Non-invasive differentiation of benign renal tumors from clear cell renal cell carcinomas using clinically translatable hyperpolarized ¹³C pyruvate magnetic resonance

Abstract

Localized renal tumors are increasingly detected incidentally at imaging. Conventional imaging cannot reliably differentiate the 20% of these tumors that are benign from malignant renal cell carcinomas (RCCs), leading to unnecessary surgical resection and resulting morbidity associated with surgery. Here, we investigated hyperpolarized ¹³C pyruvate metabolism in live patient-derived renal tumor tissue slices using a novel magnetic resonance (MR) -compatible bioreactor platform. We demonstrated for the first time that clear cell RCCs (ccRCCs), which account for 70–80% of all RCCs, have increased lactate production as well as rapid lactate efflux compared to benign renal tumors. This difference is attributed to increased lactate dehydrogenase A and monocarboxylate transporter 4 expression in ccRCCs. This distinctive metabolic phenotype can be used to differentiate RCCs from benign renal tumors using clinically translatable hyperpolarized ¹³C pyruvate MR.

Introduction

The widespread use of cross-sectional imaging has led to a significant increase in the incidental detection of renal tumors [1], many of which are localized, clinical stage 1 tumors. These tumors exhibit a wide spectrum of benign and malignant histopathology as well as aggressiveness, posing significant challenges for clinical management. Approximately 20% of the clinical stage 1 renal tumors are benign tumors such as oncocytomas or minimal fat angiomyolipomas [2–5]. These benign tumors cannot be reliably differentiated from renal cell carcinoma (RCC) preoperatively using conventional imaging [6]. Percutaneous tumor biopsy also has its limitations, including low negative predictive value of biopsy for small renal masses, and overlapping histologic features between some benign renal tumors and RCCs [7]. Because of these limitations, localized renal tumors are most frequently treated with surgical resection. This has led to greater than 10,000 unnecessary operations of benign tumors each year in the U.S. alone [8], with inherent risks of surgery, loss of renal function, and cost. Therefore, new non-invasive imaging methods are needed to distinguish benign renal tumors from RCCs in order to guide management.

Increasing evidence has shown that RCCs are strongly linked to abnormal metabolism [9–11]. In particular, increased glycolysis with lactate production (Warburg effect) is a dominant metabolic feature of RCCs. For example, clear cell RCCs, which accounts for 70–80% of all RCCs, have characteristic reprogramming of glucose and energy metabolism that promotes glycolysis and lactate production [12,13]. High expression of monocarboxylate transporters 1 and 4, which are essential for maintaining high level of glycolysis and lactate transport, are associated with more aggressive RCCs [14–16]. These studies provide the rationale for metabolic imaging as a means to differentiate benign renal tumors from RCCs.

Hyperpolarized (HP) ¹³C magnetic resonance (MR) is a powerful molecular imaging technique that allows rapid and non-invasive investigation of dynamic metabolic and physiological processes that were previously inaccessible by imaging [17]. HP ¹³C pyruvate is the most widely studied probe to date [18,19], reflecting its central role in cellular metabolism. In particular, pyruvate is reduced to lactate in a reaction catalyzed by the enzyme lactate dehydrogenase (LDH). Several previous studies have shown that in vivo HP ¹³C pyruvate to lactate flux correlates to tumor grade in preclinical cancer models [20,21].

In this study, we compared the HP ¹³C lactate levels after injection of HP ¹³C pyruvate in live patient-derived renal tissue slices maintained in a MR-compatible bioreactor. The bioreactor provides a novel platform for the assessment of tissue metabolism in a controlled and physiologic setting [22,23]. We show that clear cell RCCs (ccRCCs) have high lactate production and importantly, they have increased lactate export when compared to benign renal tumors. This suggests that such metabolic phenotype can be explored to non-invasively differentiate ccRCCs from benign renal tumors using clinically translatable hyperpolarized ¹³C pyruvate MR.

