Targeting CD47 with monoclonal antibody (mAb) therapy activates tumor-associated macrophages (TAMs). Quantitative imaging methods are important for identifying responders to this novel immunotherapy. The purpose of our study was to investigate whether the metabolic activity of osteosarcomas on 18F-FDG PET/CT changes after CD47 mAb treatment. Twenty female BALB/c mice with intratibial murine K7M2 osteosarcomas, twenty female NOD scid gamma (NSG) mice with intratibial human 143B tumors, and twenty male NSG mice with intratibial human MG63.3 tumors were treated with either phosphate-buffered saline (PBS) or murine/human CD47 mAb (n = 10 per arm) and underwent either 18F-FDG PET/CT or ferumoxytol-enhanced MRI (n = 5 per group). Differences in tumor metabolic activity (%ID/g max), tumor T2* relaxation times, TAM (%F4/80), and M1 macrophage polarization (%CD80+) between PBS and CD47 mAb-treated mice were estimated from linear regression. The tumor %ID/g max of CD47 mAb-treated K7M2 tumors (6.58 ± 2.42) was not significantly different compared to PBS-treated K7M2 tumors (8.04 ± 2.91; p = 0.17). Similarly, the tumor %ID/g max of CD47 mAb-treated 143B tumors (9.12 ± 1.68) and MG63.3 tumors (5.44 ± 1.99) were not significantly different compared to PBS-treated 143B tumors (9.38 ±  2.32; p = 0.32 ) and MG63.3 tumors (6.02 ±  0.63, p = 0.79 ). By comparison, K7M2 tumors, 143B tumors, and MG63.3 tumors all demonstrated significantly shorter T2* relaxation times after CD47 mAb treatment compared to PBS treatment (all p < 0.001). All tumors exhibited significantly higher TAM (%F4/80+) and M1 macrophage polarization (%CD80+) after CD47 mAb treatment compared to PBS treatment (all p < 0.05). The metabolic activity of osteosarcomas on 18F-FDG PET/CT does not show significant changes after CD47 mAb treatment. This lowers the risk of observing pseudoprogression and misinterpreting drug-induced inflammation, simplifying routine clinical scan interpretation.

Despite robust preclinical activity, CD47-targeting agents demonstrate limited single-agent efficacy in osteosarcomas. The purpose of our study was to investigate whether tumor size at baseline affects tumor-associated macrophage (TAM) response to CD47 mAb, as measured by ferumoxytol-MRI. Thirty female NOD SCID gamma mice and thirty BALB/c mice with small, medium-sized, and large 143B osteosarcoma xenografts or murine K7M2 tumors were treated with either CD47 mAb or PBS. All mice underwent MRI scans at baseline, one week after treatment, and after intravenous infusion of ferumoxytol nanoparticles. We measured tumor T2* relaxation times as a quantitative measure of nanoparticle retention in activated TAMs. Tumor ΔT2* was calculated as the difference between T2* post-treatment pre-contrast and T2* post-treatment post-contrast to quantify ferumoxytol enhancement. T2* and ΔT2* values were compared between treatment groups using regression models including treatment, tumor size, and their interaction. Histology served as a standard of reference. At baseline, tumors in CD47 mAb- and PBS-treated mice demonstrated no significant differences in T2* relaxation time, regardless of tumor size (all p > 0.05). After CD47 mAb therapy and ferumoxytol infusion, tumors treated with CD47 mAb demonstrated significantly shorter T2* relaxation times compared with PBS-treated tumors (all p < 0.001). The tumor ΔT2* enhancement was significantly higher in small tumors compared with large tumors (all p < 0.001), indicating higher nanoparticle accumulation in small tumors after CD47 blockade. Histopathology showed a significantly higher F4/80+CD80+ TAM staining in CD47 mAb-treated small tumors compared with large tumors (all p < 0.05). In conclusion, tumor size at baseline affects TAM response to CD47 mAb. Smaller osteosarcomas exhibit a more pronounced TAM response to CD47 mAb therapy compared with larger tumors.

