GD2 Expression Research Articles

Abstract P5-07-03: GD2-mediated FAK signaling regulates breast cancer stem cell function in triple negative breast cancer

Abstract Ganglioside GD2 identifies breast cancer stem cells (BCSCs, Battula et al., JCI, 2012) and expression of GD2 is tightly regulated by GD3 synthase (GD3S). GD3S is highly expressed in GD2+ cells and inhibition of GD3S inhibits tumor formation and metastasis of breast cancer cells. However, the mechanism of GD2-mediated regulation of BCSC function is not known. Here we hypothesize that GD2 regulates signaling pathways involved in cell adhesion, migration and invasion of breast cancer cells. To identify these signaling pathways, antibody micro-arrays were used with 850 validated antibodies specific to total or phosphorylated proteins. Interestingly, focal adhesion kinase (FAK) was the most significantly phosphorylated protein in GD2+ compared to GD2- cells (S910 and S722). In addition, expression of FAK downstream mediators including Csk, PKCq, PKCl/I, Pyk2, and p38MAPK, was up-regulated in GD2+ compared to GD2- cells. Western blot analysis of FACS sorted SUM159 cells also revealed increased phosphorylation of FAK >80% at Y397 and >25% at Y861 in GD2+ compared to GD2- cells. FAK downstream targets including paxillin, p130 Cas, pERK were also up-regulated in GD2+ cells compared to GD2- cells indicating definitive activation of FAK signaling in GD2+ BCSCs. To investigate the functional role of GD2 in FAK mediated functions, we genetically deleted GD3S using the CRISPR knock-out system in SUM159 cells. only <1% GD2expression compared to >20% in parental cells was observed. GD3S-KO cells grew 5-10% slower in cell culture mostly because of the reduction (15±5%) in adherence. Trans-well assays revealed 3-5 fold reduction in migration and invasion of GD3S-KO compared to parental cells. These data indicate that GD2 and GD3S are not only the markers of BCSCs but also regulators of their function. Finally, we tested the effect of FAK inhibitor (PF-573228) against GD2+ BCSCs and GD3S-KO SUM159 cells. PF-573228 treatment decreased the number of mammospheres generated by GD2+ cells 3-4 fold in a dose dependent manner (100nM-1µm). In addition, treatment of PF-573228, inhibited migration and invasion of GD2+ cells 2 and 3 fold, respectively. However, treatment with PF-573228 on GD3S-KO cells further reduced their ability to migrate and invade by over 70% compared to untreated cells. In addition, GD3S-KO cells failed to form any mammospheres when cultured under low adherence conditions (p<0.00001), whereas the parental cells formed 15-20 mammospheres per 1,000 cells plated. In conclusion, our data demonstrate that FAK signaling is activated in GD2+ cells but that FAK inhibition alone may not be sufficient to inhibit BCSC function. Combined FAK and GD3S inhibition may exert highly synergistic effects against BCSCs. Citation Format: Nguyen K, Sun JC, Hortobagyi GN, Andreeff M, Battula VL. GD2-mediated FAK signaling regulates breast cancer stem cell function in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-03.

Inflammatory Reactions in Microenvironments, Leading to Melanomagenesis

Malignant melanoma is resistant to various therapies, while its incidence has been dramatically increasing. Among various factors, sun exposure, particularly ultra violet (UV) irradiation is considered to induce melanomas. Gangliosides have been markers of neuro-ectoderm-derived cancers like malignant melanomas and gliomas, but they also play crucial roles in their malignant properties. GD3 regulates cell signalling transduced through membrane microdomains. Chronic inflammatory reactions toward noxious stimuli cause cumbersome diseases such as cancers and degeneration, and glycosylation is involved in those processes. Here, melanocytes did not respond to UVB, while keratinocytes responded to UVB by secreting various cytokines such as TNFα and IL-6. Furthermore, these cytokines induced expression of melanoma-associated ganglioside GD3 on melanocytes. Expression of GD3 does not necessarily induce melanomas, but may form microenvironments for the generation of melanomas after long-term continuation. Consequently, combination of DNA mutagenesis and chronic inflammatory reaction seems to be critical for the melanomagenesis. Thus, 1. Mechanisms for the induction of GD3 synthase gene by inflammatory cytokines. 2. Meaning of GD3 expression in melanocytes. 3. Linkage between GD3 expression in melanocytes and melanomagenesis. 4. Prevention of UV exposure, are proposed as urgent issues to be solved in the near future.

Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

The disialoganglioside GD3 has been considered to be involved in tumor progression or suppression in various tumor cells. However, the significance of the biological functions of GD3 in breast cancer cells is still controversial. This prompted us to study the possible relationship(s) between GD3 expression and the metastatic potential of a breast cancer MDA-MB231 cells as an estrogen receptor negative (ER-) type. The human GD3 synthase cDNA was transfected into MDA-MB231 cells, and G-418 bulk selection was used to select cells stably overexpressing the GD3 synthase. In vitro invasion potentials of the GD3 synthase over-expressing cells (pc3-GD3s) were significantly suppressed when compared with control cells. Expression of intercellular adhesion molecule-1 (ICAM-1; CD54) was down-regulated in the pc3-GD3s cells and the decrease in ICAM-I expression is directly related to the decrease in invasiveness of the pc3-GD3s cells. Another type of ER negative SK-BR3 cells exhibited the similar level of ICAM-1 expression as MDA-MB231 cells, while the ER positive MCF-7 cells (ER+) showed the increased expression level of ICAM-1. Then, we investigated signaling pathways known to control ICAM-1 expression. No difference was observed in the phosphorylation of ERK and p38 between the pc3-GD3s and control cells (pc3), but the activation of AKT was inhibited in pc3-GD3s, and not in the control (pc3). In addition, the composition of total gangliosides was changed between control (pc3) and pc3-GD3s cells, as confirmed by HPTLC. The pc3-GD3s cells had an accumulation of the GD2 instead of the GD3. RT-PCR results showed that not only GD3 synthase, but also GM2/GD2 synthase (β4-GalNc T) expression was increased in pc3-GD3s cells. Overexpression of GD3 synthase suppresses the invasive potential of human breast cancer MDA-MB-231 cells through down-regulation of ICAM-1 and the crucial pathway to allow the apoptotic effect has been attributed to accumulation of the GD2 ganglioside. ER has been linked to the ICAM-1 expression with GD3 to GD2 conversion in human breast cancer cells. This is the first finding of the endogenous sialyltransferase functions in tumor cells.

Ganglioside Profiling of the Human Retina: Comparison with Other Ocular Structures, Brain and Plasma Reveals Tissue Specificities.

Gangliosides make a wide family of glycosphingolipids, highly heterogeneous in both the ceramide moiety and the oligosaccharide chain. While ubiquitously expressed in mammalian tissues, they are particularly abundant in the brain and the peripheral nervous system. Gangliosides are known to play a crucial role in the development, maintenance and functional integrity of the nervous system. However, the expression and roles of gangliosides in the retina, although often considered as a window on the brain, has been far less studied. We performed an in-depth analysis of gangliosides of the human retina, especially using powerful LC/MS methods. We compared the pattern of ganglioside classes and ceramide molecular species of this tissue with other ocular structures and with brain and plasma in elderly human individuals. About a hundred of ganglioside molecular species among 15 distinct classes were detected illustrating the huge structural diversity of these compounds. The retina exhibited a very diverse ganglioside profile and shared several common features with the brain (prominence of tetraosylgangliosides, abundance of d20:1 long chain base and 18:0 fatty acid…). However, the retina stood out with the specific expression of GD3, GT3 and AcGT3, which further presented a peculiar molecular species distribution. The unique ganglioside pattern we observed in the human retina suggests that these ganglioside species play a specific role in the structure and function of this tissue. This lipidomic study, by highlighting retina specific ganglioside species, opens up novel research directions for a better understanding of the biological role of gangliosides in the retina.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy.

The methods described herein for activation of naïve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during naïve CD4+ T cell activation. Also, real-time PCR experiments using <40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Targeted attack: mechanisms by which ovarian cancers suppress the immune system.

Epithelial ovarian cancer (EOC) is the seventh most common cancer diagnosis among women worldwide and has the highest mortality rate of all gynecologic malignancies (1). In the United States, EOC is the fifth most common cause of cancer deaths among women (2). Despite improvements in treatment, 5-year survival rates of patients with advanced ovarian cancer remain less than 50% (3,4) because the majority of patients present with late stage disease (5). Thus, the identification of novel biomarkers and the development of innovative therapeutic approaches are urgently needed. It has been well established that evading immune surveillance is critical for tumor growth and metastasis, and other groups have shown that vascular endothelial growth factor (VEGF) expression is inversely correlated with survival in ovarian cancer patients (6,7). Therefore, we investigated the effects of ovarian cancer-associated VEGF on CD1d-mediated antigen presentation to natural killer T (NKT) cells (8). We found that inhibition of VEGF production by ovarian cancer cell lines led to a reduction in the expression of the immunosuppressive ganglioside GD3 and restored NKT cell activation. Thus, we identified a novel link between immunosuppressive ganglioside shedding and VEGF production by ovarian cancers.

