Activation of quiescent cardiac fibroblasts following myocardial infarction is essential for adequate healing, but persistent or excessive activation may be detrimental and promote adverse remodeling. Fibroblast activation protein (FAP), a key marker of activated fibroblasts, has emerged as a potential therapeutic target that can be measured by noninvasive positron emission tomography. But the temporal and regional expression of FAP is uneven and its sensitivity to intervention has not been elucidated. In cultured human cardiac fibroblasts 68Ga-FAPI-46 sensitively measured elevated FAP expression in response to pro-fibrotic transforming growth factor-β. Serial molecular imaging after coronary artery ligation and reperfusion in mice revealed increased FAP imaging signal at 3d that extended beyond the infarct territory. The signal returned to sham levels at 7d but recurred at 14d. Early FAP elevation was linked to inflammation in cultured cardiac fibroblasts, where FAP signal increased in response to inflammatory macrophage medium, and in acute MI patients, for whom increased FAP signal volume was associated with circulating inflammatory biomarkers. Treatment with angiotensin converting enzyme inhibitor enalapril lowered inflammatory cell content in the left ventricle at 3d with a parallel reduction in FAP imaging signal. The subchronic FAP signal at 14d remained elevated. Treatment using chimeric antigen receptor natural killer cells against FAP at 14d after infarction did not attenuate FAP imaging signal, though immunofluorescence of FAP was reduced. These data demonstrate dynamic fibroblast activation after myocardial infarction, where early activation is associated with cardiac inflammation. These findings support imaging guidance to optimize therapeutic intervention against FAP after ischemic injury.

Different immune cell populations influence the severity of cardiac inflammation after myocardial infarction (MI), which predicts subsequent heart failure. Concurrent neuroinflammation after MI may contribute to heightened risk of cognitive impairment, but the underlying mechanisms remain unclear. We characterized the role of macrophages and neutrophils in concurrent neuroinflammation after acute MI using PET. Methods: C57BL/6N mice underwent permanent coronary artery ligation to induce MI. To modulate specific immune populations, peripheral macrophages were depleted using clodronate-loaded liposomes, and neutrophils were inhibited using a neutralizing antibody against Ly6G. Serial molecular PET imaging of mitochondrial translocator protein (TSPO) visualized inflammation in the heart and brain over 8 wk. Ventricle geometry and contractile function were assessed by cardiac MRI. Results: Acute MI induced microglial activation identified by increased TSPO PET signal in the global brain in parallel with infarct region inflammation. Macrophage depletion before injury ablated macrophage content in the left ventricle on TSPO PET and histopathology but paradoxically increased the neuroinflammation signal in the first week after MI. Persistent neutrophil activity in the left ventricle of macrophage-depleted animals suggested a role for granulocytes in mediating heart-brain immune cell cross-talk. However, antibody-mediated inhibition of neutrophil activity before or at 24 h after MI did not reduce acute neuroinflammation but rather increased incidence of left ventricle rupture. Flow cytometry identified accelerated monocyte repopulation in the damaged left ventricle after macrophage depletion and subsequent neutrophil inhibition, underscoring the interaction between these immune cell populations in post-MI healing. Conclusion: Intact neutrophil-macrophage signaling is essential for effective myocardial repair after ischemic injury, but neutrophil persistence does not independently regulate neuroinflammation after MI. Nonetheless, PET imaging sensitively monitors response to immunomodulatory intervention and enables tracking of therapeutic efficacy at the site of injury and distant organ systems.

