Interrogation of endothelial and mural cells in brain metastasis reveals key immune-regulatory mechanisms.

Brain metastasis (BrM) is a common malignancy, predominantly originating from lung, melanoma, and breast cancers. The vasculature is a key component of the BrM tumor microenvironment with critical roles in regulating metastatic seeding and progression. However, the heterogeneity of the major BrM vascular components, namely endothelial and mural cells, is still poorly understood. We perform single-cell and bulk RNA-sequencing of sorted vascular cell types and detect multiple subtypes enriched specifically in BrM compared to non-tumor brain, including previously unrecognized immune regulatory subtypes. We integrate the human data with mouse models, creating a platform to interrogate vascular targets for the treatment of BrM. We find that the CD276 immune checkpoint molecule is significantly upregulated in the BrM vasculature, and anti-CD276 blocking antibodies prolonged survival in preclinical trials. This study provides important insights into the complex interactions between the vasculature, immune cells, and cancer cells, with translational relevance for designing therapeutic interventions.

Improved accuracy and precision of fat-suppressed isotropic 3D T2 mapping MRI of the knee with dictionary fitting and patch-based denoising.

PURPOSE: To develop an isotropic three-dimensional (3D) T2 mapping technique for the quantitative assessment of the composition of knee cartilage with high accuracy and precision. METHODS: A T2-prepared water-selective isotropic 3D gradient-echo pulse sequence was used to generate four images at 3 T. These were used for three T2 map reconstructions: standard images with an analytical T2 fit (AnT2Fit); standard images with a dictionary-based T2 fit (DictT2Fit); and patch-based-denoised images with a dictionary-based T2 fit (DenDictT2Fit). The accuracy of the three techniques was first optimized in a phantom study against spin-echo imaging, after which knee cartilage T2 values and coefficients of variation (CoV) were assessed in ten subjects in order to establish accuracy and precision in vivo. Data given as mean ± standard deviation. RESULTS: After optimization in the phantom, whole-knee cartilage T2 values of the healthy volunteers were 26.6 ± 1.6 ms (AnT2Fit), 42.8 ± 1.8 ms (DictT2Fit, p < 0.001 versus AnT2Fit), and 40.4 ± 1.7 ms (DenDictT2Fit, p = 0.009 versus DictT2Fit). The whole-knee T2 CoV reduced from 51.5% ± 5.6% to 30.5 ± 2.4 and finally to 13.1 ± 1.3%, respectively (p < 0.001 between all). The DictT2Fit improved the data reconstruction time: 48.7 ± 11.3 min (AnT2Fit) versus 7.3 ± 0.7 min (DictT2Fit, p < 0.001). Very small focal lesions were observed in maps generated with DenDictT2Fit. CONCLUSIONS: Improved accuracy and precision for isotropic 3D T2 mapping of knee cartilage were demonstrated by using patch-based image denoising and dictionary-based reconstruction. KEY POINTS: • Dictionary T2 fitting improves the accuracy of three-dimensional (3D) knee T2 mapping. • Patch-based denoising results in high precision in 3D knee T2 mapping. • Isotropic 3D knee T2 mapping enables the visualization of small anatomical details.

Multispectral fluorine-19 MRI enables longitudinal and noninvasive monitoring of tumor-associated macrophages.

High-grade gliomas, the most common and aggressive primary brain tumors, are characterized by a complex tumor microenvironment (TME). Among the immune cells infiltrating the glioma TME, tumor-associated microglia and macrophages (TAMs) constitute the major compartment. In patients with gliomas, increased TAM abundance is associated with more aggressive disease. Alterations in TAM phenotypes and functions have been reported in preclinical models of multiple cancers during tumor development and after therapeutic interventions, including radiotherapy and molecular targeted therapies. These findings indicate that it is crucial to evaluate TAM abundance and dynamics over time. Current techniques to quantify TAMs in patients rely mainly on histological staining of tumor biopsies. Although informative, these techniques require an invasive procedure to harvest the tissue sample and typically only result in a snapshot of a small region at a single point in time. Fluorine isotope 19 MRI (19F MRI) represents a powerful means to noninvasively and longitudinally monitor myeloid cells in pathological conditions by intravenously injecting perfluorocarbon-containing nanoparticles (PFC-NP). In this study, we demonstrated the feasibility and power of 19F MRI in preclinical models of gliomagenesis, breast-to-brain metastasis, and breast cancer and showed that the major cellular source of 19F signal consists of TAMs. Moreover, multispectral 19F MRI with two different PFC-NP allowed us to identify spatially and temporally distinct TAM niches in radiotherapy-recurrent murine gliomas. Together, we have imaged TAMs noninvasively and longitudinally with integrated cellular, spatial, and temporal resolution, thus revealing important biological insights into the critical functions of TAMs, including in disease recurrence.

