Method for estimation of apoptotic cell fraction of cytotherapy using in vivo fluorine-19 magnetic resonance: pilot study in a patient with head and neck carcinoma receiving tumor-infiltrating lymphocytes labeled with perfluorocarbon nanoemulsion.

BACKGROUND: Adoptive transfer of T cells is a burgeoning cancer therapeutic approach. However, the fate of the cells, once transferred, is most often unknown. We describe the first clinical experience with a non-invasive biomarker to assay the apoptotic cell fraction (ACF) after cell therapy infusion, tested in the setting of head and neck squamous cell carcinoma (HNSCC). A patient with HNSCC received autologous tumor-infiltrating lymphocytes (TILs) labeled with a perfluorocarbon (PFC) nanoemulsion cell tracer. Nanoemulsion, released from apoptotic cells, clears through the reticuloendothelial system, particularly the Kupffer cells of the liver, and fluorine-19 (19F) magnetic resonance spectroscopy (MRS) of the liver was used to non-invasively infer the ACF. METHODS: Autologous TILs were isolated from a patient in their late 50s with relapsed, refractory human papillomavirus-mediated squamous cell carcinoma of the right tonsil, metastatic to the lung. A lung metastasis was resected for T cell harvest and expansion using a rapid expansion protocol. The expanded TILs were intracellularly labeled with PFC nanoemulsion tracer by coincubation in the final 24 hours of culture, followed by a wash step. At 22 days after intravenous infusion of TILs, quantitative single-voxel liver 19F MRS was performed in vivo using a 3T MRI system. From these data, we model the apparent ACF of the initial cell inoculant. RESULTS: We show that it is feasible to PFC-label ~70×1010 TILs (F-TILs) in a single batch in a clinical cell processing facility, while maintaining >90% cell viability and standard flow cytometry-based release criteria for phenotype and function. Based on quantitative in vivo 19F MRS measurements in the liver, we estimate that ~30% cell equivalents of adoptively transferred F-TILs have become apoptotic by 22 days post-transfer. CONCLUSIONS: Survival of the primary cell therapy product is likely to vary per patient. A non-invasive assay of ACF over time could potentially provide insight into the mechanisms of response and non-response, informing future clinical studies. This information may be useful to developers of cytotherapies and clinicians as it opens an avenue to quantify cellular product survival and engraftment.

A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

Imaging Non-alcoholic Fatty Liver Disease Model Using H-1 and F-19 MRI.

PURPOSE: We explore the use of intravenously delivered perfluorocarbon (PFC) nanoemulsion and 19F MRI for detecting inflammation in a mouse model of non-alcoholic fatty liver disease (NAFLD). Correlative studies of 1H-based liver proton density fat fraction (PDFF) and T1 measurements and histology are also evaluated. PROCEDURES: C57BL/6 mice were fed standard or high-fat diet (HFD) for 6 weeks to induce NAFLD. 1H MRI measurements of PDFF and T1 relaxation time were performed at baseline to assess NAFLD onset prior to administration of a PFC nanoemulsion to enable 19F MRI of liver PFC uptake. 1H and 19F MRI biomarkers were acquired at 2, 21, and 42 days post-PFC to assess changes. Histopathology of liver tissue was performed at experimental endpoint. RESULTS: Significant increases in liver volume, PDFF, and total PFC uptake were noted in HFD mice compared to Std diet mice. Liver fluorine density and T1 relaxation time were significantly reduced in HFD mice. CONCLUSIONS: We demonstrated longitudinal quantification of multiple MRI biomarkers of disease in NAFLD mice. The changes in liver PFC uptake in HFD mice were compared with healthy mice that suggests that 19F MRI may be a viable biomarker of liver pathology.

Click-Ready Perfluorocarbon Nanoemulsion for 19F MRI and Multimodal Cellular Detection.

We describe an in vivo imaging probe platform that is readily modifiable to accommodate binding of different molecular targeting moieties and payloads for multimodal image generation. In this work, we demonstrate the utility of perfluorocarbon (PFC) nanoemulsions incorporating dibenzocyclooctyne (DBCO) by enabling postemulsification functionalization via a click reaction with azide-containing ligands. The addition of DBCO-lipid to the surfactant in PFC nanoemulsions did not affect nanoemulsion size or nanoemulsion stability. As proof-of-concept, fluorescent dye-azides were conjugated to PFC nanoemulsions, demonstrating the feasibility of functionalization the by click reaction. Uptake of the fluorescent PFC by macrophages was demonstrated both in vitro in cultured macrophages and in situ in an acute inflammation mouse model, where fluorescence imaging and 1H/19F magnetic resonance imaging (MRI) were used for in vivo detection. Overall, these data demonstrate the potential of PFC nanoemulsions incorporating DBCO as a versatile platform for generating functionalized probes.

