Background
Most primary brain tumors are classified as gliomas based on the tumor cells' resemblance with glial cell types and immunohistochemical characteristics, particularly the expression of glial fibrillary acidic protein [
1,
2]. However, the cellular origin of gliomas is still controversial [
3,
4] and the term "glioneuronal tumor" refers to gliomas which contain neuronal elements such as neurocytes or ganglion cells. In addition, several neuronal antigens have been detected in glioblastoma multiforme (GBM) [
5‐
7], diffuse astrocytomas [
8,
9], pleomorphic xanthoastrocytomas [
10], oligodendrogliomas [
11] and ependymomas [
12]. Expression of neurofilament protein (NFP) and microtubule-associated proteins in GBM cell lines have been described [
13], and increasing immunoreactivity for the class III β-tubulin has been associated with an ascending gradient of malignancy [
8]. Several studies have explored the relationship between neuronal antigens and the prognosis of gliomas. Varlet et al [
6] reported that a GBM subclass coexpressing glial fibrillary acidic protein and neurofilament protein had a lower recurrence rate at the primary site and a better prognosis. Furthermore, prognostic subclasses of high grade gliomas have been identified, including a proneural group expressing neuronal lineage markers that was associated with longer survival than the other subgroups [
3]. However, one recent study found that the prognostic significance of neuronal marker expression was limited to giant cell GBMs expressing two or more neuronal markers, and that the expression of these markers was associated with shorter survival [
5]. Thus, the role of neuronal markers in human gliomas is still unclear.
In this work, we investigated the expression and regulation of four neuronal markers class III β-tubulin, neurofilament protein (NFP), microtubule-associated protein 2 (MAP2) and neuron-specific enolase (NSE) in GBM cell lines and patient biopsies. Furthermore, we quantified their expression under different culture conditions. Since NSE was consistently upregulated under different stress conditions we performed knock-down experiments using NSE siRNA to see how this impacted on glioma cell behavior. Finally, we correlated immunopositivity for NSE with patient survival using a panel of glioma biopsies.
Methods
Cell culture
The human GBM cell line GaMG was established at the Gade Institute, University of Bergen, Norway [
14]; U87, LN229, A172, U251 were purchased from the American Tissue Culture Collection (ATCC; Manassas, VA, USA) and cultured in DMEM (Sigma, St. Louis, MO) containing 10% fetal bovine serum, supplemented with NEAA, 100 U/ml Pen/Strep, 400 μM L-glutamine, all from Cambrex (Cambrex, East Rutherford, NJ) and in stem cell medium (SCM) consisting of Neurobasal Medium (Invitrogen, Carlsbad, CA) supplemented with 20 μl/ml B27 (Invitrogen), 10 μl/ml Glutamax (Invitrogen), 20 ng/ml EGF (Sigma), 20 ng/ml FGF2 (R&D Systems, Minneapolis, MN) and 100 U/ml Pen/Strep (Cambrex). Cells were also cultured in serum-free starvation medium consisting of DMEM (Sigma) supplemented with NEAA, 100 U/ml Pen/Strep and 400 μM L-glutamine (Cambrex). The medium was changed every 2 days.
Patient biopsies
Patient biopsies were obtained from the Department of Neurosurgery, Haukeland University Hospital, Bergen, Norway. Collection of tumor biopsies and the corresponding clinical data was appoved by the Regional Ethical Committee (REK Vest). Prior to harvesting the biopsies and clinical data, informed and written consent was obtained from each patient that provided tissue. Biopsy spheroids were prepared as previously described [
15], and monolayer cells from spheroids were cultured both in DMEM medium and SCM.
Immunocytochemistry (ICC) and immunohistochemistry (IHC)
Cell suspensions were plated on 12-mm cover slips coated with poly-L-Lysine (50 μg/ml, 2 hr at 37°C). The cells were fixed with 4% PFA for 10 min followed by 4 min permeabilization in 0.5% Triton-X at room temperature, washed 4 times in PBS and then blocked with protein block (PBS with 0.5% BSA) solution for 15 min. Primary antibodies diluted in protein block solution were incubated for 45 min at 37°C or overnight at 4°C. After three washes with PBS, fluorescent conjugated secondary antibodies were applied in combination with protein block solution for 45 min at 37°C. Cover slips were washed four times with PBS, mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA) or Prolong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA) and inspected under a Nikon Eclipse TE2000-E fluorescence microscope (Nikon, Tokyo, Japan).
