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Objective-Current management of aortic aneurysms (AAs) relies primarily on size criteria to determine whether invasive repair is indicated to preempt rupture. We hypothesized that emerging molecular imaging tools could be used to more sensitively gauge local inflammation. Because macrophages are key effector cells that destabilize the extracellular matrix in the arterial wall, it seemed likely that they would represent suitable imaging targets. We here aimed to develop and validate macrophage-targeted nanoparticles labeled with fluorine-18 ((18)F) for positron emission tomography-computed tomography (PET-CT) detection of inflammation in AAs. Methods: and Results-Aneurysms were induced in apolipoprotein E(-/-) mice via systemic administration of angiotensin II. Mice were imaged using PET-CT and a monocyte/ macrophage-targeted nanoparticle. AAs were detected by contrast-enhanced micro-CT and had a mean diameter of 1.85 +/- 0.08 mm, whereas normal aortas measured 1.07 +/- 0.03 (P < 0.05). The in vivo PET signal was significantly higher in aneurysms (standard uptake value, 2.46 +/- 0.48) compared with wild-type aorta (0.82 +/- 0.05, P < 0.05). Validation with scintillation counting, autoradiography, fluorescence, and immunoreactive histology and flow cytometry demonstrated that nanoparticles localized predominantly to monocytes and macrophages within the aneurysmatic wall. Conclusion- PET-CT imaging with (18)F-labeled nanoparticles allows quantitation of macrophage content in a mouse model of AA. (Arterioscler Thromb Vasc Biol. 2011;31:750-757.), Accepted Manuscript

Purpose: A sensitive and specific imaging biomarker to monitor immune activation and quantify pharmacodynamic responses would be useful for development of immunomodulating anti-cancer agents. PF-07062119 is a T cell engaging bispecific antibody that binds to CD3 and guanylyl cyclase C, a protein that is over-expressed by colorectal cancers. Here, we used 89 Zr-Df-IAB22M2C ( 89 Zr-Df-Crefmirlimab), a human CD8-specific minibody to monitor CD8+ T cell infiltration into tumors by positron emission tomography. We investigated the ability of 89 Zr-Df-IAB22M2C to track anti-tumor activity induced by PF-07062119 in a human CRC adoptive transfer mouse model (with injected activated/expanded human T cells), as well as the correlation of tumor radiotracer uptake with CD8+ immunohistochemical staining., Procedures: NOD SCID gamma mice bearing human CRC LS1034 tumors were treated with four different doses of PF-07062119, or a non-targeted CD3 BsAb control, and imaged with 89 Zr-Df-IAB22M2C PET at days 4 and 9. Following PET/CT imaging, mice were euthanized and dissected for ex vivo distribution analysis of 89 Zr-Df-IAB22M2C in tissues on days 4 and 9, with additional data collected on day 6 (supplementary). Data were analyzed and reported as standard uptake value and %ID/g for in vivo imaging and ex vivo tissue distribution. In addition, tumor tissues were evaluated by immunohistochemistry for CD8+ T cells., Results: The results demonstrated substantial mean uptake of 89 Zr-Df-IAB22M2C (%ID/g) in PF-07062119-treated tumors, with significant increases in comparison to non-targeted BsAb-treated controls, as well as PF-07062119 dose-dependent responses over time of treatment. A moderate correlation was observed between tumor tissue radioactivity uptake and CD8+ cell density, demonstrating the value of the imaging agent for non-invasive assessment of intra-tumoral CD8+ T cells and the mechanism of action for PF-07062119., Conclusion: Immune-imaging technologies for quantitative cellular measures would be a valuable biomarker in immunotherapeutic clinical development. We demonstrated a qualification of 89 Zr-IAB22M2C PET to evaluate PD responses (mice) to a novel immunotherapeutic., (© 2021. The Author(s).)

