Perfusion and diffusion sensitive C stimulated-echo MRSI for metabolic imaging of cancer

Metabolic imaging with hyperpolarized [1-¹³C]-pyruvate can rapidly probe tissue metabolic profiles in vivo and has been shown to provide cancer imaging biomarkers for tumor detection, progression, and response to therapy. This technique uses a bolus injection followed by imaging within 1–2 minutes. The observed metabolites include vascular components and their generation is also influenced by cellular transport. These factors complicate image interpretation, especially since [1-¹³C]lactate, a metabolic product that is a biomarker of cancer, is also produced by red blood cells. It would be valuable to understand the distribution of metabolites between the vasculature, interstitial space, and intracellular compartments. The purpose of this study was to better understand this compartmentalization by using a perfusion and diffusion-sensitive stimulated-echo acquisition mode (STEAM) MRSI acquisition method tailored to hyperpolarized substrates. Our results in mouse models showed that among metabolites, the injected substrate ¹³C-pyruvate had the largest vascular fraction overall while ¹³C-alanine had the smallest vascular fraction. We observed a larger vascular fraction of pyruvate and lactate in the kidneys and liver when compared to back muscle and prostate tumor tissue. Our data suggests that ¹³C-lactate in prostate tumor tissue voxels was the most abundant labeled metabolite intracellularly. This was shown in STEAM images that highlighted abnormal cancer cell metabolism and suppressed vascular ¹³C metabolite signals.     

Pulse sequence for dynamic volumetric imaging of hyperpolarized metabolic products

Dynamic nuclear polarization and dissolution of a ¹³C-labeled substrate enables the dynamic imaging of cellular metabolism. Spectroscopic information is typically acquired, making the acquisition of dynamic volumetric data a challenge. To enable rapid volumetric imaging, a spectral-spatial excitation pulse was designed to excite a single line of the carbon spectrum. With only a single resonance present in the signal, an echo-planar readout trajectory could be used to resolve spatial information, giving full volume coverage of 32 × 32 × 16 voxels every 3.5 s. This high frame rate was used to measure the different lactate dynamics in different tissues in a normal rat model and a mouse model of prostate cancer.     

In vivo measurement of normal rat intracellular pyruvate and lactate levels after injection of hyperpolarized [1-(13)C]alanine

Hyperpolarized technology utilizing dynamic nuclear polarization has enabled rapid and high-sensitivity measurements of ¹³C metabolism in vivo. The most commonly used in vivo agent for hyperpolarized ¹³C metabolic imaging thus far has been [1-¹³C]pyruvate. In preclinical studies, not only is its uptake detected, but also its intracellular enzymatic conversion to metabolic products including [1-¹³C]lactate and [1-¹³C]alanine. However, the ratio of ¹³C-lactate/¹³C-pyruvate measured in this data does not accurately reflect cellular values since much of the [1-¹³C]pyruvate is extracellular depending on timing, vascular properties, and extracellular space and monocarboxylate transporter activity. In order to measure the relative levels of intracellular pyruvate and lactate, in this project we hyperpolarized [1-¹³C]alanine and monitored the in vivo conversion to [1-¹³C]pyruvate and then the subsequent conversion to [1-¹³C]lactate. The intracellular lactate-to-pyruvate ratio of normal rat tissue measured with hyperpolarized [1-¹³C]alanine was 4.89±0.61 (mean±S.E.) as opposed to a ratio of 0.41±0.03 when hyperpolarized [1-¹³C]pyruvate was injected.     

Quantitative O imaging towards oxygen consumption study in tumor bearing mice at 7 T

¹⁷O magnetic resonance imaging (MRI) using a conventional pulse sequence was explored as a method of quantitative imaging towards regional oxygen consumption rate measurement for tumor evaluation in mice. At 7 T, fast imaging with steady state (FISP) was the best among gradient echo, fast spin echo and FISP for the purpose. The distribution of natural abundance H₂¹⁷O in mice was visualized under spatial resolution of 2.5 × 2.5 mm² by FISP in 10 min. The signal intensity by FISP showed a linear relationship with ¹⁷O quantity both in phantom and mice. Following the injection of 5% ¹⁷O enriched saline, ¹⁷O re-distribution was monitored in temporal resolution down to 5 sec with an image quality sufficient to distinguish each organ. The image of labeled water produced from inhaled ¹⁷O₂ gas was also obtained. The present method provides quantitative ¹⁷O images under sufficient temporal and spatial resolution for the evaluation of oxygen consumption rate in each organ. Experiments using various model compounds of R-OH type clarified that the signal contribution of body constituents other than water in the present in vivo¹⁷O FISP image was negligible.     

