This study demonstrated the feasibility of detecting variations in the levels of pyruvate and lactate in human glioblastoma orthotopic xenografts using hyperpolarized [1-13C]-pyruvate as a substrate. The hyperpolarized 13C 2D MRSI data had a nominal spatial resolution of 0.25 cm3 and demonstrated significant differences in 13C metabolic profiles between tumors and normal brain tissues. The 13C lactate and pyruvate levels in the contrast-enhancing lesions of the brain in rats with tumors were much higher than those in the brains of rats without tumors.
All 6 of the 13C imaging parameters that were evaluated exhibited substantial differences between brains with and without tumors (Fig. ). The SNR of lactate, pyruvate, and total carbon, and the ratio of Lac/Pyr, Lac/tC, and Pyr/tC, were well separated without any overlap of the data between the 2 groups (P < .0004). The substantial differences between 13C cellular metabolism measurements in tumors and normal brains suggest that this method has great potential for monitoring the abnormal metabolism in brain tumors.
Elevated levels of lactate, lactate to pyruvate ratios, and lactate to total carbon ratios are consistent with the findings in previous studies that examined hyperpolarized 13
C-labeled lactate in TRAMP mice20,22
and mouse lymphomas.21
These studies suggested that hyperpolarized 13
C-labled lactate may provide an indirect method for assessing LDH activity. LDH is the enzyme catalyzing the conversion from pyruvate to lactate in glycolysis, which has been seen to have increased activity in brain tumors.35
A recent study demonstrated that the degree of upregulation correlated with the levels of hyperpolarized 13
C-labeled lactate in prostate cancer cell lines.36
Future studies will examine hyperpolarized 13
C metabolites in an ex vivo model of rat brain tumors and compare them with an assay of LDH activity.
U-251 MG and U-87 MG xenografts are well-established cell lines widely used for preclinical studies of brain tumors. Previous studies have shown that there are distinct differences in the histopathologic features of these 2 tumor types.25,26
U-251 MG tumors consist of a mixture of malignant spindle and epithelioid cells, and contain a large area of necrosis and an irregular border with adjacent brain tissue. U-87 MG tumors are relatively well circumscribed with malignant spindle cells in compact fascicles, and exhibit little or no necrosis. U-251 MG cells also contain more hypoxia than U-87 MG cells.37
The immunohistochemical data in our study were consistent with these findings (Table ). The U-251 MG model exhibited large areas of necrosis and hypoxia, whereas the U-87 MG model showed little or no necrosis and hypoxia (Fig. ). The patterns of contrast enhancement in T1 post-Gd images of rats with U-251 MG and U-87 MG xenografts also indicated that there were marked differences between these 2 tumor types (Fig. ). The U-87 MG tumors had homogenous levels of contrast enhancement with a well-delineated tumor margin, whereas the U-251 MG tumors exhibited varying levels of contrast enhancement with an irregular tumor margin.
Fig. 9. The patterns of contrast enhancement in T1 post-Gd images between rats with U-251 MG model (A) and U-87 MG model (B). The U-251 MG tumor displayed heterogeneous levels of contrast enhancement, whereas the levels of contrast enhancement of the U-87 tumor (more ...)
The SNR of lactate, pyruvate, and total carbon of U-87 MG tumors was significantly higher than those of U-251 MG tumors (P
< .02). The variation in these metabolic profiles may be due to, in part, higher necrotic fraction and thus a relatively smaller number of viable tumor cells in U-251 MG compared with U-87 MG tumors. The higher pyruvate uptake in U-87 MG tumors is consistent with a previous study that showed higher mRNA expression in epithelial tumor cell lines of a monocarboxylate transporter, which has a high affinity for the transport of pyruvate.38
In addition, low perfusion in necrotic areas of the tumors may result in inadequate delivery of pyruvate to tumor cells. Similarly, the SNR of total carbon, which can be considered as an estimate of the hyperpolarized compounds taken up by the tissue, was in agreement with the difference in necrotic fraction found between U-251 MG and U-87 MG tumors. The differences in 13
C metabolic imaging profiles between U-251 MG and U-87 MG models are consistent with the known differences in molecular characteristics between these tumors and suggest that hyperpolarized 13
C MRSI that uses pyruvate as a substrate may be useful in characterizing tumor tissues. The different features of U-87 MG and U-251 MG tumors found from this study are summarized in Table and compared with biological characteristics of the 2 tumor models.
