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Purpose: We explore the use of intravenously delivered fluorescent perfluorocarbon (PFC) nanoemulsion tracers and multi-spectral cryo-fluorescence tomography (CFT) for whole-body tracer imaging in murine inflammation models. CFT is an emerging technique that provides high-resolution, three-dimensional mapping of probe localization in intact animals and tissue samples, enabling unbiased validation of probe biodistribution and minimizes reliance on laborious histological methods employing discrete tissue panels, where disseminated populations of PFC-labeled cells may be overlooked. This methodology can be used to streamline the development of new generations of non-invasive, cellular-molecular imaging probes for in vivo imaging., Procedures: Mixtures of nanoemulsions with different fluorescent emission wavelengths were administered intravenously to naïve mice and models of acute inflammation, colitis, and solid tumor. Mice were euthanized 24 h post-injection, frozen en bloc, and imaged at high resolution (~ 50 µm voxels) using CFT at multiple wavelengths., Results: PFC nanoemulsions were visualized using CFT within tissues of the reticuloendothelial system and inflammatory lesions, consistent with immune cell (macrophage) labeling, as previously reported in in vivo magnetic resonance and nuclear imaging studies. The CFT signals show pronounced differences among fluorescence wavelengths and tissues, presumably due to autofluorescence, differential fluorescence quenching, and scattering of incident and emitted light., Conclusions: CFT is an effective and complementary methodology to in vivo imaging for validating PFC nanoemulsion biodistribution at high spatial localization, bridging the resolution gap between in vivo imaging and histology., (© 2024. The Author(s), under exclusive licence to World Molecular Imaging Society.)

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 ( 19 F) 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 19 F 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 ~70×10 10 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 19 F 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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: We explore the use of intravenously delivered perfluorocarbon (PFC) nanoemulsion and 19 F MRI for detecting inflammation in a mouse model of non-alcoholic fatty liver disease (NAFLD). Correlative studies of 1 H-based liver proton density fat fraction (PDFF) and T 1 measurements and histology are also evaluated., Procedures: C57BL/6 mice were fed standard or high-fat diet (HFD) for 6 weeks to induce NAFLD. 1 H MRI measurements of PDFF and T 1 relaxation time were performed at baseline to assess NAFLD onset prior to administration of a PFC nanoemulsion to enable 19 F MRI of liver PFC uptake. 1 H and 19 F 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 T 1 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 19 F MRI may be a viable biomarker of liver pathology., (© 2022. The Author(s), under exclusive licence to World Molecular Imaging Society.)

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.

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 T 1 relaxation rate and a commensurate gain in imaging speed and averages. However, 19 F T 2 -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 l 1 -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 T 1 . 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., (© 2022 John Wiley & Sons, Ltd.)

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 1 H/ 19 F 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., Competing Interests: The authors declare the following competing financial interest(s): E.T.A. is a founder and shareholder of Celsense, Inc. The other authors have nothing to disclose., (© 2021 The Authors. Published by American Chemical Society.)

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, Fe 3+ encapsulation imparts a strong paramagnetic relaxation effect on 19 F T 1 that dramatically accelerates 19 F MRI data acquisition times and hence sensitivity in cell tracking applications. Alternatively, 89 Zr encapsulation creates a sensitive and versatile PET probe for inflammatory macrophage detection. Adding lanthanides, such as Eu 3+ , 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.

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 19 F-containing FERM nanoemulsion encapsulates 89 Zr 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 89 Zr PET and 19 F 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 19 F and 89 Zr 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 19 F MRI. The FERM nanoemulsion has scalable production and is potentially useful for precise diagnosis, stratification, and treatment monitoring of inflammatory diseases., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: To assess the cell-specific, intracellular partial pressure of oxygen (Po 2 ) 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 Po 2 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 ( 19 F) spin-lattice relaxation time measurements of the tumor mass were used to ascertain absolute Po 2 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 Po 2 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 Po 2 over time in tumor using these same methods. Moreover, it was observed that the total 19 F tumor cell signal quenches with treatment, consistent with rapid tissue clearance of probe from apoptotic tumor cells., Conclusion: Cell-specific Po 2 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., Competing Interests: Disclosures of Conflicts of Interest: F.C. Activities related to the present article: This study was funded through National Institutes of Health grants R01-EB017271, R01-EB024015 and R01-CA139579 and the California Institute for Regenerative Medicine grant LA1-C12-06919. Activities not related to the present article: disclosed no relevant relationships. Other relationships: disclosed no relevant relationships. B.I.L. Activities related to the present article: This study was funded through National Institutes of Health grants R01-EB017271, R01-EB024015 and R01-CA139579 and the California Institute for Regenerative Medicine grant LA1-C12-06919. Activities not related to the present article: author formerly employed by Scripps Research Institute. Other relationships: disclosed no relevant relationships. R.C. disclosed no relevant relationships. D.L. disclosed no relevant relationships. K.M. disclosed no relevant relationships. H.O. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: author paid royalties fees for licensed IP as one of the inventors of the EGFRviii-CAR that has been exclusively licensed to Novartis. However, this did not influence the interpretation of the data sets in this manuscript at all because this author did not participate in the data interpretation part. Other relationships: patent US 62/653,901 Inventors: Hideho Okada et al. US 62/653,901 Trans-Antigen Targeting in Heterogeneous Cancers and Methods of Use Thereof US 62/653,929 Methods of Treating Glioblastomas US 62/654,012 Methods of Treating EGFRvIII-Expressing Glioblastomas; exclusively licensed to Novartis Pharma and royalties from Novartis Pharma. E.T.A. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: disclosed no relevant relationships. Other relationships: founder and shareholder of Celsense., (2021 by the Radiological Society of North America, Inc.)