Enhanced detection of paramagnetic fluorine-19 magnetic resonance imaging agents using zero echo time sequence and compressed sensing.

Fluorine-19 (19 F) magnetic resonance imaging (MRI) is an emerging technique offering specific detection of labeled cells in vivo. Lengthy acquisition times and modest signal-to-noise ratio (SNR) makes three-dimensional spin-density-weighted 19 F imaging challenging. Recent advances in tracer paramagnetic metallo-perfluorocarbon (MPFC) nanoemulsion probes have shown multifold SNR improvements due to an accelerated 19 F T1 relaxation rate and a commensurate gain in imaging speed and averages. However, 19 F T2 -reduction and increased linewidth limit the amount of metal additive in MPFC probes, thus constraining the ultimate SNR. To overcome these barriers, we describe a compressed sampling (CS) scheme, implemented using a "zero" echo time (ZTE) sequence, with data reconstructed via a sparsity-promoting algorithm. Our CS-ZTE scheme acquires k-space data using an undersampled spherical radial pattern and signal averaging. Image reconstruction employs off-the-shelf sparse solvers to solve a joint total variation and l1 -norm regularized least square problem. To evaluate CS-ZTE, we performed simulations and acquired 19 F MRI data at 11.7 T in phantoms and mice receiving MPFC-labeled dendritic cells. For MPFC-labeled cells in vivo, we show SNR gains of ~6.3 × with 8-fold undersampling. We show that this enhancement is due to three mechanisms including undersampling and commensurate increase in signal averaging in a fixed scan time, denoising attributes from the CS algorithm, and paramagnetic reduction of T1 . Importantly, 19 F image intensity analyses yield accurate estimates of absolute quantification of 19 F spins. Overall, the CS-ZTE method using MPFC probes achieves ultrafast imaging, a substantial boost in detection sensitivity, accurate 19 F spin quantification, and minimal image artifacts.

Emergent Fluorous Molecules and Their Uses in Molecular Imaging.

This Account summarizes recent advances in the chemistry of fluorocarbon nanoemulsion (FC NE) functionalization. We describe new families of fluorous molecules, such as chelators, fluorophores, and peptides, that are soluble in FC oils. These materials have helped transform the field of in vivo molecular imaging by enabling sensitive and cell-specific imaging using magnetic resonance imaging (MRI), positron emission tomography (PET), and fluorescence detection. FC emulsions, historically considered for artificial blood substitutes, are routinely used for ultrasound imaging in clinic and have a proven safety profile and a well-characterized biodistribution and pharmacokinetics. The inertness of fluorocarbons contributes to their low toxicity but makes functionalization difficult. The high electronegativity of fluorine imparts very low cohesive energy density and Lewis basicity to heavily fluorinated compounds, making dissolution of metal ions and organic molecules challenging. Functionalization is further complicated by colloidal instability toward heat and pH, as well as limited availability of biocompatible surfactants.We have devised new fluorous chelators that overcome solubility barriers and are able to bind a range of metal ions with high thermodynamic stability and biocompatibility. NE harboring chelators in the fluorous phase are a powerful platform for the development of multimodal imaging agents. These compositions rapidly capture metal ions added to the aqueous phase, thereby functionalizing NEs in useful ways. For example, Fe3+ encapsulation imparts a strong paramagnetic relaxation effect on 19F T1 that dramatically accelerates 19F MRI data acquisition times and hence sensitivity in cell tracking applications. Alternatively, 89Zr encapsulation creates a sensitive and versatile PET probe for inflammatory macrophage detection. Adding lanthanides, such as Eu3+, renders NE luminescent. Beyond chelators, this Account further covers our progress in formulating NEs with fluorophores, such as cyanine or BODIPY dyes, with their utility demonstrated in fluorescence imaging, biosensing, flow cytometry and histology. Fluorous dyes soluble in FC oils are also key enablers for nascent whole-body imaging technologies such as cryo-fluorescence tomography (CFT). Additionally, fluorous cell-penetrating peptides inserted on the NE surface increase the uptake of NE by ∼8-fold in weakly phagocytic stem cells and lymphocytes used in immunotherapy, resulting in significant leaps in detection sensitivity in vivo.

