Proof of concept of a multimodal intravital molecular imaging system for tumour transpathology investigation.

BACKGROUND: Transpathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation. METHODS: The proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin-eosin (HE) stained for comparison with multimodal in vivo imaging. RESULTS: The final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology. CONCLUSIONS: The established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

Dynamic 2D and 3D mapping of hyperpolarized pyruvate to lactate conversion in vivo with efficient multi-echo balanced steady-state free precession at 3 T.

The aim of this study was to acquire the transient MRI signal of hyperpolarized tracers and their metabolites efficiently, for which specialized imaging sequences are required. In this work, a multi-echo balanced steady-state free precession (me-bSSFP) sequence with Iterative Decomposition with Echo Asymmetry and Least squares estimation (IDEAL) reconstruction was implemented on a clinical 3 T positron-emission tomography/MRI system for fast 2D and 3D metabolic imaging. Simulations were conducted to obtain signal-efficient sequence protocols for the metabolic imaging of hyperpolarized biomolecules. The sequence was applied in vitro and in vivo for probing the enzymatic exchange of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate. Chemical shift resolution was achieved using a least-square, iterative chemical species separation algorithm in the reconstruction. In vitro, metabolic conversion rate measurements from me-bSSFP were compared with NMR spectroscopy and free induction decay-chemical shift imaging (FID-CSI). In vivo, a rat MAT-B-III tumor model was imaged with me-bSSFP and FID-CSI. 2D metabolite maps of [1-13 C]pyruvate and [1-13 C]lactate acquired with me-bSSFP showed the same spatial distributions as FID-CSI. The pyruvate-lactate conversion kinetics measured with me-bSSFP and NMR corresponded well. Dynamic 2D metabolite mapping with me-bSSFP enabled the acquisition of up to 420 time frames (scan time: 180-350 ms/frame) before the hyperpolarized [1-13 C]pyruvate was relaxed below noise level. 3D metabolite mapping with a large field of view (180 × 180 × 48 mm3 ) and high spatial resolution (5.6 × 5.6 × 2 mm3 ) was conducted with me-bSSFP in a scan time of 8.2 seconds. It was concluded that Me-bSSFP improves the spatial and temporal resolution for metabolic imaging of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate compared with either of the FID-CSI or EPSI methods reported at 3 T, providing new possibilities for clinical and preclinical applications.

Ab Initio Simulation of pH-Sensitive Biomarkers in Magnetic Resonance Imaging.

An ab initio simulation scheme is introduced as a theoretical prescreening approach to facilitate and enhance the research for pH-sensitive biomarkers. The proton 1H and carbon 13C nuclear magnetic resonance (NMR) chemical shifts of the recently published marker for extracellular pH, [1,5-13C2]zymonic acid (ZA), and the as yet unpublished ( Z)-4-methyl-2-oxopent-3-enedioic acid (OMPD) were calculated with ab initio methods as a function of the pH. The influence of the aqueous solvent was taken into account either by an implicit solvent model or by explicit water molecules, where the latter improved the accuracy of the calculated chemical shifts considerably. The theoretically predicted chemical shifts allowed for a reliable NMR peak assignment. The p Ka value of the first deprotonation of ZA and OMPD was simulated successfully whereas the parametrization of the implicit solvent model does not allow for an accurate description of the second p Ka. The theoretical models reproduce the pH-induced chemical shift changes and the first p Ka with sufficient accuracy to establish the ab initio prescreening approach as a valuable support to guide the experimental search for pH-sensitive biomarkers.

Hyperpolarized Amino Acid Derivatives as Multivalent Magnetic Resonance pH Sensor Molecules.

pH is a tightly regulated physiological parameter that is often altered in diseased states like cancer. The development of biosensors that can be used to non-invasively image pH with hyperpolarized (HP) magnetic resonance spectroscopic imaging has therefore recently gained tremendous interest. However, most of the known HP-sensors have only individually and not comprehensively been analyzed for their biocompatibility, their pH sensitivity under physiological conditions, and the effects of chemical derivatization on their logarithmic acid dissociation constant (pKa). Proteinogenic amino acids are biocompatible, can be hyperpolarized and have at least two pH sensitive moieties. However, they do not exhibit a pH sensitivity in the physiologically relevant pH range. Here, we developed a systematic approach to tailor the pKa of molecules using modifications of carbon chain length and derivatization rendering these molecules interesting for pH biosensing. Notably, we identified several derivatives such as [1-13C]serine amide and [1-13C]-2,3-diaminopropionic acid as novel pH sensors. They bear several spin-1/2 nuclei (13C, 15N, 31P) with high sensitivity up to 4.8 ppm/pH and we show that 13C spins can be hyperpolarized with dissolution dynamic polarization (DNP). Our findings elucidate the molecular mechanisms of chemical shift pH sensors that might help to design tailored probes for specific pH in vivo imaging applications.

