Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas
© Matsutani et al.; licensee BioMed Central Ltd. 2012
Received: 23 July 2012
Accepted: 25 September 2012
Published: 11 October 2012
Glioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable in spite of various therapeutic approaches. Clarification of the oncogenic process in its early stage is important for the diagnosis and effective therapy.
In the present study, we used the serological identification of antigens by recombinant cDNA expression cloning (SEREX) to explore the subtle changes of the protein expression in low-grade glioma. The levels of serum autoantibodies to the SEREX-identified glioma-related antigens were analyzed by ELISA, and the epitope site was identified using deletion mutants and overlap peptide array. Changes in the serum autoantibody levels were examined in the rat glioma model using C6 and 9 L glioma cell lines.
We identified 31 glioma-related antigens by SEREX. Among them, the serum level of autoantibody to src-homology 3-domain GRB2-like 1 (SH3GL1) was significantly higher in patients with low-grade glioma than healthy volunteers or high-grade gliomas. The 10 amino-acids at the C-terminal were identified as the epitope site by the overlap peptide array and the ELISA using deletion mutants. The tissue expression of SH3GL1 protein increased in proportion to glioma progression. The rat glioma models confirmed the increase of anti-SH3GL1 autoantibody level in the early stage and the suppression in the late stage.
SH3GL1 may be involved in the oncogenic process of gliomas and effectively elicit an autologous antibody response in low-grade gliomas. The immunological reaction to SH3GL1 would contribute to the establishment of a novel diagnostic and therapeutic target for gliomas.
Glioma is the most common primary malignant central nervous system (CNS) tumor in adults and arises from neuroepithelial cells, mostly astrocytes or oligodendrocytes. Glioma is divided into 4 grades according to World Health Organization (WHO) histological classification, and the prognosis of glioma is still poor[1, 2]. Glioblastoma (GB), WHO grade IV, and anaplastic astrocytoma (AA), WHO grade III, are referred to as high-grade glioma, and the median survival time of patients with AA and GB is 2–3 years and only approximately 1.5 years, respectively. In the cases of WHO grade II tumor, the median survival time of patients with diffuse astrocytoma (WHO grade II) is also limited to approximately 5–7 years. In most cases, patients with glioma present large cerebral lesion at diagnosis, which prevents effective removal without neurological deficits, and the remnant tumors relapse even though receiving post-operative treatments with radiotherapy and chemotherapy. The clarification of the oncogenic process especially in the early stage would contribute to its early diagnosis and to new molecular targets.
Serological identification of antigens by recombinant cDNA expression cloning (SEREX) is one of the powerful tools for finding novel cancer antigens and has been applied on a nationwide basis to target many cancers, including gliblastoma[6–8]. However, the specific and crucial changes in the protein expression in low-grade gliomas have not been identified yet. In contrast, it is well known that activation of the receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) is the most frequent molecular aberration found in high-grade gliomas. The receptor tyrosine kinases make the ras pathway activation through a protein-protein interaction of the adaptor protein called GRB2 with Son of Sevenless (Sos) protein through src-homology 3 (SH3) domain[10, 11]. The connection of the adaptor protein and Sos is a key step toward activating the ras-mediated oncogenic pathways in the downstream of receptor tyrosine kinases.
In the present study, the authors applied SEREX to glioma to find SH3-domain GRB2-like 1 (SH3GL1) as a novel glioma-related antigen. The levels of serum autoantibodies to SH3GL1 were significantly higher in patients with low-grade gliomas than in healthy donors by ELISA. In contrast, the serum autoantibody level was significantly depressed in high-grade glioma patients compared with low-grade gliomas patients. We identified the epitope site of SH3GL1 by overlap peptide array and an ELISA using deletion mutants. The rat glioma model using C6 and 9 L glioma cells also showed the increases of the anti-SH3GL1 autoantibody level in the early stage and decreases in the late stage. Although low-grade gliomas are not always in an early-stage of the disease, it is usually accepted that gliomas often progress from low-grade tumors to higher-grade tumors as the time proceeds. The present clinical data and the animal models suggested the immunosurveillance can work in low-grade glioma patients and the immune tolerance would occur in those with high-grade gliomas. The present findings would contribute to the knowledge of molecular basis of low-grade gliomas and the establishment of a novel diagnostic and therapeutic target.