Material and Methods

Patient-derived renal tissue slices

Fresh tissues were obtained from patients undergoing nephrectomy for renal tumors between September 2012 and August 2014 under an institutional review board-approved protocol. Tissue cores (8-mm diameter) of both tumors and adjacent uninvolved normal renal parenchyma were obtained from the nephrectomy specimen [24]. Tissues were precision-cut into 300 to 350 µm thick slices using a Krumdieck slicer (Alabama Research and Development, Mundford, AL, USA), and then cultured in an incubator at 37°C and 5% CO₂ for 12–18 hours in specialized medium in an angled rotating plate (30 degree) as previously described [25]. Subsequently, 4–6 tissue slices were loaded into a 5-mm MR-compatible bioreactor as previously described [22,23] for the hyperpolarized experiments below. Patient-derived renal slices were obtained from 10 clear cell RCCs (Furhman grade 1: n=1; Furhman grade 2: n=8; Furhman grade 3: n=1), 3 benign renal tumors (oncocytomas: n=2; angiomyolipomas: n=1), and 12 normal renal parenchyma tissues not involved with tumors. These fresh tissue slices were studied in a 3D tissue bioreactor.

MR-compatible 3-dimensional (3D) tissue culture bioreactor experiments

The tissue slices were maintained at physiological conditions in the bioreactor in circulating media at 37°C with 95% air/5% CO₂ via a gas exchanger. All bioreactor experiments were conducted using a 500 MHz Varian Inova (Agilent Technologies, Palo Alto, CA, USA) with a 5-mm, triple-tune, direct-detect, broadband probe. For the HP ¹³C pyruvate studies, 7.5 µL of 14.2 M [1-¹³C]pyruvate mixed with 15 mM of the trytl radical (GE Health, Menlo Park, CA, USA) and 2.5 mM gadolinium chelate were polarized on a Hypersense polarizer (Oxford Instruments, Oxford, UK). This was followed by dissolution in 5 mL of 50 mM phosphate buffer. Seven hundred and fifty µL of the resulting 16 mM HP ¹³C pyruvate solution was injected over 90 seconds into the bioreactor containing the tissue slices. Hyperpolarized ¹³C MR data were acquired dynamically with a 30 degree flip angle, pulse repetition time of 3 seconds, and for a duration of 300 seconds. ³¹P spectra were acquired before and after each hyperpolarized ¹³C study to assess tissue viability using a repetition time of 2 seconds, 2048 averages, and a 90 degree flip angle. The βNTP peak was quantified using the ERETIC method as described previously (26).

Immunohistochemical staining and pathological grading

At the end of the MR experiments, some of the renal tissue slices were rapidly frozen in cryo-embedding medium (Optimal Cutting Temperature Compound) for subsequent histological and immunohistochemical analyses. A clinical pathologist determined the renal tumor histology and grade (Furhman nuclear grading if RCCs) based on hemotoxylin and eosin (H&E) staining. Additionally, the slices were stained for monocarboxylate 4 (MCT4) expression.

Tissue slice mRNA expression and enzyme activity assay

Remaining tissue slices at the end of the bioreactor experiment were immediately frozen and used to measure enzymatic activity and mRNA expression as described previously [22,26]. mRNA expression of LDHA, monocarboxylate 1 (MCT1) and MCT4 was quantified using qRT-PCR. In brief, total RNA was extracted from the tissue slices using an RNAeasy kit (Qiagen, Germantown, MD, USA). Reverse transcription was performed using an iScript cDNA Synthesis kit (BioRad Laboratories, Hercules, CA, USA), and subsequently the cDNA generated was utilized for PCR in triplicate with TaqMan chemistry on the ABI 7900HT (Applied Biosystems, Foster City, CA, USA). Primers for the genes were obtained from Applied Biosystems (Foster City, CA, USA). The gene expression was calculated relative to the housekeeping gene beta-Actin.

LDH activity of tissue slices was measured spectrophotometrically by quantifying the linear decrease in NADH absorbance at varying pyruvate concentrations at 339 nm using a micro-plate reader (Tecan Group Ltd., Mannedorf, Switzerland) [22,26]. The maximum velocity (V_max) and the Michaelis–Menten constant (K_m) were estimated using the Lineweaver–Burke plot, and were normalized to the protein content.

Lactate efflux measurement of renal tissue slices

Tissue slices immediately adjacent to those used in the bioreactor experiments were incubated in 2-dimensional (2D) culture in media containing 25 mM [3-¹³C]pyruvate. The rate of lactate efflux from the tissue slices was evaluated by sampling the medium every 60–120 minutes over 8 hours. The lactate in the medium was measured by ¹H MR spectroscopy in an 800 MHz Bruker DRX spectrometer (Billerica, MA, USA) equipped with a cryo-cooled 5-mm triple-axis heteronuclear probe. The J-coupled ¹³C satellite resonance was quantified using ACD/Labs software as described below.

