. There is currently no effective medical therapy to prevent AAA rupture. The monocyte chemoattractant protein (MCP-1)/C-C chemokine receptor type 2 (CCR2) axis critically regulates AAA inflammation, matrix-metalloproteinase (MMP) production, and extracellular matrix (ECM) stability. We therefore hypothesized that a diet intervention that can modulate CCR2 axis may therapeutically impact AAA risk of rupture. Since ketone bodies (KBs) can trigger repair mechanisms in response to inflammation, we evaluated whether systemic ketosis in vivo could reduce CCR2 and AAA progression. Male Sprague-Dawley rats underwent surgical AAA formation using porcine pancreatic elastase and received daily ß-aminopropionitrile to promote AAA rupture. Rats with AAAs received either a standard diet, ketogenic diet (KD), or exogenous KBs (EKB). Rats receiving KD and EKB reached a state of ketosis and had significant reduction in AAA expansion and incidence of rupture. Ketosis also led to significantly reduced aortic CCR2 content, improved MMP balance, and reduced ECM degradation. Consistent with these findings, we also observed that Ccr2-/- mice have significantly reduced AAA expansion and rupture. In summary, this study demonstrates that CCR2 is essential for AAA expansion, and that its modulation with ketosis can reduce AAA pathology. This provides an impetus for future clinical studies that will evaluate the impact of ketosis on human AAA disease.

Toward Quantitative Multisite Preclinical Imaging Studies in Acute Myocardial Infarction: Evaluation of the Immune-Fibrosis Axis.

The immune-fibrosis axis plays a critical role in cardiac remodeling after acute myocardial infarction. Imaging approaches to monitor temporal inflammation and fibroblast activation in mice have seen wide application in recent years. However, the repeatability of quantitative measurements remains challenging, particularly across multiple imaging centers. We aimed to determine reproducibility of quantitative inflammation and fibroblast activation images acquired at 2 facilities after myocardial infarction in mice. Methods: Mice underwent coronary artery ligation and sequential imaging with 68Ga-DOTA-ECL1i to assess chemokine receptor type 2 expression at 3 d after myocardial infarction and 68Ga-FAPI-46 to assess fibroblast activation protein expression at 7 d after myocardial infarction. Images were acquired at 1 center using either a local or a consensus protocol developed with the second center; the protocols differed in the duration of isoflurane anesthesia and the injected tracer dose. A second group of animals were scanned at the second site using the consensus protocol. Image analyses performed by each site and just by 1 site were also compared. Results: The uptake of 68Ga-DOTA-ECL1i in the infarct territory tended to be higher when the consensus protocol was used (P = 0.03). No difference was observed between protocol acquisitions for 68Ga-FAPI-46. Compared with the local protocol, the consensus protocol decreased variability between individual animals. When a matched consensus protocol was used, the 68Ga-DOTA-ECL1i infarct territory percentage injected dose per gram of tissue was higher on images acquired at site B than on those acquired at site A (P = 0.006). When normalized to body weight as SUV, this difference was mitigated. Both the percentage injected dose per gram of tissue and the SUV were comparable between sites for 68Ga-FAPI-46. Image analyses at the sites differed significantly, but this difference was mitigated when all images were analyzed at site A. Conclusion: The application of a standardized acquisition protocol may lower variability within datasets and facilitate comparison of molecular radiotracer distribution between preclinical imaging centers. Like clinical studies, multicenter preclinical studies should use centralized core-based image analysis to maximize reproducibility across sites.

Expansion of Pathogenic Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

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.

Cardiac radiation improves ventricular function in mice and humans with cardiomyopathy.

