1. Introduction
Glioma or glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, with a median survival of just over 12 months and with limited effective therapy [
1]. The astrocytoma is the most aggressive form of GBM, as well as the most common. The malignant transformation of gliomas is associated with alterations in the metabolism of glucose, amino acid, and fatty acid [
2]. Understanding the metabolic variations among glioma and normal brain tissue or between different grades of glioma might provide an insight into their malignant behavior and could offer other opportunities toward therapeutic targets. Recently targeting LDH/lactate axis became a promising approach for cancer therapy [
3,
4,
5]. Lactate is produced as a result of lactate dehydrogenase (LDH) activity and was considered as a waste product of glycolysis. However, lactate plays important roles in brain energetics [
6]. LDH is a tetrameric enzyme composed of two protein subunits, forming the protein complex approximately 135 kDa [
7]. The tetramer can assemble as five separate isozymes by forming all combinations of the M (muscle) form (product of the
LDH-A gene) or the H (heart) form (product of the
LDH-B gene) producing: M
4 (A
4 = LDH
5), M
3H
1 (A
3B
1 = LDH
4), M
2H
2 (A
2B
2 = LDH
3), M
1H
3 (A
1B
3 = LDH
2), and H
4 (B
4 = LDH
1) [
8]. The expression pattern of LDH-A in GBM analyses have shown the differential mRNA expression of LDH-A between GBM tissues and normal brain tissues. The expression of LDH-A was significantly increased in GBM tissues compared with corresponding normal brain tissues, based on analyses using different databases, and indicated a deregulated expression of LDH-A in GBM [
9]. The role and impact of LDH-A expression in different cancer types has been well-explored, whereas the effect of LDH-B expression and its association with LDH-A expression is less well-understood. It is known that the LDH-B promoter is silenced in prostate, gastric, and colon cancers through the hypermethylation mechanism [
10,
11] and required for the growth of KRAS-dependent lung adenocarcinomas [
12].
LDH catalyzes the coordinated interconversion of pyruvate and lactate as well as NADH and NAD+. The properties of LDH isozymes regarding substrate affinity were investigated in in vitro experiments. M-dominated isozymes (
LDH-A, LDH-5) have 3.5–7 times higher K
m-values for pyruvate and lactate than the H-dominated forms (
LDH-B, LDH-1). LDH-1 is inhibited by pyruvate at concentrations above ~0.2 mM, while LDH-5 is minimally affected by pyruvate at concentrations as high as 5 mM [
13]. The LDH-1 isozyme is inhibited by lactate above 20–40 mM, while the LDH-5 isozyme is less inhibited by high lactate levels, pointing to functional differences between LDH-1 and LDH-5 isozymes in cellular metabolism. In the brain, astrocytes have high glycolytic metabolism and a greater proportion of the M-type LDH isozyme (LDH-5), whereas neurons have a high oxidative metabolism and a greater proportion of the H-type LDH isozyme (LDH-1) [
14].
Therefore, we decided to investigate and compare the impact of LDH-A depletion (both LDH-A shRNA knockdown (KD) and treatment with a specific LDH-A/B inhibitor (GNE-R-140)) on the expression of LDH-B in different murine glioma cell lines and corresponding i.c. tumors. We show that control intracranial GL261 tumors shift from an LDH-A (LDH4 dominant) to an LDH-B (LDH1 dominant) pattern and phenotype following LDH-A KD and treatment with GNE-R-140. These changes in the LDH isoenzyme profile and changes in tumor phenotype were not observed in CT2A or ALTS1C1 NC and LDH-A KD tumors. The level of LDH-A expression and its interplay with LDH-B can lead to metabolic changes and complex interactions between tumor cells and their environment. These interactions need to be carefully assessed, since the inhibition of glycolysis in tumor cells may lead to the activation of other metabolic pathways (e.g., lipid, fatty acid, oxidative) and to phenotypic changes, including increased tumor aggressiveness.
