Bertram L. Koelsch

Hyperpolarized 13C-pyruvate magnetic resonance reveals rapid lactate export in metastatic renal cell carcinomas

Renal cell carcinomas (RCC) are a heterogeneous group of tumors with a wide range of aggressiveness. Noninvasive methods to confidently predict the tumor biologic behavior and select appropriate treatment are lacking. Here, we investigate the dynamic metabolic flux in living RCC cells using hyperpolarized (13)C-pyruvate magnetic resonance spectroscopy (MRS) combined with a bioreactor platform and interrogated the biochemical basis of the MRS data with respect to cancer aggressiveness. RCC cells have significantly higher pyruvate-to-lactate flux than the normal renal tubule cells. Furthermore, a key feature distinguishing the localized from the metastatic RCC cells is the lactate efflux rate, mediated by the monocarboxylate transporter 4 (MCT4). The metastatic RCC cells have significantly higher MCT4 expression and corresponding higher lactate efflux, which is essential for maintaining a high rate of glycolysis. We show that such differential cellular transporter expression and associated metabolic phenotype can be noninvasively assessed via real-time monitoring of hyperpolarized (13)C-pyruvate-to-lactate flux.

High Resolution

(13)C steady state free precession (SSFP) magnetic resonance imaging and effective spin-spin relaxation time (T2) mapping were performed using hyperpolarized [(13)C] urea and [(13) C,(15)N2] urea injected intravenously in rats. (15)N labeling gave large T2 increases both in solution and in vivo due to the elimination of a strong scalar relaxation pathway. The T2 increase was pronounced in the kidney, with [(13) C,(15) N2] urea giving T2 values of 6.3±1.3 s in the cortex and medulla, and 11±2 s in the renal pelvis. The measured T2 in the aorta was 1.3±0.3 s. [(13)C] urea showed shortened T2 values in the kidney of 0.23±0.03 s compared to 0.28±0.03 s measured in the aorta. The enhanced T2 of [(13)C,(15)N2] urea was utilized to generate large signal enhancement by SSFP acquisitions with flip angles approaching the fully refocused regime. Projection images at 0.94 mm in-plane resolution were acquired with both urea isotopes, with [(13)C,(15) N2] urea giving a greater than four-fold increase in signal-to-noise ratio over [(13)C] urea.

^{13}

We combined the high MR signal enhancement achieved using dissolution dynamic nuclear polarization (DNP) with a pulsed gradient double spin echo diffusion MR sequence to rapidly and accurately measure the diffusion coefficients of various hyperpolarized (13)C molecules in solution. Furthermore, with a diffusion-weighted imaging sequence we generate diffusion coefficient maps of multiple hyperpolarized metabolites simultaneously. While hyperpolarized experiments can measure rapid, non-equilibrium processes by avoiding signal averaging, continuous signal loss due to longitudinal relaxation (T(1)) complicates quantitation. By correcting for this signal loss, we demonstrate the feasibility of using hyperpolarized (13)C diffusion-weighted MR to accurately measure real-time (seconds) molecular transport phenomena. Potential applications include rapidly measuring molecular binding, cellular membrane transport, in vivo metabolite distribution and establishing a magnetic field independent hyperpolarized parameter.

C MRI With Hyperpolarized Urea: In Vivo

Purpose Hyperpolarized 13C MR allows for the study of real-time metabolism in vivo, including significant hyperpolarized 13C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized 13C lactate but also understand its compartmentalization using diffusion weighted MR will provide unique information for improved tumor characterization.Hyperpolarized 13C magnetic resonance allows for the study of real‐time metabolism in vivo, including significant hyperpolarized 13C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized 13C lactate but also understand its compartmentalization using diffusion‐weighted MR will provide unique information for improved tumor characterization.

T_{2}

In this study, in vivo T2 heterogeneity of hyperpolarized [13C,15N2]urea in rat kidney has been investigated. Selective quenching of the vascular hyperpolarized 13C signal with a macromolecular relaxation agent revealed that a long T2 component of the [13C,15N2]urea signal originated from the renal extravascular space, thus allowing the vascular and renal filtrate contrast agent pools of the [13C,15N2]urea to be distinguished via multiexponential analysis. The T2 response to induced diuresis and antidiuresis was determined using 2 imaging agents—hyperpolarized [13C,15N2]urea and hyperpolarized bis-1,1-(hydroxymethyl)-1-13C-cyclopropane-2H8 (control agent). During antidiuresis, large T2 increases in the inner medulla and papilla were observed using the former agent only. Therefore, [13C,15N2]urea relaxometry is sensitive to the following 2 steps of the renal urea handling process: glomerular filtration process and inner medullary urea transporter-A1- and urea transporter-A3-mediated urea concentrating process. To aid multiexponential data analysis, simple motion correction and subspace denoising algorithms are presented. Furthermore, a T2-edited, ultralong echo time sequence was developed for sub-2 mm3 resolution 3-dimensional encoding of urea by exploiting relaxation differences in the vascular and filtrate pools.