and Purpose. Activation of tumor-associated macrophages (TAMs) by CD47 mAb therapy can be monitored with ferumoxytol-enhanced magnetic resonance imaging (MRI). The purpose of our study was to investigate if tumor size at baseline affects TAM response to CD47 mAb, as measured with ferumoxytol-MRI. Thirty female NOD scid gamma (NSG) mice with small-sized (tumor volume = 30 mm3), medium-sized (tumor volume = 500 mm3), and large (tumor volume = 1200 mm3) 143B osteosarcoma xenografts were treated with human CD47 mAb (10 mg/kg, day 1, 3, and 5 for 1 week) or PBS (control group). All mice underwent MRI scans at baseline and at one week after CD47 mAb or sham treatment. Post-treatment scans were obtained before and after intravenous infusion of ferumoxytol nanoparticles (30 Fe mg/kg). We measured tumor T2* relaxation times as a quantitive measure of nanoparticle retention in TAM. The tumor ferumoxytol enhancement, calculated as: ΔT2* = 100 * (T2*precontrast - T2*postcontrast) / T2*precontrast, was compared between experimental groups using exact two-sided Wilcoxon rank-sum tests. Histology served as standard of reference. At baseline, tumors of CD47 mAb and PBS-treated mice demonstrated no significant difference in T2* relaxation time, regardless of tumor size (all p>0.05). After CD47 mAb therapy, tumors treated with CD47 mAb demonstrated significantly shorter T2* relaxation times compared to PBS-treated mice (all p<0.001). The tumor T2* enhancement was significantly higher in small tumors (ΔT2* = 41.3% +/- 2.182%) compared to large tumors (ΔT2* = 2.93% +/- 2.474%; p=0.001), indicating stronger nanoparticle phagocytosis in small sized tumors, apparently due to increased TAM activation. There was not statistically significance difference between T2* relaxation time of small and medium-sized tumors in all time points (all p> 0.05). Histopathological correlations confirmed a significantly higher number of CD80-positive TAMs in small sized tumors after CD47 mAb therapy (61.28% +/- 1.77%) compared to large sized tumors (3.38% +/- 0.81%, p<0.001). Tumor size at baseline affects TAM response to CD47 mAb. Smaller osteosarcomas exhibit a more pronounced TAM response to CD47 mAb therapy compared to larger tumors. Raheleh Roudi, Iryna Vasylkiv, Laura Pisani, Tie Liang, Raya Saab, Heike Daldrup-Link. Impact of baseline tumor size on response to anti-CD47 immunotherapy in an osteosarcoma mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5797.

Senescent cells promote osteoarthritis progression through the secretion of inflammatory mediators. Preclinical studies have identified senescence-associated beta-galactosidase (β-gal) as a biomarker of senescence, but in vivo detection remains challenging. Here, we evaluated whether a β-gal responsive gadolinium (Gd) chelate can non-invasively detect β-gal expressing senescent cells with standard clinical magnetic resonance imaging (MRI) technology in vitro, ex vivo, and in vivo in porcine joints. In vitro studies showed that senescent mesenchymal stromal cells (MSCs) exhibited significant MRI signal enhancement upon incubation with the β-gal responsive Gd-chelate compared to viable control cells. In vivo, intraarticular injection of the probe into pig knee joints revealed its retention and activation by senescent cells in cartilage defects, evidenced by a significant increase in R1 relaxation rate. MRI-based senescent cell detection holds promise for identifying patients amenable to senolytic therapies, tailoring treatment plans, and monitoring therapy response in real-time.

Based on conventional somatic mutation theory (SMT), the goal of cancer therapy is complete tumor cell eradication. In glioblastoma, this has proven very difficult to achieve without irreparably damaging normal tissue. We believe that the missing link to more effective treatment lies in increased attention to tissue healing and have been investigating a non-toxic compound that both eradicates tumor cells without damaging and perhaps strengthening normal ones. BPM31510 is a nanoparticle incorporating water insoluble oxidized CoQ10 into a lipid emulsion allowing delivery of supraphysiological levels of CoQ10. In a unique long-term in vitro coculture competition model, we noted that at the correct dosage, robust normal growth occurs while cancer cells are near or totally eradicated in three glioma/normal cell pairs from human, mouse and rat cell lines. In vivo, we can increase survival and in effect cure over 50% of animals treated in three different tumor models. Focusing on the C6 glioma model, where we can serially deliver iv BPM31510, cures were obtained in 100% at a dose of 400 mg/kg BPM31510. Notable in our in vivo studies was the observation that tumor size markedly increased on MRI in the early stages of administration, only to start shrinking after 2 weeks. During this time, rats do not appear ill. Therefore, in order to optimally use this medication in clinic, we will need non-invasive measurement tools and have noted that magnetic resonance spectroscopy (MRS) can detect major changes in the tumor environment, suggesting substantial metabolic reprogramming is occurring while the tumors are in the post-treatment growth phase. These findings demonstrate that BPM31510 has the capacity to be an effective, non-toxic agent with great clinical potential. We hypothesize that unlike most conventional anticancer agents that much of its effect results from its capacity to restore tissue homeostasis and enhance healing.