2. Materials and Methods
2.1. Cells and Culture Conditions
The GL261 murine glioblastoma cell line was originally obtained from NCI depository. The ALTS1C1 murine glioblastoma cell line was kindly provided by Dr. Chiang (Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Taiwan) and the CT2A murine glioblastoma cell line was kindly provided by Dr. Seyfried (Biology Department, Boston College, Boston, MA, USA). These cell lines were cultured in DMEM media supplemented with 25 mM of glucose, 10% FCS, 4 mM glutamine, and penicillin/streptomycin. LDH-A KD (knock-down) and NC (control) cells, derived from each murine glioblastoma cell, were grown in the media described above containing 2.5 mg/L of puromycin.
2.2. Generation of LDH-A Knockdown and Control Cell Lines
GL261, CT2A, and ALTS1C1 cells were transfected with SureSilencing shRNA plasmids (QIAGEN, Frederick, MD, USA) to specifically knock down expression of the mouse
LDH-A gene as we described previously [
15,
16,
17,
18,
19]. Stably transduced clones (KD cell lines) were developed, along with a control (NC) cell line bearing a scrambled shRNA. Based on our previous experience, we decided to use the most effective shRNA (shRNA-2) from the set of 4 shRNAs to develop LDH-A KD in murine glioma cells. Our previous experience in other cell lines determined that shRNA-2 resulted in the best
LDH-A knockdown function in murine cells. Although shRNA-3
LDH-A knockdown was less effective, the phenotypic changes in cells and tumors were comparable to that obtained with shRNA-2 [
15,
18]. The transfection of GL261 cancer cells with shRNA-2 resulted in a significant knockdown effect for LDH-A (approximately 30% of that in wild type cells), while bulk CT2A and ALTS1C1 cells transfected with shRNA-2 had a less profound LDH-A knockdown (40–60%) detected by mRNA, proteins levels. To enrich the level of LDH-A knockdown we used a subcloning strategy for CT2A and ALTS1C1 cell lines, while LDH-A GL261 knockdown cells were used as a bulk [
19].
2.3. Western Blotting
Western blotting for protein expression was performed as described previously [
15,
16]. RIPA Buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (Thermo Scientific, Waltham, MA, USA) and protease and phosphatase inhibitor cocktail (1:100, Thermo Scientific Halt Protease and Phosphatase Inhibitor Single-Use Cocktail) was used to lyse cell pellets. Bicinchoninic acid assay (BCA Protein Assay Kit, Thermo Scientific) was performed to assess protein concentrations. 10–30 µg of proteins were separated by electrophoresis using a NuPAGE 4–12% Bis-Tris Gradient Gel or NuPAGE 10% Bis-Tris Gradient Gel (Invitrogen, Waltham, MA, USA) and transferred to an Immuno-Blot PVDF Membrane (BioRad, Hercules, CA, USA). Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline—Tween20 buffer and immunoblotted with anti-LDH-A antibody (Cell Signaling, #2012S) at a 1:1000 dilution; LDH-B (Proteintech #14824-1-AP) at a dilution 1:2000 and antiactin antibody (Sigma life science, #A2103) at a 1:5000 dilution. Bound primary antibodies were visualized with Eu-labeled antibody using ScanLater Western Blot Assay kit and SpectraMax ID5 (Molecular Devices, San Jose, CA, USA).
2.4. LDH Enzyme Activity and Lactate-Glo Assay
Total LDH enzyme activity was assessed using the Cytotoxicity Detection Kit PLUS (LDH) (Roche Diagnostics). 10,000 cells were plated in 96-well plates and incubated (37 °C, 5% CO2, humidified incubator) for 1 h. LDH enzyme activity from lysed cells was measured using absorbance assessed at 490 nm using Spectramax ID5 (Molecular Devices, USA).
For other biochemical assays, 20,000 or 200,000 cells were seeded in 96-well or 6-well plates correspondingly and incubated (37 °C, 5% CO2, humidified incubator) for 2–4 h for their attachment. After the attachment, the media was changed to DMEM media containing 0 or 5 mM glucose, 0 or 2 mM pyruvate, and 0 or 10 mM Na-lactate. After the 24 h incubation, media was aspirated, and cells were washed with PBS and collected by centrifugation. Lactate was extracted by using 50 mM Tris (pH-7.4) solution with following 0.6 N HCl. In order to measure the intracellular lactate level from cells we used the Lactate-Glo kit (Promega, Madison, WI, USA).