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 vivo 19 F/ 1 H 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 β-diketones can be formulated for intravenous delivery and inflammation detection in vivo.

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 ~10 12 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., (© 2019 International Society for Magnetic Resonance in Medicine.)

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.

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( β -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 19 F longitudinal relaxation time ( T 1 ) to <100 ms with modest line broadening and displays superior properties for 19 F 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 19 F image sensitivity per time via signal averaging. Additionally, 19 F 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 19 F in nanoemulsion, which makes them potentially useful for multichromatic 19 F imaging. Formulated nanoemulsions have a shelf life >200 days. Free β -diketonate or its iron complex in formed PFC nanoemulsion did not induce cytotoxicity in intracellularly labeled macrophages. Overall, ferric tris( β -diketonate) chelate provides a scalable approach for boosting sensitivity of PFC-based 19 F MRI probes. More generally, it can functionalize PFC oil, whose chemical modification remains challenging., Competing Interests: The authors declare the following competing financial interest(s): E.T.A. is founder, consultant, member of the advisory board, and shareholder of Celsense, Inc.

Fluorine-19 MRI is an emerging cellular imaging approach, enabling lucid, quantitative "hot-spot" imaging with no background signal. The utility of 19 F-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 19 F longitudinal relaxation time ( T 1 ). Shortening T 1 can increase the 19 F 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. Mn 4+ , Fe 3+ , Co 3+ , and Ga 3+ cations formed stable and separable chelates with SALTAME, but only Fe 3+ yielded superior T 1 shortening with modest line broadening at 3 and 9.4 T. We mixed Fe 3+ 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 19 F 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 19 F probes.

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 19 F 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.

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 × 10 19 and 2.0 × 10 19 19 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., (© 2017 International Society for Magnetic Resonance in Medicine.)

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 ( 19 F) 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, 19 F NMR cytometry is a rapid and quantitative method to evaluate cell biodistribution, tumor homing, and fate in preclinical studies.

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.

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 β-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-β-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.

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.

Purpose: The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI., Methods: We characterized (19)F-NMR relaxation properties of PFC-labeled cells in the presence of SPIO and imaged cells both ex vivo and in vivo in a rodent inflammation model to demonstrate selective visualization of cell populations., Results: We show that with UTE3D, RARE, and FLASH (19) F images one can uniquely identify PFC-labeled cells, colocalized PFC- and SPIO-labeled cells, and PFC/SPIO-colabeled cells., Conclusion: This new methodology has the ability to improve and expand applications of MRI cell tracking. Combining PFC and SPIO strategies can potentially provide a method to quench PFC signal transferred from dead cells to macrophages, thereby eliminating false positives. In addition, combining these techniques could also be used to track two cell types simultaneously and probe cell-cell proximity in vivo with MRI., (© 2014 Wiley Periodicals, Inc.)

Purpose: Cellular therapeutics are emerging as a treatment option for a host of serious human diseases. To accelerate clinical translation, noninvasive imaging of cell grafts in clinical trials can potentially be used to assess the initial delivery and behavior of cells., Methods: The use of a perfluorocarbon (PFC) tracer agent for clinical fluorine-19 ((19) F) MRI cell detection is described. This technology was used to detect immunotherapeutic dendritic cells (DCs) delivered to colorectal adenocarcinoma patients. Autologous DC vaccines were labeled with a PFC MRI agent ex vivo. Patients received DCs intradermally, and (19) F spin-density-weighted MRI at 3 Tesla (T) was used to observe cells., Results: Spin-density-weighted (19) F images at the injection site displayed DCs as background-free "hot-spot" images. (19) F images were acquired in clinically relevant scan times (<10 min). Apparent DC numbers could be quantified in two patients from the (19) F hot-spots and were observed to decrease by ∼50% at injection site by 24 h. From 3T phantom studies, the sensitivity limit for DC detection is estimated to be on the order of ∼10(5) cells/voxel in this study., Conclusion: These results help to establish a clinically applicable means to track a broad range of cell types used in cell therapy., (© 2014 The Authors. Magnetic Resonance in Medicine Published by Wiley Periodicals, Inc. on behalf of International Society of Medicine in Resonance.)