Efficient Killing of High Risk Neuroblastoma Using Natural Killer Cells Activated by Plasmacytoid Dendritic Cells.

High-risk neuroblastoma (NB) remains a major therapeutic challenge despite the recent advent of disialoganglioside (GD2)-antibody treatment combined with interleukin (IL)-2 and granulocyte monocyte-colony stimulating factor (GM-CSF). Indeed, more than one third of the patients still die from this disease. Here, we developed a novel approach to improve the current anti-GD2 immunotherapy based on NK cell stimulation using toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDCs). We demonstrated that this strategy led to the efficient killing of NB cells. When the expression of GD2 was heterogeneous on NB cells, the combination of pDC-mediated NK-cell activation and anti-GD2 treatment significantly increased the cytotoxicity of NK cells against NB cells. Activation by pDCs led to a unique NK-cell phenotype characterized by increased surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with increased expression of CD69 on CD56dim cytotoxic cells, and strong interferon-γ production. Additionally, NB-cell killing was mediated by the TRAIL death-receptor pathway, as well as by the release of cytolytic granules via the DNAX accessory molecule 1 pathway. NK-cell activation and lytic activity against NB was independent of cell contact, depended upon type I IFN produced by TLR-9-activated pDCs, but was not reproduced by IFN-α stimulation alone. Collectively, these results highlighted the therapeutic potential of activated pDCs for patients with high-risk NB.

Identification and Tumour-Binding Properties of a Peptide with High Affinity to the Disialoganglioside GD2.

Neuroectodermal tumours are characterized by aberrant processing of disialogangliosides concomitant with high expression of GD2 or GD3 on cell surfaces. Antibodies targeting GD2 are already in clinical use for therapy of neuroblastoma, a solid tumour of early childhood. Here, we set out to identify peptides with high affinity to human disialoganglioside GD2. To this end, we performed a combined in vivo and in vitro screen using a recombinant phage displayed peptide library. We isolated a phage displaying the peptide sequence WHWRLPS that specifically binds to the human disialoganglioside GD2. Binding specificity was confirmed by mutational scanning and by comparative analyses using structurally related disialogangliosides. In vivo, significant enrichment of phage binding to xenografts of human neuroblastoma cells in mice was observed. Tumour-specific phage accumulation could be blocked by intravenous coinjection of the corresponding peptide. Comparative pharmacokinetic analyses revealed higher specific accumulation of 68Ga-labelled GD2-binding peptide compared to 111In-labelled peptide in xenografts of human neuroblastoma. In contrast to 124I-MIBG, which is currently evaluated as a neuroblastoma marker in PET/CT, 68Ga-labelled GD2-specific peptide spared the thyroid but was enriched in the kidneys, which could be partially blocked by infusion of amino acids.In summary, we here report on a novel tumour-homing peptide that specifically binds to the disialoganglioside GD2, accumulates in xenografts of neuroblastoma cells in mice and bears the potential for tumour detection using PET/CT. Thus, this peptide may serve as a new scaffold for diagnosing GD2-positive tumours of neuroectodermal origin.

Expression of B4GALNT1, an essential glycosyltransferase for the synthesis of complex gangliosides, suppresses BACE1 degradation and modulates APP processing