BACKGROUND: Rapidly dividing cells are more sensitive to radiation therapy (RT) than quiescent cells. In the failing myocardium, macrophages and fibroblasts mediate collateral tissue injury, leading to progressive myocardial remodeling, fibrosis, and pump failure. Because these cells divide more rapidly than cardiomyocytes, we hypothesized that macrophages and fibroblasts would be more susceptible to lower doses of radiation and that cardiac radiation could therefore attenuate myocardial remodeling. METHODS: In three independent murine heart failure models, including models of metabolic stress, ischemia, and pressure overload, mice underwent 5 Gy cardiac radiation or sham treatment followed by echocardiography. Immunofluorescence, flow cytometry, and non-invasive PET imaging were employed to evaluate cardiac macrophages and fibroblasts. Serial cardiac magnetic resonance imaging (cMRI) from patients with cardiomyopathy treated with 25 Gy cardiac RT for ventricular tachycardia (VT) was evaluated to determine changes in cardiac function. FINDINGS: In murine heart failure models, cardiac radiation significantly increased LV ejection fraction and reduced end-diastolic volume vs. sham. Radiation resulted in reduced mRNA abundance of B-type natriuretic peptide and fibrotic genes, and histological assessment of the LV showed reduced fibrosis. PET and flow cytometry demonstrated reductions in pro-inflammatory macrophages, and immunofluorescence demonstrated reduced proliferation of macrophages and fibroblasts with RT. In patients who were treated with RT for VT, cMRI demonstrated decreases in LV end-diastolic volume and improvements in LV ejection fraction early after treatment. CONCLUSIONS: These results suggest that 5 Gy cardiac radiation attenuates cardiac remodeling in mice and humans with heart failure. FUNDING: NIH, ASTRO, AHA, Longer Life Foundation.

Chemokine Receptor 2 Is A Theranostic Biomarker for Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a degenerative vascular disease impacting aging populations with a high mortality upon rupture. There are no effective medical therapies to prevent AAA expansion and rupture. We previously demonstrated the role of the monocyte chemoattractant protein-1 (MCP-1) / C-C chemokine receptor type 2 (CCR2) axis in rodent AAA pathogenesis via positron emission tomography/computed tomography (PET/CT) using CCR2 targeted radiotracer 64 Cu-DOTA-ECL1i. We have since translated this radiotracer into patients with AAA. CCR2 PET showed intense radiotracer uptake along the AAA wall in patients while little signal was observed in healthy volunteers. AAA tissues collected from individuals scanned with 64 Cu-DOTA-ECL1i and underwent open-repair later demonstrated more abundant CCR2+ cells compared to non-diseased aortas. We then used a CCR2 inhibitor (CCR2i) as targeted therapy in our established male and female rat AAA rupture models. We observed that CCR2i completely prevented AAA rupture in male rats and significantly decreased rupture rate in female AAA rats. PET/CT revealed substantial reduction of 64 Cu-DOTA-ECL1i uptake following CCR2i treatment in both rat models. Characterization of AAA tissues demonstrated decreased expression of CCR2+ cells and improved histopathological features. Taken together, our results indicate the potential of CCR2 as a theranostic biomarker for AAA management.

Ketosis Prevents Abdominal Aortic Aneurysm Rupture Through C-C Chemokine Receptor Type 2 Downregulation and Enhanced MMP Balance.

Abdominal aortic aneurysms (AAAs) are prevelant with aging, and AAA rupture is associated with high mortality. There is currently no effective medical therapy for AAA rupture. Previous work demonstrated that the monocyte chemoattractant protein (MCP-1) / C-C chemokine receptor type 2 (CCR2) axis critically regulates AAA inflammation, matrix-metalloproteinase (MMP) production, and extracellular matrix (ECM) stability. Here we similarly observed that Ccr2-/- mice have significantly reduced AAA expansion and rupture. We therefore hypothesized that a dietary modulation of the CCR2 axis may therapeutically impact AAA risk of rupture. Since ketone bodies (KBs) can trigger repair mechanisms in response to inflammation, we specifically evaluated whether systemic ketosis in vivo can reduce CCR2 and AAA progression. Male Sprague-Dawley rats underwent surgical AAA formation using porcine pancreatic elastase (PPE), and received daily ß-aminopropionitrile (BAPN) to promote AAA rupture. Animals with AAAs received either a standard diet (SD), ketogenic diet (KD), or exogenous KBs (EKB). Animals recieving KD and EKB reached a state of ketosis, and had significant reduction in AAA expansion and incidence of rupture. Ketosis also led to significantly reduced aortic CCR2 content, improved MMP balance, and reduced ECM degradation. In summary, this study demonstrates that ketosis plays a crucial role in AAA pathobiology, and provides the impetus for future clinical studies investigating the potential benefit of ketosis for prevention of AAA expansion and rupture.