Frequency-specific SSFP for hyperpolarized C metabolic imaging at 14.1 T

Metabolic imaging of hyperpolarized [1-¹³C] pyruvate co-polarized with [¹³C]urea by dynamic nuclear polarization with rapid dissolution is a promising new method for assessing tumor metabolism and perfusion simultaneously in vivo. Novel pulse sequences are required to enable dynamic imaging of multiple ¹³C spectral lines with high spatiotemporal resolution. The goal of this study was to investigate a new frequency-specific approach for rapid metabolic imaging of multiple ¹³C resonances using the spectral selectivity of steady-state free precession pulse (SSFP) trains. Methods developed in simulations were implemented in a dynamic frequency-cycled balanced SSFP pulse sequence on a 14.1-T animal magnetic resonance imaging scanner. This acquisition was tested in thermal and hyperpolarized phantom imaging studies and in a transgenic mouse with prostate cancer.     

Design of spectral-spatial outer volume suppression RF pulses for tissue specific metabolic characterization with hyperpolarized C pyruvate

[1-¹³C] pyruvate pre-polarized via DNP has been used in animal models to probe changes in metabolic enzyme activities in vivo. To more accurately assess the metabolic state and its change from disease progression or therapy in a specific region or tissue in vivo, it may be desirable to separate the downstream ¹³C metabolite signals resulting from the metabolic activity within the tissue of interest and those brought into the tissue by perfusion. In this study, a spectral-spatial saturation pulse that selectively saturates the signal from the metabolic products [1-¹³C] lactate and [1-¹³C] alanine was designed and implemented as outer volume suppression for localized MRSI acquisition. Preliminary in vivo results showed that the suppression pulse did not prevent the pre-polarized pyruvate from being delivered throughout the animal while it saturated the metabolites within the targeted saturation region.     

Kinetic modeling of hyperpolarized 13C1-pyruvate metabolism in normal rats and TRAMP mice

PurposeTo investigate metabolic exchange between ¹³C₁-pyruvate, ¹³C₁-lactate, and ¹³C₁-alanine in pre-clinical model systems using kinetic modeling of dynamic hyperpolarized ¹³C spectroscopic data and to examine the relationship between fitted parameters and dose–response.Materials and methodsDynamic ¹³C spectroscopy data were acquired in normal rats, wild type mice, and mice with transgenic prostate tumors (TRAMP) either within a single slice or using a one-dimensional echo-planar spectroscopic imaging (1D-EPSI) encoding technique. Rate constants were estimated by fitting a set of exponential equations to the dynamic data. Variations in fitted parameters were used to determine model robustness in 15 mm slices centered on normal rat kidneys. Parameter values were used to investigate differences in metabolism between and within TRAMP and wild type mice.ResultsThe kinetic model was shown here to be robust when fitting data from a rat given similar doses. In normal rats, Michaelis–Menten kinetics were able to describe the dose–response of the fitted exchange rate constants with a 13.65% and 16.75% scaled fitting error (SFE) for k_pyr→lac and k_pyr→ala, respectively. In TRAMP mice, k_pyr→lac increased an average of 94% after up to 23 days of disease progression, whether the mice were untreated or treated with casodex. Parameters estimated from dynamic ¹³C 1D-EPSI data were able to differentiate anatomical structures within both wild type and TRAMP mice.ConclusionsThe metabolic parameters estimated using this approach may be useful for in vivo monitoring of tumor progression and treatment efficacy, as well as to distinguish between various tissues based on metabolic activity.     