Acute bacterial endocarditis is a rapid, difficult to manage, and frequently lethal disease. Potent antibiotics often cannot efficiently kill Staphylococcus aureus that colonizes the heart's valves. S. aureus relies on virulence factors to evade therapeutics and the host's immune response, usurping the host's clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor-binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance. Targeting virulence factors may provide previously unidentified avenues to better diagnose and treat endocarditis. To tap into this unused therapeutic opportunity, we codeveloped therapeutics and multimodal molecular imaging to probe the host-pathogen interface. We introduced and validated a family of small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies. The imaging agents, based on the clinical thrombin inhibitor dabigatran, are bound to heart valve vegetations in mice. Using optical imaging, we monitored therapy with antibodies neutralizing staphylocoagulase and von Willebrand factor-binding protein in mice with S. aureus endocarditis. This treatment deactivated bacterial defenses against innate immune cells, decreased in vivo imaging signal, and improved survival. Aortic or tricuspid S. aureus endocarditis in piglets was also successfully imaged with clinical PET/magnetic resonance imaging. Our data map a route toward adjuvant immunotherapy for endocarditis and provide efficient tools to monitor this drug class for infectious diseases., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Myeloperoxidase (MPO) is a critical proinflammatory enzyme implicated in cardiovascular, neurological, and rheumatological diseases. Emerging therapies targeting inflammation have raised interest in tracking MPO activity in patients. We describe 18 F-MAPP, an activatable MPO activity radioprobe for positron emission tomography (PET) imaging. The activated radioprobe binds to proteins and accumulates at sites of MPO activity. The radioprobe 18 F-MAPP has a short blood half-life, remains stable in plasma, does not demonstrate cytotoxicity, and crosses the intact blood-brain barrier. The 18 F-MAPP imaging detected sites of elevated MPO activity in living mice embedded with human MPO and in mice induced with chemical inflammation or myocardial infarction. The 18 F-MAPP PET imaging noninvasively differentiated varying amounts of MPO activity, competitive inhibition, and MPO deficiency in living animals, confirming specificity and showing that the radioprobe can quantify changes in in vivo MPO activity. The radiosynthesis has been optimized and automated, an important step in translation. These data indicate that 18 F-MAPP is a promising translational candidate to noninvasively monitor MPO activity and inflammation in patients., Competing Interests: Conflict of interest statement: This study was partly funded by a research grant from Pfizer. L.B., J.C., and K.M. are employees of Pfizer. However, Pfizer did not influence the analysis or interpretation of the data. C.W., E.K., and J.W.C. have filed a patent application on the imaging technology described in the article.

Currently, there is no stable and flexible method to label and track cytotoxic T lymphocytes (CTLs) in vivo in CTL immunotherapy. We aimed to evaluate whether the sulfo-hydroxysuccinimide (NHS)-biotin-streptavidin (SA) platform could chemically modify the cell surface of CTLs for in vivo tracking. CD8+ T lymphocytes were labeled with sulfo-NHS-biotin under different conditions and then incubated with SA-Alexa647. Labeling efficiency was proportional to sulfo-NHS-biotin concentration. CD8+ T lymphocytes could be labeled with higher efficiency with sulfo-NHS-biotin in DPBS than in RPMI (P < 0.05). Incubation temperature was not a key factor. CTLs maintained sufficient labeling for at least 72 h (P < 0.05), without altering cell viability. After co-culturing labeled CTLs with mouse glioma stem cells (GSCs) engineered to present biotin on their surface, targeting CTLs could specifically target biotin-presenting GSCs and inhibited cell proliferation (P < 0.01) and tumor spheres formation. In a biotin-presenting GSC brain tumor model, targeting CTLs could be detected in biotin-presenting gliomas in mouse brains but not in the non-tumor-bearing contralateral hemispheres (P < 0.05). In vivo fluorescent molecular tomography imaging in a subcutaneous U87 mouse model confirmed that targeting CTLs homed in on the biotin-presenting U87 tumors but not the control U87 tumors. PET imaging with 89 Zr-deferoxamine-biotin and SA showed a rapid clearance of the PET signal over 24 h in the control tumor, while only minimally decreased in the targeted tumor. Thus, sulfo-NHS-biotin-SA labeling is an efficient method to noninvasively track the migration of adoptive transferred CTLs and does not alter CTL viability or interfere with CTL-mediated cytotoxic activity., Competing Interests: The authors declare that they have no conflict of interest.