Abstract Background New immunotherapies activate tumor-associated macrophages (TAMs) in the osteosarcoma microenvironment. Iron oxide nanoparticles (IONPs) are phagocytosed by TAMs and, therefore, enable TAM detection on T2*- and T2-weighted magnetic resonance images. We assessed the repeatability and reproducibility of T2*- and T2-mapping of osteosarcomas in a mouse model. Methods Fifteen BALB/c mice bearing-murine osteosarcomas underwent magnetic resonance imaging (MRI) on 3-T and 7-T scanners before and after intravenous IONP infusion, using T2*-weighted multi-gradient-echo, T2-weighted fast spin-echo, and T2-weighted multi-echo sequences. Each sequence was repeated twice. Tumor T2 and T2* relaxation times were measured twice by two independent investigators. Repeatability and reproducibility of measurements were assessed. Results We found excellent agreement between duplicate acquisitions for both T2* and T2 measurements at either magnetic field strength, by the same individual (repeatability), and between individuals (reproducibility). The repeatability concordance correlation coefficient (CCC) for T2* values were 0.99 (coefficients of variation (CoV) 4.43%) for reader 1 and 0.98 (CoV 5.82%) for reader 2. The reproducibility of T2* values between the two readers was 0.99 (CoV 3.32%) for the first acquisitions and 0.99 (CoV 6.30%) for the second acquisitions. Regarding T2 values, the repeatability of CCC was similar for both readers, 0.98 (CoV 3.64% for reader 1 and 4.45% for reader 2). The CCC of the reproducibility of T2 was 0.99 (CoV 3.1%) for the first acquisition and 0.98 (CoV 4.38%) for the second acquisition. Conclusions Our results demonstrated high repeatability and reproducibility of quantitative T2* and T2 mapping for monitoring the presence of TAMs in osteosarcomas. Relevance statement T2* and T2 measurements of osteosarcomas on IONP-enhanced MRI could allow identifying patients who may benefit from TAM-modulating immunotherapies and for monitoring treatment response. The technique described here could be also applied across a wide range of other solid tumors. Key points • Optimal integration of TAM-modulating immunotherapies with conventional chemotherapy remains poorly elucidated. • We found high repeatability of T2* and T2 measurements of osteosarcomas in a mouse model, both with and without IONPs contrast, at 3-T and 7-T MRI field strengths. • T2 and T2* mapping may be used to determine response to macrophage-modulating cancer immunotherapies. Graphical Abstract