Quantitative Evaluation of the Lactate Signal Loss and Its Spatial Dependence in PRESS Localized H NMR Spectroscopy

Localized ¹H NMR spectroscopy using the 90°−t₁−180°−t₁+t₂−180°−t₂−Acq. PRESS sequence can lead to a signal loss for the lactate doublet compared with signals from uncoupled nuclei which is dependent on the choice of t₁ and t₂. The most striking signal loss of up to 78% of the total signal occurs with the symmetrical PRESS sequence (t₁=t₂) at an echo time of 2/J (290 ms). Calculations have shown that this signal loss is related to the pulse angle distributions produced by the two refocusing pulses which leads to the creation of single quantum polarization transfer (PT) as well as to not directly observable states (NDOS) of the lactate AX₃ spin system: zero- and multiple-quantum coherences, and longitudinal spin orders. In addition, the chemical shift dependent voxel displacement (VOD) leads to further signal loss. By calculating the density operator for various of the echo times TE=n/J, n=1, 2, 3, …, we calculated quantitatively the contributions of these effects to the signal loss as well as their spatial distribution. A maximum signal loss of 75% can be expected from theory for the symmetrical PRESS sequence and TE=2/J for Hamming filtered sinc pulses, whereby 47% are due to the creation of NDOS and up to 28% arise from PT. Taking also the VOD effect into account (2 mT/m slice selection gradients, 20-mm slices) leads to 54% signal loss from NDOS and up to 24% from PT, leading to a maximum signal loss of 78%. Using RE-BURP pulses with their more rectangular pulse angle distributions reduces the maximum signal loss to 44%. Experiments at 1.5 T using a lactate solution demonstrated a maximum lactate signal loss for sinc pulses of 82% (52% NDOS, 30% PT) at TE=290 ms using the symmetrical PRESS sequence. The great signal loss and its spatial distribution is of importance for investigations using a symmetrical PRESS sequence at TE=2/J.     

Detecting peritoneal dissemination of ovarian cancer in mice by DWIBS

Diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) is a relatively new diffusion-based pulse sequence that produces positron emission tomography (PET) with 2-[fluorine-18]-fluoro-2-deoxy-D-glucose (¹⁸F-FDG)-like images. We tested the feasibility of DWIBS in detecting peritoneal ovarian cancer in a syngeneic mouse model. Female C57BL/6 mice were injected intraperitoneally with ID8 murine ovarian carcinoma cells. After 11 weeks, the abdomen was imaged by DWIBS. A respiratory gating diffusion-weighted spin-echo echo-planar imaging in abdomen was used (imaging parameters of field of view of 47×47 mm², matrix size of 64×64 zero-filled to 256×256 and b-value of 1500 s/mm²). We also performed FDG microPET as the reference standard. For comparison of the correlating surface areas of tumor foci on both DWIBS and FDG microPET imaging, two-dimensional region-of-interest (ROI) analysis was performed, and correlation between the two modalities was determined. Mice were also subjected to macroscopic examination for tumor location and pathology after imaging. DWIBS in all mice depicted the tumors as abnormal high signal intensity. The results show that the ROI analysis of correlating lesions reveals relatively high correlation (r²=0.7296) and significant difference (P = .021) between DWIBS and FDG microPET. These results demonstrate that DWIBS has the potential for detecting peritoneal dissemination of ovarian cancer. Nonetheless, due to low ratios of image signal-to-noise and motion artifacts, DWIBS can be limited for lesions near the liver.     

Selective F MR imaging of 5-fluorouracil and α-fluoro-β-alanine

A ¹⁹F MR chemical shift imaging (CSI) technique is presented which enables selective imaging of the antineoplastic drug 5-fluorouracil (5-FU) and its major catabolite α-fluoro-β-alanine (FBAL). The CSI sequence employs a chemical shift selective (CHESS) saturation pulse to suppress either the 5-FU or the FBAL resonance before the other component of the two-line ¹⁹F MR spectrum is measured. Because the transmitter frequency can always be set to the Larmor frequency of the ¹⁹F resonance to be imaged, this approach yields 5-FU and FBAL MR images free of chemical shift artifacts in read-out and slice-selection direction. In phantom experiments, selective 5-FU and FBAL images with a spatial resolution of 15 × 15 × 20 mm³ (4.5 ml) were obtained in 30 min from a model solution, whose drug and catabolite concentrations were similar to those estimated in the liver of tumor patients undergoing IV chemotherapy with 5-FU. The drug-specific MR imaging technique developed is, therefore, well-suited for the direct and noninvasive monitoring of the up-take and trapping of 5-FU in liver tumors in vivo.     