The primary antibodies were diluted as follows: chicken polyclonal anti-class III β-tubulin, 1:200 (Abcam, Cambridge, UK); mouse monoclonal anti-neurofilament 70 kDa, 1:50 (Chemicon, Tamecula, CA, USA); chicken polyclonal anti-MAP2, 1:100 (Abcam); rabbit polyclonal anti-NSE, 1:100 (Abcam). The secondary antibodies were diluted as follows: goat anti-chicken IgY-TR, 1:800 (Santa Cruz Biotechnology, CA, USA); goat anti-rabbit IgG, 1:800 (Southern Biotech, Birmingham, Alabama, USA); goat anti-mouse IgG1, 1:200 (Southern Biotech).
For IHC, paraffin sections were rehydrated through a procedure of 2 × 5 min in Xylene, 2 × 3 min in 100% EtOH, 2 × 3 min in 96% EtOH and finally in ddH2O for 5 min. Heat induced epitope retrieval (HIER) was done by heating sections to 95°C for 25 min in 10 mM Na-Citrate buffer pH = 6. The sections were blocked with peroxidase block buffer (DAKO, Denmark) for 5 min, washed three times with TBS Tween 0.05% and blocked with protein block (DAKO) for 30 min at room temperature. Then the sections were incubated with the rabbit anti-NSE antibody (Abcam) diluted 1:200 in buffer (25 mM Tris-HCl, 75 mM NaCl, 1% BSA, pH 7.4) overnight at 4°C, washed three times with TBS Tween 0.05% and incubated with HRP labeled polymer anti-rabbit secondary antibody (DAKO) for 40 min at room temperature. After 4 washes, the sections were developed with DAB+ (Dako) for 4 min, counterstained with haematoxylin, dehydrated and mounted with Entellan (Merck KGaA, Darmstadt, Germany). The sections were examined and photographed using a light microscope equipped with a digital camera (DXM1200, Nikon, Japan).
For each slide, ten different microscopic fields were selected and photomicrographs were captured at a magnification of 40×. Immunoreactivity was scored as area fractions calculated as the ratio of immunopositive area to the total area of the microscopic field by an investigator as described before using a morphometry software (NIS-Elements BR 3.2, Nikon, Japan) [
16]. Immunostaining intensity higher than average background was set as pixel threshold for positive staining, which included both nuclear and cytoplasmic stainings. The investigator was blinded to the patient identities, with no prior knowledge of clinical and pathological parameters. Only after the scoring was completed, a clinician retrieved the patient data from the journals at the hospital.
Immunoblots
Cultured cells were washed in PBS 2 × and subsequently homogenized in lysis buffer (20 mM MOPS, 5 mM EDTA, 2 mM EGTA, 30 mM NaF, 0.5% Triton X, 40 mM b-Glycerophosphate, 20 mM Na-Pyrophosphate, 1 mM Na-Orthovanadate, 3 mM Benzamidine, 5 uM Pepstatin, 10 uM Leupeptin, 1 mM PMSF, pH 7.2) by sonication 3 × 2 sec using Sonics Vibra Cell TM (Cole-Parmer Instruments, Vernon Hills, IL). Whole lysate was centrifuged for 30 min at 13,000 rpm and used for the subsequent analysis. Protein (10-20 μg) was added in each well and run on SDS-PAGE using NuPage precast Gels (Invitrogen). After blotting the nitrocellulose membrane for 80 min and subsequent treatment with blocking solution (TBS with 0.1% Tween, 5% milk powder) for 1 hour at room temperature the membrane was incubated overnight at 4°C in blocking solution containing chicken anti-class III β-tubulin (Abcam) diluted 1:25000, chicken anti-MAP2 (Abcam) diluted 1:100000, rabbit anti-NSE (Abcam) diluted 1:1000, mouse anti-NFL (Chemicon) diluted 1:1000 and rabbit anti-GAPDH (Abcam) diluted 1:2000. The primary antibodies were detected using a horseradish peroxidase (HRP)-conjugated rabbit anti-chicken secondary antibody diluted 1:3000 (Abcam), goat anti-rabbit secondary antibody diluted 1:100000 (Beckman Coulter), goat anti-mouse secondary antibody diluted 1:2500 (Santa Cruz Biotechnology). The Western blot was developed using Supersignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL) and detected with Fuji LAS 3000 Imager (Fuji Photo Film, Tokyo, Japan). Densitometric analysis was performed using Multi Gauge V2.3 software (Fuji Photo Film, Tokyo, Japan) and the levels of NSE were normalized to GAPDH levels.