RATIONALE:Mutations in the KRAS gene are some of the most frequent drivers in cancer, including non-small cell lung and colorectal cancer. Covalent inhibitors such as adagrasib, targeting the inactive, GDP-bound form of KRAS-G12C, have shown clinical efficacy but patient selection still depends on invasive biopsies. This study aimed to develop novel fluorine-18 labeled KRAS-G12C inhibitors for noninvasive positron emission tomography (PET) imaging of KRAS mutation status. METHODS:Three KRAS-G12C inhibitors were synthesized by modifying the adagrasib scaffold to allow for radiofluorination. Compounds were evaluated for target affinity and specificity using pERK inhibition assays in KRAS-G12C positive MiaPaCa-2 cells and KRAS protein binding assays. Finally, in vivo PET imaging and biodistribution studies were performed in MiaPaCa-2 xenograft-bearing mice under baseline and blocking conditions to assess tumor uptake, specificity, and tracer pharmacokinetics. RESULTS:In µPET, the tracers showed distinct tumor uptake and biodistribution profiles. Among them, [18F]KRAS490 demonstrated the most promising characteristics, with clear tumor accumulation that was significantly reduced under blocking conditions, indicating specific target binding. In contrast, [18F]KRAS8125 and [18F]KRAS3776 showed limited or non-replaceable tumor uptake. The tracers showed varied clearance patterns, with [18F]KRAS490 showing combined hepatobiliary and renal clearance. Notably, [18F]KRAS490 also entered the brain, suggesting the potential for central nervous system imaging. CONCLUSIONS:[18F]KRAS490 showed specific, blockable tumor uptake and favorable pharmacokinetics, making it a promising tracer for noninvasive imaging of KRAS-G12C mutant tumors. Its ability to penetrate the CNS supports potential application in imaging both peripheral and brain lesions.

Macrophage depletion before myocardial infarction (MI) in mice disrupts the inflammatory healing response and leads to left ventricular thrombus formation and tissue calcification. Here, we aimed to determine the effect of clodronate-loaded liposome-mediated macrophage depletion on C-C chemokine receptor 2 (CCR2)-expressing immune cells after MI in male and female mice. Further, we sought to discern the relationship of clodronate liposomes with thrombus calcification and functional outcome using multitracer imaging. Methods: Male and female C57BL/6N mice underwent 60-min ischemia/reperfusion MI or sham surgery after a single or repeated injection of clodronate liposomes for macrophage depletion or injection with liposomes containing phosphate-buffered saline. Macrophage depletion was verified in peripheral blood. PET imaging was performed to assess infarct CCR2 signal at 2 or 4 d after MI and fibroblast activation protein expression at 7 d after MI. Microcalcification was assessed by 18F-NaF PET/CT over 4 wk. Cardiac function was assessed at 6 wk by MRI. Histopathology validated inflammation and calcification in the heart. Results: Macrophage depletion significantly increased susceptibility to early death after MI, especially in male mice. Molecular imaging revealed an early increase in cardiac CCR2 content after MI compared with sham, which persisted despite macrophage depletion. Histology verified reduced CD68 cell content but sustained CCR2 signal that partially overlapped CD11c, a marker of dendritic cells among other cell types. Ly6G-positive cell content increased in the myocardium of macrophage-depleted mice after MI but did not overlap with CCR2. All macrophage-depleted male mice and 64% of macrophage-depleted female mice developed a dense intracavity thrombus overlying endocardial damage, with gradual microcalcification starting from 2 wk and reaching a maximum at 4 wk after MI. Macrophage depletion did not impact infarct size or contractile function at 6 wk after MI in surviving mice. Conclusion: Macrophage depletion before MI elevated the risk of acute death, particularly in male mice. A persistent CCR2 PET signal suggested that non-CD68-positive immune cells contribute to tracer substrate and altered proportions of other immune cell subtypes during healing. These findings suggest that extreme antiinflammatory interventions after MI may have unintended consequences for scar formation and remodeling, which may be monitored by molecular imaging.

Purpose:After cochlear implantation, molecular processes at the electrode-nerve interface significantly influence the variability in clinical outcomes. The present study investigates molecular processes in a guinea pig model of cochlear implant (CI) using positron emission tomography/computed tomography (PET/CT) and correlates the imaging findings with histological analyses. Methods:Animals were examined with PET in the 3 weeks and 9-12 months post-implantation using the inflammation marker [18F]FDG and, at the later time points, [68Ga]FAPI-46 as a marker for fibrosis. Tracer accumulation in the cochlea was determined from PET imaging based on the co-registered CT. Nine animals (seven with unilateral CI) were included. Uptake in non-implanted cochleae served as reference. Tissue growth around the implant was evaluated histologically. Results:Post-implantation, [18F]FDG uptake was significantly increased when pooling early and late in investigation time points, while after 1 year, [68Ga]FAPI-46 uptake was increased inside the cochlear. Cochlear volumes measured by CT did not show significant differences between compared groups. Tissue growth around the implant was observed in all animals, with a trend toward increased growth associated with insertion depth. However, no clear correlation was observed between the extent of tissue growth and the uptake intensities of FDG and FAPI. Discussion:The data indicate that increased accumulation of PET biomarkers in the cochlea after implantation can be detected in guinea pigs using a dedicated PET/CT. Given the high resolution of current clinical PET/CT devices, this method is expected to be suitable for use in patients, particularly for assessing the effect of anti-inflammatory or anti-fibrotic therapies.