2.5. LDH Zymography
LDH zymography was used to detect tissue-specific differences in LDH isoenzymes. We were able to observe 5 isozyme bands in the active state as previously described [
20]. Based on the different electrophoretic motilities of the isoenzymes, they can be identified as LDH1 (B4 or H4), LDH2 (B3A1 or H3M1), LDH3 (B2A2 or H2M2), LDH4 (B1A3 or H1M3), and LDH5 (A4 or M4). We used a buffer at pH 8.6 for the best separation of the five LDH isoenzymes [
13,
14].
2.6. Metabolic Extracellular Flux Analysis
Glycolytic and mitochondrial activity of cells was measured using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Seahorse XF Technology, Billerica, MA, USA). Cells were seeded at 25,000–30,000 cells per well with standard growth media using Seahorse XF96 96-well plates; the cells were allowed to attach over 6 h at 37 °C in an incubator (95% air/5% CO2). Total proton efflux rate (PER) was measured by plotting proton efflux as a function of time (pmol/min). Oxygen consumption rate (OCR) was measured as the change in oxygen content of the media as a function of time (pmol/min). Data was normalized to the number of cells in each well. Data from 3 independent experiments were analyzed using Seahorse Wave Desktop Software and compiled together using GraphPad Prism 7.
2.7. mRNA Gene Expression Profile Analysis
LDH-A knockdown was verified by two approaches. First, a quantitative digital droplet PCR (ddPCR) was performed for LDH-A and LDH-B by the Genomics Core Laboratory at MSKCC. For RNA purification, cells were grown for 48 h (exponential growth phase). RNA was isolated using the RNeasy total RNA isolation kit (QIAGEN, catalog No. 74104), following the manufacturer’s protocol. Second, RNA sequencing was performed after RNA extraction, library preparations, and RNA-sequencing reactions conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). Total RNA was extracted from frozen cell-pellet samples using Qiagen RNeasy Plus Universal mini kit following manufacturer’s instructions (Qiagen, Hilden, Germany). RNA Sample QC, DNase treatment, library preparations, sequencing reactions, and read mapping and alignment were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA) and are described in the
Supplemental Information. After extraction of gene hit counts, the gene hit counts table was used for downstreaming differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate
p-values and Log2 fold changes. Genes with adjusted
p-values < 0.05 and absolute log2 fold changes > 1 were called as differentially expressed genes for each comparison. Significantly differentially expressed genes were used for Gene Set Enrichment Analysis (GSEA) using the fgsea package in the R statistical software (v4.0). Gene sets (pathways) used were downloaded from the Broad Molecular Signature Database and only pathways from Gene Ontology (GO), Reactome, or KEGG were used.
2.8. Proliferation Assay In Vitro
Tumor cells were cultured in their respective culture media. On day 0, 200,000 or 1,000,000 cells were seeded in 6-well plates in 3 mL DMEM media (25 mM glucose, 4 mM glutamine, 10% FCS and penicillin/streptomycin). The proliferation of cells was tracked over 72–96 h. At each timepoint, cells were collected via trypsinization and counted using a Countess Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA).
2.9. Animal Models
The animal protocol was approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center (protocol number: 08-07-011; Approval Date for data presented: 19 September 2014). To develop an orthotopic i.c. tumor model, 200,000 cells suspended in 2 μL of PBS were injected using a 30-gauge needle syringe into the right frontal cortex [stereotactic coordinates: bregma +1.7 mm (anterior), lateral −0.5 mm (right), and at a depth of 2.5 mm] of immunocompetent C57BL/6 female (6–7 weeks old) mice (Charles River Laboratories, Wilmington, MA, USA) and immunocompromised Hsd: Athymic Nude Foxn/nu female (6–7 weeks old) (Envigo, Indianapolis, IN, USA). Animal health was monitored by weighing mice at regular intervals. Intracranial tumor volume (V) was calculated from MRI measurements (described later). Kaplan–Meier survival curves were generated, and mean survival time calculated in analysis of Prism GraphPad. The blood samples were collected by retro-orbital venipuncture using standard heparinized microhematocrit capillary tubes. Samples were used to assess LDH enzyme activity in red blood cells. Mice bearing i.c. gliomas were euthanized for H&E, IHC staining when they became lethargic and were noted to have weight loss.