We generated 18 F-labeled antibody fragments for PET imaging using a sortase-mediated reaction to install a transcyclooctene (TCO)-functionalized short peptide onto proteins of interest, followed by reaction with a tetrazine-labeled- 18 F-2-deoxyfluoroglucose (FDG). The method is rapid, robust, and site-specific (radiochemical yields >25%, not decay corrected). The availability of 18 F-2-deoxyfluoroglucose avoids the need for more complicated chemistries used to generate carbon-fluorine bonds. We demonstrate the utility of the method by detecting heterotopic pancreatic tumors in mice by PET, using anti-Class II MHC single domain antibodies. We correlate macroscopic PET images with microscopic two-photon visualization of the tumor. Our approach provides easy access to 18 F-labeled antibodies and their fragments at a level of molecular specificity that complements conventional 18 F-FDG imaging.

Background: Macrophages predominate among the inflammatory cells in rejecting allografts. These innate immune cells, in addition to allospecific T cells, can damage cardiomyocytes directly., Methods and Results: We explored whether sensitive positron emission tomography-computed tomography (PET-CT) imaging of macrophages-avid nanoparticles detects rejection of heart allografts in mice. In addition, we used the imaging method to follow the immunomodulatory impact of angiotensin-converting enzyme inhibitor therapy on myeloid cells in allografts. Dextran nanoparticles were derivatized with the PET isotope copper-64 and imaged 7 days after transplantation. C57BL/6 recipients of BALB/c allografts displayed robust positron emission tomography signal (standard uptake value allograft, 2.8±0.3; isograft control, 1.7±0.2; P<0.05). Autoradiography and scintillation counting confirmed the in vivo findings. We then imaged the effects of angiotensin-converting enzyme inhibitor (5 mg/kg enalapril). Angiotensin-converting enzyme inhibitor significantly decreased nanoparticle signal (P<0.05). Histology and flow cytometry showed a reduced number of myeloid cells in the graft, blood, and lymph nodes and diminished antigen presentation (P<0.05 versus untreated allografts). Angiotensin-converting enzyme inhibitor also significantly prolonged allograft survival (12 versus 7 days; P<0.0001)., Conclusions: Nanoparticle macrophage PET-CT detects heart transplant rejection and predicts organ survival by reporting on myeloid cells.

We have developed a multifaceted, highly specific reporter for multimodal in vivo imaging and applied it for detection of brain tumors. A metabolically biotinylated, membrane-bound form of Gaussia luciferase was synthesized, termed mbGluc-biotin. We engineered glioma cells to express this reporter and showed that brain tumor formation can be temporally imaged by bioluminescence following systemic administration of coelenterazine. Brain tumors expressing this reporter had high sensitivity for detection by magnetic resonance and fluorescence tomographic imaging upon injection of streptavidin conjugated to magnetic nanoparticles or fluorophore, respectively. Moreover, single photon emission computed tomography showed enhanced imaging of these tumors upon injection with streptavidin complexed to (111)In-DTPA-biotin. This work shows for the first time a single small reporter (∼40 kDa) which can be monitored with most available molecular imaging modalities and can be extended for single cell imaging using intravital microscopy, allowing real-time tracking of any cell expressing it in vivo.

Monocytes (Mo) and macrophages (MΦ) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MΦ and their effector functions without compromising innate immunity's critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MΦ in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (20 h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by IL-1β; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MΦ exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke. These findings expand our knowledge of Mo/MΦ flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure.

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.