Isolation of total RNA
Total RNA was extracted using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). The cell lines were washed twice in PBS, added RLT buffer and scraped off using a cell scraper. The remaining procedure was performed according to the manufacturer's instructions, including treatment with DNase I (Qiagen GmbH, Hilden, Germany).
Real-time qPCR
250 ng total RNA was reverse transcribed in a total volume of 10 μl using iScriptTM cDNA Synthesis Kit (BioRad Laboratories, Hercules, CA) according to the manufacturer's instructions. The resulting cDNA reaction mix was then diluted 20 times in ddH2O. Real time qPCR was subsequently performed using iQ SYBR Green Supermix (Bio-Rad Laboratories) according to the manufacturer's instructions, using 0.5 μl of the diluted cDNA reaction mix in a total volume of 5 μl. The following parameters were used for the qPCR reaction: initial denaturation at 95°C for 3 min, 45 cycles of 20 sec at 95°C, 20 sec at 58°C and 20 sec at 72°C using Light Cycler 480 (Roche). Amplicon purity and size were verified by melt curve analysis. Primers directed against GAPDH were used as an internal control. Primers were designed using OligoPerfect™ Designer (Invitrogen). Human-specific primers were as follows:
Class III β-tubulin forward 5'-GCGAGATGTACGAAGACGAC-3', reverse 5'-TTTAGACACTGCTGGCTTCG-3'; NFL forward 5'-GAGGCAGCTGAAGAGGAAGA-3', reverse 5'-AAGGAAATGGGGGTTCAATC-3'; NFM forward 5'-AAGGGATCCAGGAAGGAAGA-3', reverse 5'-TGACAACGCCTTTCTCCTCT-3'; NFH forward 5'-GAGGAACACCAAGTGGGAGA-3', reverse 5'-TTCTGGAAGCGAGAAAGGAA-3'; MAP2 forward 5'-CCAATGGATTCCCATACAGG-3', reverse 5'-TCCTTGCAGACACCTCCTCT-3'; NSE forward 5'-CTGATGCTGGAGTTGGATGG-3', reverse 5'-CCATTGATCACGTTGAAGGC-3'; GAPDH forward 5'-GAGTCAACGGATTTGGTCGT-3', reverse 5'-GACAAGCTTCCCGTTCTCAG-3'. Negative controls were performed without reverse transcriptase in the reaction. In those cases, no amplification was found, ruling out contamination of the RNA samples with genomic DNA (data not shown). Fold changes were calculated using the comparative CT (2-ΔΔCT) method.
RNA interference
ENO2 (NSE) silencing siRNA and nonsilencing control siRNA were purchased from Ambion. Two validated silencer select siRNAs targeting ENO2 at exon 7 or 9 were used separately in the experiment. SiRNA duplex (s4685, Ambion, Foster City, CA, USA) with sense senquence: 5'-GGUGCAGAGGUCUACCAUATT-3' and antisense sequence: 5'-UAUGGUAGACCUCUGCACCTA-3' targets exon 7. SiRNA duplex (s4684, Ambion) with sense sequence: 5'-GUGACCAACCCAAAACGUATT-3' and antisense sequence: 5'-UACGUUUUGGGUUGGUCACTG-3' targets exon 9. Cells were transfected with siRNA oligonucleotides by using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol.