Compressed sensing with signal averaging for improved sensitivity and motion artifact reduction in fluorine-19 MRI.

Fluorine-19 (19 F) MRI of injected perfluorocarbon emulsions (PFCs) allows for the non-invasive quantification of inflammation and cell tracking, but suffers from a low signal-to-noise ratio and extended scan time. To address this limitation, we tested the hypotheses that a 19 F MRI pulse sequence that combines a specific undersampling regime with signal averaging has both increased sensitivity and robustness against motion artifacts compared with a non-averaged fully sampled pulse sequence, when both datasets are reconstructed with compressed sensing. As a proof of principle, numerical simulations and phantom experiments were performed on selected variable ranges to characterize the point spread function of undersampling patterns, as well as the vulnerability to noise of undersampling and reconstruction parameters with paired numbers of x signal averages and acceleration factor x (NAx-AFx). The numerical simulations demonstrated that a probability density function that uses 25% of the samples to fully sample the k-space central area allowed for an optimal balance between limited blurring and artifact incoherence. At all investigated noise levels, the Dice similarity coefficient (DSC) strongly depended on the regularization parameters and acceleration factor. In phantoms, the motion robustness of an NA8-AF8 undersampling pattern versus NA1-AF1 was evaluated with simulated and real motion patterns. Differences were assessed with the DSC, which was consistently higher for the NA8-AF8 compared with the NA1-AF1 strategy, for both simulated and real cyclic motion patterns (P < 0.001). Both strategies were validated in vivo in mice (n = 2) injected with perfluoropolyether. Here, the images displayed a sharper delineation of the liver with the NA8-AF8 strategy than with the NA1-AF1 strategy. In conclusion, we validated the hypotheses that in 19 F MRI the combination of undersampling and averaging improves both the sensitivity and the robustness against motion artifacts.

Free-running 5D coronary MR angiography at 1.5T using LIBRE water excitation pulses.

PURPOSE: To implement, optimize, and characterize lipid-insensitive binomial off-resonant RF excitation (LIBRE) pulses for fat-suppressed fully self-gated free-running 5D cardiac MRI. METHODS: Bloch equation simulations were used to optimize LIBRE parameter settings in non-interrupted bSSFP prior to in vitro validation. Thus, optimized LIBRE pulses were subsequently applied to free-running coronary MRA in 20 human adult subjects, where resulting images were quantitatively compared to those obtained with non-fat-suppressing excitation (SP), conventional 1-2-1 water excitation (WE), and a previously published interrupted free-running (IFR) sequence. SAR and scan times were recorded. Respiratory-and-cardiac-motion-resolved images were reconstructed with XD-GRASP, and contrast ratios, coronary artery detection rate, vessel length, and vessel sharpness were computed. RESULTS: The numerically optimized LIBRE parameters were successfully validated in vitro. In vivo, LIBRE had the lowest SAR and a scan time that was similar to that of WE yet 18% shorter than that of IFR. LIBRE improved blood-fat contrast when compared to SP, WE, and IFR, vessel detection relative to SP and IFR, and vessel sharpness when compared to WE and IFR (for example, for the left main and anterior descending coronary artery, 51.5% ± 10.2% [LIBRE] versus 42.1% ± 6.8% [IFR]). Vessel length measurements remained unchanged for all investigated methods. CONCLUSION: LIBRE enabled fully self-gated non-interrupted free-running 5D bSSFP imaging of the heart at 1.5T with suppressed fat signal. Measures of image quality, vessel conspicuity, and scan time compared favorably to those obtained with the more conventional non-interrupted WE and the previously published IFR, while SAR reduction offers added flexibility.