Data analysis

The MR data was processed and analyzed using ACD/Labs software (Toronto, Canada). The ¹³C data were processed with minimal line broadening and the dynamic data were summed and expressed as a ratio of the hyperpolarized ¹³C lactate peak area to that of ¹³C pyruvate in order to normalize any differences in polarization across experiments. All data are represented as mean ± standard deviation. Two-tailed Student’s t-test was used to assess the difference between groups.

Results

Bioenergetics and viability of renal tissue slices in the MR-compatible 3D tissue culture bioreactor

In a prior study, we demonstrated that the MR-compatible 3D bioreactor maintains tissue viability and provides reproducible HP ¹³C MR data [22]. In this study, it allowed the metabolic evaluation of 60–90 mg of living tissue and had excellent B₀ field homogeneity (average water line width at half maximum was 12.2 ± 0.68 Hz). ³¹P MR spectroscopy was employed to monitor changes in renal tissue slice bioenergetics during the bioreactor studies. Figure 1A shows a representative ³¹P spectrum of ccRCC tissue slices. NMR signals for the nucleoside triphosphates (NTPs: γNTP, αNTP, and βNTP), nicotinamide adenine dinucleotide/uridine diphosphates (NAD/UDP), phosphocholine (PC), inorganic phosphate (P_i), and glycerol phosphocholine (GPC) were readily visible. The βNTP content was unchanged following the injection of HP ¹³C pyruvate, indicating maintenance of tissue bioenergetics during the course of hyperpolarized experiments.

Figure 1.

Bioenergetics of renal tissue slices. A) ³¹P spectrum of tissue slices (~80mg) from a grade 2 ccRCC. The inset shows the maintenance of tissue viability with unchanged βNTP concentration of tissue slices continuously perfused in the bioreactor for over 24 hours. B) Bar graph of the varying levels of phospholipids in renal tissue slices (n=10 for normal renal parenchymal tissue, n=10 for ccRCCs, and n=3 for benign renal tumors). PC concentration in the benign renal tumors is significantly higher than both the normal renal parenchyma (p=0.019) and ccRCC (p=0.008) tissues. GPC, on the other hand, was significantly higher in both benign renal tumors and ccRCCs compared to normal renal parenchyma tissue (p=0.027 and 0.003, respectively). White bars=normal renal parenchymal tissue, gray bars=benign renal tumors, and black bars=ccRCCs, with standard deviation error bars.

Figure 1B shows the varying levels of phospholipids in the renal tissue slices. Interestingly, the PC level in the benign renal tumors was significantly higher than levels in either the normal renal parenchyma (p=0.019) or ccRCC (p=0.008) tissues. This finding is similar to that from a prior ¹H high-resolution study of renal tissue extracts [27]. It suggests that, while PC has been used as a biomarker of proliferation and aggressiveness in other types of cancer [28], it has limited value in stratifying renal tumor aggressiveness. PC is converted from choline by the enzyme choline kinase-alpha (CHKA) in the phosphatidylcholine synthesis (Kennedy) pathway. A prior study reported that functional interaction between CHKA, epidermal growth factor receptor (EGFR) and c-Src is required for cell proliferation [29]. Such functional interactions may explain the lack of direct correlation between the PC level and renal tumor aggressiveness in our study. GPC, on the other hand, was significantly higher in both benign renal tumors and ccRCCs compared to normal renal parenchyma tissue (p=0.027 and 0.003, respectively). While GPC is an osmolyte in the renal medulla, it is also involved in cell membrane recycling [30]. The biological basis of elevated GPC levels in the renal tumor tissues requires further investigation.

Hyperpolarized ¹³C pyruvate metabolism of renal tissue slices in the 3D MR-compatible bioreactor

Figure 2A illustrates the scheme of ¹³C-labeled carbon flux used to detect [1-¹³C]pyruvate metabolism during the HP MR experiment. After injection of hyperpolarized [1-¹³C]pyruvate into the bioreactor, the ¹³C lactate level in the renal tissue slices was assessed in real time. The ¹³C lactate spectrum had excellent signal to noise ratio (SNR) of 15 ± 2 (Figure 2B). The benign renal tumors and ccRCCs showed 2.7-fold and 1.7-fold higher hyperpolarized ¹³C lactate levels (Figure 2C), consistent with increased aerobic glycolysis, when compared to normal renal parenchymal tissues (p=0.023 and 0.017, respectively). However, the observed ¹³C lactate level was 59% lower in ccRCCs than in benign renal tumors. Prior studies of human RCC cells in a similar continuous perfusion system showed that rapidly exported ¹³C lactate quickly flows out of the MR-sensitive volume without contributing to the measured hyperpolarized ¹³C lactate signal [26]. Therefore, we hypothesized that the apparent lower ¹³C lactate level in ccRCC compared to benign renal tumors may be a result of rapid lactate efflux in ccRCCs, and tested this hypothesis in the lactate efflux measurements described below.