Assessing Oximetry Response to Chimeric Antigen Receptor T-cell Therapy against Glioma with 19F MRI in a Murine Model.

Purpose: To assess the cell-specific, intracellular partial pressure of oxygen (Po2) dynamics of both tumor and chimeric antigen receptor (CAR) T cells in a murine immunotherapy model. Materials and Methods: Human glioblastoma cells or human T cells were intracellularly labeled with perfluorocarbon nanoemulsion droplet sensors prior to in vivo injection in severe combined immunodeficient mice to measure Po2 in the two cell types in response to treatment. Two main sets of experiments were performed: (a) mice were injected in the flank with perfluorocarbon-labeled human glioblastoma cells and were then inoculated with either CAR T cells or untransduced T cells or were untreated 5 days after tumor inoculation; and (b) mice with unlabeled glioblastoma tumors were inoculated with perfluorocarbon-labeled CAR T cells or untransduced T cells 5 days after tumor inoculation. Longitudinal fluorine 19 (19F) spin-lattice relaxation time measurements of the tumor mass were used to ascertain absolute Po2 in vivo. Results were analyzed for significance using an analysis of variance, a linear mixed-effect model, and a Pearson correlation coefficient test, as appropriate. Results: The intracellular tumor cell Po2 temporal dynamics exhibited delayed, transient hyperoxia at 3 days after infusion of CAR T cells, commensurate with significant tumor cell killing and CAR T-cell infiltration, as observed by bioluminescence imaging and histologic findings. Conversely, no significant changes were detected in CAR or untransduced T-cell intracellular Po2 over time in tumor using these same methods. Moreover, it was observed that the total 19F tumor cell signal quenches with treatment, consistent with rapid tissue clearance of probe from apoptotic tumor cells. Conclusion: Cell-specific Po2 measurements using perfluorocarbon probes can provide insights into effector cell function and tumor response in cellular immunotherapeutic cancer models.Keywords: Animal Studies, MR-Imaging, MR-Spectroscopy, Molecular Imaging-Cancer, Molecular Imaging-Immunotherapy Supplemental material is available for this article. © RSNA, 2021See also commentary by Bulte in this issue.

Metallofluorocarbon Nanoemulsion for Inflammatory Macrophage Detection via PET and MRI.

Inflammation is associated with a range of serious human conditions, including autoimmune and cardiovascular diseases and cancer. The ability to image active inflammatory processes greatly enhances our ability to diagnose and treat these diseases at an early stage. We describe molecular compositions enabling sensitive and precise imaging of inflammatory hotspots in vivo. Methods: A functionalized nanoemulsion with a fluorocarbon-encapsulated radiometal chelate (FERM) was developed to serve as a platform for multimodal imaging probe development. The 19F-containing FERM nanoemulsion encapsulates 89Zr in the fluorous oil via a fluorinated hydroxamic acid chelate. Simple mixing of the radiometal with the preformed aqueous nanoemulsion before use yields FERM, a stable in vivo cell tracer, enabling whole-body 89Zr PET and 19F MRI after a single intravenous injection. Results: The FERM nanoemulsion was intrinsically taken up by phagocytic immune cells, particularly macrophages, with high specificity. FERM stability was demonstrated by a high correlation between the 19F and 89Zr content in the blood (correlation coefficient > 0.99). Image sensitivity at a low dose (37 kBq) was observed in a rodent model of acute infection. The versatility of FERM was further demonstrated in models of inflammatory bowel disease and 4T1 tumor. Conclusion: Multimodal detection using FERM yields robust whole-body lesion detection and leverages the strengths of combined PET and 19F MRI. The FERM nanoemulsion has scalable production and is potentially useful for precise diagnosis, stratification, and treatment monitoring of inflammatory diseases.

Paramagnetic Fluorinated Nanoemulsions for in vivo F-19 MRI.

