Healthy prostate tissue has been described as having a low rate of respiration, high anaerobic glycolysis, and considerable aerobic glycolysis (36
). This is due to the unique function of healthy prostatic epithelial cells to synthesize and secrete large amounts of citrate rather than use it for energy production or lipogenesis (36
). It has been proposed that this inefficient and low level of energy production is insufficient for cells to perform the synthetic and bioenergetic requirements that are essential for the growth and proliferation of prostate cancer (36
). In general, cancer has been associated with an increase in glycolytic flux, and high lactate concentrations have been associated with more aggressive disease and metastasis while lower lactate concentrations have shown an overall longer and disease-free survival (38
). The amino acid alanine is another important end-product of glucose utilization, which is required in cytosolic amino acid transformations and in protein synthesis (42
). Alanine and citrate are also end-products of glutamate oxidation, which has been found to be a major source of respiratory energy in cancer (42
). It has been suggested that this alternative pathway is required to maintain lipogenesis/cholesterogenesis, which is especially needed for the formation of cellular membranes in proliferating cancer cells (36
Because of the earlier detection and down-staging of prostate cancer that has occurred in recent years, new biomarkers are necessary to better identify and distinguish potentially aggressive cancers from those that are not as aggressive. In this study, lactate and alanine were investigated as potential biomarkers for prostate adenocarcinoma. 1
H HR-MAS spectroscopy provided high-quality spectra from TRUS-guided prostate biopsies, and the reference method ERETIC was robust for absolute quantification of lactate and alanine concentrations in intact biopsy tissue. Compared to previously reported lactate concentrations in surgical samples (~45 mmol/kg) (27
), very low concentrations (<1.0 mmol/kg) were observed in benign prostate biopsies. This indicates that minimal anaerobic glycolysis occurs during biopsy collection (≤15 s), and biopsies therefore provide a better “snapshot” of in vivo metabolism. Additionally, the minimal changes in lactate (3.6%) and alanine (0.6%) during the experiments shows that biopsy tissues are well suited for studies on glycolysis.
The significant increase in lactate concentration found in prostate cancer biopsies is in agreement with previous studies of lactate in other human cancers (38
). It was concluded that high lactate concentrations in solid tumors are not just a substitute for hypoxia, but a balance between a permanent generation of lactate and inefficient microcirculation in the tumor (41
). This is explained by the “Warburg effect,” where high lactate production is observed in malignant cells even in the presence of oxygen. An elevation in aerobic glycolysis might therefore be a fundamental property of cancer cells. Lactate has also been found to enhance the degree of tumor malignancy by activation of hyaluronan synthesis (43
), upregulation of the growth factor VEGF (44
), and the hypoxia-inducible factor HIF-1alpha (45
). These mechanisms generate favorable conditions for metastatic spread. Thus, lactate accumulation in prostate cancer tissue may reflect the degree of tumor malignancy and may be related to the outcome of the disease. The isoenzymes of lactate dehydrogenase (LDH) catalyze the interconversion of lactate and pyruvate in the pathway of glycolysis. Several studies have shown that LDH is upregulated in other cancer types, such as ovarian cancer (46
), colorectal cancer (47
), and lung cancer (48
). Therefore, an elevation in LDH levels in prostate cancer tissue might contribute to the elevated concentration of lactate.
The observation of significantly higher alanine concentrations in prostate cancer tissue supports the theory that tumor malignancy is associated with an increase in glycolytic flux, and is also consistent with the need for increased protein synthesis in tumors (36
). Only a few previous studies have investigated alanine levels related to cancer, but the present results are in agreement with a study suggesting that glutamate oxidation may be a major source of respiratory energy for tumor cells (42
). Glutamate is transaminated primarily to pyruvate to form alanine, and increased alanine concentrations may in this case be a result of the need for membranogenesis (lipogenesis) in proliferating cancer cells (36
). Additionally, the transamination to alanine from pyruvate is catalyzed by the enzyme alanine transaminase (ALT); however, further studies are needed to establish the activity of ALT in prostate cancer tissue. The increase in alanine concentration could also be derived from catabolism of cellular proteins caused by the cancer. This study shows that alanine is an important biomarker in prostate cancer tissue, but further studies are needed to find the underlying biochemical pathways for alanine utilization in prostate cancer.