Comparison of biological, immunohistochemical, and MR features between U-251 MG and U-87 MG model
Uncontrolled proliferation is one of the major properties of cancer cells. In gliomas, a number of previous studies have correlated proliferation markers with clinical outcome and have validated the MIB-1/Ki-67 index as one of the major predictors of patient survival.39,40
The strong correlation between the MIB-1 index and the level of 13
C-labeled lactate that is converted from hyperpolarized 13
C-labeled pyruvate suggests that this methodology may provide an indirect method for characterizing tumor activity and be a potential surrogate marker for prognosis.
Although several imaging techniques such as 1
H MRSI, 13
C MRS, and PET using FDG as substrate have been applied to evaluate in vivo tumor metabolism,4–7,12–17
MR metabolic imaging using hyperpolarized 13
C substrates has several advantages. With the increase in sensitivity of these 13
C substrates, the detection of metabolites is possible at high spatial resolution (0.25 cm3
) in a very short acquisition time (11 seconds). These provide critical improvements over conventional 13
C MRS, which is limited by low sensitivity and prohibitively long acquisition time. Unlike 1
H MRSI, where the overlapping lipid peaks provide a technical challenge for quantifying lactate, the spectrum obtained using hyperpolarized 13
C metabolic imaging lacks a background signal. More importantly, while the interpretation of the steady-state 1
H lactate signal is complicated due to the various potential sources of 1
the lactate signal using 13
C MRS reflects only the metabolically active lactate that is generated from hyperpolarized 13
C-labeled pyruvate via LDH activity during the experiment. The high reproducibility in the difference in lactate uptake between the tumor and normal brain tissue suggests that this technique will be valuable in assessing prognosis and monitoring brain tumors' response to therapy.
Although the dynamic hyperpolarized 13
C data contain important biological information, the results of these studies were primarily used to determine the timing of subsequent 2D MRSI exams. They were limited by the 15 mm-thick slice, which included contributions from the brain, muscle, and vasculature (Fig. C). The application of a time-resolved MRSI method with an appropriate spatial resolution20,41
may be expected to provide more specific information about the dynamics of tumor and normal brain tissue. It should be noted that despite the presence of the BBB, the hyperpolarized 13
C 2D MRSI data provided ample SNR for evaluating lactate and pyruvate in both normal brain and tumors (Fig. ). The high SNR means that it may be possible to obtain an increased coverage with higher spatial resolution in future experiments, and that the application of a fast acquisition technique incorporating a flyback echo-planar readout20,29
could allow the acquisition of 3D MRSI data that would cover the entire rat brain in a scan time of 10–17 seconds.
The brain tissue in normal rats demonstrated much lower pyruvate and lactate uptake compared with the malignant tumor tissue (Figs. C and 3C). The low uptake of pyruvate in the normal brain tissue is thought to be due to the selective permeability of the BBB. It is known that lipid-soluble molecules readily penetrate the BBB whereas charged molecules, which are not lipid-soluble, cross the BBB slowly, or not at all.42
Since pyruvate is a negatively charged molecule in solution, the crossing of pyruvate molecules through the BBB is restricted. It is impossible to determine the degree of pyruvate signal contribution between the brain tissue and the blood vessels in this study because some of the pyruvate and lactate signal in the brain may come from the capillaries in the endothelial cells of the brain. However, the hyperpolarized lactate-to-pyruvate ratio in normal brain tissue was found to be notably different from that in the voxels located around blood vessels (Fig. ). In addition, the fact that the rats with U-87 MG and U-251 MG tumors exhibited dissimilar metabolic profiles (Fig. ) suggests that the pyruvate was delivered and metabolized in the tumor tissue and the conversion of pyruvate to lactate before its delivery to the tumor tissue was not the major source of the observed hyperpolarized signals. The results from this study suggest that hyperpolarized 13
C MRSI is capable of discriminating cancerous and normal brain tissues.