Wounding experiment using Electric cell-substrate impedance sensing (ECIS)
In ECIS, the cells are grown on the surface of small and planar gold-film electrodes, then the AC impedance of the cell-covered electrode is measured continuously at a frequency of 64 kHz. Due to the insulating properties of cell membranes, the impedance increases with increasing coverage of the electrode until a confluent layer of cells is established. 40000 A172 glioma cells were seeded out in an 8E1W ECIS array (Applied Biophysics, Troy, NY, USA), each well containing a single 250μM electrode in the middle. Cells in four of the wells were treated with 20nM NSE siRNA, whereas two wells were treated with scrambled siRNA at the same concentration (both from Ambion, Foster City, CA, USA). Upon confluence a high field current with frequency 48 kHz and amplitude 5 was applied for 10 seconds which killed cells overgrowing the electrode, creating "wounds" in the wells devoid of cells. Successful wounding was confirmed with a rapid drop in impedance and absence of cells on the electrode. Migration of surrounding glioma cells into the area overlying the electrode could then be monitored by measuring changes in impedance over time. The migration was compared between control and knockdown groups at the end of each experiment, which was presented as the ratio of impedance before and after the wounding until the end of each experiment. The experiments were performed 3 times.
MTS assays and hypoxia, irradiation and temozolomide (TMZ) treatment
MTS assays (Promega) were performed as previously described [
17]. Briefly A172 and U251 cells were seeded into 96-well plates at a density of 2000 per well and were subsequently treated with NSE or control siRNA. 18 hr after the preparation, the plates were incubated under 0.5% oxygen in a hypoxia chamber (BioSpherix, Lacona, New York, USA) or radiated with 4 Gy or treated with 10 μM (A172) or 50 μM (U251) TMZ (Tocris Bioscience, UK) and cultured in a standard tissue culture incubator with 5% CO
2 in air and 100% relative humidity at 37°C. Each treatment condition was tested in 30 wells, excluding wells at the edge. 5 or 6 days after treatment, medium was removed and fresh medium with MTS substrate (Promega) was added to each well. Following 3 hr of incubation with MTS, the absorbance at 490 nm was measured for each well with a scanning multiwell spectrophotometer (Biochrom Asys UVM 340 Microplate Reader; Biochrom Ltd, UK). The experiments were performed 3 times.
Patient data
The study population consisted of 28 consecutive glioma patients admitted to the Department of Neurosurgery, Haukeland University Hospital, Bergen, Norway, between January 2005 and December 2007. These patients were diagnosed with different glioma types and patient characteristics and treatment parameters are provided in Table
1.
Table 1
Glioma patients listed according to the NSE expression level
1 | GBM | 58 | M | 60 | T |
2 | Oligo (III) | 59 | M | 54 | T+PCV |
3 | Oligoastro(II) | 33 | M | 54 | T |
4 | GBM | 66 | M | 36 | T |
5 | GBM | 58 | M | 60 | T |
6 | GBM | 58 | M | 60 | T |
7 | Astro (II) | 49 | M | 54 | T |
8 | Astro (II) | 41 | M | - | - |
9 | GBM | 42 | M | 60 | T |
10 | GBM | 58 | F | 60 | T |
11 | GBM | 67 | F | 60 | T |
12 | Astro (II) | 33 | M | 54 | T+PCV |
13 | GBM | 38 | M | 90 | T+PCV+GK |
14 | GBM | 22 | F | 60 | T+PCV |
15 | Oligo (III) | 59 | M | 54 | T+PCV |
16 | GBM | 58 | F | 76 | T |
17 | GBM | 67 | M | 39 | - |
18 | GBM | 67 | F | 60 | T |
19 | GBM | 82 | M | 39 | - |
20 | GBM | 55 | M | 60 | T |
21 | GBM | 56 | M | 76 | T |
22 | GBM | 74 | F | 60 | - |
23 | GBM | 48 | M | 58 | T |
24 | GBM | 66 | F | 60 | T |
25 | GBM | 71 | F | - | - |
26 | GBM | 58 | M | 60 | T+GL |
27 | GBM | 64 | M | 60 | T |
28 | Oligo (II) | 49 | M | 54 | T |
Statistical analysis
Student's t-test was performed using a 2-tailed distribution analysis. The duration of survival for patients with gliomas was measured from the time of diagnosis to the time of death or last follow-up. Survival data were analyzed using the log-rank test.