Materials and methods
Sera and glioma tissue
Sera were obtained from patients with various types of glioma and from healthy volunteers after they had provided written informed consent. Patients with glioma underwent surgery and the tumor was histologically diagnosed as grade II–IV glioma at Chiba University Hospital in 1998–2008; healthy donors were confirmed to have no cerebral diseases using radiological imaging such as computed tomography or magnetic resonance imaging. No patient received steroid therapy at the time of blood sampling. Each sample was centrifuged at 3 000 × g for 5 min and then frozen at –80°C until use. Glioma tissue was collected from the tumor tissue during surgical treatment. Normal brain tissue, which did not show any glioma cell infiltration under microscopic examination, was isolated from the circumference of the glioma specimen and from non-neoplastic CNS tissues that were obtained during a lesionectomy from a patient with intractable epilepsy or during a lobectomy from patients with benign CNS tumors, such as meningioma. The Local Ethical Review Board of the Graduate School of Medicine, Chiba University approved the studies in this issue, and we obtained written informed consent from the patients and healthy volunteers concerning the use of material for scientific research.
Phage cDNA library
A total RNA was prepared from the human glioblastoma cell-line U-87 MG (ATCC, HTB-14) using the acid guanidium thiocyanate-phenol-chloroform method with an mRNA purification kit (AquaPure RNA isolation kit, BioRad, Hercules, CA) used in accordance with the manufacturer’s instructions. Double-stranded cDNA was synthesized through conventional procedures and ligated into the EcoRI-XhoI site of λZAP II phage. The library size was over 1.0 × 106 PFU/ml.
Immunological screening using SEREX
Ecoli XL1-Blue MRF’ was infected with λZAP II phages containing a cDNA library and the expression of cDNA was induced by blotting on nitrocellulose membranes, pretreated with 10 mM isopropyl-β-D-thiogalactoside (IPTG; Wako Pure Chemicals, Osaka, Japan). After washing and blocking, the membranes were exposed in 1:2000-diluted serum for 1 h. The membranes were treated with 1:5000-diluted alkalinephosphatase-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). After incubation in a color development solution containing 0.3 mg/ml of nitroblue tetrazolium chloride (Wako Pure Chemicals) and 0.15 mg/ml of 5-bromo-4-chloro-3-indolylphosphate (Wako Pure Chemicals), positive reactions were detected. Positive clones were re-cloned twice to obtain monoclonality.
Sequence analysis of identified clones
Monoclonalized phage cDNA clones were converted to pBluescript phagemids through in vivo excision using ExAssist helper phage (Stratagene, La Jolla, CA). Plasmid DNA was obtained from an E. coli SOLR strain transformed by the phagemid. The inserted cDNAs were sequenced using the dideoxy chain termination method and the sequences were analyzed for homology with a public database provided by the National Center for Biotechnology Information (NCBI).
Production of glutathione S-transferase (GST) fusion proteins
cDNA inserts of these clones incorporated in pBluescript were cleaved by EcoRI and XhoI generally and cloned into the EcoRI-XhoI site of pGEX-4 T-3, pGEX-4 T-2, and pGEX-4 T-1 vectors (Amersham Bioscience, Piscataway, NJ) that express recombinant GST fusion proteins. E. coli JM109 cells containing pGEX clones (A600 = 0.3–0.5) were cultured in 200 ml of Luria broth (LB), and lysed through sonication. The lysate was then centrifuged and the GST-fusion proteins in the supernatants were purified by glutathione-Sepharose. These samples were centrifuged and affinity-purified with glutathione-Sepharose.
