Eric T. Ahrens

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.

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.

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.

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. Magn Reson Med, 2013.

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. Magn Reson Med, 2011.

Preclinical development of therapeutic agents against cancer could greatly benefit from noninvasive markers of tumor killing. Potentially, the intracellular partial pressure of oxygen (pO₂) can be used as an early marker of antitumor efficacy. Here, the feasibility of measuring intracellular pO₂ of central nervous system glioma cells in vivo using ¹⁹F 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. ¹⁹F MRI was used to visualize tumor location in vivo. The mean ¹⁹F T₁ of the implanted cells was measured using localized, single‐voxel spectroscopy. The intracellular pO₂ in tumor cells was determined from an in vitro calibration curve. The basal pO₂ 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 pO₂ were monitored. The pO₂ 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 ¹⁹F magnetic resonance techniques may provide a valuable preclinical tool for monitoring therapeutic response against central nervous system or other deep‐seated tumors. Magn Reson Med, 2010.

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.

Iron oxide–based MRI contrast agents are increasingly being used to noninvasively track cells, target molecular epitopes, and monitor gene expression in vivo. Detecting regions of contrast agent accumulation can be challenging if resulting contrast is subtle relative to endogenous tissue hypointensities. A postprocessing method is presented that yields enhanced positive‐contrast images from the phase map associated with T₂*‐weighted MRI data. As examples, the method was applied to an agarose gel phantom doped with superparamagnetic iron‐oxide nanoparticles and in vivo and ex vivo mouse brains inoculated with recombinant viruses delivering transgenes that induce overexpression of paramagnetic ferritin. Overall, this approach generates images that exhibit a 1‐ to 8‐fold improvement in contrast‐to‐noise ratio in regions where paramagnetic agents are present compared to conventional magnitude images. This approach can be used in conjunction with conventional T₂* pulse sequences, requires no prescans or increased scan time, and can be applied retrospectively to previously acquired data. Magn Reson Med, 2009.