2.10. Alzet Pump
A subcutaneous Alzet pump (Model 1007D, Durect Corporation, Cupertino, CA, USA) with an infusion rate 0.5 µL/h and an infusion duration of 7 days was implanted on the back mice, following the MSKCC IACUC protocol. Two groups of mice (treated and control) were established, and both groups were implanted with the Alzet pumps. In the treated group of mice, the Alzet pumps were loaded with 18 mg GNE-R-140 in 100 µL of 100% DMSO, delivering 100 mg/kg/day to ensure a blood concentration of 10 mM over the duration of the experiment. In the control group, the Alzet pumps were loaded with DMSO only.
2.11. MR Imaging
Intracranial tumors were imaged using T2-weighted MR imaging; 3 mice were randomly picked up from each group. MRI was performed on a Bruker AV NEO 9.4T scanner equipped with a high-power ID 115 mm gradient capable of a maximum strength of 640 mT/m. An ID 40 mm Bruker quadrature volume coil was used for both RF excitation and detection. The mouse was anesthetized by 2% isoflurane in air, and mouse breathing was monitored by a small-animal physiological monitoring unit. Tumor volume (V) was calculated by V = (π/6) × long diameter × short diameter × height, which were measured based on MR imaging.
2.12. Histological Staining and Image Analysis
Excised tumors were processed with 4% paraformaldehyde followed by paraffin embedding for H&E histology and immunohistochemistry (IHC) studies. IHC staining was performed by the Molecular Cytology Core Facility (MCCF) of MSKCC, using a Discovery XT processor (Ventana Medical Systems, Roche—AZ), in accordance with their established protocols. After 32 min of heat and CC1 (Cell Conditioning 1, Ventana cat#950–500) retrieval, the tissue sections were blocked first for 30 min in Background Blocking reagent (Innovex, catalog#: NB306). The incubation with the primary antibody was performed for 6 h, followed by 60 min incubation with biotinylated goat antirabbit IgG (Vector labs, cat#:PK6101) in 5.75 μg/mL. Blocker D, Streptavidin-HRP and DAB detection kit (Ventana Medical Systems) were used according to the manufacturer instructions. Primary antibodies for LDH-A were obtained from Cell Signaling (#2012), and for LDH-B from Proteintech (#14824-1-AP). The optimal concentration of the primary antibody was determined to be 0.2 µg/mL for LDH-A and 2.0 µg/mL for LDH-B. The slides were counterstained with hematoxylin and cover-slipped with Permount (Fisher Scientific). Quantification of the LDH-A and LDH-B stained sections was performed using FIJI and trainable Weka Segmentation (Image J segmentation plugin) [
17,
18].
2.13. Statistical Analysis
Results are presented as mean ± standard error. Statistical significance was determined by a two-tailed Student t-test. A p-value of <0.05 was considered significant. All data presented were analyzed using GraphPad Prism (version 7.0; GraphPad Software) and are presented as mean +/− SEM/SD.
4. Discussion
Three intracranial murine gliomas were studied and we compared the effects of genetic-shRNA LDH-A knockdown [
19,
25,
26] and LDH drug-targeted inhibition [
19,
25,
26] on tumor-cell metabolism, nutrient dependence, tumor growth, and animal survival time. We show that murine glioma cells (i) can engage different metabolic pathways to support their proliferation; (ii) have a varied dependence on specific nutrients and nutrient combinations (glucose, pyruvate, and lactate); and (iii) have differences with respect to lactate production vs. consumption. We also compared the responses to genetic-shRNA LDH-A knockdown vs. LDH drug-targeted inhibition [
19,
25,
26]. We show that LDH drug-targeted inhibition with GNE-R-140 reproduces the effects of LDH-A shRNA knockdown on tumor-cell metabolism, tumor growth, and animal survival.