Deuteration of Hyperpolarized 13 C-Labeled Zymonic Acid Enables Sensitivity-Enhanced Dynamic MRI of pH.

Aberrant pH is characteristic of many pathologies such as ischemia, inflammation or cancer. Therefore, a non-invasive and spatially resolved pH determination is valuable for disease diagnosis, characterization of response to treatment and the design of pH-sensitive drug-delivery systems. We recently introduced hyperpolarized [1,5-13 C2 ]zymonic acid (ZA) as a novel MRI probe of extracellular pH utilizing dissolution dynamic polarization (DNP) for a more than 10000-fold signal enhancement of the MRI signal. Here we present a strategy to enhance the sensitivity of this approach by deuteration of ZA yielding [1,5-13 C2 , 3,6,6,6-D4 ]zymonic acid (ZAd ), which prolongs the liquid state spin lattice relaxation time (T1 ) by up to 39 % in vitro. Measurements with ZA and ZAd on subcutaneous MAT B III adenocarcinoma in rats show that deuteration increases the signal-to-noise ratio (SNR) by up to 46 % in vivo. Furthermore, we demonstrate a proof of concept for real-time imaging of dynamic pH changes in vitro using ZAd , potentially allowing for the characterization of rapid acidification/basification processes in vivo.

Multiparametric human hepatocellular carcinoma characterization and therapy response evaluation by hyperpolarized (13) C MRSI.

Individual tumor characterization and treatment response monitoring based on current medical imaging methods remain challenging. This work investigates hyperpolarized (13) C compounds in an orthotopic rat hepatocellular carcinoma (HCC) model system before and after transcatheter arterial embolization (TAE). HCC ranks amongst the top six most common cancer types in humans and accounts for one-third of cancer-related deaths worldwide. Early therapy response monitoring could aid in the development of personalized therapy approaches and novel therapeutic concepts. Measurements with selectively (13) C-labeled and hyperpolarized urea, pyruvate and fumarate were performed in tumor-bearing rats before and after TAE. Two-dimensional, slice-selective MRSI was used to obtain spatially resolved maps of tumor perfusion, cell energy metabolic conversion rates and necrosis, which were additionally correlated with immunohistochemistry. All three injected compounds, taken together with their respective metabolites, exhibited similar signal distributions. TAE induced a decrease in blood flow into the tumor and thus a decrease in tumor to muscle and tumor to liver ratios of urea, pyruvate and its metabolites, alanine and lactate, whereas conversion rates remained stable or increased on TAE in tumor, muscle and liver tissue. Conversion from fumarate to malate successfully indicated individual levels of necrosis, and global malate signals after TAE suggested the washout of fumarase or malate itself on necrosis. This study presents a combination of three (13) C compounds as novel candidate biomarkers for a comprehensive characterization of genetically and molecularly diverse HCC using hyperpolarized MRSI, enabling the simultaneous detection of differences in tumor perfusion, metabolism and necrosis. If, as in this study, bolus dynamics are not required and qualitative perfusion information is sufficient, the desired information could be extracted from hyperpolarized fumarate and pyruvate alone, acquired at higher fields with better spectral separation. Copyright © 2016 John Wiley & Sons, Ltd.

Diffusion of hyperpolarized (13) C-metabolites in tumor cell spheroids using real-time NMR spectroscopy.

The detection of tumors noninvasively, the characterization of their progression by defined markers and the monitoring of response to treatment are goals of medical imaging techniques. In this article, a method which measures the apparent diffusion coefficients (ADCs) of metabolites using hyperpolarized (13) C diffusion-weighted spectroscopy is presented. A pulse sequence based on the pulsed gradient spin echo (PGSE) was developed that encodes both kinetics and diffusion information. In experiments with MCF-7 human breast cancer cells, we detected an ADC of intracellularly produced lactate of 1.06 ± 0.15 µm(2) /ms, which is about one-half of the value measured with pyruvate in extracellular culture medium. When monitoring tumor cell spheroids during progressive membrane permeabilization with Triton X-100, the ratio of lactate ADC to pyruvate ADC increases as the fraction of dead cells increases. Therefore, (13) C ADC detection can yield sensitive information on changes in membrane permeability and subsequent cell death. Our results suggest that both metabolic label exchange and (13) C ADCs can be acquired simultaneously, and may potentially serve as noninvasive biomarkers for pathological changes in tumor cells.