Fluorine ((19)F) MRI of perfluorocarbon-labeled cells has become a powerful technique to track the migration and accumulation of cells in living organisms. It is common to label cells for (19)F MRI with nanoemulsions of perfluoropolyethers that contain a large number of chemically equivalent fluorine atoms. Understanding the mechanisms of (19)F nuclear relaxation, and in particular the spin-lattice relaxation of these molecules, is critical to improving experimental sensitivity. To date, the temperature and magnetic field strength dependence of spin-lattice relaxation rate constant (R1) for perfluoropolyethers has not been described in detail. In this study, we evaluated the R1 of linear perfluoropolyether (PFPE) and cyclic perfluoro-15-crown-5 ether (PCE) at three magnetic field strengths (7.0, 9.4, and 14.1T) and at temperatures ranging from 256-323K. Our results show that R1 of perfluoropolyethers is dominated by dipole-dipole interactions and chemical shift anisotropy. R1 increased with magnetic field strength for both PCE and PFPE. In the temperature range studied, PCE was in the fast motion regime (ωτc<1) at all field strengths, but for PFPE, R1 passed through a maximum, from which the rotational correlation time was estimated. The importance of these measurements for the rational design of new (19)F MRI agents and methods is discussed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon coincubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions were characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry and displayed a steady decrease in pH to a level of 5.5 over 3 h, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using (19)F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH-sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics, nanoemulsion stability and cell viability over time.

We report the design of a MRI reporter gene with applications to non-invasive molecular imaging. We modified mitochondrial ferritin to localize to the cell cytoplasm. We confirmed the efficient cellular processing of this engineered protein and demonstrated high iron loading in mammalian cells. The reporter's intracellular localization appears as distinct clusters that deliver robust MRI contrast. We used this new reporter to image in vivo and ex vivo the gene expression in native olfactory sensory neurons in the mouse epithelium. This robust MRI reporter can facilitate the study of the molecular mechanisms of olfaction and to monitor intranasal gene therapy delivery, as well as a wide range of cell tracking and gene expression studies in living subjects.

We report results of an analysis of embolism formation and subsequent refilling observed in stems of Acer rubrum L. using magnetic resonance imaging (MRI). MRI is one of the very few techniques that can provide direct non-destructive observations of the water content within opaque biological materials at a micrometer resolution. Thus, it has been used to determine temporal dynamics and water distributions within xylem tissue. In this study, we found good agreement between MRI measures of pixel brightness to assess xylem liquid water content and the percent loss in hydraulic conductivity (PLC) in response to water stress (P50 values of 2.51 and 2.70 for MRI and PLC, respectively). These data provide strong support that pixel brightness is well correlated to PLC and can be used as a proxy of PLC even when single vessels cannot be resolved on the image. Pressure induced embolism in moderately stressed plants resulted in initial drop of pixel brightness. This drop was followed by brightness gain over 100 min following pressure application suggesting that plants can restore water content in stem after induced embolism. This recovery was limited only to current-year wood ring; older wood did not show signs of recovery within the length of experiment (16 h). In vivo MRI observations of the xylem of moderately stressed (~-0.5 MPa) A. rubrum stems revealed evidence of a spontaneous embolism formation followed by rapid refilling (~30 min). Spontaneous (not induced) embolism formation was observed only once, despite over 60 h of continuous MRI observations made on several plants. Thus this observation provide evidence for the presence of naturally occurring embolism-refilling cycle in A. rubrum, but it is impossible to infer any conclusions in relation to its frequency in nature.

This article presents a brief review of preclinical in vivo cell-tracking methods and applications using perfluorocarbon (PFC) probes and fluorine-19 ((19) F) MRI detection. Detection of the (19) F signal offers high cell specificity and quantification ability in spin density-weighted MR images. We discuss the compositions of matter, methods and applications of PFC-based cell tracking using ex vivo and in situ PFC labeling in preclinical studies of inflammation and cellular therapeutics. We also address the potential applicability of (19) F cell tracking to clinical trials., (Copyright © 2013 John Wiley & Sons, Ltd.)