Expansion of Disease Specific Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

Background: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1/PD-L1 or CTLA4 have revolutionized cancer management but are associated with devastating immune-related adverse events (irAEs) 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. While much has been learned regarding the role of T-cells in ICI-myocarditis, little is understood regarding the identity, transcriptional diversity, and functions of infiltrating macrophages. Methods: We employed 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 and molecular imaging and antibody neutralization studies. Results: We observed marked increases in CCR2 + 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 , Cxcl10 , Gbp2b , and Fcgr4 that originated from CCR2 + monocytes. Importantly, a similar macrophage population expressing CXCL9 , CXCL10 , and CD16α (human homologue of mouse FcgR4) was found selectively expanded in patients with ICI myocarditis compared to other forms of heart failure and myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9 + Cxcl10 + macrophages via IFN-γ and CXCR3 signaling pathways. Depleting CD8 + T-cells, 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. Conclusion: 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.

Ultrasmall, elementary and highly translational nanoparticle X-ray contrast media from amphiphilic iodinated statistical copolymers.

To expand the single-dose duration over which noninvasive clinical and preclinical cancer imaging can be conducted with high sensitivity, and well-defined spatial and temporal resolutions, a facile strategy to prepare ultrasmall nanoparticulate X-ray contrast media (nano-XRCM) as dual-modality imaging agents for positron emission tomography (PET) and computed tomography (CT) has been established. Synthesized from controlled copolymerization of triiodobenzoyl ethyl acrylate and oligo(ethylene oxide) acrylate monomers, the amphiphilic statistical iodocopolymers (ICPs) could directly dissolve in water to afford thermodynamically stable solutions with high aqueous iodine concentrations (>140 mg iodine/mL water) and comparable viscosities to conventional small molecule XRCM. The formation of ultrasmall iodinated nanoparticles with hydrodynamic diameters of ca. 10 nm in water was confirmed by dynamic and static light scattering techniques. In a breast cancer mouse model, in vivo biodistribution studies revealed that the 64Cu-chelator-functionalized iodinated nano-XRCM exhibited extended blood residency and higher tumor accumulation compared to typical small molecule imaging agents. PET/CT imaging of tumor over 3 days showed good correlation between PET and CT signals, while CT imaging allowed continuous observation of tumor retention even after 10 days post-injection, enabling longitudinal monitoring of tumor retention for imaging or potentially therapeutic effect after a single administration of nano-XRCM.

Positron Emission Tomography Imaging of Vessel Wall Matrix Metalloproteinase Activity in Abdominal Aortic Aneurysm.

