In vivo tracking of [89Zr]Zr-labeled engineered extracellular vesicles by PET reveals organ-specific biodistribution based upon the route of administration.

Extracellular vesicles (EVs) have garnered increasing interest as delivery vehicles for multiple classes of therapeutics based on their role as mediators in an important, natural intercellular communication system. We recently described a platform to allow the design, production and in vivo study of human EVs with specific properties (drug or tropism modifiers). This article seeks to compare and expand upon historical biodistribution and kinetic data by comparing systemically and compartmentally administered labeled engineered EVs using in vivo and ex vivo techniques. METHODS: EVs were surface-labeled to high radiochemical purity and specific activity with 89Zirconium deferoxamine ([89Zr]Zr-DFO) and/or cy7-scrambled antisense oligonucleotide (Cy7-ExoASOscr), or luminally loaded with GFP for in vivo tracking in rodents and non-human primates (NHPs). Positron Emission Tomography (PET) and subsequent immunohistochemistry (IHC) and autoradiography (ARG) cross-validation enabled assessment of the anatomical and cellular distribution of labeled EVs both spatially and temporally. RESULTS: Over time, systemic administration of engineered EVs distributed preferentially to the liver and spleen (Intravenous, IV), gastrointestinal tract and lymph nodes (Intraperitoneal, IP) and local/regional lymph nodes (Subcutaneous, SC). Immunostaining of dissected organs displaying PET signal revealed co-localization of an EV marker (PTGFRN) with a subset of macrophage markers (CD206, F4/80, IBA1). Compartmental dosing into NHP cerebrospinal fluid (CSF) resulted in a heterogenous distribution of labeled EVs depending upon whether the route was intrathecal (ITH), intracisterna magna (ICM) or intracerebroventricular (ICV), compared to the homogeneous distribution observed in rodents. Thus anatomically, ITH administration in NHP revealed meningeal distribution along the neuraxis to the base of the skull. In contrast ICM and ICV dosing resulted in meningeal distribution around the skull and to the cervical and thoracic spinal column. Further characterization using IHC shows uptake in a subset of meningeal macrophages. CONCLUSIONS: The present studies provide a comprehensive assessment of the fate of robustly and reproducibly labeled engineered EVs across several mammalian species. The in vivo distribution was observed to be both spatially and temporally dependent upon the route of administration providing insight into potential targeting opportunities for engineered EVs carrying a therapeutic payload.

Mitochondrial Complex 1, Sigma 1, and Synaptic Vesicle 2A in Early Drug-Naive Parkinson's Disease.

BACKGROUND: Dysfunction of mitochondrial energy generation may contribute to neurodegeneration, leading to synaptic loss in Parkinson's disease (PD). The objective of this study was to find cross-sectional and longitudinal changes in PET markers of synaptic vesicle protein 2A, sigma 1 receptor, and mitochondrial complex 1 in drug-naive PD patients. METHODS: Twelve early drug-naive PD patients and 16 healthy controls underwent a 3-Tesla MRI and PET imaging to quantify volume of distribution of [11 C]UCB-J, [11 C]SA-4503, and [18 F]BCPP-EF for synaptic vesicle protein 2A, sigma 1 receptor, and mitochondrial complex 1, respectively. Nine PD patients completed approximately 1-year follow-up assessments. RESULTS: Reduced [11 C]UCB-J volume of distribution in the caudate, putamen, thalamus, brain stem, and dorsal raphe and across cortical regions was observed in drug-naive PD patients compared with healthy controls. [11 C]UCB-J volume of distribution was reduced in the locus coeruleus and substantia nigra but did not reach statistical significance. No significant differences were found in [11 C]SA-4503 and [18 F]BCPP-EF volume of distribution in PD compared with healthy controls. Lower brain stem [11 C]UCB-J volume of distribution correlated with Movement Disorder Society Unified Parkinson's Disease Rating Scale part III and total scores. No significant longitudinal changes were identified in PD patients at follow-up compared with baseline. CONCLUSIONS: Our findings represent the first in vivo evidence of mitochondrial, endoplasmic reticulum, and synaptic dysfunction in drug-naive PD patients. Synaptic dysfunction likely occurs early in disease pathophysiology and has relevance to symptomatology. Mitochondrial complex 1 and sigma 1 receptor pathology warrants further investigations in PD. Studies in larger cohorts with longer follow-up will determine the validity of these PET markers to track disease progression. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Imaging of Activated T Cells as an Early Predictor of Immune Response to Anti-PD-1 Therapy.