PURPOSE: We aim to develop perfluorocarbon-based nanoemulsions with improved sensitivity for detection of inflammatory macrophages in situ using F-19 MRI. Towards this goal, we evaluate the feasibility of nanoemulsion formulation incorporating a metal chelate in the fluorous phase which shortens the F-19 longitudinal relaxation rate and image acquisition time. PROCEDURES: Perfluorinated linear polymers were conjugated to metal-binding tris-diketonate, blended with unconjugated polymers, and emulsified in water. Phospholipid-based surfactant was used to stabilize nanoemulsion and provide biocompatibility. Nanoemulsions were metalated with the addition of ferric salt to the buffer. Physical stability of surfactant and nanoemulsion was evaluated by mass spectrometry and dynamic light scattering measurements. Nanoemulsions were injected intravenously into a murine granuloma inflammation model, and in vivo19F/1H MRI at 11.7 T was performed. RESULTS: We demonstrated stability and biocompatibility of lipid-based paramagnetic nanoemulsions. We investigated potential oxidation of lipid in the presence of metal chelate. As a proof of concept, we performed non-invasive monitoring of macrophage burden in a murine inflammation model following intravenous injection of nanoemulsion using in vivo F-19 MRI. CONCLUSION: Lipid-based nanoemulsion probes of perfluorocarbon synthesized with iron-binding fluorinated ß-diketones can be formulated for intravenous delivery and inflammation detection in vivo.

Cell penetrating peptide functionalized perfluorocarbon nanoemulsions for targeted cell labeling and enhanced fluorine-19 MRI detection.

PURPOSE: A bottleneck in developing cell therapies for cancer is assaying cell biodistribution, persistence, and survival in vivo. Ex vivo cell labeling using perfluorocarbon (PFC) nanoemulsions, paired with 19 F MRI detection, is a non-invasive approach for cell product detection in vivo. Lymphocytes are small and weakly phagocytic limiting PFC labeling levels and MRI sensitivity. To boost labeling, we designed PFC nanoemulsion imaging probes displaying a cell-penetrating peptide, namely the transactivating transcription sequence (TAT) of the human immunodeficiency virus. We report optimized synthesis schemes for preparing TAT co-surfactant to complement the common surfactants used in PFC nanoemulsion preparations. METHODS: We performed ex vivo labeling of primary human chimeric antigen receptor (CAR) T cells with nanoemulsion. Intracellular labeling was validated using electron microscopy and confocal imaging. To detect signal enhancement in vivo, labeled CAR T cells were intra-tumorally injected into mice bearing flank glioma tumors. RESULTS: By incorporating TAT into the nanoemulsion, a labeling efficiency of ~1012 fluorine atoms per CAR T cell was achieved that is a >8-fold increase compared to nanoemulsion without TAT while retaining high cell viability (~84%). Flow cytometry phenotypic assays show that CAR T cells are unaltered after labeling with TAT nanoemulsion, and in vitro tumor cell killing assays display intact cytotoxic function. The 19 F MRI signal detected from TAT-labeled CAR T cells was 8 times higher than cells labeled with PFC without TAT. CONCLUSION: The peptide-PFC nanoemulsion synthesis scheme presented can significantly enhance cell labeling and imaging sensitivity and is generalizable for other targeted imaging probes.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

ß­Diketonate-Iron(III) Complex: A Versatile Fluorine-19 MRI Signal Enhancement Agent.

Fluorine-19 magnetic resonance imaging (MRI) has gained considerable momentum as a promising imaging modality for in vivo tracking of cellular therapies and as a diagnostic for inflammatory disease. To further the utility of this technique, we increase imaging probe sensitivity by merging paramagnetic metal chelates with aqueous perfluorocarbon (PFC) nanoemulsions. We prepared a highly fluorinated ferric tris(ß-diketonate) chelate (MW = 1265.2 g/mol) at gram scale. This iron chelate is soluble in multiple PFC oils used for MRI and readily reduces the 19F longitudinal relaxation time (T 1) to <100 ms with modest line broadening and displays superior properties for 19F MRI applications. The sensitivity enhancement by Fe(III) laden PFC nanoemulsion was confirmed in MRI phantom studies, where reduced T 1 speeds data acquisition thereby increasing the 19F image sensitivity per time via signal averaging. Additionally, 19F relaxivity of nanoemulsions incorporating other metal ions, including Gd, Er, Ho, Dy, Mn, Cr, and Ni, were evaluated. High-moment lanthanide ions, such as Gd(III), display severe line broadening, but other ions [e.g., Ho(III)] induce pseudocontact chemical shifts (up to 0.5 ppm) of 19F in nanoemulsion, which makes them potentially useful for multichromatic 19F imaging. Formulated nanoemulsions have a shelf life >200 days. Free ß-diketonate or its iron complex in formed PFC nanoemulsion did not induce cytotoxicity in intracellularly labeled macrophages. Overall, ferric tris(ß-diketonate) chelate provides a scalable approach for boosting sensitivity of PFC-based 19F MRI probes. More generally, it can functionalize PFC oil, whose chemical modification remains challenging.