Mapping and

Metabolic shifts in disease are of great interest for the development of novel therapeutics. In cancer treatment, these therapies exploit the metabolic phenotype associated with oncogenesis and cancer progression. One recent strategy involves the depletion of the cofactors needed to maintain the high rate of glycolysis seen with the Warburg effect. Specifically, blocking nicotinamide adenine dinucleotide (NAD) biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) inhibition depletes cancer cells of the NAD needed for glycolysis. To characterize this metabolic phenotype in vivo and describe changes in flux with treatment, non‐invasive biomarkers are necessary. One such biomarker is hyperpolarized (HP) [1‐13C] pyruvate, a clinically translatable probe that allows real‐time assessment of metabolism.

^{15}

This work demonstrates the separation of extra- and intracellular components of glycolytic metabolites with diffusion weighted hyperpolarized (13)C magnetic resonance spectroscopy. Using b-values of up to 15,000smm(-2), a multi-exponential signal response was measured for hyperpolarized [1-(13)C] pyruvate and lactate. By fitting the fast and slow asymptotes of these curves, their extra- and intracellular weighted diffusion coefficients were determined in cells perfused in a MR compatible bioreactor. In addition to measuring intracellular weighted diffusion, extra- and intracellular weighted hyperpolarized (13)C metabolites pools are assessed in real-time, including their modulation with inhibition of monocarboxylate transporters. These studies demonstrate the ability to simultaneously assess membrane transport in addition to enzymatic activity with the use of diffusion weighted hyperpolarized (13)C MR. This technique could be an indispensible tool to evaluate the impact of microenvironment on the presence, aggressiveness and metastatic potential of a variety of cancers.

N Labeling Effects

In this work, we present a new ultrafast method for acquiring dynamic 2D EXchange SpectroscopY (EXSY) within a single acquisition. This technique reconstructs two-dimensional EXSY spectra from one-dimensional spectra based on the phase accrual during echo times. The Ultrafast-EXSY acquisition overcomes long acquisition times typically needed to acquire 2D NMR data by utilizing sparsity and phase dependence to dramatically undersample in the indirect time dimension. This allows for the acquisition of the 2D spectrum within a single shot. We have validated this method in simulations and hyperpolarized enzyme assay experiments separating the dehydration of pyruvate and lactate-to-pyruvate conversion. In a renal cell carcinoma cell (RCC) line, bidirectional exchange was observed. This new technique revealed decreased conversion of lactate-to-pyruvate with high expression of monocarboxylate transporter 4 (MCT4), known to correlate with aggressive cancer phenotypes. We also showed feasibility of this technique in vivo in a RCC model where bidirectional exchange was observed for pyruvate-lactate, pyruvate-alanine, and pyruvate-hydrate and were resolved in time. Broadly, the technique is well suited to investigate the dynamics of multiple exchange pathways and applicable to hyperpolarized substrates where chemical exchange has shown great promise across a range of disciplines.

Diffusion MR of hyperpolarized 13C molecules in solution

Purpose Hyperpolarized 13C MR allows for the study of real-time metabolism in vivo, including significant hyperpolarized 13C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized 13C lactate but also understand its compartmentalization using diffusion weighted MR will provide unique information for improved tumor characterization.Hyperpolarized 13C magnetic resonance allows for the study of real‐time metabolism in vivo, including significant hyperpolarized 13C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized 13C lactate but also understand its compartmentalization using diffusion‐weighted MR will provide unique information for improved tumor characterization.

Rapid in vivo apparent diffusion coefficient mapping of hyperpolarized 13C metabolites

Purpose Hyperpolarized 13C MR allows for the study of real-time metabolism in vivo, including significant hyperpolarized 13C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized 13C lactate but also understand its compartmentalization using diffusion weighted MR will provide unique information for improved tumor characterization.Hyperpolarized 13C magnetic resonance allows for the study of real‐time metabolism in vivo, including significant hyperpolarized 13C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized 13C lactate but also understand its compartmentalization using diffusion‐weighted MR will provide unique information for improved tumor characterization.