OBJECTIVE:X-linked adrenoleukodystrophy (ALD) is caused by mutations in ABCD1, a peroxisomal gene. More than half of males with an ABCD1 mutation develop inflammatory cerebral demyelination (cALD), but underlying mechanisms remain unknown and therapies are limited. We sought to develop and characterize a mouse model of cALD to facilitate study of disease mechanisms and therapy development. METHODS:We used immunoassays and immunohistochemistry to assess novel (interleukin 18 [IL-18]) and established molecular markers in cerebrospinal fluid (CSF) and postmortem brain tissue from cALD patients. We generated a cALD phenotype in Abcd1-knockout mice using a 2-hit method that combines cuprizone and experimental autoimmune encephalomyelitis models. We then used magnetic resonance imaging (MRI) and immunohistochemistry to assess the fidelity of cALD molecular markers in the mice. RESULTS:Human and mouse cALD lesions shared histologic features of myelin phagocytosis, myelin loss, abundant microglial activation, T and B-cell infiltration, and astrogliosis. Compared to wild-type controls, Abcd1-knockout mice displayed more cerebral demyelination, blood-brain barrier disruption, and perivascular immune cell infiltration. This enhanced inflammatory response was associated with higher levels of fibrin deposition, oxidative stress, demyelination, and axonal injury. IL-18 immunoreactivity co-localized with perivascular monocytes/macrophages in both human and mouse brain tissue. In cALD patients, CSF IL-18 levels correlated with MRI lesion severity. INTERPRETATION:Our results suggest loss of Abcd1 function in mice predisposes to more severe blood-brain barrier disruption, cerebral inflammation driven by the infiltration of peripheral immune cells, demyelination, and axonal damage, replicating human cALD features. This novel mouse model could shed light on cALD mechanisms and accelerate cALD therapy development. ANN NEUROL 2025;97:296-312.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood -brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescentlabeled nanoparticles with two -photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM). The protocol contains a multi -step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post -surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications. This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Rationale: As a cancer, Glioblastoma (GBM) is a highly lethal and difficult-to-treat. With the aim of improving therapies to GBM, we developed novel and target-specific theranostic nanoparticles (TNPs) that can be selectively cleaved by cathepsin B (Cat B) to release the potent toxin monomethyl auristatin E (MMAE). Methods: We synthesized TNPs composed of a ferumoxytol-based nanoparticle carrier and a peptide prodrug with a Cat-B-responsive linker and the tubulin inhibitor MMAE. We hypothesized that intratumoral Cat B can cleave our TNPs and release MMAE to kill GBM cells. The ferumoxytol core enables in vivo drug tracking with magnetic resonance imaging (MRI). We incubated U87-MG GBM cells with TNPs or ferumoxytol and evaluated the TNP content in the cells with transmission electron microscopy and Prussian blue staining. In addition, we stereotaxically implanted 6- to 8-week-old nude mice with U87-MG with U87-MG GBM cells that express a fusion protein of Green Fluorescence Protein and firefly Luciferase (U87-MG/GFP-fLuc). We then treated the animals with an intravenous dose of TNPs (25 mg/kg of ferumoxytol, 0.3 mg/kg of MMAE) or control. We also evaluated the combination of TNP treatment with radiation therapy. We performed MRI before and after TNP injection. We compared the results for tumor and normal brain tissue between the TNP and control groups. We also monitored tumor growth for a period of 21 days. Results: We successfully synthesized TNPs with a hydrodynamic size of 41 ± 5 nm and a zeta potential of 6 ± 3 mV. TNP-treated cells demonstrated a significantly higher iron content than ferumoxytol-treated cells (98 ± 1% vs. 3 ± 1% of cells were iron-positive, respectively). We also found significantly fewer live attached cells in the TNP-treated group (3.8 ± 2.0 px2) than in the ferumoxytol-treated group (80.0 ± 14.5 px2, p < 0001). In vivo MRI studies demonstrated a decline in the tumor signal after TNP (T2= 28 ms) but not control (T2= 32 ms) injections. When TNP injection was combined with radiation therapy, the tumor signals dropped further (T2 = 24 ms). The combination therapy of radiation therapy and TNPs extended the median survival from 14.5 days for the control group to 45 days for the combination therapy group. Conclusion: The new cleavable TNPs reported in this work accumulate in GBM, cause tumor cell death, and have synergistic effects with radiation therapy.

T2* relaxometry is one of the established methods to measure the effect of superparamagnetic iron oxide nanoparticles on tumor tissues with magnetic resonance imaging (MRI). Iron oxide nanoparticles shorten the T1, T2, and T2* relaxation times of tumors. While the T1 effect is variable based on the size and composition of the nanoparticles, the T2 and T2* effects are usually predominant, and T2* measurements are the most time-efficient in a clinical context. Here, we present our approach to measuring tumor T2* relaxation times, using multi-echo gradient echo sequences, external software, and a standardized protocol for creating a T2* map with scanner-independent software. This facilitates the comparison of imaging data from different clinical scanners, different vendors, and co-clinical research work (i.e., tumor T2* data obtained in mouse models and patients). Once the software is installed, the T2 Fit Map plugin needs to be installed from the plugin manager. This protocol provides step-by-step procedural details, from importing the multi-echo gradient echo sequences into the software, to creating color-coded T2* maps and measuring tumor T2* relaxation times. The protocol can be applied to solid tumors in any body part and has been validated based on preclinical imaging data and clinical data in patients. This could facilitate tumor T2* measurements for multi-center clinical trials and improve the standardization and reproducibility of tumor T2* measurements in co-clinical and multi-center data analyses.