Cell tracking using perfluorocarbon labels and fluorine-19 (19F) MRI is a noninvasive approach to visualize and quantify cell populations in vivo. In this study, we investigated three-dimensional compressed sensing methods to accelerate 19F MRI data acquisition for cell tracking and evaluate the impact of acceleration on 19F signal quantification. We show that a greater than 8-fold reduction in imaging time was feasible without pronounced image degradation and with minimal impact on the image signal-to-noise ratio and 19F quantification accuracy. In 19F phantom studies, we show that apparent feature topology is maintained with compressed sensing reconstruction, and false positive signals do not appear in areas devoid of fluorine. We apply the three-dimensional compressed sensing 19F MRI methods to quantify the macrophage burden in a localized wounding-inflammation mouse model in vivo; at 8-fold image acceleration, the 19F signal distribution was accurately reproduced, with no loss in signal-to-noise ratio. Our results demonstrate that three-dimensional compressed sensing methods have potential for advancing in vivo 19F cell tracking for a wide range of preclinical and translational applications., (Copyright © 2012 Wiley Periodicals, Inc.)

The development of effective strategies for gene therapy has been hampered by difficulties verifying transgene delivery in vivo and quantifying gene expression non-invasively. Magnetic resonance imaging (MRI) offers high spatial resolution and three-dimensional views, without tissue depth limitations. The iron-storage protein ferritin is a prototype MRI gene reporter. Ferritin forms a paramagnetic ferrihydrite core that can be detected by MRI via its effect on the local magnetic field experienced by water protons. In an effort to better characterize the ferritin reporter for central nervous system applications, we expressed ferritin in the mouse brain in vivo using a neurotropic herpes simplex virus type 1 (HSV-1). We computed three-dimensional maps of MRI transverse relaxation rates in the mouse brain with ascending doses of ferritin-expressing HSV-1. We established that the transverse relaxation rates correlate significantly to the number of inoculated infectious particles. Our results are potentially useful for quantitatively assessing limitations of ferritin reporters for gene therapy applications.

Noninvasive biomarkers of anti-tumoral efficacy are of great importance to the development of therapeutic agents. Tumor oxygenation has been shown to be an important indicator of therapeutic response. We report the use of intracellular labeling of tumor cells with perfluorocarbon (PFC) molecules, combined with quantitative ¹⁹F spin-lattice relaxation rate (R₁) measurements, to assay tumor cell oxygen dynamics in situ. In a murine central nervous system (CNS) GL261 glioma model, we visualized the impact of Pmel-1 cytotoxic T cell immunotherapy, delivered intravenously, on intracellular tumor oxygen levels. GL261 glioma cells were labeled ex vivo with PFC and inoculated into the mouse striatum. The R₁ of ¹⁹F labeled cells was measured using localized single-voxel magnetic resonance spectroscopy, and the absolute intracellular partial pressure of oxygen (pO₂) was ascertained. Three days after tumor implantation, mice were treated with 2×10⁷ cytotoxic T cells intravenously. At day five, a transient spike in pO₂ was observed indicating an influx of T cells into the CNS and putative tumor cell apoptosis. Immunohistochemistry and quantitative flow cytometry analysis confirmed that the pO₂ was causally related to the T cells infiltration. Surprisingly, the pO₂ spike was detected even though few (∼4×10⁴) T cells actually ingress into the CNS and with minimal tumor shrinkage. These results indicate the high sensitivity of this approach and its utility as a non-invasive surrogate biomarker of anti-cancer immunotherapeutic response in preclinical models.

Background: Non-invasive imaging of inflammation to measure the progression of autoimmune diseases, such as rheumatoid arthritis (RA), and to monitor responses to therapy is critically needed. V-Sense, a perfluorocarbon (PFC) contrast agent that preferentially labels inflammatory cells, which are then recruited out of systemic circulation to sites of inflammation, enables detection by 19F MRI. With no 19F background in the host, detection is highly-specific and can act as a proxy biomarker of the degree of inflammation present., Methods: Collagen-induced arthritis in rats, a model with many similarities to human RA, was used to study the ability of the PFC contrast agent to reveal the accumulation of inflammation over time using 19F MRI. Disease progression in the rat hind limbs was monitored by caliper measurements and 19F MRI on days 15, 22 and 29, including the height of clinically symptomatic disease. Naïve rats served as controls. The capacity of the PFC contrast agent and 19F MRI to assess the effectiveness of therapy was studied in a cohort of rats administered oral prednisolone on days 14 to 28., Results: Quantification of 19F signal measured by MRI in affected limbs was linearly correlated with disease severity. In animals with progressive disease, increases in 19F signal reflected the ongoing recruitment of inflammatory cells to the site, while no increase in 19F signal was observed in animals receiving treatment which resulted in clinical resolution of disease., Conclusion: These results indicate that 19F MRI may be used to quantitatively and qualitatively evaluate longitudinal responses to a therapeutic regimen, while additionally revealing the recruitment of monocytic cells involved in the inflammatory process to the anatomical site. This study may support the use of 19F MRI to clinically quantify and monitor the severity of inflammation, and to assess the effectiveness of treatments in RA and other diseases with an inflammatory component.