Abstract Introduction Following myocardial infarction (MI), orchestrated infiltration of diverse leukocyte subtypes contributes to local repair. Systemic and concurrent neuroinflammation is thought to contribute to cognitive impairment after MI. We have demonstrated that macrophage depletion impairs ventricle healing and exacerbates neuroinflammation early after MI, with persistence of cardiac neutrophils. Purpose We aimed to explore the role of neutrophils in neuroinflammation post-MI in macrophage-depleted mice using timed anti-neutrophil antibody dosing and multiparametric imaging. Methods Male C57Bl6N mice received two distinct treatments: initially clodronate-loaded liposomes intravenously (n=27) to deplete peripheral macrophages. Subsequently, 24h later all mice underwent permanent ligation of the left coronary artery. These mice also received treatment at 24h after MI– with anti-Ly6G therapeutic antibody (n=15) for neutrophil depletion or with an isotype control antibody (n=12). Longitudinal TSPO PET/CT with 18F-GE180 at 3d, 7d, and 8w after MI measured cardiac and brain inflammation. SPECT/CT defined infarct size and CMR calculated contractile function at 8w. Correlative analyses including flow cytometry, immunostaining, and behavioural experiments validated PET data. Results Neutrophil inhibition 24h post-MI reduced incidence of LV rupture compared to clodronate alone (33% vs. 66%). However, isotype and Ly6G treated survivors at 8w showed similar contractile functional impairment (%EF 25±9 vs 16±7%, p=0.071) and perfusion defects (%LV 37±8 vs 40±9, p=0.64). Fluorescence associated cell sorting demonstrated successful neutrophil depletion in the infarct territory (cells x103 39±11 vs 9±4, p<0.01), and a higher proportion of anti-inflammatory Ly6Clow monocytes (cells x103 32±19 vs 70±10, p<0.05). In anti-Ly6G and isotype control treatment groups, the 18F-GE180 signal was lower in the infarct region 3d after MI (%ID/g 5.3±1.5 vs 5.8±2.4, p=0.55), reflecting selectivity to macrophages. Despite successful neutrophil inhibition in the myocardium, neuroinflammation was not significantly lower in anti-Ly6G treated compared to isotype control macrophage depleted mice post-MI (%ID/g 2.3±0.5 vs 2.7±1.0, p=0.27). This may suggest severity of functional impairment, rather than persistent neutrophil activity, is a more prominent contributor to microglial activation after MI. Conclusion Unrestricted neutrophil activity impedes the acute healing process of the infarct, resulting in increased ventricle rupture- a phenomenon reminiscent of infarct expansion observed in patients. The healing process after MI and the associated neuroinflammation constitute a multifaceted phenomenon that cannot be solely attributed to a single proinflammatory leukocyte subpopulation i.e. neutrophils. Effective therapeutic interventions may need to target entire systems rather than focusing on individual leukocyte cell types for the prevention of cardiac and cognitive dysfunction.Figure 1.Schematic timeline Figure 2.Supporting results

