Use of Magnetotactic Bacteria as an MRI Contrast Agent for In Vivo Tracking of Adoptively Transferred Immune Cells.

PURPOSE: In vivo immune cell tracking using MRI can be a valuable tool for studying the mechanisms underlying successful cancer therapies. Current cell labeling methods using superparamagnetic iron oxide (SPIO) lack the persistence to track the fate and location of transplanted cells long-term. Magnetospirillum magneticum is a commercially available, iron-producing bacterium that can be taken up by and live harmoniously within mammalian cells as magneto-endosymbionts (MEs). MEs have shown promise as labeling agents for in vivo stem and cancer cell tracking but have yet to be evaluated in immune cells. This pilot study examined ME labeling in myeloid-derived suppressor cells (MDSCs), cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) and its effects on cell purity, function, and MRI contrast. PROCEDURES: MDSCs, CTLs, and DCs were incubated with MEs at various ME labeling ratios (MLR), and various biological metrics and iron uptake were assessed. For in vivo imaging, MDSCs were labeled overnight with either MEs or SPIO (Molday ION Rhodamine B) and injected into C3 tumor-bearing mice via tail vein injection 24 days post-implant and scanned daily with MRI for 1 week to assess cellular quantification. RESULTS: Following incubations, MDSCs contained > 0.6 pg Fe/cell. CTLs achieved Fe loading of < 0.5 pg/cell, and DCs achieved Fe loading of ~ 1.4 pg/cell. The suppressive functionality of MDSCs at 1000 MLR was not affected by ME labeling but was affected at 2000 MLR. Markers of CTL dysfunction were not markedly affected by ME labeling nor were DC markers. In vivo data demonstrated that the MDSCs labeled with MEs generated sufficient contrast to be detectable using TurboSPI, similar to SPIO-labeled cells. CONCLUSIONS: Cells can be labeled with sufficient numbers of MEs to be detectable with MRI without compromising cell viability. Care must be taken at higher concentrations of MEs, which may affect some cell types' functional activity and/or morphology. Immune cells with minimal phagocytic behavior have much lower iron content per cell after incubation with MEs vs SPIO; however, MEs can successfully be used as a contrast agent for phagocytic immune cells.

Mapping of fatty acid composition with free-breathing MR spectroscopic imaging and compressed sensing.

Nonalcoholic fatty liver disease (NAFLD) is a growing health problem, and a major challenge in NAFLD management is identifying which patients are at risk of progression to more serious disease. Simple measurements of liver fat content are not strong predictors of clinical outcome, but biomarkers related to fatty acid composition (ie, saturated vs. unsaturated fat) may be more effective. MR spectroscopic imaging (MRSI) methods allow spatially resolved, whole-liver measurements of chemical composition but are traditionally limited by slow acquisition times. In this work we present an accelerated MRSI acquisition based on spin echo single point imaging (SE-SPI), which, using appropriate sampling and compressed sensing reconstruction, allows free-breathing acquisition in a mouse model of fatty liver disease. After validating the technique's performance in oil/water phantoms, we imaged mice that had received a normal diet or a methionine and choline deficient (MCD) diet, some of which also received supplemental injections of iron to mimic hepatic iron overload. SE-SPI was more resistant to the line-broadening effects of iron than single-voxel spectroscopy measurements, and was consistently able to measure the amplitudes of low-intensity spectral peaks that are important to characterizing fatty acid composition. In particular, in the mice receiving the MCD diet, SE-SPI showed a significant decrease in a metric associated with unsaturated fat, which is consistent with the literature. This or other related metrics may therefore offer more a specific biomarker of liver health than fat content alone. This preclinical study is an important precursor to clinical testing of the proposed method. MR-based quantification of fatty acid composition may allow for improved characterization of non-alcoholic fatty liver disease. A spectroscopic imaging method with appropriate sampling strategy allows whole-liver mapping of fat composition metrics in a free-breathing mouse model. Changes in metrics like the surrogate unsaturation index (UIs) are visible in mice receiving a diet which induces fat accumulation in the liver, as compared to a normal diet; such metrics may prove useful in future clinical studies of liver disease.

Quantitative MRI cell tracking of immune cell recruitment to tumors and draining lymph nodes in response to anti-PD-1 and a DPX-based immunotherapy.