Figure 2.

Hyperpolarized [1-¹³C]pyruvate metabolism of renal tissue slices. A) Schematic illustrating the metabolism of hyperpolarized [1-¹³C]pyruvate to [1-¹³C] lactate and [1-¹³C] alanine, catalyzed by lactate dehydrogenase (LDH) and alanine aminotransaminase (ALT), respectively. ¹³C pyruvate is transported intracellularly via monocarboxylate transporter 1 (MCT1), and ¹³C lactate is exported out of the cells via monocarboxylate transporter 4 (MCT4). B) Representative hyperpolarized ¹³C spectrum of grade 2 ccRCC tissue slices. Inset shows the lactate kinetics over 5 minutes. C) Bar graphs of normalized hyperpolarized [1-¹³C] lactate and [1-¹³C] alanine to the injected pyruvate in the tissue slices. Benign renal tumors and ccRCCs show 2.7 fold and 1.7 fold higher hyperpolarized ¹³C lactate levels, consistent with increased aerobic glycolysis, when compared to normal renal parenchymal tissues. The observed ¹³C lactate level is 59% lower in ccRCCs than benign renal tumors. White bars = normal renal tissue, gray bars = benign renal tumors and black bars=ccRCC, with standard deviation error bars.

HP ¹³C alanine was consistently detected in the normal renal tissues with a SNR of at least 3. Alanine was not detectable in the benign renal tumor tissues, and it was occasionally observed (in ~30% of the cases) at low levels in the ccRCC tissues. The low alanine level observed in the RCC tissues is consistent with a prior study by our group that demonstrated lower alanine levels in immortalized human RCC cells compared to normal renal tubular cells [26].

Tissue analysis confirms that ccRCCs have higher lactate production and efflux than benign renal tumors

To test the hypothesis that ccRCCs have higher lactate production and efflux than benign renal tumors, we assayed mRNA expression and enzymatic activity of LDH, and mRNA expression of MCT1 and MCT4, in the tissue slices. The LDHA gene encodes the M subunits of LDH, which catalyzes the conversion between pyruvate and lactate. MCT1 mediates pyruvate transport into cells, and MCT4 mediates efflux of lactate out of cells [31,32]. Mean LDHA mRNA expression was significantly higher in ccRCCs (171 ± 106) compared to either normal renal tissues (41 ± 25) or benign tumors (50 ± 36) (p=0.001 and 0.016, respectively) (Figure 3A). Correspondingly LDH activity was significantly higher in ccRCC compared to normal renal tissues (p=0.020) and benign tumors (p=0.030), by 2.4- and 1.7-fold, respectively (Figure 3A). Similarly, mRNA expression of MCT1 (figure 3B) was significantly higher in ccRCCs compared to normal renal tissues (3-fold higher, p=0.006) and benign tumors (8-fold higher, p=0.002). The higher expression of LDHA and MCT1 in ccRCCs compared to benign renal tumors is consistent with a higher level of glycolysis and lactate production in ccRCCs.

Figure 3.

LDHA expression and LDH activity, and MCT1 expression in renal tissue slices. A) Mean LDHA mRNA expression is higher in ccRCCs compared to either normal renal tissues or benign tumors (p=0.001 and 0.016, respectively), and the LDH activity was also correspondingly higher in ccRCC than normal renal tissues (p=0.020) and benign tumors (p=0.030). B) MCT1 expression is significantly higher in ccRCCs than both normal renal tissues (p=0.006) and benign tumors (p=0.002). The sample size of the tissues used for mRNA analyses is as follows: n=11 for normal renal tissue, n=3 for benign renal tumors, and n=7 for ccRCCs. White bars=normal renal tissue, gray bars=benign renal tumors, and black bars=ccRCC, with standard deviation error bars.