Discussion
Enolase is one of the glycolytic enzymes, and exists as a dimer of two subunits. There are three kinds of subunits, α-, β- and γ- subunits. NSE is a γγ- isozyme, also called ENO2, and was reportedly only detected in neurons and neuroendocrine cells under physiological conditions [
19,
20]. Glycolysis is considered the main source of energy for cancer cells [
21]. Interestingly, astrocytic tumor cells also stain positively for NSE [
22], suggesting that NSE-negative cells can acquire the ability to produce NSE. This may reflect a form of adaptation to the increased metabolic demands associated with a neoplastic state [
23]. Furthermore, others have reported that NSE has a neurotrophic and neuroprotective effect on neurons in the CNS mediated by specific binding to the neuronal surface [
24]. Thus, NSE may also display non-glycolytic functions, possibly acting as a survival factor. Concordant with previously reported studies [
22,
25], we found that NSE was aberrantly expressed in all the five GBM cell lines and two patient biopsies, and was upregulated both in serum-starvation medium and under hypoxic conditions. Thus, NSE may play a role when glioma cells adjust to a niche dominated by hypoxia and lack of nutrients as tumors outgrow their blood supply. Interestingly, NSE was also upregulated in long-term cultures in SCM.
Moreover, the involvement of NSE in glycolysis and its role as a possible survival factor may also imply that it represent a therapeutic target [
26]. Indeed, knockdown of NSE inhibited the proliferation by 17% in U251, although not significantly (p = 0.08), NSE knockdown also reduced the migration of A172 in the wounding experiments. Furthermore, NSE knockdown significantly reduced the viability of GBM cell lines in hypoxia. Since hypoxia, has been linked to chemo- and radioresistance [
27], overall tumor aggressiveness, as well as upregulation of NSE in our experiments, it also suggests that expression levels of NSE may correlate with prognosis in glioma patients. Notably, overexpression of ENO1, another enolase isozyme, in hepatocellular carcinoma [
28] and head and neck cancer [
29] has been associated with poor clinical outcomes. In our stuy, glioma patients displaying higher NSE expression levels had a significantly shorter survival. Our findings are in accordance with a previous study [
5]. This may result from the effect of NSE upon the response to radiotherapy and chemotherapy, as we found that knockdown of NSE sensitized these GBM cell lines to irradiation and TMZ. It should be emphasized however, that the mean age in the NSE low expression group was lower than in the NSE high expression group (52.5 vs 64.5). Moreover, 4 patients in the NSE high expression group did not receive chemotherapy. As such, our study suffers from the same limitations as other retrospective studies, with a biased selection of patients which influences the results. Therefore, controlled studies will be needed to validate the clinical significance of NSE in a greater number of glioma patients. In fact, the expression and prognostic value of NSE has been investigated in several other tumors, including non small cell lung cancer [
30], breast cancer [
31] and prostate cancer [
32]; however, the expression of NSE in lung cancer and breast cancer is associated with better survival, while NSE staining was associated with shorter survival [
32] or was of no practical value as an independent prognostic indicator in patients with prostate cancer [
33]. Vos et al [
34] also reported there was no prognostic value of serum NSE levels in brain tumor patients.
Due to the cytoplasmic staining of NSE, it is difficult to identify single positive cells in the field. For this reason, we applied the area fraction of immunopositivity method as previously described [
16,
35]. Previously, we have used this method to obtain accurate estimates of immunopositivity with cytoplasmic stainings [
16].
In our study, both the hmw-MAP2 and lmw-MAP2 could be detected in GBM cells. However, different quantities of the isoforms were expressed in different cell lines. Interestingly, different expression levels were noted under different culture conditions. Class III β-tubulin was significantly downregulated in four GBM cell lines in hypoxia, which contrasts previous studies. Previously, increased expression of class III β-tubulin has been observed in GBMs bordering geographic areas of ischemic necrosis [
8,
36], the discrepancy between in vivo and in vitro observations warrants further investigation.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TY carried out all of the experiments except for ECIS and wrote this manuscript. KOS helped qPCR and contributed to data analysis. LL performed cell culture, including hypoxia experiments, immunocytochemistry, RNA isolation and Western blot. LS carried out ECIS experiment. JW, XL and PØE participated in study design and manuscript preparation. All authors read and approved the final manuscript.