BACKGROUND: Matrix metalloproteinases (MMPs) play a key role in the pathogenesis of abdominal aortic aneurysm (AAA). Imaging aortic MMP activity, especially using positron emission tomography to access high sensitivity, quantitative data, could potentially improve AAA risk stratification. Here, we describe the design, synthesis, characterization, and evaluation in murine AAA and human aortic tissue of a first-in-class MMP-targeted positron emission tomography radioligand, 64Cu-RYM2. METHODS: The broad spectrum MMP inhibitor, RYM2 was synthetized, and its potency as an MMP inhibitor was evaluated by a competitive inhibition assay. Toxicology studies were performed. Tracer biodistribution was evaluated in a murine model of AAA induced by angiotensin II infusion in Apolipoprotein E-deficient mice. 64Cu-RYM2 binding to normal and aneurysmal human aortic tissues was assessed by autoradiography. RESULTS: RYM2 functioned as an MMP inhibitor with nanomolar affinities. Toxicology studies showed no adverse reaction in mice. Upon radiolabeling with Cu-64, the resulting tracer was stable in murine and human blood in vitro. Biodistribution and metabolite analysis in mice showed rapid renal clearance and acceptable in vivo stability. In vivo positron emission tomography/computed tomography in a murine model of AAA showed a specific aortic signal, which correlated with ex vivo measured MMP activity and Cd68 gene expression. 64Cu-RYM2 specifically bound to normal and aneurysmal human aortic tissues in correlation with MMP activity. CONCLUSIONS: 64Cu-RYM2 is a first-in-class MMP-targeted positron emission tomography tracer with favorable stability, biodistribution, performance in preclinical AAA, and importantly, specific binding to human tissues. These data set the stage for 64Cu-RYM2-based translational imaging studies of vessel wall MMP activity, and indirectly, inflammation, in AAA.

Cardiac immune cell infiltration associates with abnormal lipid metabolism.

CD36 mediates the uptake of long-chain fatty acids (FAs), a major energy substrate for the myocardium. Under excessive FA supply, CD36 can cause cardiac lipid accumulation and inflammation while its deletion reduces heart FA uptake and lipid content and increases glucose utilization. As a result, CD36 was proposed as a therapeutic target for obesity-associated heart disease. However, more recent reports have shown that CD36 deficiency suppresses myocardial flexibility in fuel preference between glucose and FAs, impairing tissue energy balance, while CD36 absence in tissue macrophages reduces efferocytosis and myocardial repair after injury. In line with the latter homeostatic functions, we had previously reported that CD36-/- mice have chronic subclinical inflammation. Lipids are important for the maintenance of tissue homeostasis and there is limited information on heart lipid metabolism in CD36 deficiency. Here, we document in the hearts of unchallenged CD36-/- mice abnormalities in the metabolism of triglycerides, plasmalogens, cardiolipins, acylcarnitines, and arachidonic acid, and the altered remodeling of these lipids in response to an overnight fast. The hearts were examined for evidence of inflammation by monitoring the presence of neutrophils and pro-inflammatory monocytes/macrophages using the respective positron emission tomography (PET) tracers, 64Cu-AMD3100 and 68Ga-DOTA-ECL1i. We detected significant immune cell infiltration in unchallenged CD36-/- hearts as compared with controls and immune infiltration was also observed in hearts of mice with cardiomyocyte-specific CD36 deficiency. Together, the data show that the CD36-/- heart is in a non-homeostatic state that could compromise its stress response. Non-invasive immune cell monitoring in humans with partial or total CD36 deficiency could help evaluate the risk of impaired heart remodeling and disease.

C-X-C Chemokine Receptor Type 4-Targeted Imaging in Glioblastoma Multiforme Using 64Cu-Radiolabeled Ultrasmall Gold Nanoclusters.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary malignant brain cancer in adults, and it carries a poor prognosis. Despite the current multimodality treatment, including surgery, radiation, and chemotherapy, the overall survival is still poor. Neurooncological imaging plays an important role in the initial diagnosis and prediction of the treatment response of GBM. Positron emission tomography (PET) imaging using radiotracers that target disease-specific hallmarks, which are both noninvasive and specific, has drawn much attention. C-X-C chemokine receptor 4 (CXCR4) plays an important role in neoangiogenesis and vasculogenesis, and, moreover, it is reported to be overexpressed in GBM, which is associated with poor patient survival; thus, CXCR4 can be an ideal candidate for PET imaging of GBM. Nanomaterials, which possess multifunctional capabilities, effective drug delivery, and favorable pharmacokinetics, are now being applied to improve the diagnosis and therapy of the most difficult-to-treat cancers. Herein, we engineered an ultrasmall, renal-clearable gold nanoclusters intrinsically radiolabeled with 64Cu (64Cu-AuNCs-FC131) for targeted PET imaging of CXCR4 in a U87 intracranial GBM mouse model. These targeted nanoclusters demonstrated specific binding to U87 cells with minimal cytotoxicity. The in vivo biodistribution showed favorable pharmacokinetics and efficient renal clearance. PET/computed tomography imaging of the U87 model revealed the effective delivery of 64Cu-AuNCs-FC131 into the tumors. In vivo toxicity studies demonstrated insignificant safety concerns at various dosages, indicating its potential as a useful platform for GBM imaging and drug delivery.