Immunofluorescent confocal images for MMP-14 expression in pcGBM39 tumors

Objectives: A novel clinically translatable iron oxide nanoparticle (IOP) is currently being tested in phase 2 clinical trials as a magnetic resonance imaging (MRI) contrast agent for hepatocellular carcinoma diagnosis. The purpose of our study is to evaluate if this IOP can detect activation of tumor-associated macrophages (TAMs) due to CD47 mAb-targeted immunotherapy in 2 mouse models of osteosarcoma. Materials and Methods: The toxicity, biodistribution, and pharmacokinetics of IOP were evaluated in 77 female and 77 male rats. Then, 24 female BALB/c mice with intratibial murine K7M2 tumors and 24 female NOD scid gamma mice with intratibial human 143B osteosarcoma xenografts were treated with either CD47 mAb (n = 12) or control antibody (n = 12). In each treatment group, 6 mice underwent MRI scans before and after intravenous infusion of either IOP or ferumoxytol (30 mg Fe/kg). Tumor T2* values and TAM markers F4/80, CD80, CD206, and Prussian blue staining were compared between different experimental groups using exact 2-sided Wilcoxon rank sum tests. Results: Biodistribution and safety evaluations of IOP were favorable for doses of less than 50 mg Fe/kg body weight in female and male rats. Both IOP and ferumoxytol caused negative enhancement (darkening) of the tumor tissue. Both murine and human osteosarcoma tumors treated with CD47 mAb demonstrated significantly shortened T2* relaxation times after infusion of IOP or ferumoxytol compared with controls (all P's < 0.05). Higher levels of F4/80+CD80+ were found in murine and human osteosarcomas treated with CD47 mAb compared with sham-treated controls (all P's < 0.05). In addition, murine CD47 mAb-treated tumors after infusion of either IOP or ferumoxytol showed significantly higher numbers of Prussian blue-positive cells compared with controls (P < 0.05). There was no significant difference of F4/80+CD206+ cells among any of the groups (all P's > 0.05). Conclusions: Iron oxide nanoparticle-enhanced MRI can be used to diagnose CD47 mAb-mediated TAM-activation in osteosarcomas.

Cellular senescence has been implicated in age-related pathophysiologies, including osteoarthritis (OA). The targeted removal of senescent cells can ameliorate the development of OA. To advance our understanding of the role of senescent cells in OA and monitor novel senolytic therapies, there is a pressing need for imaging biomarkers that can detect senescence in arthritic joints. Senescent cells overexpress β-galactosidase. Our objective was to detect senescent cells in knee joints of small (mice) and large (pigs) animal models using a novel β-galactosidase-based radiotracer, [18F]-PyGal, using PET imaging. We hypothesized that senescent cells will demonstrate increased [18F]-PyGal radiotracer uptake compared to viable cells, in vitro and in vivo. Triplicate samples of murine primary chondrocytes incubated with 400 nM doxorubicin (doxo) to induce senescence, followed by incubation with 20 µM [18F]-PyGal radiotracer for one hour and PET-CT imaging. Non-doxo-exposed cells served as controls. Next, senescence was induced in the left knee joint of 30 C57BL/6 mice (5, 12, and 23-month-old mice, both sexes) by intra-articular doxorubicin injection. The contralateral knee served as control. All knees underwent PET/CT and PET/MRI imaging at 1 hour after intravenous injection of 250uCi [18F]-PyGal. The radiotracer signal of senescent and viable joints was measured using operator defined regions of interest (ROI; %ID/g) and compared with a student's t-test. Next, senescent mesenchymal stromal cells (MSC) or viable controls were implanted into twelve cartilage defects of six knees of three Yucatan pigs. 24 hours later, 250uCi [18F]-PyGal was injected into the knee joint, followed by integrated PET-MRI. The standardized uptake value (SUV) of senescent and viable cell implants was compared with a t-test. IL-6, CXCL5 and β-galactosidase immunostains served as reference standards for all experiments. Compared to untreated controls, doxorubicin-exposed chondrocytes demonstrated significantly increased expression of IL6 (Control: 18.39±5.10 pg/mL; senescence 58.85±1.43pg/mL, p<0.005), CXCL5 (Control 153.8162±38.14pg/mL, senescence:1163.24±169.09pg/mL p<0.005) and beta-gal (Control 9.16±0.7%, Senescence 90±2%, p<0.0005). Senescent cells exhibited a 2.28-fold higher uptake of [18F]-PyGal compared to controls (Control 1754±247.22, Senescence 3704.23±570.76, p=0.005). In 12-month-old mice, senescent knees demonstrated significantly higher retention of [18F]-PyGal (0.47±0.18 %ID/g, 0.47±0.12 %ID/g), compared to control knees (0.22±0.03%ID/g, 0.29±0.08, p=0.02, p=0.03 female and male respectively). Accordingly senescent knees demonstrated significantly increased p-16 staining, p-21 staining, and β-gal staining compared to normal controls. In the large animal model, cartilage defects implanted with senescent cells showed significantly higher [18F]-PyGal uptake (39.835±7.3 SUVmax) compared to control knees (19.69±0.69 SUVmax, p<0.05). Histopathologic correlations of large animal studies are ongoing. [18F]-PyGal can detect senescent cells in knee joints of small (mice) and large (pigs) animal models using a novel β-galactosidase-based radiotracer, [18F]-PyGal and PET imaging. Senescent cells demonstrated significantly increased [18F]-PyGal radiotracer uptake compared to viable cells, in vitro and in vivo, in small and large animal models of OA.