A characterization of ABL-101 as a potential tracer for clinical fluorine-19 MRI.

The two main challenges that prevent the translation of fluorine-19 (19 F) MRI for inflammation monitoring or cell tracking into clinical practice are (i) the relatively low signal-to-noise ratio generated by the injected perfluorocarbon (PFC), which necessitates long scan times, and (ii) the need for regulatory approval and a high biocompatibility of PFCs that are also suitable for MRI. ABL-101, an emulsion of perfluoro(t-butylcyclohexane), is a third-generation PFC that is already used in clinical trials, but has not yet been used for 19 F MRI. The objective of this study was therefore to assess the performance of ABL-101 as a 19 F MRI tracer. At magnetic field strengths of 3, 9.4 and 14.1 T, the CF3 groups of ABL-101 generated a large well-separated singlet with T2 /T1 ratios of >0.27, >0.14 and > 0.05, respectively. All relaxation times decreased with the increase in magnetic field strength. The detection limit of ABL-101 in a 0.25 mm3 voxel at 3 T, 37°C and with a 3-minute acquisition time was 7.21mM. After intravenous injection, the clearance half-lives of the ABL-101 19 F MR signal in mouse (n = 3) spleen and liver were 6.85 ± 0.45 and 3.20 ± 0.35 days, respectively. These results demonstrate that ABL-101 has 19 F MR characteristics that are similar to those of PFCs developed specifically for MRI, while it has clearance half-lives similar to PFCs that have previously been used in large doses in non-MRI clinical trials. Overall, ABL-101 is thus a very promising candidate tracer for future clinical trials that use 19 F MRI for cell tracking or the monitoring of inflammation.

Towards Quantification of Inflammation in Atherosclerotic Plaque in the Clinic - Characterization and Optimization of Fluorine-19 MRI in Mice at 3 T.

Fluorine-19 (19F) magnetic resonance imaging (MRI) of injected perfluorocarbons (PFCs) can be used for the quantification and monitoring of inflammation in diseases such as atherosclerosis. To advance the translation of this technique to the clinical setting, we aimed to 1) demonstrate the feasibility of quantitative 19F MRI in small inflammation foci on a clinical scanner, and 2) to characterize the PFC-incorporating leukocyte populations and plaques. To this end, thirteen atherosclerotic apolipoprotein-E-knockout mice received 2 × 200 µL PFC, and were scanned on a 3 T clinical MR system. 19F MR signal was detected in the aortic arch and its branches in all mice, with a signal-to-noise ratio of 11.1 (interquartile range IQR = 9.5-13.1) and a PFC concentration of 1.15 mM (IQR = 0.79-1.28). Imaging flow cytometry was used on another ten animals and indicated that PFC-labeled leukocytes in the aortic arch and it branches were mainly dendritic cells, macrophages and neutrophils (ratio 9:1:1). Finally, immunohistochemistry analysis confirmed the presence of those cells in the plaques. We thus successfully used 19F MRI for the noninvasive quantification of PFC in atherosclerotic plaque in mice on a clinical scanner, demonstrating the feasibility of detecting very small inflammation foci at 3 T, and advancing the translation of 19F MRI to the human setting.

Accelerated and high-resolution cardiac T2 mapping through peripheral k-space sharing.

PURPOSE: To develop high-spatial-resolution cardiac T2 mapping that allows for a reduced acquisition time while maintaining its precision. We implemented and optimized a new golden-angle radial T2 mapping technique named SKRATCH (Shared k-space Radial T2 Characterization of the Heart) that shares k-space peripheries of T2 -weighted images while preserving their contrasts. METHODS: Six SKRATCH variants (gradient-recalled echo and balanced SSFP, free-breathing and breath-held, with and without a saturation preparation) were implemented, and their precision was compared with a navigator-gated reference technique in phantoms and 22 healthy volunteers at 3 T. The optimal breath-held SKRATCH technique was applied in a small cohort of patients with subacute myocardial infarction. RESULTS: The faster free-breathing SKRATCH technique reduced the acquisition time by 52.4%, while maintaining the precision and spatial resolution of the reference technique. Similarly, the most precise and robust breath-held SKRATCH technique demonstrated homogenous T2 values that did not significantly differ from the navigator-gated reference (T2 = 39.9 ± 3.4 ms versus 39.5 ± 3.4 ms, P > .20, respectively). All infarct patients demonstrated a large T2 elevation in the ischemic regions of the myocardium. CONCLUSION: The optimized SKRATCH technique enabled the accelerated acquisition of high-spatial-resolution T2 maps, was validated in healthy adult volunteers, and was successfully applied to a small initial group of patients.