MCT4 mRNA expression in the ccRCCs was significantly higher than in normal renal tissues (4-fold higher, p=0.021) and benign tumors (11-fold higher, p=0.045) (Figure 4B). Corresponding immunohistochemical staining also showed progressively increased MCT4 staining from normal renal tissues to ccRCCs (Figure 4A). These data suggest that ccRCCs have a high rate of lactate efflux. To further verify that the higher MCT4 expression in ccRCCs resulted in increased lactate efflux, we quantified the rate of lactate efflux in the tissue slices in a 2D culture by labeling with [3-¹³C]pyruvate (Figure 4C). The incubating medium was sampled intermittently for up to 8 hours and the [3-¹³C]lactate was measured using high-resolution NMR spectroscopy. The normal renal tissues and benign tumors had similar lactate efflux rates of 1.83 ± 1.98 nmols/min and 2.32 ± 0.89 nmols/min, respectively, while ccRCCs had a significantly higher efflux rate of 5.04 ± 1.82 nmols/min (p=0.013 and 0.002, respectively). Taken together, these observations support the hypothesis that ccRCCs have the highest lactate production and efflux compared to benign renal tumors and normal renal tissues. The rapid lactate efflux out of the cells is the dominant factor resulting in the observed apparent lower HP ¹³C lactate level in ccRCCs than benign renal tumors.

Figure 4.

Renal tissue MCT4 expression and lactate efflux. A) Representative images of MCT4 immunohistochemical staining show increased MCT4 staining (brown staining) in ccRCCs compared to benign renal tumors and normal renal parenchymal tissues. B) ccRCCs show the highest MCT4 mRNA expression compared to normal renal parenchymal tissues (p=0.021), and benign renal tumors (p=0.045). C) ccRCCs show the highest rate of lactate efflux compared to normal renal parenchymal tissues (p=0.013), and benign renal tumors (p=0.002). N=11 for normal renal tissue, n=3 for benign renal tumors, and n=7 for ccRCCs. White bars = normal renal tissue, gray bars= benign renal tumors and black bars=ccRCC, with standard deviation error bars.

Discussion and Conclusion

An unmet need in the management of patients with localized renal tumors is the lack of imaging biomarkers that can reliably discriminate benign tumors from RCCs. In this study, we investigated pyruvate metabolism in live patient-derived renal tumor tissues using hyperpolarized [1-¹³C]pyruvate, a HP ¹³C MR probe that has been used in patient studies [33]. We showed that high lactate production and rapid lactate efflux are characteristic features of clear cell RCC, which comprise the majority of RCCs, and that this metabolic feature may be used to differentiate cancers from benign renal tumors.

Increasing evidence has shown that RCCs are strongly linked to abnormal metabolism [9,10,34]. In particular, increased glycolysis with lactate production is a dominant metabolic feature of many RCCs. Lactate is exported out of the cells, predominantly mediated by MCT4, a proton-coupled lactate transporter. Rapid lactate export plays a key role in maintaining high levels of lactate production, acidifying the tumor interstitium and promoting invasion and metastasis, all key features of cancers [35,36]. The potential importance of MCT4 in RCCs was suggested by a recent study which showed that MCT4 protein expression in clear cell RCCs was associated with poorer relapse-free survival, and correlated with Fuhrman nuclear grade [15]. In our current work, we showed that live patient–derived clear cell RCC tissues have more rapid lactate efflux, as a result of high MCT4 expression, compared to benign renal tumors. Such differential expression and the resultant lactate efflux rate may be explored noninvasively using HP ¹³C MR. In our current pre-clinical study which utilized an ex vivo system, the higher lactate export in RCCs compared to benign tumors was inferred from a combination of HP ¹³C MR and steady-state labeling experiments. However, it is possible to discriminate the local environment of HP metabolites using diffusion-weighted HP ¹³C MR in vivo [37]. Our findings provide rationale for such in vivo studies, and work is ongoing to utilize in vivo diffusion-weighted HP ¹³C MR to directly interrogate the relative amount of intracellular versus extracellular lactate. Additionally, in vivo HP imaging will also permit the assessment of total lactate as a marker of lactate production in renal tumors. During the time frame of the HP studies, the lactate exported out of the renal tumor cells is likely to remain within the tumor interstitium, and the combined intra- and extracellular lactate can be measured. The lactate production, in addition to the rate of lactate efflux, can provide complementary biomarkers of renal tumor aggressiveness.