The regulation and function of LDH-A and lactate in human gliomas and other solid tumors has gained increased attention over the past decade [
27]. Serum LDH and lactate are well-known markers of aggressive systemic tumors, and it was recently shown that serum lactate levels are also associated with the grade of gliomas [
28]. It is also known that MiR-200b is a regulator of tumor progression and metabolism targeting LDH-A in human malignant gliomas [
29], and that upregulation of KLHDC8A (Kelch domain-containing 8A) is induced by lactate and contributes to the proliferation, migration, and apoptosis of human glioma cells [
20]. Furthermore, LDH-A silencing occurs in IDH mutant gliomas, and likely contributes to their characteristically slower progression [
30]. These studies (and others) demonstrate the important role of LDH-A and lactate in human gliomas, and suggest that LDH-A may be a potential therapeutic target [
27]. However, our studies show that not all murine gliomas are alike, and that the level of LDH-A expression and its interplay with LDH-B can lead to changes in the activity of different metabolic pathways and complex interactions between tumor cells and their environment. These changes and interactions need to be carefully assessed, since the inhibition of glycolysis in tumor cells may lead to undesirable metabolic and phenotypic effects, including increased tumor aggressiveness.
The isoenzyme profiles of i.c. GL261 NC and LDH-A KD tumors were clearly different, whereas the isoenzyme profiles of i.c. CT2A NC and LDH-A KD were similar. Although the LDH-A and LDH-B immunohistochemistry and a Weka analysis were generally consistent with the in vitro metabolic analyses and the LDH zymography, our analyses clearly demonstrate an intratumor variability in the spatial and level of LDH-A and LDH-B expression in these tumors. GL261 LDH-A KD i.c. tumors grew more rapidly and resulted in shorter animal survival times than control i.c. GL261 NC tumors. In contrast, LDH-A KD had little or no effect on the growth of CT2A and ALTS1C1 i.c. tumors. The differences in tumor-cell metabolism, LDH isoenzyme, LDH-A/LDH-B immunohistochemistry, and tumor-growth and animal survival patterns were clearly shown to be related to LDH-A depletion and an associated increase in LDH-B expression. Interestingly, it has been suggested that the sole (or high) expression of LDH-B could identify an important biological marker of glioma cells that is critical for their progression, and it might afford a new target for anticancer drugs [
31].
The metabolic patterns, we observed in GL261 LDHA-depleted cells strongly suggest that they are able to utilize lactate and/or fatty acids to support an active TCA cycle and an increase in oxidative phosphorylation (accompanying manuscript [
17,
18]). Our RNA-seq bioinformatics analysis provides additional evidence that GL261 LDH-A KD cells may have an improved ability to metabolize fatty acids and lipids through enhancement of the PPAR pathway and upregulation of fatty-acid catabolism genes (specifically in GL261 LDH-A depleted cells). The PPAR signaling pathway involves a receptor-activated nuclear transcription factor superfamily that is activated by a diverse number of ligands, and it functions as a transcriptional regulator of many biological processes, including lipid/fatty-acid transport and metabolism, cell proliferation, differentiation, apoptosis, and inflammatory responses, among others. These patterns strongly suggest that GL261 LDH-A depleted cells may be able to utilize lactate and fatty acids to support their TCA cycle and increased oxidative phosphorylation.
These data are consistent with recent observations that show increased mitochondrial biogenesis induced by activation of the CREB-PGC1a pathway, which triggers a metabolic shift and differentiation in glioma cells [
32]. Blocking mitochondrial biogenesis by silencing PGC1a abrogates differentiation; conversely, overexpression of PGC1a elicits differentiation, showing that mitochondrial biogenesis and the metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells [
32]. Interestingly, others have recently shown a novel carbohydrate-metabolism regulation through protein–metabolite interactions [
33]. For example, both ATP accumulated during oxidative phosphorylation and long-chain fatty acetyl-CoAs inhibit LDH-A, but not LDH-B, and long-chain fatty acids caused a loss of pyruvate/lactate interconversion only in cells dependent on LDH-A [
33].