Purified recombinant proteins diluted at 10 μg protein/ml in PBS were added to each well of 96-well plates and incubated at room temperature overnight. As a control, the same amount of GST was applied. Sera diluted at 1:100 in PBS with 10% FBS were added to the wells and incubated for 1 h. The wells were exposed to 1:2 000-diluted horseradish peroxidase-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Then, 100 μl of a peroxidase substrate (o-phenylenediamine, 0.4 mg/ml) containing 0.02% (v/v) H2O2 were added. Absorbance at 490 nm was determined using a microplate reader (Emax, Molecular Devices, Sunnyvale, CA).
Construction of SH3GL1 deletion mutants
Some deletion constructs of SH3GL1 were obtained through digestion with restriction enzymes or the inverse PCR method. The SEREX-identified phage clone was containing a full-length coding sequence of SH3GL1 (1–368 amino acids), that comprised Bin-Amphiphysin-Rvs (BAR) domain (amino acid positions between 5 and 242) in the N-terminal portion, coiled-coil (CC) domain (amino acid proteins between 180 and 250) at the middle, and the SH3 domain (amino acid positions between 309 and 364) in the C-terminal portion. The region of SH3GL1 cDNA corresponding to amino acids between 260 and 368 was cleaved by SmaI and XhoI and subcloned into the pGEX 4 T-3 vector at the SmaI-XhoI digestion sites (SH3GL1 mut-1). Amino acids 316–368, 260–289 and 354–368 were deleted through the inverse PCR method with the KOD–Plus Mutagenesis Kit (Toyobo, Osaka, Japan) using the SH3GL1 mut-1 cDNA as a template (SH3GL1 mut-2, 3 and 4, respectively). The primers for SH3GL1 mut-2 were forward 5′-CCAGTCTTCCGACAAGCCCATC-3′, reverse 5′-TGGGGATCCACGCGGAACCAG-3′; for SH3GL1 mut-3 were forward 5′-TCGAGCGGCCGCATCGTGAC-3′, reverse 5′-GCCCGACTGGCCGTCCAGCATG-3′; and for SH3GL1 mut-4 were; forward 5′-TCGAGCGGCCGCATCGTGAC-3′, reverse 5′-GCCCGACTGGCCGTCCAGCATG-3′.
Overlap peptide array
Peptides spanning amino acid residues 1–368 of SH3GL1 were synthesized on cellulose membranes as a series of peptides with the overlapping by 12 amino acids using F-moc amino acids according to the manufacturer’s protocol (Auto spot robot ASP222; ABIMED Analysen-Technik GmbH, Langenfeld, Germany) as previously described. Membranes were incubated with the sera of patients at 1:200 dilutions for more than 12 h. Then, the antigen-antibody complexes were detected with FITC-conjugated goat anti-human IgG (109-095-098; Jackson ImmunoResearch, West Grove, PA) at 1:10000 dilutions. The fluorescence of the peptide spots were detected using Typhoon 9400 (Amersham Biosciences, Stockholm, Sweden) with a 488 nm/520 nm filter. The scanned image was also analyzed with CS analyzer ver. 3.0 (Atto & Rise Corporation, Tokyo, Japan) and fluorescent intensity of each spot was calculated.
Immunohistochemical staining for SH3GL1 protein
Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Tokyo, Japan), and according to the manufacturer’s recommendations. This antibody was confirmed to be cross-reactive for human, mouse, and rat SH3GL1. Sections were counterstained with hematoxylin, then dehydrated and mounted.
Staining of tissue specimens was observed in 100 × fields with approximately all fields presenting glioma cells. The staining intensity in cytosole was classified into 5 groups, absent (−), light partial staining (±), homogeneous light staining (+), partly strong positive staining (++) and homogeneous strong positive staining (+++).
Brain Tumor Model, Monitoring of Tumor Size, and Serum Sampling
Rat C6 glioma cells and 9 L gliosarcoma cells were originally obtained from ATCC and maintained in Dulbecco's modified Eagle medium (D-MEM) supplemented with 10% fetal calf serum in a humidified atmosphere of 5% CO2. Male Wister rats for C6 cells and Fisher rats for 9 L cells, weighing between 200 and 240 g (7–8 weeks old) were used. The animals were anesthetized and placed in a stereotaxic apparatus. A burr hole was made at 4 mm posterior to bregma and 3 mm right to midline. A 25-gauge needle was inserted to the point of 3 mm ventral from dura where 1 × 105 syngeneic C6 or 9 L tumor cells in 10 μl medium were slowly injected. To estimate i.c. tumor volume sequentially, all the animals were examined with a 7 tesla MRI every 7 days started on day 7 after the tumor inoculation. The sera were obtained from tail vein every 7 days. The animal experimentation was reviewed and approved by the Institutional Animal Care and Use Committee of National Institute of Radiological Science.