In vivo detection of distal tumor glycolytic flux stimulated by hepatic ablation in a breast cancer model using hyperpolarized 13C MRI

PurposeHepatic thermal ablation therapy can result in c-Met-mediated off-target stimulation of distal tumor growth. The purpose of this study was to determine if a similar effect on tumor metabolism could be detected in vivo with hyperpolarized ¹³C MRI.Materials and methodsIn this prospective study, female Fisher rats (n = 28, 120–150 g) were implanted with R3230 rat breast adenocarcinoma cells and assigned to either: sham surgery, hepatic radiofrequency ablation (RFA), or hepatic RFA + adjuvant c-Met inhibition with PHA-665752 (RFA + PHA). PHA-665752 was administered at 0.83 mg/kg at 24 h post-RFA. Tumor growth was measured daily. MRI was performed 24 h before and 72 h after treatment on 14 rats, and the conversion of ¹³C-pyruvate into ¹³C-lactate within each tumor was quantified as lactate:pyruvate ratio (LPR). Comparisons of tumor growth and LPR were performed using paired and unpaired t-tests.ResultsHepatic RFA alone resulted in increased growth of the distant tumor compared to sham treatment (0.50 ± 0.13 mm/day versus 0.11 ± 0.07 mm/day; p < 0.001), whereas RFA + PHA (0.06 ± 0.13 mm/day) resulted in no significant change from sham treatment (p = 0.28). A significant increase in LPR was seen following hepatic RFA (+0.016 ± 0.010, p = 0.02), while LPR was unchanged for sham treatment (−0.048 ± 0.051, p = 0.10) or RFA + PHA (0.003 ± 0.041, p = 0.90).ConclusionIn vivo hyperpolarized ¹³C MRI can detect hepatic RFA-induced increase in lactate flux within a distant R3230 tumor, which correlates with increased tumor growth. Adjuvant inhibition of c-Met suppresses these off-target effects, supporting a role for the HGF/c-Met signaling axis in these tumorigenic responses.     

Development of high resolution 3D hyperpolarized carbon-13 MR molecular imaging techniques

The goal of this project was to develop and apply techniques for T₂ mapping and 3D high resolution (1.5 mm isotropic; 0.003 cm³) ¹³C imaging of hyperpolarized (HP) probes [1-¹³C]lactate, [1-¹³C]pyruvate, [2-¹³C]pyruvate, and [¹³C,¹⁵N₂]urea in vivo. A specialized 2D bSSFP sequence was implemented on a clinical 3T scanner and used to obtain the first high resolution T₂ maps of these different hyperpolarized compounds in both rats and tumor-bearing mice. These maps were first used to optimize timings for highest SNR for single time-point 3D bSSFP acquisitions with a 1.5 mm isotropic spatial resolution of normal rats. This 3D acquisition approach was extended to serial dynamic imaging with 2-fold compressed sensing acceleration without changing spatial resolution. The T₂ mapping experiments yielded measurements of T₂ values of > 1 s for all compounds within rat kidneys/vasculature and TRAMP tumors, except for [2-¹³C]pyruvate which was ~ 730 ms and ~ 320 ms, respectively. The high resolution 3D imaging enabled visualization the biodistribution of [1-¹³C]lactate, [1-¹³C]pyruvate, and [2-¹³C]pyruvate within different kidney compartments as well as in the vasculature. While the mouse anatomy is smaller, the resolution was also sufficient to image the distribution of all compounds within kidney, vasculature, and tumor. The development of the specialized 3D sequence with compressed sensing provided improved structural and functional assessments at a high (0.003 cm³) spatial and 2 s temporal resolution in vivo utilizing HP ¹³C substrates by exploiting their long T₂ values. This 1.5 mm isotropic resolution is comparable to ¹H imaging and application of this approach could be extended to future studies of uptake, metabolism, and perfusion in cancer and other disease models and may ultimately be of value for clinical imaging.     