Objective: Current management of aortic aneurysms (AAs) relies primarily on size criteria to determine whether invasive repair is indicated to preempt rupture. We hypothesized that emerging molecular imaging tools could be used to more sensitively gauge local inflammation. Because macrophages are key effector cells that destabilize the extracellular matrix in the arterial wall, it seemed likely that they would represent suitable imaging targets. We here aimed to develop and validate macrophage-targeted nanoparticles labeled with fluorine-18 ((18)F) for positron emission tomography-computed tomography (PET-CT) detection of inflammation in AAs., Methods and Results: Aneurysms were induced in apolipoprotein E-/- mice via systemic administration of angiotensin II. Mice were imaged using PET-CT and a monocyte/macrophage-targeted nanoparticle. AAs were detected by contrast-enhanced micro-CT and had a mean diameter of 1.85 ± 0.08 mm, whereas normal aortas measured 1.07 ± 0.03 (P < 0.05). The in vivo PET signal was significantly higher in aneurysms (standard uptake value, 2.46 ± 0.48) compared with wild-type aorta (0.82 ± 0.05, P < 0.05). Validation with scintillation counting, autoradiography, fluorescence, and immunoreactive histology and flow cytometry demonstrated that nanoparticles localized predominantly to monocytes and macrophages within the aneurysmatic wall., Conclusions: PET-CT imaging with (18)F-labeled nanoparticles allows quantitation of macrophage content in a mouse model of AA.

Fusion imaging of radionuclide-based molecular (PET) and structural data [x-ray computed tomography (CT)] has been firmly established. Here we show that optical measurements [fluorescence-mediated tomography (FMT)] show exquisite congruence to radionuclide measurements and that information can be seamlessly integrated and visualized. Using biocompatible nanoparticles as a generic platform (containing a (18)F isotope and a far red fluorochrome), we show good correlations between FMT and PET in probe concentration (r(2) > 0.99) and spatial signal distribution (r(2) > 0.85). Using a mouse model of cancer and different imaging probes to measure tumoral proteases, macrophage content and integrin expression simultaneously, we demonstrate the distinct tumoral locations of probes in multiple channels in vivo. The findings also suggest that FMT can serve as a surrogate modality for the screening and development of radionuclide-based imaging agents.

Objectives: The aim of this study was to iteratively develop and validate an (18)F-labeled small vascular cell adhesion molecule (VCAM)-1 affinity ligand and demonstrate the feasibility of imaging VCAM-1 expression by positron emission tomography-computed tomography (PET-CT) in murine atherosclerotic arteries., Background: Hybrid PET-CT imaging allows simultaneous assessment of atherosclerotic lesion morphology (CT) and may facilitate early risk assessment in individual patients. The early induction, confinement of expression to atherosclerotic lesions, and accessible position in proximity to the blood pool render the adhesion molecule VCAM-1 an attractive imaging biomarker for inflamed atheroma prone to complication., Methods: A cyclic, a linear, and an oligomer affinity peptide, internalized into endothelial cells by VCAM-1-mediated binding, were initially derivatized with DOTA to determine their binding profiles and pharmacokinetics. The lead compound was then (18)F-labeled and tested in atherosclerotic apoE(-/-) mice receiving a high-cholesterol diet as well as wild type murine models of myocardial infarction and heart transplant rejection., Results: The tetrameric peptide had the highest affinity and specificity for VCAM-1 (97% inhibition with soluble VCAM-1 in vitro). In vivo PET-CT imaging using (18)F-4V showed 0.31 +/- 0.02 SUV in murine atheroma (ex vivo %IDGT 5.9 +/- 1.5). (18)F-4V uptake colocalized with atherosclerotic plaques on Oil Red O staining and correlated to mRNA levels of VCAM-1 measured by quantitative reverse transcription polymerase chain reaction (R = 0.79, p = 0.03). Atherosclerotic mice receiving an atorvastatin-enriched diet had significantly lower lesional uptake (p < 0.05). Furthermore, (18)F-4V imaging in myocardial ischemia after coronary ligation and in transplanted cardiac allografts undergoing rejection showed high in vivo PET signal in inflamed myocardium and good correlation with ex vivo measurement of VCAM-1 mRNA by quantitative polymerase chain reaction., Conclusions: (18)F-4V allows noninvasive PET-CT imaging of VCAM-1 in inflammatory atherosclerosis, has the dynamic range to quantify treatment effects, and correlates with inflammatory gene expression.