Macrophages have an important role in the pathogenesis of most chronic inflammatory diseases. A means of non-invasively quantifying macrophage migration would contribute significantly towards our understanding of chronic inflammatory processes and aid the evaluation of novel therapeutic strategies. We describe the use of a perfluorocarbon tracer reagent and in vivo (19)F magnetic resonance imaging (MRI) to quantify macrophage burden longitudinally. We apply these methods to evaluate the severity and three-dimensional distribution of macrophages in a murine model of inflammatory bowel disease (IBD). MRI results were validated by histological analysis, immunofluorescence and quantitative real-time polymerase chain reaction. Selective depletion of macrophages in vivo was also performed, further validating that macrophage accumulation of perfluorocarbon tracers was the basis of (19)F MRI signals observed in the bowel. We tested the effects of two common clinical drugs, dexamethasone and cyclosporine A, on IBD progression. Whereas cyclosporine A provided mild therapeutic effect, unexpectedly dexamethasone enhanced colon inflammation, especially in the descending colon. Overall, (19)F MRI can be used to evaluate early-stage inflammation in IBD and is suitable for evaluating putative therapeutics. Due to its high macrophage specificity and quantitative ability, we envisage (19)F MRI having an important role in evaluating a wide range of chronic inflammatory conditions mediated by macrophages.

Transplantation of human neural stem cells (hNSCs) is emerging as a viable treatment for stroke related brain injury. However, intraparenchymal grafts do not regenerate lost tissue, but rather integrate into the host parenchyma without significantly affecting the lesion cavity. Providing a structural support for the delivered cells appears important for cell based therapeutic approaches. The non-invasive monitoring of therapeutic methods would provide valuable information regarding therapeutic strategies but remains a challenge. Labeling transplanted cells with metal-based (1)H-magnetic resonance imaging (MRI) contrast agents affects the visualization of the lesion cavity. Herein, we demonstrate that a (19)F-MRI contrast agent can adequately monitor the distribution of transplanted cells, whilst allowing an evaluation of the lesion cavity and the formation of new tissue on (1)H-MRI scans. Twenty percent of cells labeled with the (19)F agent were of host origin, potentially reflecting the re-uptake of label from dead transplanted cells. Both T(2)- and diffusion-weighted MRI scans indicated that transplantation of hNSCs suspended in a gel form of a xenogeneic extracellular matrix (ECM) bioscaffold resulted in uniformly distributed cells throughout the lesion cavity. However, diffusion MRI indicated that the injected materials did not yet establish diffusion barriers (i.e. cellular network, fiber tracts) normally found within striatal tissue. The ECM bioscaffold therefore provides an important support to hNSCs for the creation of de novo tissue and multi-nuclei MRI represents an adept method for the visualization of some aspects of this process. However, significant developments of both the transplantation paradigm, as well as regenerative imaging, are required to successfully create new tissue in the lesion cavity and to monitor this process non-invasively., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Adult neurogenesis research in mammals presents a challenge as most stem cells and progenitors are located deep in opaque brain tissues. Here, we describe an efficient ferritin-based magnetic resonance imaging (MRI) reporter and its use to label mouse subventricular zone progenitors, enabling in vivo visualization of endogenous neuroblast migration toward the olfactory bulb. We quantify the effect of the ferritin transgene expression on cellular iron transport proteins such as transferrin receptor, divalent metal transporter and STEAP reductase. Based on these data, we elucidate key aspects of the cellular pathways that the reporter utilizes to load iron and form its superparamagnetic core. This MRI reporter gene platform can facilitate the non-invasive study of native or transplanted stem cell migration and associated neurogenic or therapeutic molecular events in live animals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Understanding how individual cells behave inside living systems will help enable new diagnostic tools and cellular therapies. Superparamagnetic iron oxide particles can be used to label cells and theranostic capsules for noninvasive tracking using MRI. Contrast changes from superparamagnetic iron oxide are often subtle relative to intrinsic sources of contrast, presenting a detection challenge. Here, we describe a versatile postprocessing method, called Phase map cross-correlation Detection and Quantification (PDQ), that automatically identifies localized deposits of superparamagnetic iron oxide, estimating their volume magnetic susceptibility and magnetic moment. To demonstrate applicability, PDQ was used to detect and characterize superparamagnetic iron oxide-labeled magnetocapsules implanted in porcine liver and suspended in agarose gel. PDQ was also applied to mouse brains infiltrated by MPIO-labeled macrophages following traumatic brain injury; longitudinal, in vivo studies tracked individual MPIO clusters over 3 days, and tracked clusters were corroborated in ex vivo brain scans. Additionally, we applied PDQ to rat hearts infiltrated by MPIO-labeled macrophages in a transplant model of organ rejection. PDQ magnetic measurements were signal-to-noise ratio invariant for images with signal-to-noise ratio > 11. PDQ can be used with conventional gradient-echo pulse sequences, requiring no extra scan time. The method is useful for visualizing biodistribution of cells and theranostic magnetocapsules and for measuring their relative iron content., (Copyright © 2011 Wiley-Liss, Inc.)