The significance of differences among healthy donors, patients with low-grade glioma, and patients with high-grade glioma was calculated using the Kruskal Wallis H-test and the Mann–Whitney U-test with Bonferroni correction. Differences were considered significant only if p < 0.05. The overall survivals from the date of initial diagnosis were estimated using Kaplan-Meier methodology and compared by the Log rank test to estimate the clinical significance of production of autoantibody for SH3GL1.
Serological screening of cDNA library
Genes identified by SEREX
NCBI accession no.
cDNA inserts of recombinant protein†
amplified in breast cancer 1
anillin, actin binding protein (scraps homolog, Drosophilia)
ATP synthase, H + transporting, mitochondrial F1complex, beta polypeptide, nuclear gene encoding mitochondrial protein
catenin (cadherin-associated protein), alpha-like 1
CDV3 homolog (mouse)
centromere protein F, 350/400 ka (mitosin)
chromosome 14 open reading frame 145
coagulation factor III (thromboplastin, tissue factor)
coiled-coil domain containing 86
cyclin G1, transcript variant 2
eukaryotic translation elongation factor 1 alpha 1
ferritin, heavy polypeptide 1
ferritin, light polypeptide
heterogeneous nuclear ribonucleoprotein C (C1/C2), transcript variant 4
Homo sapiens mRNA for KIAA0146 gene, partial cds.
macrophage migration inhibitory factor
myosin phosphatase-Rho interacting protein, transcript variant 1
nucleolar protein 8
oral-facial-digital syndrome 1
postmeiotic segregation increased 1(S.cerevisiae)
retinoblastoma binding protein 8, transcript variant 1
ribosomal protein, large, P0, transcript variant 1
RNA export 1 homolog (S.pombe), transcript variant 1
serine/threonine kinase 3(STE20 homolog, yeast)
SH3-domain GRB2-like 1
synaptonemal complex protein SC65
TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 55 kDa
transforming growth factor, beta-induced, 68 kDa
unc-45 homolog A (C.elegans), transcript variant 2 or 3
ELISA to detect serum antibodies
Characteristics of screening serum sets
1st sampling test
No. of patients
Healthy donors, n (%)
Age (mean ± SD), range
48.1 ± 18.3, 16-76
51.3 ± 15.2, 16-76
Low-grade glioma, n(%)
Age (mean ± SD), range
45.8 ± 14.8, 20-74
44.2 ± 14.1, 22-78
Pilocytic astrocytoma, n (%)
Diffuse astrocytoma, n (%)
Oligodendroglioma, n (%)
Oligoastrocytoma, n (%)
Ependymoma, n (%)
Ganglioglioma, n (%)
High-grade glioma, n (%)
Age (mean ± SD), range
49.7 ± 18.3, 8-78
49.8 ± 15.5, 28-78
Glioblastoma, n (%)
Anaplastic astrocytoma, n (%)
Anaplastic oligodendroglioma, n (%)
Anaplastic oligoastrocytoma, n (%)
Anaplastic ependymoma, n (%)
Choroid plexus carcinoma, n (%)
The levels of serum antibodies of CENPF, MIF, M-RIP, RPLP0, TGFBI and UNC45A were significantly lower in patients with high-grade glioma than in those with low-grade glioma (Figure1A-C, E, H and I) and, moreover, the levels of anti-M-RIP and anti-RPLP0 antibodies in patients with high-grade glioma were also significantly lower than in healthy volunteers (Figure1C and E).