Simultaneous characterization of tumor cellularity and the Warburg effect with PET, MRI and hyperpolarized 13C-MRSI.

Modern oncology aims at patient-specific therapy approaches, which triggered the development of biomedical imaging techniques to synergistically address tumor biology at the cellular and molecular level. PET/MR is a new hybrid modality that allows acquisition of high-resolution anatomic images and quantification of functional and metabolic information at the same time. Key steps of the Warburg effect-one of the hallmarks of tumors-can be measured non-invasively with this emerging technique. The aim of this study was to quantify and compare simultaneously imaged augmented glucose uptake and LDH activity in a subcutaneous breast cancer model in rats (MAT-B-III) and to study the effect of varying tumor cellularity on image-derived metabolic information. Methods: For this purpose, we established and validated a multimodal imaging workflow for a clinical PET/MR system including proton magnetic resonance (MR) imaging to acquire accurate morphologic information and diffusion-weighted imaging (DWI) to address tumor cellularity. Metabolic data were measured with dynamic [18F]FDG-PET and hyperpolarized (HP) 13C-pyruvate MR spectroscopic imaging (MRSI). We applied our workflow in a longitudinal study and analyzed the effect of growth dependent variations of cellular density on glycolytic parameters. Results: Tumors of similar cellularity with similar apparent diffusion coefficients (ADC) showed a significant positive correlation of FDG uptake and pyruvate-to-lactate exchange. Longitudinal DWI data indicated a decreasing tumor cellularity with tumor growth, while ADCs exhibited a significant inverse correlation with PET standard uptake values (SUV). Similar but not significant trends were observed with HP-13C-MRSI, but we found that partial volume effects and point spread function artifacts are major confounders for the quantification of 13C-data when the spatial resolution is limited and major blood vessels are close to the tumor. Nevertheless, analysis of longitudinal data with varying tumor cellularity further detected a positive correlation between quantitative PET and 13C-data. Conclusions: Our workflow allows the quantification of simultaneously acquired PET, MRSI and DWI data in rodents on a clinical PET/MR scanner. The correlations and findings suggest that a major portion of consumed glucose is metabolized by aerobic glycolysis in the investigated tumor model. Furthermore, we conclude that variations in cell density affect PET and 13C-data in a similar manner and correlations of longitudinal metabolic data appear to reflect both biochemical processes and tumor cellularity.

Imaging of pH in vivo using hyperpolarized 13C-labelled zymonic acid.

Natural pH regulatory mechanisms can be overruled during several pathologies such as cancer, inflammation and ischaemia, leading to local pH changes in the human body. Here we demonstrate that 13C-labelled zymonic acid (ZA) can be used as hyperpolarized magnetic resonance pH imaging sensor. ZA is synthesized from [1-13C]pyruvic acid and its 13C resonance frequencies shift up to 3.0 p.p.m. per pH unit in the physiological pH range. The long lifetime of the hyperpolarized signal enhancement enables monitoring of pH, independent of concentration, temperature, ionic strength and protein concentration. We show in vivo pH maps within rat kidneys and subcutaneously inoculated tumours derived from a mammary adenocarcinoma cell line and characterize ZA as non-toxic compound predominantly present in the extracellular space. We suggest that ZA represents a reliable and non-invasive extracellular imaging sensor to localize and quantify pH, with the potential to improve understanding, diagnosis and therapy of diseases characterized by aberrant acid-base balance.

Earth's magnetic field enabled scalar coupling relaxation of 13C nuclei bound to fast-relaxing quadrupolar 14N in amide groups.

Scalar coupling relaxation, which is usually only associated with closely resonant nuclei (e.g., (79)Br-(13)C), can be a very effective relaxation mechanism. While working on hyperpolarized [5-(13)C]glutamine, fast liquid-state polarization decay during transfer to the MRI scanner was observed. This behavior could hypothetically be explained by substantial T(1) shortening due to a scalar coupling contribution (type II) to the relaxation caused by the fast-relaxing quadrupolar (14)N adjacent to the (13)C nucleus in the amide group. This contribution is only effective in low magnetic fields (i.e., less than 800 µT) and prevents the use of molecules bearing the (13)C-amide group as hyperpolarized MRS/MRI probes. In the present work, this hypothesis is explored both theoretically and experimentally. The results show that high hyperpolarization levels can be retained using either a (15)N-labeled amide or by applying a magnetic field during transfer of the sample from the polarizer to the MRI scanner.