Imaging of the H NMR Second Moment with C Chemical-Shift Resolution

A method of ¹³C chemical-shift-resolved ¹H second moment imaging is proposed for molecular mobility imaging of heterogeneous materials. For evaluating the ¹H second moment, the method relies on the curve fitting procedure using spin-echo shapes indirectly: The information of ¹H echo shapes is transferred to the ¹³C signal amplitude through ¹H–¹³C cross polarization and then the curve fitting is made using the ¹³C signal amplitude. The ¹³C signal is detected under ¹H dipolar decoupling and magic angle spinning, resulting in the incorporation of ¹³C chemical-shift resolution. Imaging information is included in the ¹³C signal by application of phase-encoding gradients. The second moment images obtained can reflect the molecular mobility at every molecular site separated by ¹³C chemical shifts, yielding detailed information on the molecular mobility. The method is demonstrated by spatially 1D experiments performed on a model sample.     

13C/12C-Untersuchungen an Tumorzellen

Abstract

The metabolism of tumor-cells differs in many ways from normal (healthy) cells. One of the major differences is the high glycolytic activity in tumor-cells with the subsequent formation of lactate from glucose, even in the presence of oxygen. The question whether this high rate of glycolysis has any effect on the ¹³C/¹²C-relation of the cells is examined in experiments with a tumor-cell line (HT29) and in specimens of human breast cancer.

The HT29 cells show a clear decrease in ¹³C content compared to their culture medium (Δδ = 3.28‰).

Tissue from human breast cancer has more ¹³C than normal breast tissue taken from the same patient ((Δδ = 2.74‰). But the content of fat is much higher in the normal tissue and its δ-value is negatively correlated with its fat content. It is concluded that the difference between normal and tumor tissue is due to the heterogeneous composition of the normal tissue samples.     

Fast volumetric spatial-spectral MR imaging of hyperpolarized C-labeled compounds using multiple echo 3D bSSFP

Purpose

The goal of this work was to develop a fast 3D chemical shift imaging technique for the noninvasive measurement of hyperpolarized ¹³C-labeled substrates and metabolic products at low concentration.

Materials and Methods

Multiple echo 3D balanced steady state magnetic resonance imaging (ME-3DbSSFP) was performed in vitro on a syringe containing hyperpolarized [1,3,3-2H3; 1-¹³C]2-hydroxyethylpropionate (HEP) adjacent to a ¹³C-enriched acetate phantom, and in vivo on a rat before and after intravenous injection of hyperpolarized HEP at 1.5 T. Chemical shift images of the hyperpolarized HEP were derived from the multiple echo data by Fourier transformation along the echoes on a voxel by voxel basis for each slice of the 3D data set.

Results

ME-3DbSSFP imaging was able to provide chemical shift images of hyperpolarized HEP in vitro, and in a rat with isotropic 7-mm spatial resolution, 93 Hz spectral resolution and 16-s temporal resolution for a period greater than 45 s.

Conclusion

Multiple echo 3D bSSFP imaging can provide chemical shift images of hyperpolarized ¹³C-labeled compounds in vivo with relatively high spatial resolution and moderate spectral resolution. The increased signal-to-noise ratio of this 3D technique will enable the detection of hyperpolarized ¹³C-labeled metabolites at lower concentrations as compared to a 2D technique.     

Diode-pumped doubly passively Q-switched c-cut Nd:GdVO4 1.34 μm laser with V:YAG and Co:LMA saturable absorbers

By simultaneously using both V³⁺:YAG and Co:LMA saturable absorbers in the cavity, a diode-pumped doubly passively Q-switched c-cut Nd:GdVO₄ laser at 1.34 μm is demonstrated for the first time. The average output power, the pulse width and the pulse repetition rate have been measured. The experimental results show that the doubly passively Q-switched laser can generate shorter pulse width with higher peak power in comparison to the singly passively Q-switched laser only with V³⁺:YAG or Co:LMA saturable absorber. At the pump power 13 W, the pulse width has been compressed 83% and the peak power has been improved 15 times, respectively.     