The development of novel imaging markers of RCC presence and aggressiveness has been impeded by the lack of robust models that recapitulate human disease. Available preclinical models are predominantly based on immortalized aggressive RCC cells either grown in culture or implanted in animals. We previously studied pyruvate metabolism in immortalized RCC cells [26], but were not able to investigate the metabolism of benign renal tumors as there are no existing preclinical models of benign human renal tumors. Immortalized RCC cells also have unusually high proliferation indices compared to patient-derived renal tumor tissues, which may be reflected in the observed metabolism. Furthermore, immortalized cell models do not capture the complex tumor cell-matrix interactions which occur in human renal tumors, and which are likely important for tumor metabolism. To overcome these difficulties, we utilized in this study a patient-derived renal tumor slice model for the metabolic assessment of intact living human tissues. The novel MR-compatible micro-engineered bioreactor permits evaluation of living tissue metabolism in a physiological environment, and has been previously validated by our group in prostate cancer studies [22]. The combination of primary human renal tumor tissue slices and MR-compatible bioreactor provides a unique and realistic model for HP ¹³C biomarker discovery in renal tumors prior to patient studies.

The main limitation of our study is the small number of benign renal tumors. Benign tumors tend to be smaller in size, and limited tissues were available for this research study without potentially compromising the tissues required for clinical diagnosis. Nonetheless, we have shown a significant difference in both the hyperpolarized ¹³C data and the tissue correlative findings between benign tumors and RCCs. The results of the study provide motivation for future clinical studies of HP ¹³C pyruvate MR in patients with renal tumors. Another limitation of our study is that we have only included clear cell RCCs, and not other subtypes of RCCs. This in part reflects the fact that ccRCCs comprise the majority of RCCs (70–80%), and we did not obtain sufficient numbers of other subtypes of RCCs to include in our analysis. Along the same line, the majority of the ccRCC tissues obtained were grade 1 or 2. Accordingly, there was an insufficient number of higher grade (grade 3 or 4) ccRCCs to perform separate analysis based on grade. Future studies are warranted to assess any potential grade-dependent findings. Development of imaging markers that can reliably differentiate low from high grade RCCs is of great clinical interest given the increasing recognition that low grade indolent RCCs may be treated conservatively via active surveillance rather than surgical resection [38]. Another limitation is that the renal tumor tissue slices used in the 2D culture experiments were not the same ones used in the 3D bioreactor experiments. Although renal tumors do have intra-tumoral heterogeneity, the tumor tissue slices were from the same core, and were immediately adjacent to each other. Therefore, we believe any potential heterogeneity is not likely to affect the overall results.

Notwithstanding these limitations, we showed that high lactate production and rapid lactate efflux are dominant features of ccRCCs. These features can be explored to noninvasively differentiate cancers from benign renal tumors using hyperpolarized ¹³C pyruvate MR, especially when combined with diffusion weighting. These initial findings provide strong motivation for developing hyperpolarized ¹³C MR for clinical evaluation of renal tumors. Notably, the safety and feasibility of HP ¹³C pyruvate has already been demonstrated in the phase I clinical trial in prostate cancer patients [33], which opens doors for potential clinical translation of this technology to other diseases. Additionally, work is ongoing to develop multi-channel ¹³C MR coil to provide optimal signal reception in abdominal organs for clinical studies. These technical advances will facilitate the translation of this emerging molecular imaging tool for assessment of renal tumors, a disease of increasing frequency, with the ultimate goal of guiding treatment selection.

Abbreviations used

2D

2-dimensional

3D

3-dimensional

HP

hyperpolarized

LDH

lactate dehydrogenase

MCT1

monocarboxylate 1

MCT4

monocarboxylate 4

MR

magnetic resonance

MRS

magnetic resonance spectroscopy

DNP

dynamic nuclear polarization

RCC

renal cell carcinoma

ccRCC

clear cell renal cell carcinoma

NTP

nucleoside triose phosphate

MCT

monocarboxylate transporter

LDH

lactate dehydrogenase

TCA

tricarboxylic acid

H & E

hemotoxylin and eosin

mRNA

messenger ribonucleic acid

cDNA

complimentary deoxyribonucleic acid

qRT-PCR

quantitative reverse transcription polymerase chain reaction

NADH

nicotinamide adenine dinucleotide

PC

phosphocholine

Pi

inorganic phosphate

GPC

glycerol phosphocholine

CHKA

choline kinase-alphs

EGFR

epidermal growth factor receptor

SNR

signal to noise ratio