Simultaneous fat-free isotropic 3D anatomical imaging and T2 mapping of knee cartilage with lipid-insensitive binomial off-resonant RF excitation (LIBRE) pulses.

BACKGROUND: Improved knee cartilage morphological delineation and T2 mapping precision necessitates isotropic 3D high-resolution and efficient fat suppression. PURPOSE: To develop and assess an isotropic 3D lipid-insensitive T2 mapping technique of the knee for improved cartilage delineation and precise measurement of T2 relaxation times. STUDY TYPE: Prospective. PHANTOM/SUBJECTS: Phantoms (n = 6) used in this study were designed to mimic the T1 and T2 relaxation times of cartilage and fat. The study cohort comprised healthy volunteers (n = 7) for morphometry and T2 relaxation time measurements. FIELD STRENGTH/SEQUENCE: A high-resolution isotropic 3D T2 mapping technique that uses sequential T2 -prepared segmented gradient-recalled echo (Iso3DGRE) images and lipid-insensitive binomial off-resonant radiofrequency (RF) excitation (LIBRE) at 3T. ASSESSMENT: Numerical simulations and phantom experiments were performed to optimize the LIBRE pulse. Phantom studies were carried out to test the accuracy of the technique against reference standard spin-echo (SE) T2 mapping. Subsequently, T2 maps with and without LIBRE pulses were acquired in knees of healthy volunteers and the T2 relaxation time values in different cartilage compartments were compared. STATISTICAL TESTS: A two-tailed paired Student's t-test was used to compare the average T2 values and the relative standard deviations (inverse measurement of the precision) obtained with and without LIBRE pulses. RESULTS: A LIBRE pulse of 1 msec suppressed fat with an RF excitation frequency offset of 1560 Hz and optimal RF excitation angle of 35°. These results were corroborated by phantom and knee experiments. Robust and homogeneous fat suppression was obtained (a fat signal-to-noise ratio (SNR) decrease of 86.4 ± 2.4%). In phantoms, T2 values were found in good agreement when comparing LIBRE-Iso3DGRE with SE (slope 0.93 ± 0.04, intercept 0.11 ± 1.6 msec, R2 >0.99). In vivo, LIBRE excitation resulted in more precise T2 estimation (23.7 ± 7.4%) than normal excitation (30.5 ± 9.9%, P < 0.0001). DATA CONCLUSION: Homogeneous LIBRE fat signal suppression was achieved with a total RF pulse duration of 1 msec, allowing for the removal of chemical shift artifacts and resulting in improved cartilage delineation and precise T2 values. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;49:1275-1284.

Isotropic three-dimensional T2 mapping of knee cartilage: Development and validation.

PURPOSE: 1) To implement a higher-resolution isotropic 3D T2 mapping technique that uses sequential T2 -prepared segmented gradient-recalled echo (Iso3DGRE) images for knee cartilage evaluation, and 2) to validate it both in vitro and in vivo in healthy volunteers and patients with knee osteoarthritis. MATERIALS AND METHODS: The Iso3DGRE sequence with an isotropic 0.6 mm spatial resolution was developed on a clinical 3T MR scanner. Numerical simulations were performed to optimize the pulse sequence parameters. A phantom study was performed to validate the T2 estimation accuracy. The repeatability of the sequence was assessed in healthy volunteers (n = 7). T2 values were compared with those from a clinical standard 2D multislice multiecho (MSME) T2 mapping sequence in knees of healthy volunteers (n = 13) and in patients with knee osteoarthritis (OA, n = 5). RESULTS: The numerical simulations resulted in 100 excitations per segment and an optimal radiofrequency (RF) excitation angle of 15°. The phantom study demonstrated a good correlation of the technique with the reference standard (slope 0.9 ± 0.05, intercept 0.2 ± 1.7 msec, R2 ≥ 0.99). Repeated measurements of cartilage T2 values in healthy volunteers showed a coefficient of variation of 5.6%. Both Iso3DGRE and MSME techniques found significantly higher cartilage T2 values (P < 0.03) in OA patients. Iso3DGRE precision was equal to that of the MSME T2 mapping in healthy volunteers, and significantly higher in OA (P = 0.01). CONCLUSION: This study successfully demonstrated that high-resolution isotropic 3D T2 mapping for knee cartilage characterization is feasible, accurate, repeatable, and precise. The technique allows for multiplanar reformatting and thus T2 quantification in any plane of interest. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:362-371.

