Imaging of branched chain amino acid metabolism in tumors with hyperpolarized ¹³C ketoisocaproate

Abstract

Powerful analytical tools are vital for characterizing the complex molecular changes underlying oncogenesis and cancer treatment. This is particularly true, if information is to be collected in vivo by noninvasive approaches. In the recent past, hyperpolarized 13 C magnetic resonance (MR) spectroscopy has been employed to quickly collect detailed spectral information on the chemical fate of tracer molecules in different tissues at high sensitivity. Here, we report a preclinical study showing that a-ketoisocaproic acid (KIC) can be used to assess molecular signatures of tumors with hyperpolarized MR spectroscopy. KIC is metabolized to leucine by the enzyme branched chain amino acid transferase (BCAT), which is found upregulated in some tumors. BCAT is a putative marker for metastasis and a target of the proto-oncogene c-myc. Very different fluxes through the BCAT-catalyzed reaction can be detected for murine lymphoma (EL4) and rat mammary adenocarcinoma (R3230AC) tumors in vivo. EL4 tumors show a more than 7-fold higher hyperpolarized 13 C leucine signal relative to the surrounding healthy tissue. In R3230AC tumor on the other hand branched chain amino acid metabolism is not enhanced relative to surrounding tissues. The distinct molecular signatures of branched chain amino acid metabolism in EL4 and R3230AC tumors correlate well with ex vivo assays of BCAT activity.Modern genetic tools have markedly improved the understanding of cancer as a genetic disease by linking the development, progression and remission of cancer to underlying genetic changes. 1,2 These approaches have also revealed the genetic heterogeneity of tumors. Thus, an understanding of the molecular signatures of the disease with noninvasive techniques would be highly desirable in order to define molecular targets for a tumor-specific or even personalized diagnosis and treatment.3 This can be achieved by hyperpolarized chemical shift imaging (CSI), which is a recently devised imaging modality for the visualization of molecular processes in vivo. [4][5][6] The method relies on a signal enhancement of the inherently weak nuclear magnetic resonance (NMR) signal by several orders of magnitude in a process termed dynamic nuclear polarization (DNP). This process increases the magnetization of nuclear spins ex situ to generate a 'hyperpolarized' molecule, which is injected as a nonradionuclide imaging marker. 7 The enhancement of detectable NMR signal by several orders of magnitude renders imaging experiments with a variety of substrates possible. As NMR is a high-resolution spectral technique, the method bears a unique potential to monitor chemical modifications of the hyperpolarized molecule in vivo. Previous studies using hyperpolarized pyruvate have detected cancer tissues by their increased anaerobic metabolism. 4,5,8 Notably, the method has also shown the potential to measure early tumor responses to therapy. 9 Cellular amino acid metabolism is regulated by the activity, organ distribution and cellular compartmentalization of metabolic enzymes incl...

Metastatic Melanoma Induced Metabolic Changes in C57BL/6J Mouse Stomach Measured by 1H NMR Spectroscopy

“…Increased glutamate concentration has also been reported by previous studies as a common phenomenon in tumors [41]. Furthermore, Magnus Karlsson and coworkers have reported that increased glutamate pools in tumors leads to increased leucine formation, a consistent result reflected in this study [42]. The blockade of glutamate receptors has been proposed as a promising novel therapy for treating melanoma [43].…”

Tumor imaging using hyperpolarized 13C magnetic resonance spectroscopy

“…This 13 CO 2 rapidly In many cases, higher unpublished levels of polarization have been achieved with these molecules, and in some cases a gadolinium chelate has also been added to improve polarization (47). Results obtained at higher polarization field show further increase in polarization levels (33,43). Abbreviations: ALT, alanine transaminase; BCAT, branched-chain amino acid transferase; GLUT5, glucose transporter; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PPP, pentose phosphate pathway.…”

“…Importantly, a large part of the pulse sequence development has been performed at 3 T on clinical systems, which is likely to speed up their availability in the clinic. Several studies have used a simple spectroscopic imaging sequence (19,26,40,43), with phase-encoding gradients in two or three directions. The sequence is widely available in a clinical setting, has relatively lowgradient amplitude requirements, and detects all the resonances simultaneously with high spectral quality.…”

“…Branched-chain amino acid transferase enzyme activity has been shown to be upregulated in some tumors (61). The production of [1- 13 C]leucine has been demonstrated in murine lymphoma in vivo following the injection of hyperpolarized [1][2][3][4][5][6][7][8][9][10][11][12][13] C]ketoisocaproate (43). An association between the branched-chain amino acid transferase activity and the appearance of leucine was observed, so it is possible that this could be used as a method to probe branched-chain amino acid transferase activity as well as the concentration of glutamate within the cell.…”

Detection of tumor glutamate metabolism in vivo using 13C magnetic resonance spectroscopy and hyperpolarized [1‐13C]glutamate

“…The activity of ALT has been detected previously using hyperpolarized [1][2][3][4][5][6][7][8][9][10][11][12][13] C]pyruvate in tumors, skeletal muscle, and in liver, where the formation of [1-13 C]alanine has been detected (2,6). ALT activity has also been detected in heart and liver from the formation of [1][2][3][4][5][6][7][8][9][10][11][12][13] C]pyruvate following the administration of hyperpolarized [1-13 C]alanine (12). Glutamate is an important nitrogen source and is involved in nitrogen transport within the body and in the central nervous system.…”