The levels of serum antibodies to SH3GL1 were significantly higher in patients with low-grade glioma than those with high-grade glioma (P = 0.0243) and healthy volunteers (P = 0.0045) (Figure2A). When the antibody levels were divided into 2 groups with a cut-off value of 0.383 corresponding to the mean + 2 standard deviations (SD) of SH3GL1 antibodies in healthy volunteers, the positive rate of patients with low-grade glioma was 62.5% (25 of 40), whereas those of patients with high-grade glioma and healthy volunteers were 8.9% (5 of 56) and 15.6% (5 of 32), respectively.
Independent validation test for the levels of antibodies to SH3GL1
To verify the generality of low-grade glioma-specific increase in serum antibodies to SH3GL1, an independent validation test was carried out using other serum set. In validations, consecutive serum samples that were collected in 2005–2008 after the first serum sampling, were enrolled, and no apparent differences in the characteristics were observed between the 2 serum sets (Table 2). The results of the ELISA based on the newly collected serum set showed that the levels of serum autoantibodies to SH3GL1 were significantly higher than those of healthy donors (P = 0.0189) (Figure2B). Although there was no statistical significance in the levels of antigens between patients with low- and high-grade glioma, similar distribution was recognized. In the combined population of the first sampling test and the validation test, there was a significant difference between low-grade gliomas and high-grade gliomas (p = 0.0351).
The same results of both ELISA based on the independent serum sets support the possibility that SH3GL1 is aberrantly expressed and efficiently elicits a systemic immune response in low-grade glioma patients. The level of anti-SH3GL1 autoantibody could be a novel low-grade glioma-specific serum marker. In contrast, the lower serum autoantibody levels against these determined SEREX-antigens in patients with high-grade glioma as opposed to those with low-grade glioma and healthy volunteers suggest that the existence of some immunosuppressive mechanisms in high-grade gliomas.
Search for epitope sites of SH3GL1
Immunohistochemical staining for SH3GL1 protein
It is known that glioma cells are commonly localized in white matter and progress along neural fibers. Therefore, we compare the immunostaining levels between normal glial cells in white matter and glioma cells. In glioma tissues, strong positive staining of SH3GL1 was observed in the cytoplasms but not in the nucleus (Figure6B). The levels of stain in white matter increased according to the malignancy of tumors; that is, high-grade glioma tissues were most heavily stained while normal glial cells were barely stained (Figures6C). These results indicated that the protein levels of SH3GL1 were much higher in glioma cells than in normal glial cells in white matter.
Alteration of anti-SH3GL1 autoantibody level in rat glioma model
The molecular pathogenesis of glioblastoma has been well characterized and involves both gain and loss of a number of genes participating in proliferative or mitogenic signals. One of the most prevalent molecular changes consists of aberrant activation of EGFR, which occur in 50% of glioblastoma, but not seen in low-grade astrocytomas[12, 15]. We have shown in this study that the SH3-domain of GRB2-like protein, which links the receptor tyrosine kinases activation to the ras pathway, had already overexpressed in low-grade gliomas and strongly induced a humoral immune response. In high-grade gliomas, the tissue expression of SH3GL1 was further increased, but the immune response was suppressed. Although there are few reports describing overexpression of this protein in human cancers, SH3GL1 protein is related to the activation of MLL proto-oncogene by chromosomal translocation. Solitary SH3GL1 overexpression in NIH3T3 cells also reported to do some oncogenic behaviors in vivo[17, 18]. It is not clear whether the overexpression is a result of amplification of receptor tyrosine kinases or not. However, the net result of these signaling complexes induces the shift of ras-GDP to its activated form ras-GTP, and may lead to activate the MAPK cascade and resultant alteration in gene expression concerning cell proliferation.