Chemical shift encoding (CSE) for sensitive fluorine-19 MRI of perfluorocarbons with complex spectra.

PURPOSE: To implement a fluorine-19 (19 F) chemical shift encoding (CSE) approach for the sensitive imaging of molecules with multi-resonance spectra to remove their chemical shift displacement (CSD) artifacts, and to characterize its sensitivity versus established pulse sequences. METHODS: The feasibility of CSE spoiled gradient echo (GRE) and balanced steady-state free precession (bSSFP) was first demonstrated in a phantom study. The dependence of the sensitivity of CSE-bSSFP on several pulse sequence parameters was then established, after which the occurrence of out-of-plane excitation was assessed for 2D and 3D techniques. Next, the sensitivity (in mm-3 s-0.5 ) of both CSE techniques was compared to bSSFP ultrashort echo time (bSSFP-UTE) imaging and multi-chemical-shift-selective turbo spin echo (MCSS-TSE) in a second phantom study. Finally, the sensitivity of the CSE-bSSFP, bSSFP-UTE, and MCSS-TSE pulse sequences was compared in a preliminary in vivo mouse study. RESULTS: Both CSE approaches were successfully implemented and resulted in negligible residual CSD artifacts, while large-volume 3D acquisitions should be considered to reduce problems related to out-of-plane excitation. CSE-bSSFP was shown to have a higher sensitivity than the bSSFP-UTE and MCSS-TSE pulse sequences (15.8 ± 1.3 vs. 11.7 ± 1.0 vs. 13.3 ± 0.9 mm-3 s-0.5 , respectively, P < 0.001), whereas CSE-GRE technique had a lower sensitivity (4.8 ± 1.1 mm-3 s-0.5 ). CONCLUSION: CSE 19 F MR imaging enables the unambiguous visualization of compounds with complex spectra, and provides high sensitivity both in vitro and in vivo. Magn Reson Med 79:2724-2730, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Characterization of perfluorocarbon relaxation times and their influence on the optimization of fluorine-19 MRI at 3 tesla.

PURPOSE: To characterize and optimize 19 F MRI for different perfluorocarbons (PFCs) at 3T and quantify the loss of acquisition efficiency as a function of different temperature and cellular conditions. METHODS: The T1 and T2 relaxation times of the commonly used PFCs perfluoropolyether (PFPE), perfluoro-15-crown-5-ether (PFCE), and perfluorooctyl bromide (PFOB) were measured in phantoms and in several different conditions (cell types, presence of fixation agent, and temperatures). These relaxation times were used to optimize pulse sequences through numerical simulations. The acquisition efficiency in each cellular condition was then determined as the ratio of the signal after optimization with the reference relaxation times and after optimization with its proper relaxation times. Finally, PFC detection limits were determined. RESULTS: The loss of acquisition efficiency due to parameter settings optimized for the wrong temperature and cellular condition was limited to 13%. The detection limits of all PFCs were lower at 24 °C than at 37 °C and varied from 11.8 ± 3.0 mM for PFCE at 24 °C to 379.9 ± 51.8 mM for PFOB at 37 °C. CONCLUSION: Optimizing 19 F pulse sequences with a known phantom only leads to moderate loss in acquisition efficiency in cellular conditions that might be encountered in in vivo and in vitro experiments. Magn Reson Med 77:2263-2271, 2017. © 2016 International Society for Magnetic Resonance in Medicine.