A growing amount of published data indicate that the metabolic information provided by MRSI combined with the anatomical information provided by MRI can significantly improve the clinical assessment of prostate cancer extent and location (1
), extracapsular spread (7
) and aggressiveness (9
). The presence of thousands of whole-body 1.5T MRI scanners in hospitals worldwide and the availability of commercial MRI/MRSI packages will allow the routine clinical use of these techniques. Despite its value, combined MRI/MRSI has recognized limitations at 1.5T, including the potential for false-positive and false-negative results, particularly in patients with early-stage cancer (15
). New biomarkers such as lactate and alanine could reduce the number of false-positives and -negatives in 1.5T MRI/MRSI studies of prostate cancer patients. In current commercial 1.5T MRI/MRSI packages, the lactate and alanine region of the spectrum is not excited to minimize periprostatic lipid contamination. However, with improved volume selection, outer voxel suppression techniques, and higher-field (3T) scanners, it is becoming feasible to use spectral-spatial RF editing sequences to detect lactate and alanine (21
Additionally, new hyperpolarized 13
C MR techniques could potentially exploit lactate and alanine as biomarkers in future clinical investigations. Because of the unprecedented NMR signal enhancement (~50,000-fold) provided by hyperpolarized 13
C techniques, the transformation of pyruvate into lactate and alanine can be detected noninvasively in less than a minute (49
), and the glycolytic status of the cell and the metabolic difference between normal and cancer tissue can therefore be quantified and mapped (49
). Therefore, the changes in lactate and alanine observed in the present study could be used to improve the clinical diagnosis and characterization of human prostate cancer using lactate and alanine edited 1
H or hyperpolarized 13
C SI sequences.
Prostate cancer is found to have a broad range of biological malignancies, and histopathological heterogeneity-(both regional and cellular) is a challenge in characterizing the cancer and the different tumor grades. The Gleason scoring system evaluates this heterogeneity by scoring how effectively cancer cells are able to structure themselves into glands resembling those of the normal prostate. However, there are often discrepancies in pathologic scoring, and the present study is therefore based on average readings from two pathologists. For prostate biopsy tissue, there was good consensus among the pathology scores. Benign prostate tissue is also heterogeneous and confounding factors such as chronic inflammation (prostatitis) and BPH were accounted for in this study. By collecting larger numbers of samples, these confounding factors can potentially be separated by their metabolic patterns. In future studies, the concentrations of lactate and/or alanine may be of importance as biomarkers for the diagnosis of these benign conditions. Likewise, metabolic studies of different prostate zones would be of interest in understanding regional metabolic and pathologic relationships.
There were a few notable limitations in the present study. Although biopsy tissues provided a more accurate snapshot of in vivo metabolism, there were an increased number of contaminants compared to surgical samples. These contaminants arose from periprostatic lipid contamination and the topical anesthetic (Hurricaine®; Beutlich) that was applied prior to the TRUS procedure. This resulted in 32 (~25%) of the biopsy samples being unusable in the current study. Additionally, only two samples could be obtained per patient and the sample sizes were much smaller than what can be obtained from surgical specimens. Finally, the relatively small number of biopsies containing cancer in this study, which is consistent with the down-staging of prostate cancer in the PSA era, limits our ability to assess changes in lactate and alanine concentration with cancer grade. Nevertheless, the use of biopsy tissues was critical for this study in order to minimize the impact of anaerobic glycolysis, which occurs in surgical specimens, on lactate and alanine concentrations.
In summary, using quantitative HR-MAS spectroscopy of snap-frozen prostate biopsies, it was determined that lactate and alanine concentrations were very low in benign prostate biopsy samples, and were significantly elevated in biopsy samples containing prostate cancer. The magnitude of elevation in the concentration of these metabolites is most likely a function of both the percentage of the biopsy core that is cancer and the pathologic grade of the cancer, but this will need to be determined in a study involving a larger number of malignant biopsies. The significant increase in the concentration of both lactate and alanine in biopsy samples containing as little as 5% cancer, and the minimal overlap of lactate concentrations between benign and malignant biopsies suggest that lactate and possibly alanine will be useful biomarkers that could be utilized in hyperpolarized 13C MRSI staging exams of prostate cancer patients.