DPX is a unique T cell activating formulation that generates robust immune responses (both clinically and preclinically) which can be tailored to various cancers via the use of tumor-specific antigens and adjuvants. While DPX-based immunotherapies may act complementary with checkpoint inhibitors, combination therapy is not always easily predictable based on individual therapeutic responses. Optimizing these combinations can be improved by understanding the mechanism of action underlying the individual therapies. Magnetic Resonance Imaging (MRI) allows tracking of cells labeled with superparamagnetic iron oxide (SPIO), which can yield valuable information about the localization of crucial immune cell subsets. In this work, we evaluated the use of a multi-echo, single point MRI pulse sequence, TurboSPI, for tracking and quantifying cytotoxic T lymphocytes (CTLs) and myeloid lineage cells (MLCs). In a subcutaneous cervical cancer model (C3) we compared untreated mice to mice treated with either a single therapy (anti-PD-1 or DPX-R9F) or a combination of both therapies. We were able to detect, using TurboSPI, significant increases in CTL recruitment dynamics in response to combination therapy. We also observed differences in MLC recruitment to therapy-draining (DPX-R9F) lymph nodes in response to treatment with DPX-R9F (alone or in combination with anti-PD-1). We demonstrated that the therapies presented herein induced time-varying changes in cell recruitment. This work establishes that these quantitative molecular MRI techniques can be expanded to study a number of cancer and immunotherapy combinations to improve our understanding of longitudinal immunological changes and mechanisms of action.

3D single point imaging with compressed sensing provides high temporal resolution R 2* mapping for in vivo preclinical applications.

OBJECTIVE: Purely phase-encoded techniques such as single point imaging (SPI) are generally unsuitable for in vivo imaging due to lengthy acquisition times. Reconstruction of highly undersampled data using compressed sensing allows SPI data to be quickly obtained from animal models, enabling applications in preclinical cellular and molecular imaging. MATERIALS AND METHODS: TurboSPI is a multi-echo single point technique that acquires hundreds of images with microsecond spacing, enabling high temporal resolution relaxometry of large-R 2* systems such as iron-loaded cells. TurboSPI acquisitions can be pseudo-randomly undersampled in all three dimensions to increase artifact incoherence, and can provide prior information to improve reconstruction. We evaluated the performance of CS-TurboSPI in phantoms, a rat ex vivo, and a mouse in vivo. RESULTS: An algorithm for iterative reconstruction of TurboSPI relaxometry time courses does not affect image quality or R 2* mapping in vitro at acceleration factors up to 10. Imaging ex vivo is possible at similar acceleration factors, and in vivo imaging is demonstrated at an acceleration factor of 8, such that acquisition time is under 1 h. CONCLUSIONS: Accelerated TurboSPI enables preclinical R 2* mapping without loss of data quality, and may show increased specificity to iron oxide compared to other sequences.

Biexponential longitudinal relaxation in white matter: Characterization and impact on T1 mapping with IR-FSE and MP2RAGE.

PURPOSE: Magnetization transfer in white matter (WM) causes biexponential relaxation, but most quantitative T1 measurements fit data assuming monoexponential relaxation. The resulting monoexponential T1 estimate varies based on scan parameters and represents a source of variation between studies, especially at high fields. In this study, we characterized WM T1 relaxation and performed simulations to determine how to minimize this deviation. METHODS: To characterize biexponential relaxation, four volunteers were scanned at 3T and 7T using inversion recovery fast spin echo (IR-FSE) with 13 inversion times (TIs). Three volunteers were scanned with IR-FSE using TIs chosen by simulations to reduce T1 deviation, and with MP2RAGE. RESULTS: At 3T, the biexponential relaxation has a short component of T1 = 48 ms (9%) and a long component of T1 = 939 ms. At 7T the short component is T1 = 57 ms (11%) and the long component is 1349 ms (89%). For IR-FSE, acquiring four TIs with a minimum of 150 ms (3T) or 200 ms (7T) yielded monoexponential T1 estimates that match the long component to within 10 ms. For MP2RAGE, significant differences (90 ms at 3T, 125 ms at 7T) remain at all parameter values. CONCLUSION: Many T1 mapping sequences yield robust estimates of the long T1 component with suitable choice of TIs, allowing reproducible, sequence-independent T1 values to be measured. However, this is not true of MP2RAGE in its current implementation. Magn Reson Med 75:2265-2277, 2016. © 2015 Wiley Periodicals, Inc.

Signal displacement in spiral-in acquisitions: simulations and implications for imaging in SFG regions.