Abstract Background/Introduction Myeloid-derived inflammatory macrophages expressing CCR2 are crucial for scar formation and repair after myocardial infarction (MI). Interference with macrophage dynamics impairs healing and promotes intraventricular thrombus formation, but the precise dynamics of CCR2 macrophages in the left ventricle (LV) under these conditions remain unclear. Purpose We investigated the impact of macrophage depletion on infarct CCR2 expression, microcalcification, and cardiac functional outcome in mice after reperfused MI. Methods Male and female C57BL/6N mice underwent 60 min ischemia/reperfusion MI (n=24) or sham (n=8) 24h after injection of clodronate-liposomes (n=8m, 8f) for macrophage depletion, or PBS-liposomes (n=8m, 8f). Infarct CCR2 expression was measured on MI+4d by 68Ga-ECL1i PET. Microcalcification was assessed by 18F-NaF PET/CT at 4wks. MRI and 99mTc-tetrofosmin SPECT calculated LV function and infarct sizes at 6wks. Results Acute mortality was markedly higher after macrophage depletion within 7d of MI compared to PBS (%, 37.5 vs 0). Infarct CCR2 expression was significantly increased in macrophage depletion MI vs sham on d4 (%injected dose (ID)/g; 0.39±0.13 vs 0.25±0.05; p=0.005), but similar between PBS MI and sham (ID/g; 0.31±0.06, p=0.1), indicating rapid repopulation of peripheral monocytes after single time macrophage depletion, and delayed infiltration into injured myocardium. There were no sex dependent differences in infarct CCR2 expression. Infarct size after MI was 25.9±9.3 %LV with no difference between treatments. LV ejection fraction at 6wks was significantly reduced after MI vs sham, but comparable between treatments (%; clodronate: 33±9 vs PBS: 37±8 vs sham 66±5, p<0.001). Heart weight at 6wks was significantly increased only in macrophage depleted MI compared to sham (heart weight/body weight (mg/g); 5.5±0.8 vs 4.6±0.4, p=0.02). Male macrophage-depleted mice and 50% of female mice exhibited a dense intracavity thrombus adherent to the infarct wall after MI, which showed significantly higher 18F-NaF uptake at 4wks compared to PBS MI (%ID/g; 6.0±4.9 vs 0.4±0.3; p<0.001), highlighting necessity of timely infiltration of the infarct with inflammatory macrophages for adequate cardiac repair. Conclusion By 4d after reperfused MI, the myocardium exhibits signs of inflammation resolution and decline of CCR2+ cell content in mice. Single time macrophage depletion prior to MI delays but does not prevent infiltration of CCR2+ inflammatory macrophages into infarct territories. Male mice are at higher risk of LV thrombus formation, demonstrating sex dependent differences in cardiac repair, which seem to be independent from the amount of CCR+ macrophages.

Background Implant infections caused by biofilm forming bacteria are a major threat in orthopedic surgery. Delivering antibiotics directly to an implant affected by a bacterial biofilm via superparamagnetic nanoporous silica nanoparticles could present a promising approach. Nevertheless, short blood circulation half-life because of rapid interactions of nanoparticles with the host’s immune system hinder them from being clinically used. The aim of this study was to determine the temporal in vivo resolution of magnetic nanoporous silica nanoparticle (MNPSNP) distribution and the effect of PEGylation and clodronate application using PET/CT imaging and gamma counting in an implant mouse model. Methods PEGylated and non-PEGylated MNPSNPs were radiolabeled with gallium-68 ( 68 Ga), implementing the chelator tris(hydroxypyridinone). 36 mice were included in the study, 24 mice received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. MNPSNP pharmacokinetics and implant accumulation was analyzed in dependence on PEGylation and additional clodronate application. Subsequently gamma counting was performed for further final analysis. Results The pharmacokinetics and biodistribution of all radiolabeled nanoparticles could clearly be visualized and followed by dynamic PET/CT imaging. Both variants of 68 Ga-labeled MNPSNP accumulated mainly in liver and spleen. PEGylation of the nanoparticles already resulted in lower liver uptakes. Combination with macrophage depletion led to a highly significant effect whereas macrophage depletion alone could not reveal significant differences. Although MNPSNP accumulation around implants was low in comparison to the inner organs in PET/CT imaging, gamma counting displayed a significantly higher %I.D./g for the tissue surrounding the magnetic implants compared to the titanium control. Additional PEGylation and/or macrophage depletion revealed no significant differences regarding nanoparticle accumulation at the implantation site. Conclusion Tracking of 68 Ga-labeled nanoparticles in a mouse model in the first critical hours post-injection by PET/CT imaging provided a better understanding of MNPSNP distribution, elimination and accumulation. Although PEGylation increases circulation time, nanoparticle accumulation at the implantation site was still insufficient for infection treatment and additional efforts are needed to increase local accumulation.