We have demonstrated previously that BM-derived DCs can prevent diabetes development and halt progression of insulitis in NOD mice, the mouse model of type 1 diabetes. The DC population that was most effective in this therapy had a mature phenotype, expressed high levels of costimulatory molecules, and secreted low levels of IL-12p70. The protective DC therapy induced Treg and Th2 cells in vitro and in vivo. Microarray analysis of therapeutic and nontherapeutic DC populations revealed differences in the expression of OX40L, CD200, Ym-1, CCL2, and CCL5, which could play important roles in the observed DC-mediated therapy. The unique pattern of costimulatory molecules and chemokines expressed by the therapeutic DCs was confirmed by flow cytometry and ELISA. Using a novel cell-labeling and (19)F NMR, we observed that the chemokines secreted by the therapeutic DCs altered the migration of diabetogenic Th1 cells in vivo and attracted Th2 cells. These results suggest that the therapeutic function of DCs is mediated by a combination of costimulatory and chemokine properties that results in the attraction of diabetogenic Th1 and the induction of Th2 and/or Treg differentiation.

Monitoring of cell therapeutics in vivo is of major importance to estimate its efficacy. Here, we present a novel intracellular label for (19)F magnetic resonance imaging (MRI)-based cell tracking, which allows for noninvasive, longitudinal cell tracking without the use of radioisotopes. A key advantage of (19)F MRI is that it allows for absolute quantification of cell numbers directly from the MRI data. The (19)F label was tested in primary human monocyte-derived dendritic cells. These cells took up label effectively, resulting in a labeling of 1.7 ± 0.1 × 10(13) (19)F atoms per cell, with a viability of 80 ± 6%, without the need for electroporation or transfection agents. This results in a minimum detection sensitivity of about 2,000 cells/voxel at 7 T, comparable with gadolinium-labeled cells. Comparison of the detection sensitivity of cells labeled with (19)F, iron oxide and gadolinium over typical tissue background showed that unambiguous detection of the (19)F-labeled cells was simpler than with the contrast agents. The effect of the (19)F agent on cell function was minimal in the context of cell-based vaccines. From these data, we calculate that detection of 30,000 cells in vivo at 3 T with a reasonable signal to noise ratio for (19)F images would require less than 30 min with a conventional fast spin echo sequence, given a coil similar to the one used in this study. This is well within acceptable limits for clinical studies, and thus, we conclude that (19)F MRI for quantitative cell tracking in a clinical setting has great potential., (Copyright © 2010 UICC.)

Quantification of inflammation in tissue samples can be a time-intensive bottleneck in therapeutic discovery and preclinical endeavors. We describe a versatile and rapid approach to quantitatively assay macrophage burden in intact tissue samples. Perfluorocarbon (PFC) emulsion is injected intravenously, and the emulsion droplets are effectively taken up by monocytes and macrophages. These 'in situ' labeled cells participate in inflammatory events in vivo resulting in PFC accumulation at inflammatory loci. Necropsied tissues or intact organs are subjected to conventional fluorine-19 ((19)F) NMR spectroscopy to quantify the total fluorine content per sample, proportional to the macrophage burden. We applied these methods to a rat model of experimental allergic encephalomyelitis (EAE) exhibiting extensive inflammation and demyelination in the central nervous system (CNS), particularly in the spinal cord. In a cohort of EAE rats, we used (19)F NMR to derive an inflammation index (IFI) in intact CNS tissues. Immunohistochemistry was used to confirm intracellular colocalization of the PFC droplets within CNS CD68+ cells having macrophage morphology. The IFI linearly correlated to mRNA levels of CD68 via real-time PCR analysis. This (19)F NMR approach can accelerate tissue analysis by at least an order of magnitude compared with histological approaches.