Fluorous-Soluble Metal Chelate for Sensitive Fluorine-19 Magnetic Resonance Imaging Nanoemulsion Probes.

Fluorine-19 MRI is an emerging cellular imaging approach, enabling lucid, quantitative "hot-spot" imaging with no background signal. The utility of 19F-MRI to detect inflammation and cell therapy products in vivo could be expanded by improving the intrinsic sensitivity of the probe by molecular design. We describe a metal chelate based on a salicylidene-tris(aminomethyl)ethane core, with solubility in perfluorocarbon (PFC) oils, and a potent accelerator of the 19F longitudinal relaxation time ( T1). Shortening T1 can increase the 19F image sensitivity per time and decrease the minimum number of detectable cells. We used the condensation between the tripodal ligand tris-1,1,1-(aminomethyl)ethane and salicylaldehyde to form the salicylidene-tris(aminomethyl)ethane chelating agent (SALTAME). We purified four isomers of SALTAME, elucidated structures using X-ray scattering and NMR, and identified a single isomer with high PFC solubility. Mn4+, Fe3+, Co3+, and Ga3+ cations formed stable and separable chelates with SALTAME, but only Fe3+ yielded superior T1 shortening with modest line broadening at 3 and 9.4 T. We mixed Fe3+ chelate with perfluorooctyl bromide (PFOB) to formulate a stable paramagnetic nanoemulsion imaging probe and assessed its biocompatibility in macrophages in vitro using proliferation, cytotoxicity, and phenotypic cell assays. Signal-to-noise modeling of paramagnetic PFOB shows that sensitivity enhancement of nearly 4-fold is feasible at clinical magnetic field strengths using a 19F spin-density-weighted gradient-echo pulse sequence. We demonstrate the utility of this paramagnetic nanoemulsion as an in vivo MRI probe for detecting inflammation macrophages in mice. Overall, these paramagnetic PFC compounds represent a platform for the development of sensitive 19F probes.

Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer.

Over the past two decades, immune cell therapy has emerged as a potent treatment for multiple cancers, first through groundbreaking leukemia therapy, and more recently, by tackling solid tumors. Developing successful therapeutic strategies using live cells could benefit from the ability to rapidly determine their in vivo biodistribution and persistence. Assaying cell biodistribution is unconventional compared to traditional small molecule drug pharmacokinetic readouts used in the pharmaceutical pipeline, yet this information is critical towards understanding putative therapeutic outcomes and modes of action. Towards this goal, efforts are underway to visualize and quantify immune cell therapy in vivo using advanced magnetic resonance imaging (MRI) techniques. Cell labeling probes based on perfluorocarbon nanoemulsions, paired with fluorine-19 MRI detection, enables background-free quantification of cell localization and survival. Here, we highlight recent preclinical and clinical uses of perfluorocarbon probes and 19F MRI for adoptive cell transfer (ACT) studies employing experimental T lymphocytes, NK, PBMC, and dendritic cell therapies. We assess the forward looking potential of this emerging imaging technology to aid discovery and preclinical phases, as well as clinical trials. The limitations and barriers towards widespread adoption of this technology, as well as alternative imaging strategies, are discussed.

Visualization of macrophage recruitment in head and neck carcinoma model using fluorine-19 magnetic resonance imaging.

PURPOSE: To evaluate the role of infiltrating macrophages in murine models of single and double mutation head and neck tumors using a novel fluorine-19 (19 F) MRI technology. METHODS: Tumor cell lines single-hit/SCC4 or double-hit/Cal27, with mutations of TP53 and TP53 & FHIT, respectively, were injected bilaterally into the flanks of (n = 10) female mice. With tumors established, perfluorocarbon nanoemulsion was injected intravenously, which labels in situ predominantly monocytes and macrophages. Longitudinal spin density-weighted 19 F MRI data enabled quantification of the macrophage burden in tumor and surrounding tissue. RESULTS: The average number of 19 F atoms within the tumors was twice as high in the Cal27 group compared with SCC4 (3.9 × 1019 and 2.0 × 101919 F/tumor, respectively; P = 0.0034) two days after contrast injection, signifying increased tumor-associated macrophages in double-hit tumors. The difference was still significant 10 days after injection. Histology stains correlated with in vivo results, exhibiting numerous perfluorocarbon-labeled macrophages in double-hit tumors and to a lesser extent in single-hit tumors. CONCLUSIONS: This study helps to establish 19 F MRI as a method for quantifying immune cells in the tumor microenvironment, allowing distinction between double and single-hit head and neck tumors. This technique would be extremely valuable in the clinic for pretreatment planning, prognostics, and post-treatment surveillance. Magn Reson Med 79:1972-1980, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Fluorine-19 nuclear magnetic resonance of chimeric antigen receptor T cell biodistribution in murine cancer model.