Brown adipose tissue monocytes support tissue expansion.

Monocytes are part of the mononuclear phagocytic system. Monocytes play a central role during inflammatory conditions and a better understanding of their dynamics might open therapeutic opportunities. In the present study, we focused on the characterization and impact of monocytes on brown adipose tissue (BAT) functions during tissue remodeling. Single-cell RNA sequencing analysis of BAT immune cells uncovered a large diversity in monocyte and macrophage populations. Fate-mapping experiments demonstrated that the BAT macrophage pool requires constant replenishment from monocytes. Using a genetic model of BAT expansion, we found that brown fat monocyte numbers were selectively increased in this scenario. This observation was confirmed using a CCR2-binding radiotracer and positron emission tomography. Importantly, in line with their tissue recruitment, blood monocyte counts were decreased while bone marrow hematopoiesis was not affected. Monocyte depletion prevented brown adipose tissue expansion and altered its architecture. Podoplanin engagement is strictly required for BAT expansion. Together, these data redefine the diversity of immune cells in the BAT and emphasize the role of monocyte recruitment for tissue remodeling.

Resident cardiac macrophages mediate adaptive myocardial remodeling.

Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2+) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2-) macrophages. Herein, we identified an essential role for CCR2- macrophages in the chronically failing heart. Depletion of CCR2- macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2- macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4)-dependent pathway that controlled growth factor expression. These findings establish a role for tissue-resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation.

Assessment of ultrasmall nanocluster for early and accurate detection of atherosclerosis using positron emission tomography/computed tomography.

The development of atherosclerosis therapy is hampered by the lack of molecular imaging tools to identify the relevant biomarkers and determine the dynamic variation in vivo. Here, we show that a chemokine receptor 2 (CCR2) targeted gold nanocluster conjugated with extracellular loop 1 inverso peptide (AuNC-ECL1i) determines the initiation, progression and regression of atherosclerosis in apolipoprotein E knock-out (ApoE-/-) mouse models. The CCR2 targeted 64Cu-AuNC-ECL1i reveals sensitive detection of early atherosclerotic lesions and progression of plaques in ApoE-/- mice. CCR2 targeting specificity was confirmed by the competitive receptor blocking studies. In a mouse model of aortic arch transplantation, 64Cu-AuNC-ECL1i accurately detects the regression of plaques. Human atherosclerotic tissues show high expression of CCR2 related to the status of the disease. This study confirms CCR2 as a useful marker for atherosclerosis and points to the potential of 64Cu-AuNC-ECL1i as a targeted molecular imaging probe for future clinical translation.

CC Chemokine Receptor 5 Targeted Nanoparticles Imaging the Progression and Regression of Atherosclerosis Using Positron Emission Tomography/Computed Tomography.