Objectives Iron oxide nanoparticles have been used to track the accumulation of chimeric antigen receptor (CAR) T cells with magnetic resonance imaging (MRI). However, the only nanoparticle available for clinical applications to date, ferumoxytol, has caused rare but severe anaphylactic reactions. MegaPro nanoparticles (MegaPro-NPs) provide an improved safety profile. We evaluated whether MegaPro-NPs can be applied for in vivo tracking of CAR T cells in a mouse model of glioblastoma multiforme. Materials and Methods We labeled tumor-targeted CD70CAR (8R-70CAR) T cells and non–tumor-targeted controls with MegaPro-NPs, followed by inductively coupled plasma optical emission spectroscopy, Prussian blue staining, and cell viability assays. Next, we treated 42 NRG mice bearing U87-MG/eGFP-fLuc glioblastoma multiforme xenografts with MegaPro-NP-labeled/unlabeled CAR T cells or labeled untargeted T cells and performed serial MRI, magnetic particle imaging, and histology studies. The Kruskal-Wallis test was conducted to evaluate overall group differences, and the Mann-Whitney U test was applied to compare the pairs of groups. Results MegaPro-NP-labeled CAR T cells demonstrated significantly increased iron uptake compared with unlabeled controls ( P < 0.01). Cell viability, activation, and exhaustion markers were not significantly different between the 2 groups ( P > 0.05). In vivo, tumor T2* relaxation times were significantly lower after treatment with MegaPro-NP-labeled CAR T cells compared with untargeted T cells ( P < 0.01). There is no significant difference in tumor growth inhibition between mice injected with labeled and unlabeled CAR T cells. Conclusions MegaPro-NPs can be used for in vivo tracking of CAR T cells. Because MegaPro-NPs recently completed phase II clinical trial investigation as an MRI contrast agent, MegaPro-NP is expected to be applied to track CAR T cells in cancer immunotherapy trials in the near future.

Fragile X syndrome (FXS) is the leading monogenetic cause of autism spectrum disorder and inherited cause of intellectual disability that affects approximately one in 7,000 males and one in 11,000 females. In FXS, the Fmr1 gene is silenced and prevents the expression of the fragile X mental retardation protein (FMRP) that directly targets mRNA transcripts of multiple GABAA subunits. Therefore, FMRP loss adversely impacts the neuronal firing of the GABAergic system which creates an imbalance in the excitatory/inhibitory ratio within the brain. Current FXS treatment strategies focus on curing symptoms, such as anxiety or decreased social function. While treating symptoms can be helpful, incorporating non-invasive imaging to evaluate how treatments change the brain's biology may explain what molecular aberrations are associated with disease pathology. Thus, the GABAergic system is suitable to explore developing novel therapeutic strategies for FXS. To understand how the GABAergic system may be affected by this loss-of-function mutation, GABA concentrations were examined within the frontal cortex and thalamus of 5-day-old wild type and Fmr1 knockout mice using both 1H magnetic resonance imaging (1H-MRS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our objective was to develop a reliable scanning method for neonatal mice in vivo and evaluate whether 1H-MRS is suitable to capture regional GABA concentration differences at the front end of the critical cortical period where abnormal neurodevelopment occurs due to FMRP loss is first detected. 1H-MRS quantified GABA concentrations in both frontal cortex and thalamus of wild type and Fmr1 knockout mice. To substantiate the results of our 1H-MRS studies, in vitro LC-MS/MS was also performed on brain homogenates from age-matched mice. We found significant changes in GABA concentration between the frontal cortex and thalamus within each mouse from both wild type and Fmr1 knockout mice using 1H-MRS and LC-MS/MS. Significant GABA levels were also detected in these same regions between wild type and Fmr1 knockout mice by LC-MS/MS, validating that FMRP loss directly affects the GABAergic system. Thus, these new findings support the need to develop an effective non-invasive imaging method to monitor novel GABAergic strategies aimed at treating patients with FXS.