Current diagnosis of organ rejection following transplantation relies on tissue biopsy, which is not ideal due to sampling limitations and risks associated with the invasive procedure.We have previously shown that cellular magnetic resonance imaging (MRI) of iron-oxide labeled immune-cell infiltration can provide a noninvasive measure of rejection status by detecting areas of hypointensity on T 2*-weighted images. In this study, we tested the feasibility of using a fluorine-based cellular tracer agent to detect macrophage accumulation in rodent models of acute allograft rejection by fluorine-19 ((19) F) MRI and magnetic resonance spectroscopy. This study used two rat models of acute rejection, including abdominal heterotopic cardiac transplant and orthotopic kidney transplant models. Following in vivo labeling of monocytes and macrophages with a commercially available agent containing perfluoro-15-crown-5-ether, we observed (19) F-signal intensity in the organs experiencing rejection by (19) F MRI, and conventional (1) H MRI was used for anatomical context. Immunofluorescence and histology confirmed macrophage labeling. These results are consistent with our previous studies and show the complementary nature of the two cellular imaging techniques. With no background signal, (19) F MRI/magnetic resonance spectroscopy can provide unambiguous detection of fluorine labeled cells, and may be a useful technique for detecting and quantifying rejection grade in patients., (Copyright © 2010 Wiley-Liss, Inc.)

Preclinical development of therapeutic agents against cancer could greatly benefit from noninvasive markers of tumor killing. Potentially, the intracellular partial pressure of oxygen (pO(2) ) can be used as an early marker of antitumor efficacy. Here, the feasibility of measuring intracellular pO(2) of central nervous system glioma cells in vivo using (19) F magnetic resonance techniques is examined. Rat 9L glioma cells were labeled with perfluoro-15-crown-5-ether ex vivo and then implanted into the rat striatum. (19) F MRI was used to visualize tumor location in vivo. The mean (19) F T(1) of the implanted cells was measured using localized, single-voxel spectroscopy. The intracellular pO(2) in tumor cells was determined from an in vitro calibration curve. The basal pO(2) of 9L cells (day 3) was determined to be 45.3 ± 5 mmHg (n = 6). Rats were then treated with a 1 × LD10 dose of bischloroethylnitrosourea intravenously and changes in intracellular pO(2) were monitored. The pO(2) increased significantly (P = 0.042, paired T-test) to 141.8 ± 3 mmHg within 18 h after bischloroethylnitrosourea treatment (day 4) and remained elevated (165 ± 24 mmHg) for at least 72 h (day 6). Intracellular localization of the perfluoro-15-crown-5-ether emulsion in 9L cells before and after bischloroethylnitrosourea treatment was confirmed by histological examination and fluorescence microscopy. Overall, noninvasive (19) F magnetic resonance techniques may provide a valuable preclinical tool for monitoring therapeutic response against central nervous system or other deep-seated tumors., (Copyright © 2010 Wiley-Liss, Inc.)

Teratoma formation in xenografts is a sufficiently stringent pluripotency assay for stem cells. However, little is known about the composition and spatial relationships of tissues within teratomas that may provide clues about development and platforms for studying organ development. Additionally, teratoma formation and analysis lack standards for reporting as assays of pluripotency. Three of 27 total teratomas derived from pedigreed primate embryonic stem cells underwent quantitative three-dimensional high-resolution magnetic resonance microscopy (MRM). Teratomas were subsequently serially sectioned and tissue types identified, semiquantitated, and correlated with MRM images. All teratomas demonstrated tissue derivatives from the three germ layers and approximately 23 different tissue types were identified. Certain tissue groups attempted to form organs more frequently (e.g., trachea/bronchi, small intestine). MRM discriminated some tissues readily (e.g., bone, adipose, cartilage) while other tissue types with like MR intensities could not be distinguished. Semiquantitative histopathological analysis of teratomas demonstrates the ability to delineate multiple tissues as derived from ectoderm, mesoderm, or endoderm and to use this information for comparison to other teratomas. MRM provides rapid quantitative imaging of intact teratomas that complements histology and identifies sites of interest for additional biological studies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This paper describes the design and characterization of a novel ferritin chimera. The iron storage protein ferritin forms a paramagnetic ferrihydrite core. This biomineral, when placed in a magnetic field, can decrease the transverse NMR relaxation times (T (2) and T (2)*) of nearby mobile water protons. Ferritin nucleic acid constructs have recently been studied as "probeless" magnetic resonance imaging (MRI) reporters. Following reporter expression, ferritin sequesters endogenous iron and imparts hypointensity to T (2)- and T (2)*-weighted images in an amount proportional to the ferritin iron load. Wild-type ferritin consists of various ratios of heavy H and light L subunits, and their ratio affects ferritin's stability and iron storage capacity. We report a novel chimeric ferritin with a fixed subunit stoichiometry obtained by fusion of the L and the H subunits (L*H and H*L) using a flexible linker. We characterize these supramolecular ferritins expressed in human cells, including their iron loading characteristics, hydrodynamic size, subcellular localization, and effect on solvent water T (2) relaxation rate. Interestingly, we found that the L*H chimera exhibits a significantly enhanced iron loading ability and T (2) relaxation compared to wild-type ferritin. We suggest that the L*H chimera may be useful as a sensitive MRI reporter molecule.