Chemokines and chemokine receptors play an important role in the initiation and progression of atherosclerosis by mediating the trafficking of inflammatory cells. Chemokine receptor 5 (CCR5) has major implications in promoting the development of plaques to advanced stage and related vulnerability. CCR5 antagonist has demonstrated the effective inhibition of atherosclerotic progression in mice, making it a potential biomarker for atherosclerosis management. To accurately determine CCR5 in vivo, we synthesized CCR5 targeted Comb nanoparticles through a modular design and construction strategy with control over the physiochemical properties and functionalization of CCR5 targeting peptide d-Ala-peptide T-amide (DAPTA-Comb). In vivo pharmacokinetic evaluation through 64Cu radiolabeling showed extended blood circulation of 64Cu-DAPTA-Combs conjugated with 10%, 25%, and 40% DAPTA. The different organ distribution profiles of the three nanoparticles demonstrated the effect of DAPTA on not only physicochemical properties but also targeting efficiency. In vivo positron emission tomography/computed tomography (PET/CT) imaging in an apolipoprotein E knockout mouse atherosclerosis model (ApoE-/-) showed that the three 64Cu-DAPTA-Combs could sensitively and specifically detect CCR5 along the progression of atherosclerotic lesions. In an ApoE-encoding adenoviral vector (AAV) induced plaque regression ApoE-/- mouse model, decreased monocyte recruitment, CD68+ macrophages, CCR5 expression, and plaque size were all associated with reduced PET signals, which not only further confirmed the targeting efficiency of 64Cu-DAPTA-Combs but also highlighted the potential of these targeted nanoparticles for atherosclerosis imaging. Moreover, the up-regulation of CCR5 and colocalization with CD68+ macrophages in the necrotic core of ex vivo human plaque specimens warrant further investigation for atherosclerosis prognosis.

CC Chemokine Receptor 2-Targeting Copper Nanoparticles for Positron Emission Tomography-Guided Delivery of Gemcitabine for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with dire prognosis due to aggressive biology, lack of effective tools for diagnosis at an early stage, and limited treatment options. Detection of PDAC using conventional radiographic imaging is limited by the dense, hypovascular stromal component and relatively scarce neoplastic cells within the tumor microenvironment (TME). The CC motif chemokine 2 (CCL2) and its cognate receptor CCR2 (CCL2/CCR2) axis are critical in fostering and maintaining this kind of TME by recruiting immunosuppressive myeloid cells such as the tumor-associated macrophages, thereby presenting an opportunity to exploit this axis for both diagnostic and therapeutic purposes. We engineered CCR2-targeting ultrasmall copper nanoparticles (Cu@CuOx) as nanovehicles not only for targeted positron emission tomography imaging by intrinsic radiolabeling with 64Cu but also for loading and delivery of the chemotherapy drug gemcitabine to PDAC. This 64Cu-radiolabeled nanovehicle allowed sensitive and accurate detection of PDAC malignancy in autochthonous genetically engineered mouse models. The ultrasmall Cu@CuOx showed efficient renal clearance, favorable pharmacokinetics, and minimal in vivo toxicity. Systemic administration of gemcitabine-loaded Cu@CuOx effectively suppressed the progression of PDAC tumors in a syngeneic xenograft mouse model and prolonged survival. These CCR2-targeted ultrasmall nanoparticles offer a promising image-guided therapeutic agent and show great potential for translation.

Targeted PET Imaging of Chemokine Receptor 2-Positive Monocytes and Macrophages in the Injured Heart.

Proinflammatory macrophages are important mediators of inflammation after myocardial infarction and of allograft injury after heart transplantation. The aim of this study was to image the recruitment of proinflammatory chemokine receptor 2-positive (CCR2+) cells in multiple heart injury models. Methods:64Cu-DOTA-extracellular loop 1 inverso (ECL1i) PET was used to image CCR2+ monocytes and macrophages in a heart transplantation mouse model. Flow cytometry was performed to characterize CCR2+ cells. Autoradiography on a human heart specimen was conducted to confirm binding specificity. 64Cu- and 68Ga-DOTA-ECL1i were compared in an ischemia-reperfusion injury mouse model. Results:64Cu-DOTA-ECL1i showed sensitive and specific detection of CCR2+ cells in all tested mouse models, with efficacy comparable to that of 68Ga-DOTA-ECL1i. Flow cytometry demonstrated specific expression of CCR2 on monocytes and macrophages. The tracer binds to human CCR2. Conclusion: This work establishes the utility of 64Cu-DOTA-ECL1i to image CCR2+ monocytes and macrophages in mouse models and provides the requisite preclinical information to translate the targeted clinical-grade CCR2 imaging probe for clinical investigation of heart diseases.