Compelling evidence points to immune cell infiltration as a critical component of successful immunotherapy. However, there are currently no clinically available, noninvasive methods capable of evaluating immune contexture prior to or during immunotherapy. In this study, we evaluate a T-cell-specific PET agent, [18F]F-AraG, as an imaging biomarker predictive of response to checkpoint inhibitor therapy. We determined the specificity of the tracer for activated T cells in vitro and in a virally induced model of rhabdomyosarcoma. Of all immune cells tested, activated human CD8+ effector cells showed the highest accumulation of [18F]F-AraG. Isolation of lymphocytes from the rhabdomyosarcoma tumors showed that more than 80% of the intratumoral signal came from accumulation of [18F]F-AraG in immune cells, primarily CD8+ and CD4+. Longitudinal monitoring of MC38 tumor-bearing mice undergoing anti-PD-1 treatment revealed differences in signal between PD-1 and isotype antibody-treated mice early into treatment. The differences in [18F]F-AraG signal were also apparent between responders and nonresponders to anti-PD-1 therapy. Importantly, we found that the signal in the tumor-draining lymph nodes provides key information about response to anti-PD-1 therapy. Overall, [18F]F-AraG has potential to serve as a much needed immunomonitoring clinical tool for timely evaluation of immunotherapy. SIGNIFICANCE: These findings reveal differences in T-cell activation between responders and nonresponders early into anti-PD-1 treatment, which may impact many facets of immuno-oncology, including patient selection, management, and development of novel combinatorial approaches.

Differential responses in CBF and CBV to cocaine as measured by fMRI: implications for pharmacological MRI signals derived oxygen metabolism assessment.

INTRODUCTION: Cognitive performance-induced brain oxygen metabolism has been successfully measured by functional magnetic resonance imaging (fMRI) in human studies. The measurement of the cerebral metabolic rate of oxygen consumption (CMRO2) is typically achieved by assuming a fixed coupling of cerebral blood flow (CBF) and cerebral blood volume (CBV) and by performing a separate experiment to assess the vascular response to a hypercapnic challenge. Psychoactive drugs may have directly effect on the cerebral vasculature, potentially confounding the interpretation of pharmacological MRI (phMRI) data. In this study, we tested the assumptions of the standard CMRO2 calculation following the administration of cocaine, in order to test the validity of this measurement in phMRI studies. The initial transient state and later steady state CBF and CBV responses to a hypercapnic challenge were measured. METHODS: CBF and CBV responses were directly measured by fMRI using continuous arterial spin-labeling (ASL) and contrast-enhanced fMRI, respectively. The coupling between changes in CBF and CBV during a hypercapnic challenge was examined under normal conditions and following the administration of cocaine. RESULTS: A decoupling of changes in CBF and CBV was observed during the transient state immediately following the administration of cocaine, and an altered coupling of CBF and CBV was found during the steady state after cocaine injection. DISCUSSION: These data suggest caution in interpreting CMRO2 measurements from phMRI studies and may also lead to an improved understanding of the complex neuronal and vascular mechanisms of drug action.

Neuroimaging revolutionizes therapeutic approaches to chronic pain.

An understanding of how the brain changes in chronic pain or responds to pharmacological or other therapeutic interventions has been significantly changed as a result of developments in neuroimaging of the CNS. These developments have occurred in 3 domains : (1) Anatomical Imaging which has demonstrated changes in brain volume in chronic pain; (2) Functional Imaging (fMRI) that has demonstrated an altered state in the brain in chronic pain conditions including back pain, neuropathic pain, and complex regional pain syndromes. In addition the response of the brain to drugs has provided new insights into how these may modify normal and abnormal circuits (phMRI or pharmacological MRI); (3) Chemical Imaging (Magnetic Resonance Spectroscopy or MRS) has helped our understanding of measures of chemical changes in chronic pain. Taken together these three domains have already changed the way in which we think of pain - it should now be considered an altered brain state in which there may be altered functional connections or systems and a state that has components of degenerative aspects of the CNS.

Hemodynamic and metabolic changes induced by cocaine in anesthetized rat observed with multimodal functional MRI.

RATIONALE: Physiological changes (such as heart rate and respiration rate) associated with strong pharmacological stimuli could change the blood-oxygenation-level-dependent (BOLD) functional magnetic resonance imaging (fMRI) mapping signals, independent of neural activity. OBJECTIVES: This study investigates whether the physiological changes per se associated with systemic cocaine administration (1 mg/kg) contaminate the BOLD fMRI signals by measuring BOLD and cerebral blood flow (CBF) fMRI and estimating the cerebral metabolic rate of oxygen (CMRO(2)) changes. MATERIALS AND METHODS: BOLD and CBF fMRI was performed, and changes in CMRO(2) were estimated using the BOLD biophysical model. RESULTS: After systemic cocaine administration, blood pressure, heart rate, and respiration rate increased, fMRI signals remained elevated after physiological parameters had returned to baseline. Cocaine induced changes in the BOLD signal within regions of the reward pathway that were heterogeneous and ranged from -1.2 to 5.4%, and negative changes in BOLD were observed along the cortical surface. Changes in CBF and estimated CMRO(2) were heterogeneous and positive throughout the brain, ranging from 14 to 150% and 10 to 55%, respectively. CONCLUSIONS: This study demonstrates a valuable tool to investigate the physiological and biophysical basis of drug action on the central nervous system, offering the means to distinguish the physiological from neural sources of the BOLD fMRI signal.

Comparison of ischemic lesion evolution in embolic versus mechanical middle cerebral artery occlusion in Sprague Dawley rats using diffusion and perfusion imaging.