Recent evidence of gadolinium deposition in the brain has raised safety concerns. Iron oxide nanoparticles are re-emerging as promising alternative MR contrast agents, because the iron core can be metabolized. However, long-term follow up studies of the brain after intravenous iron oxide administration have not been reported thus far. In this study, we investigated, if intravenously administered ferumoxytol nanoparticles are deposited in porcine brains. Methods: In an animal care and use committee-approved prospective case-control study, ten Göttingen minipigs received either intravenous ferumoxytol injections at a dose of 5 mg Fe/kg (n=4) or remained untreated (n=6). Nine to twelve months later, pigs were sacrificed and the brains of all pigs underwent ex vivo MRI at 7T with T2 and T2*-weighted sequences. MRI scans were evaluated by measuring R2* values (R2*=1000/T2*) of the bilateral caudate nucleus, lentiform nucleus, thalamus, dentate nucleus, and choroid plexus. Pig brains were sectioned and stained with Prussian blue and evaluated for iron deposition using a semiquantitative scoring system. Data of ferumoxytol exposed and unexposed groups were compared with an unpaired t-test and a Mann-Whitney U test. Results: T2 and T2* signal of the different brain regions was not visually different between ferumoxytol exposed and unexposed controls. There were no significant differences in R2* values of the different brain regions in the ferumoxytol exposed group compared to controls (p>0.05). Prussian blue stains of the same brain regions, scored according to a semiquantitative score, were not significantly different either between the ferumoxytol exposed group and unexposed controls (p>0.05). Conclusions: Our study shows that intravenous ferumoxytol doses of 5-10 mg Fe/kg do not lead to iron deposition in the brain of pigs. We suggest iron oxide nanoparticles as a promising alternative for gadolinium-enhanced MRI.

SUMMARY: Magnetic particle imaging is an emerging tomographic technique with the potential for simultaneous high-resolution, high-sensitivity, and real-time imaging. Magnetic particle imaging is based on the unique behavior of superparamagnetic iron oxide nanoparticles modeled by the Langevin theory, with the ability to track and quantify nanoparticle concentrations without tissue background noise. It is a promising new imaging technique for multiple applications, including vascular and perfusion imaging, oncology imaging, cell tracking, inflammation imaging, and trauma imaging. In particular, many neuroimaging applications may be enabled and enhanced with magnetic particle imaging. In this review, we will provide an overview of magnetic particle imaging principles and implementation, current applications, promising neuroimaging applications, and practical considerations.

CD47 monoclonal antibodies (mAbs) activate tumor-associated macrophages (TAMs) in sarcomas to phagocytose and eliminate cancer cells. Though CD47 mAbs have entered clinical trials, diagnostic tests for monitoring therapy response in vivo are currently lacking. Ferumoxytol is an FDA-approved iron supplement which can be used "off label" as a contrast agent: the nanoparticle-based drug is phagocytosed by TAM and can be detected with magnetic resonance imaging (MRI). We evaluated if ferumoxytol-enhanced MRI can monitor TAM response to CD47 mAb therapy in osteosarcomas. Forty-eight osteosarcoma-bearing mice were treated with CD47 mAb or control IgG and underwent pre- and post-treatment ferumoxytol-MRI scans. Tumor enhancement, quantified as T2 relaxation times, was compared with the quantity of TAMs as determined by immunofluorescence microscopy and flow cytometry. Quantitative data were compared between experimental groups using exact two-sided Wilcoxon rank-sum tests. Compared to IgG-treated controls, CD47 mAb-treated tumors demonstrated significantly shortened T2 relaxation times on ferumoxytol-MRI scans (p < 0.01) and significantly increased F4/80+CD80+ M1 macrophages on histopathology (p < 0.01). CD47 mAb-treated F4/80+ macrophages demonstrated significantly augmented phagocytosis of ferumoxytol nanoparticles (p < 0.01). Thus, we conclude that ferumoxytol-MRI can detect TAM response to CD47 mAb in mouse models of osteosarcoma. The ferumoxytol-MRI imaging test could be immediately applied to monitor CD47 mAb therapies in clinical trials.