Discovery of effective cell therapies against cancer can be accelerated by the adaptation of tools to rapidly quantitate cell biodistribution and survival after delivery. Here, we describe the use of nuclear magnetic resonance (NMR) 'cytometry' to quantify the biodistribution of immunotherapeutic T cells in intact tissue samples. In this study, chimeric antigen receptor (CAR) T cells expressing EGFRvIII targeting transgene were labeled with a perfluorocarbon (PFC) emulsion ex vivo and infused into immunocompromised mice bearing subcutaneous human U87 glioblastomas expressing EGFRvIII and luciferase. Intact organs were harvested at day 2, 7 and 14 for whole-sample fluorine-19 (19F) NMR to quantitatively measure the presence of PFC-labeled CAR T cells, followed by histological validation. NMR measurements showed greater CAR T cell homing and persistence in the tumors and spleen compared to untransduced T cells. Tumor growth was monitored with bioluminescence imaging, showing that CAR T cell treatment resulted in significant tumor regression compared to untransduced T cells. Overall, 19F NMR cytometry is a rapid and quantitative method to evaluate cell biodistribution, tumor homing, and fate in preclinical studies.

Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.

Paramagnetic fluorinated nanoemulsions for sensitive cellular fluorine-19 magnetic resonance imaging.

Fluorine-19 magnetic resonance imaging ((19)F MRI) probes enable quantitative in vivo detection of cell therapies and inflammatory cells. Here, we describe the formulation of perfluorocarbon-based nanoemulsions with improved sensitivity for cellular MRI. Reduction of the (19)F spin-lattice relaxation time (T1) enables rapid imaging and an improved signal-to-noise ratio, thereby improving cell detection sensitivity. We synthesized metal-binding ß-diketones conjugated to linear perfluoropolyether (PFPE), formulated these fluorinated ligands as aqueous nanoemulsions, and then metallated them with various transition and lanthanide ions in the fluorous phase. Iron(III) tris-ß-diketonate ('FETRIS') nanoemulsions with PFPE have low cytotoxicity (<20%) and superior MRI properties. Moreover, the (19)F T1 can readily be reduced by an order of magnitude and tuned by stoichiometric modulation of the iron concentration. The resulting (19)F MRI detection sensitivity is enhanced by three- to fivefold over previously used tracers at 11.7 T, and is predicted to increase by at least eightfold at the clinical field strength of 3 T.

In Vivo Quantification of Inflammation in Experimental Autoimmune Encephalomyelitis Rats Using Fluorine-19 Magnetic Resonance Imaging Reveals Immune Cell Recruitment outside the Nervous System.

Progress in identifying new therapies for multiple sclerosis (MS) can be accelerated by using imaging biomarkers of disease progression or abatement in model systems. In this study, we evaluate the ability to noninvasively image and quantitate disease pathology using emerging "hot-spot" 19F MRI methods in an experimental autoimmune encephalomyelitis (EAE) rat, a model of MS. Rats with clinical symptoms of EAE were compared to control rats without EAE, as well as to EAE rats that received daily prophylactic treatments with cyclophosphamide. Perfluorocarbon (PFC) nanoemulsion was injected intravenously, which labels predominately monocytes and macrophages in situ. Analysis of the spin-density weighted 19F MRI data enabled quantification of the apparent macrophage burden in the central nervous system and other tissues. The in vivo MRI results were confirmed by extremely high-resolution 19F/1H magnetic resonance microscopy in excised tissue samples and histopathologic analyses. Additionally, 19F nuclear magnetic resonance spectroscopy of intact tissue samples was used to assay the PFC biodistribution in EAE and control rats. In vivo hot-spot 19F signals were detected predominantly in the EAE spinal cord, consistent with the presence of inflammatory infiltrates. Surprising, prominent 19F hot-spots were observed in bone-marrow cavities adjacent to spinal cord lesions; these were not observed in control animals. Quantitative evaluation of cohorts receiving cyclophosphamide treatment displayed significant reduction in 19F signal within the spinal cord and bone marrow of EAE rats. Overall, 19F MRI can be used to quantitatively monitored EAE disease burden, discover unexpected sites of inflammatory activity, and may serve as a sensitive biomarker for the discovery and preclinical assessment of novel MS therapeutic interventions.