BACKGROUND AND PURPOSE: Differences among models in the temporal evolution of ischemia after middle cerebral artery occlusion (MCAO) in rats may considerably influence the results of experimental stroke research. Using diffusion and perfusion imaging, we compared the spatiotemporal evolution of ischemia in Sprague Dawley rats after permanent suture MCAO (sMCAO; n=8) and embolic MCAO (eMCAO; n=8). METHODS: Serial measurements of quantitative cerebral blood flow (CBF) and the apparent diffusion coefficient (ADC) were performed up to 180 minutes after MCAO. ADC and CBF values within 5 different brain regions were analyzed. ADC and CBF lesion volumes were calculated by using previously established viability thresholds and correlated with infarct volume defined by 2,3,5-triphenyltetrazolium chloride staining 24 hours after MCAO. RESULTS: Compared with sMCAO animals, the threshold-derived CBF lesion volume was significantly larger in eMCAO at all time points (P<0.01), remained relatively constant over time, and was highly correlated with the 2,3,5-triphenyltetrazolium chloride-defined infarct size. The ADC lesion volume did not differ between models at any time point. A diffusion/perfusion mismatch was present significantly longer in eMCAO animals (P<0.05), and these rats demonstrated larger absolute mismatch volumes that were statistically significant at 30, 60, and 90 minutes (P<0.05). In both models, CBF and ADC declines were highly correlated. CONCLUSIONS: This study demonstrated substantial differences in acute ischemic lesion evolution between the eMCAO and sMCAO models.

Volume reconstruction techniques improve the correlation between histological and in vivo tumor volume measurements in mouse models of human gliomas.

Assessment of therapy efficacy using animal models of tumorigenic cancer requires the ability to accurately measure changes in tumor volume over the duration of disease course. In order to be meaningful, in vivo tumor volume measurements by non-invasive techniques must correlate with tumor volume measurements from endpoint histological analysis. Tumor volume is frequently assessed by endpoint histological analyses approximating the tumor volume with geometric primitives such as spheroids and ellipsoids. In this study we investigated alternative techniques for quantifying histological volume measurements of tumors in a xenograft orthotopic mouse model of human glioblastoma multiforme, and compared these to in vivo tumor volume measurements based on magnetic resonance imaging (MRI) data. Two techniques leveraging three-dimensional (3D) image analysis methods were investigated. The first technique involves the reconstruction of a smoothed polygonal model representing the tumor volume from histological section images and is intended for accuracy and qualitative assessment of tumor burden by visualization, while a second technique which approximates the tumor volume as a series of slabs is presented as an abbreviated process intended to produce quantitatively similar volume measurements with a minimum of effort required on behalf of the investigator. New software (QuickVol) designed for use in the first technique, is also discussed. In cases where tumor growth is asymmetric and invasive, we found that 3D analysis techniques using histological section images produced volume measurements more consistent with in vivo volume measurements based on MRI data, than approximation of tumor volume using geometric primitives. Visualizations of the volumes represented by each of these techniques qualitatively support this finding, and suggest that future research using mouse models of glioblastoma multiforme (genetically engineered or xenograft) will benefit from the use of these or similar alternative tumor volume measurement techniques.

Imaging oxygen consumption in forepaw somatosensory stimulation in rats under isoflurane anesthesia.

The cerebral metabolic rate of oxygen (CMRO2) was dynamically evaluated on a pixel-by-pixel basis in isoflurane-anesthetized and spontaneously breathing rats following graded electrical somatosensory forepaw stimulations (4, 6, and 8 mA). In contrast to alpha-chloralose, which is the most widely used anesthetic in forepaw-stimulation fMRI studies of rats under mechanical ventilation, isoflurane (1.1-1.2%) provided a stable anesthesia level over a prolonged period, without the need to adjust the ventilation volume/rate or sample blood gases. Combined cerebral blood flow signals (CBF) and blood oxygenation level-dependent (BOLD) fMRI signals were simultaneously measured with the use of a multislice continuous arterial spin labeling (CASL) technique (two-coil setup). CMRO2 was calculated using the biophysical BOLD model of Ogawa et al. (Proc Natl Acad Sci USA 1992;89:5951-5955). The stimulus-evoked BOLD percent changes at 4, 6, and 8 A were, respectively, 0.5% +/- 0.2%, 1.4% +/- 0.3%, and 2.0% +/- 0.3% (mean +/- SD, N = 6). The CBF percent changes were 23% +/- 6%, 58% +/- 9%, and 87% +/- 14%. The CMRO2 percent changes were 14% +/- 4%, 24% +/- 6%, and 43% +/- 11%. BOLD, CBF, and CMRO2 activations were localized to the forepaw somatosensory cortices without evidence of plateau for oxygen consumption, indicative of partial coupling of CBF and CMRO2. This study describes a useful forepaw-stimulation model for fMRI, and demonstrate that CMRO2 changes can be dynamically imaged on a pixel-by-pixel basis in a single setting with high spatiotemporal resolution.