Cellular therapy, including stem cell transplants and dendritic cell vaccines, is typically monitored for dosage optimization, accurate delivery, and localization using noninvasive imaging, of which magnetic resonance imaging (MRI) is a key modality. (19)F MRI retains the advantages of MRI as an imaging modality, and also allows direct detection of labeled cells for unambiguous identification and quantification, unlike typical metal-based contrast agents. Recent developments in (19)F MRI-based in vivo cell quantification, the existing clinical use of (19)F compounds and current explosive interest in cellular therapeutics have brought (19)F imaging technology closer to clinical application. We review the application of (19)F MRI to cell tracking, discussing intracellular (19)F labels, cell labeling and in vivo quantification, as well as the potential clinical uses of (19)F MRI.

Objective: Pancreatitis occurs as an extraintestinal complication of inflammatory bowel disease (IBD), but the cause is poorly understood. Mucin 1 (MUC1) is overexpressed in an abnormal, hypoglycosylated form on the colonic epithelium in human IBD where it contributes to inflammation. MUC1 is also expressed on pancreatic ductal epithelia. We tested the possibility that in IBD, MUC1 expression on pancreatic ducts is also abnormal leading to inflammation and pancreatitis., Methods: We used MUC1/interleukin-10 mice that develop IBD. We imaged abnormal MUC1 expression in these mice by adoptively transferring T cells from T cell receptor transgenic mice specific for abnormal MUC1. Cells were labeled with a novel perfluorocarbon tracer reagent and quantified and visualized in vivo using high-throughput F nuclear magnetic resonance spectroscopy and magnetic resonance imaging., Results: MUC1-specific T cells migrated to the colon in mice with IBD and also to the pancreas. Immunohistochemistry confirmed increased expression on the pancreatic ducts of the abnormal MUC1 seen in the colon and the presence of cellular infiltrate., Conclusions: Migration of MUC1-specific T cells to the colon and the pancreas in diseased mice suggests that pancreatitis is an extraintestinal site of IBD, characterized by proinflammatory abnormal expression of MUC1. Therapies directed against abnormal MUC1 have the potential of targeting the disease in both sites.

Background Aims: Dendritic cells (DC) are increasingly being used as cellular vaccines to treat cancer and infectious diseases. While there have been some promising results in early clinical trials using DC-based vaccines, the inability to visualize non-invasively the location, migration and fate of cells once adoptively transferred into patients is often cited as a limiting factor in the advancement of these therapies. A novel perflouropolyether (PFPE) tracer agent was used to label human DC ex vivo for the purpose of tracking the cells in vivo by (19)F magnetic resonance imaging (MRI). We provide an assessment of this technology and examine its impact on the health and function of the DC., Methods: Monocyte-derived DC were labeled with PFPE and then assessed. Cell viability was determined by examining cell membrane integrity and mitochondrial lipid content. Immunostaining and flow cytometry were used to measure surface antigen expression of DC maturation markers. Functional tests included bioassays for interleukin (IL)-12p70 production, T-cell stimulatory function and chemotaxis. MRI efficacy was demonstrated by inoculation of PFPE-labeled human DC into NOD-SCID mice., Results: DC were effectively labeled with PFPE without significant impact on cell viability, phenotype or function. The PFPE-labeled DC were clearly detected in vivo by (19)F MRI, with mature DC being shown to migrate selectively towards draining lymph node regions within 18 h., Conclusions: This study is the first application of PFPE cell labeling and MRI cell tracking using human immunotherapeutic cells. These techniques may have significant potential for tracking therapeutic cells in future clinical trials.

Imaging gene expression non-invasively and deep into opaque tissues has been a long-standing goal of molecular science. Optical gene reporters such as green fluorescent protein and luciferase have revolutionized cellular and molecular biology, however their in vivo application is limited, due to poor tissue penetration of visible light. The iron storage protein ferritin forms a paramagnetic ferrihydrite core that affects the relaxation rate of surrounding nuclear spins. Ferritin has recently emerged as an MRI gene reporter for molecular applications, however its detection with MRI still has relatively low sensitivity. In this work we present an improved ferritin chimera, genetically engineered to exhibit stronger paramagnetic properties.