Susceptibility field gradients (SFGs) cause problems for functional magnetic resonance imaging (fMRI) in regions like the orbital frontal lobes, leading to signal loss and image artifacts (signal displacement and "pile-up"). Pulse sequences with spiral-in k-space trajectories are often used when acquiring fMRI in SFG regions such as inferior/medial temporal cortex because it is believed that they have improved signal recovery and decreased signal displacement properties. Previously postulated theories explain differing reasons why spiral-in appears to perform better than spiral-out; however it is clear that multiple mechanisms are occurring in parallel. This study explores differences in spiral-in and spiral-out images using human and phantom empirical data, as well as simulations consistent with the phantom model. Using image simulations, the displacement of signal was characterized using point spread functions (PSFs) and target maps, the latter of which are conceptually inverse PSFs describing which spatial locations contribute signal to a particular voxel. The magnitude of both PSFs and target maps was found to be identical for spiral-out and spiral-in acquisitions, with signal in target maps being displaced from distant regions in both cases. However, differences in the phase of the signal displacement patterns that consequently lead to changes in the intervoxel phase coherence were found to be a significant mechanism explaining differences between the spiral sequences. The results demonstrate that spiral-in trajectories do preserve more total signal in SFG regions than spiral-out; however, spiral-in does not in fact exhibit decreased signal displacement. Given that this signal can be displaced by significant distances, its recovery may not be preferable for all fMRI applications.

Quantification of superparamagnetic iron oxide with large dynamic range using TurboSPI.

This work proposes the use of TurboSPI, a multi-echo single point imaging sequence, for the quantification of labeled cells containing moderate to high concentrations of iron oxide contrast agent. At each k-space location, TurboSPI acquires several hundred time points during a spin echo, permitting reliable relaxation rate mapping of large-R(2)(∗) materials. An automatic calibration routine optimizes image quality by promoting coherent alignment of spin and stimulated echoes throughout the multi-echo train, and this calibration is sufficiently robust for in vivo applications. In vitro relaxation rate measurements of SPIO-loaded cervical cancer cells exhibit behavior consistent with theoretical predictions of the static dephasing regime in the spin echo case; the relaxivity measured with TurboSPI was 10.47±2.3 s(-1)/mG, comparable to the theoretical value of 10.78 s(-1)/mG. Similar measurements of micron-sized iron oxide particles (0.96 µm and 1.63 µm diameter) show a reduced relaxivity of 8.06±0.68 s(-1)/mG and 7.13±0.31 s(-1)/mG respectively, indicating that the static dephasing criterion was not met. Nonetheless, accurate quantification of such particles is demonstrated up to R(2)(∗)=900 s(-1), with a potentially higher upper limit for loaded cells having a more favorable R(2)('):R(2) ratio. Based on the cells used in this study, reliable quantification of cells loaded with 10 pg of iron per cell should be possible up to a density of 27 million cells/mL. Such quantification will be of crucial importance to the development of longitudinal monitoring for cellular therapy and other procedures using iron-labeled cells.

Asymmetric spin-echo (ASE) spiral improves BOLD fMRI in inhomogeneous regions.

Functional MRI (fMRI) is of limited use in areas such as the orbitofrontal and inferior temporal lobes due to the presence of local susceptibility-induced field gradients (SFGs), which result in severe image artifacts. Several techniques have been developed to reduce these artifacts, the most common being the dual-echo spiral sequences (spiral-in/out and spiral-in/in). In this study, a new multiple spiral acquisition technique was developed, in which the later spiral acquisitions are acquired asymmetrically with the peak of a spin-echo causing increased R(2)-weighting but matched R(2)'-weighting. This sequence, called asymmetric spin-echo (ASE) spiral, has demonstrated significant improvements in minimizing the signal loss and increasing the image quality as well as optimal blood-oxygen-level-dependent (BOLD)-weighting. The ASE spiral is compared to conventional spiral-out using both signal-to-noise ratio (SNR) and whole brain fMRI activation volumes from a breath-hold task acquired at 4 Tesla. The ASE dual spiral has exhibited SNR increases of up to 300% in areas where strong SFGs are present. As a result, the ASE spiral is highly efficient for recovering lost activation in areas of SFGs, as demonstrated by a 16% increase in the total number of activated voxels over the whole brain. Post spin-echo ASE spiral images have decreasing SNR due to R(2) signal losses, however the increase in R(2)-weighting leads to a higher percentage of signal changes producing ASE spiral images with equivalent contrast-to-noise ratio (CNR) for each echo. The use of this sequence allows for recovery of BOLD activation in areas of SFG without sacrificing the CNR over the whole brain.

Velocity imaging of highly turbulent gas flow.

We introduce a noninvasive, quantitative magnetic resonance imaging (MRI) wind-tunnel measurement in flowing gas (>10 m s(-1)) at high Reynolds numbers (Re>10(5)). The method pertains to liquids and gases, is inherently three dimensional, and extends the range of Re to which MRI is applicable by orders of magnitude. There is potential for clear time savings over traditional pointwise techniques. The mean velocity and turbulent diffusivity of gas flowing past a bluff obstruction and a wing section at realistic stall speeds were measured. The MRI data are compared with computational fluid dynamics.