It has also been shown that lactate is a primary circulating TCA substrate in many tissues and tumors [
34], and that mitochondria play a central and multifunctional role in malignant tumor progression [
35]. However, different tumors manage lactate differently; some tumors are lactate producers/excreters and some tumors are lactate consumers/utilizers. Furthermore, within a single heterogenous tumor there may be shuttling of lactate between different cell types [
36]. Some cancer cells cannot utilize lactate [
37], while other tumors can easily use lactate as a fuel, and respond to supplemental lactate with increased proliferation and vascularity [
37]. The detection of labeled lactate in the TME can reflect two processes: (1) exogenous lactate uptake from the circulation or (2) glycolysis-derived lactate (produced by tumor and/or stromal cells), which are dependent on MCTs and LDH, respectively. Tumor perfusion can also be an important factor that influences lactate accumulation from the circulation. If perfusion or MCT activity is limited, then glucose-derived lactate will predominantly accumulate in the tumor, and circulation-derived lactate will be limited. Recently, the infusion [1,2-C
13] glucose in patients bearing triple-negative breast tumors detected more labeled pyruvate and lactate in tumors than in the circulation, indicating the predominance of locally synthesized lactate from glucose in these highly glycolytic breast tumors [
38]. We clearly show that GL261 LDH-A KD cells consume lactate and that 10 mM Na-lactate (in the absence of glucose) supports cell proliferation, whereas this is not the case for GL261 NC and the other cell lines we studied (
Figure S4). These results in GL261 LDH-A KD cells (but not in the other cell lines) are consistent with findings that lactate can serves as a carbon source in tumors [
34,
39], particularly in a nutrient (glucose) deficient TME [
39]. Recently, circulating lactate has been suggested as a “universal fuel” [
34]. In several mouse models of lung and pancreatic cancers, C
13-lactate infusion contributed significantly to TCA cycle metabolites [
34]. Our in vitro nutrient modulation studies support this hypothesis for GL261 LDH-A KD cells.
Our results do not completely correspond to the findings of Rabinowitz et al. [
34,
40], who showed that lactate exchange between the tumor and the circulation is rapid via MCT transporters. Our RNA-Seq data show that MCT1 (
SLC16A1) is lowest in GL261 NC and LDH-A KD cells (accompanying manuscript [
17]), compared to the other glioma cell lines. Furthermore, there was a significant reduction in the expression of the lactate exporter MCT4 (
SCL16A3) only in GL261 LDH-A KD cells. This raises the question as to whether a rapid intra/extracellular exchange of lactate is taking place in these cells and tumors. Furthermore, these findings support our hypothesis that inhibition of glycolysis in LDH-A KD GL261 tumor cells leads to increased fatty-acid catabolism.
Recently, there have been several studies focusing on the targeting of cancer metabolism through genetic knockout (or knockdown) of LDH-A and by using small-molecule inhibitors of LDH-A [
25,
41,
42,
43]. In human colon adenocarcinoma and murine melanoma cells, neither
LDH-A nor
LDH-B knockout strongly reduced lactate secretion, whereas the double knockout (
LDHA/B-DKO) fully suppressed LDH activity and lactate secretion [
25]. These results were reproduced pharmacologically by treating WT cells with the LDHA/B-specific inhibitor GNE-R-140. Pancreatic cell lines that predominantly utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-R-140 with the OXPHOS inhibitor phenformin [
41]. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, whereas inhibitors targeting this pathway prevented resistance. Peptides sequences with high affinity to the β-sheet region of LDH-A (LDH5) were shown to inhibit enzymatic activity; a lead peptide (cGmC9) inhibited LDH-A activity in vitro in the low-micromolar range and was more efficient than GNE-R-140 [
42]. In addition, the coregulation of LDH and the heat shock response with respect to radiation resistance was presented [
43]. It was shown that that inhibition of LDH, either pharmacologically (oxamate or GNE-R-140) or by gene knockout of
LDHA and
LDHB, significantly increases the radiosensitivity in tumor cells by a global impairment of the stress response. The authors suggest that inhibition of the lactate metabolism might provide a promising strategy in the future to improve the clinical outcome of patients with highly aggressive, therapy-resistant tumors.
What is clear from our study and those discussed above is that tumors are not all alike, and that the level of LDH-A expression and the interplay with LDH-B can lead to metabolic changes and complex interactions between tumor cells and their environment. These interactions need to be carefully assessed, since the inhibition of glycolysis in tumor cells may lead to undesirable metabolic and phenotypic changes, including increased tumor aggressiveness.