SH3GL1 is known to be predominantly localized in the nuclei of haematopoietic cells and fibroblasts in contrast to cytoplasmic localization in neurons and osteoblasts[19, 20]. In the adult brain, SH3GL1 is highest in the neurons of the granular layer of the cerebral cortex and known to be involved in the development of central nervous system[22, 23]. These published data were compatible with our results of immunohistochemical staining with SH3GL1 antibody. In glioma tissues, strong positive staining of SH3GL1 was obtained in the cytoplasms but not in the nucleus, and the levels of staining in white matter increased according to the advance of its malignancy. These results suggested that the SH3GL1 overexpression might have some oncogenic roles in gliomas. However, the levels of serum autoantibodies to SH3GL1 in the patients with high-grade glioma were not increased in our study, while the levels in the patients with low-grade glioma were increased. It is believed that the abnormal cytoplasmic SH3GL1 overexpression in glioma cell has a potential to induce immune responses, but various mechanisms of immunosuppression prevent the reaction in high-grade glioma[24–27]. All the other candidate genes identified in this study showed the same low immunoreactivity in patients with high-grade gliomas. The suppression of the immunosurveillance mechanism in high-grade glioma would attenuate the recognition of SEREX-derived antigens in antigen presenting cells (APC). In fact, it has been known that various immunosuppressive molecules, such as TGF-β, IL-10, and prostaglandins, are highly expressed in cancers including high-grade glioma[24, 25], and these molecules could inhibit the maturation of professional APCs. Such an evading immune destruction has now added to the hallmark of cancer.
The major cause of the lower level of anti-SH3GL1 autoantibody in high-grade glioma patients would be the non-specific immunosuppression caused by increased immunosuppressive cytokines[24, 25]. However, the animal experiment provides an additional hypothesis that the depressed autoantibody levels could be partly due to the antigen-specific immune tolerance induced by the existence of large tumor and long-term antigen exposure. The early stage of the rat glioma models indicates a relatively small tumor and short-term antigen exposure, and the late stage indicates a large tumor and long-term antigen exposure to the immune system. The long-term antigen exposure from a large tumor could generally induce immune tolerance through development of immune resistant tumor variants and the tumor microenvironment inducing immune cell anergy or death[26, 27]. It is usually accepted that gliomas often progress from low-grade tumors to higher-grade tumors as the time proceeds, although low-grade gliomas are not always in an early-stage of the disease and secondary glioblastoma is less frequent than de novo glioblastoma. The possible contribution of antigen-specific immune tolerance to the depressed autoantibody levels in high-grade glioma patients remains to be elucidated.
SEREX is one of the most powerful tools to find novel tumor antigen for various cancers, and some autologous antigens to esophageal cancer have been identified in our groups[29–31]. Compared with other screening techniques such as transcriptomics or proteomics, SEREX offers a crucial advantage that subtle changes in the protein expressions can be detected through immunological reactions[32, 33]. Several authors have already applied SEREX to glioma, and some antigens, including glioma-expressed antigen 2 (GLEA2), PHD finger protein 3 (PHF3)[7, 34], and SRY-box 6 (SOX6) have been identified. It should be noted that we found autologous antibodies against SH3GL1 to be a low-grade glioma-specific marker with similar experimental systems to others. Our unique approach was the quantitative comparison of the levels of serum antibodies using the ELISA, while the approach of others was qualitative analysis. The application of ELISA in the validation step could lead to the discovery of a low-grade glioma-specific high titer of the autoantibody and the decrease in high-grade gliomas. Although some candidates of glioma biomarkers have been identified by various screening methods[6–8, 34–37], no serum marker for early diagnosis has been found yet. Therefore, it is quite valuable to find a novel serum biomarker for its early diagnosis, prediction of the prognosis in each patient, and development of a new molecular target. Indeed,
The results of an overlap peptide array and ELISA using deletion mutants of SH3GL1 showed that 12 amino acids in the C-terminal portion, FPLSYVEVLVPL, were indicated as a major epitope site. By using a synthetic peptide corresponding to the epitope as an antigen, a more accurate screening for the patients with low-grade gliomas and a specific peptide vaccine therapy would be achieved in the future.
1Departments of Neurological Surgery, Chiba University, Graduate School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. 2Genetics and Biochemistry, Chiba University, Graduate School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. 3Diagnostic Pathology, Chiba University, Graduate School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan. 4Department of Biochemistry, Graduate School of Life Science, Nagoya Women’s University, 3-40, Shioji-cho, Mizuho-ku, Nagoya 467-8610, Japan.
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