Detection of intracellular lactate with localized diffusion {1H-13C}-spectroscopy in rat glioma in vivo

The aim of this study was to compare the diffusion characteristic of lactate and alanine in a brain tumor model to that of normal brain metabolites known to be mainly intracellular such as N-acetylaspartate or creatine. The diffusion of (13)C-labeled metabolites was measured in vivo with localized NMR spectroscopy at 9.4 T (400 MHz) using a previously described localization and editing pulse sequence known as ACED-STEAM ('adiabatic carbon editing and decoupling'). (13)C-labeled glucose was administered and the apparent diffusion coefficients of the glycolytic products, {(1)H-(13)C}-lactate and {(1)H-(13)C}-alanine, were determined in rat intracerebral 9L glioma. To obtain insights into {(1)H-(13)C}-lactate compartmentation (intra- versus extracellular), the pulse sequence used very large diffusion weighting (50 ms/microm(2)). Multi-exponential diffusion attenuation of the lactate metabolite signals was observed. The persistence of a lactate signal at very large diffusion weighting provided direct experimental evidence of significant intracellular lactate concentration. To investigate the spatial distribution of lactate and other metabolites, (1)H spectroscopic images were also acquired. Lactate and choline-containing compounds were consistently elevated in tumor tissue, but not in necrotic regions and surrounding normal-appearing brain. Overall, these findings suggest that lactate is mainly associated with tumor tissue and that within the time-frame of these experiments at least some of the glycolytic product ([(13)C] lactate) originates from an intracellular compartment.     

In vivo C magnetic resonance spectroscopy of a human brain tumor after application of C-1-enriched glucose

Objectives

As a unique tool to assess metabolic fluxes noninvasively, ¹³C magnetic resonance spectroscopy (MRS) could help to characterize and understand malignancy in human tumors. However, its low sensitivity has hampered applications in patients. The aim of this study was to demonstrate that with sensitivity-optimized localized ¹³C MRS and intravenous infusion of [1-¹³C]glucose under euglycemia, it is possible to assess the dynamic conversion of glucose into its metabolic products in vivo in human glioma tissue.

Materials and Methods

Measurements were done at 3 T with a broadband single RF channel and a quadrature ¹³C surface coil inserted in a ¹H volume coil. A ¹H/¹³C polarization transfer sequence was applied, modified for localized acquisition, alternatively in two (50 ml) voxels, one encompassing the tumor and the other normal brain tissue.

Results

After about 20 min of [1-¹³C]glucose infusion, a [3-¹³C]lactate signal appeared among several resonances of metabolic products of glucose in MR spectra of the tumor voxel. The resonance of [3-¹³C]lactate was absent in MR spectra from contralateral tissue. In addition, the intensity of [1-¹³C]glucose signals in the tumor area was about 50% higher than that in normal tissue, likely reflecting more glucose in extracellular space due to a defective blood–brain barrier. The signal intensity for metabolites produced in or via the tricarboxylic acid (TCA) cycle was lower in the tumor than in the contralateral area, albeit that the ratios of isotopomer signals were comparable.

Conclusion

With an improved ¹³C MRS approach, the uptake of glucose and its conversion into metabolites such as lactate can be monitored noninvasively in vivo in human brain tumors. This opens the way to assessing metabolic activity in human tumor tissue.     

17O and 13C NMR Studies of Isobenzopyrylium Salts

¹³C and ¹⁷O NMR chemical shifts for a series of isobenzopyrylium salts are reported. The oxygen signal range from 300 to 270 ppm as a double bonded carboxylic oxygen, From the ¹⁷O and ¹³C data valuable informations on the conjugative and substituent effects of isobenzopyrylium salts were obtained.     

In Vivo3D LocalizedC Spectroscopy Using Modified INEPT and DEPT

The 3D localized¹³C spectroscopy methods LINEPT and LODEPT, which are modifications of INEPT and DEPT, are proposed. As long as a¹³C inversion pulse (180-degree pulse) is applied at 1/(4J) before the proton echo time in LINEPT and a¹³C excitation pulse (90-degree pulse) is applied at 1/(2J) before the proton echo time in LODEPT, the proton echo time can be set to any value longer than 1/(2J) in LINEPT and longer than 1/Jin LODEPT. As a result, the proton and the¹³C pulses can be applied separately and these proton pulses can be made slice-selective pulses. These localization features of LINEPT and LODEPT were evaluated using a phantom consisting of a cylinder filled with ethanol placed inside another cylinder filled with oil, and localized ethanol spectra could be obtained.In vivo3D localized¹³C spectra from the brain of a monkey could be obtained using decoupled LINEPT, and glutamate C-4 appeared directly after the administration of glucose C-1, followed by the appearance of glutamate C-2, C-3 and glutamine C-2, C-3, C-4.