Alzheimer’s disease (AD) is the most prevalent form of dementia characterized by the extracellular accumulation of amyloid β (Aβ) peptides, which are produced by proteolytic cleavages of amyloid precursor protein (APP). Gangliosides are involved in AD pathophysiology including Aβ deposition and APP processing, yet the detailed mechanisms are not fully understood. Here we examined how changes in the carbohydrate moiety of gangliosides alter APP processing in human melanoma cells, neuroectoderm-derived cells. We showed that forced expression of GD2, GM2 or GM1 (by introducing B4GALNT1 cDNA into cells not expressing this glycosyltransferase) results in increases of α- and β-site cleavages of APP with a prominent increase in β-cleavage. We also showed that β-site APP cleaving enzyme 1 (BACE1) protein is highly protected from the degradation in cells expressing these gangliosides, thereby increasing the expression of this protein. Unexpectedly, adding gangliosides exogenously altered neither BACE1 levels nor β-site cleavage. The stabilisation of BACE1 protein led to the increase of this protein in lipid rafts, where BACE1 processes APP. Based on the current results, we propose a hitherto undisclosed link between ganglioside expression and AD; the expression of B4GALNT1 positively regulates the β-site cleavage by mainly inhibiting the lysosomal degradation of BACE1 protein.

Lack of immunocytological GD2 expression on neuroblastoma cells in bone marrow at diagnosis, during treatment, and at recurrence.

Loss of disialoganglioside 2 (GD2) expression in neuroblastoma (NB) bone marrow cells has been reported in rare cases. This study investigated prospectively the frequency and the patterns of visible GD2 loss at diagnosis, during treatment, and at recurrence. Bone marrow aspirates of patients with new or recurrent stage 4 and 4S NB diagnosed between January 1, 2002 and August 31, 2013 were investigated in parallel by cytology and GD2 immunocytology. Complete negative immunostaining was defined if staining was absent in all and partial if absent in a portion and/or in case of atypical faint staining. Of 1,261 investigated trial patients of all stages, 474 had unequivocal cytological bone marrow infiltration at initial diagnosis. Thirty-seven patients had tumor cells with complete or partial negative GD2 staining at initial diagnosis, nine during chemotherapy, and 11 at recurrence (altogether 12.0%). The percentage of GD2 negativity in stages 4 and 4S were similar (13% and 9%, respectively). Complete negativity was seen in 14 and partial in 43 cases. Twenty-one cases changed from positive to negative (15 to partial and six to complete) and three cases from negative to positive staining (two to partial and one to complete). The GD2 negative and positive groups were not different regarding tumor sites, molecular characteristics, histology, and tumor markers. Children with stage 4 and GD2 negativity tended to be older at diagnosis (42 vs. 32 months, P = 0.056). Event-free survival and overall survival comparing negative versus positive staining did not show any differences. Complete or partial lack of GD2 staining on NB cells in bone marrow is more frequent than currently recognized.

Neogenin, Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells

To investigate mechanisms for increased malignant properties in malignant melanomas by ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3- cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a γ-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3- cells. Overexpression of Ne-ICD in GD3- cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with γ-secretase. γ-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and γ-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits γ-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.

Abstract 2303: The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients

Abstract Background: Chimeric antigen receptor (CAR)-engineered T cells have demonstrated potent clinical efficacy in patients with B cell lymphoma. However, the application of CAR-T cell therapy targeting other solid tumors has been limited. The GD2 gangliosides are sialic acid-containing glycosphingolipids that play a role in signal transduction and cell-cell recognition. 1st generation gangliosides GD2 chimeric antigen receptor T cells in some patients with refractory neuroblastoma already studied in a phase I clinical trials, indicate that T cells expressing 1st generation anti-GD2 chimeric antigen receptors were safe and mediated modest antitumor activity. In a phase I study, the murine IgG3 monoclonal antibody (MoAb) 3F8, specific for the ganglioside GD2, was administered intravenously to 17 patients with metastatic GD2 positive neuroblastoma or malignant melanoma. Antitumor responses occurred in 7 of 17 patients. However, the expression of the gangliosides GD2 in Asian melanoma populations has not been reported. Methods: This study involved tumor samples from 228 melanoma patients (129 acral melanomas, 65 mucosal melanomas, 13 melanomas on skin with chronic sun-induced damage, 21 melanomas on skin without chronic sun-induced damage) in Peking University Cancer Hospital &amp; Institute. Clinical data were collected. Immunohistochemistry assays using antibodies against gangliosides GD2 were performed on formalin-fixed, paraffin-embedded specimens. The ability of gangliosides GD2 chimeric antigen receptor T cells to kill gangliosides GD2+ melanoma cell was evaluated by MTT in vitro. Results: Among the 228 samples,40.3% of the cases(92/228) demonstrated positive staining with gangliosides GD2. Expression of gangliosides GD2 were relatively more frequent in acral (48.1%) and mucosal (36.9%) melanomas than in CSD (15.4%) and Non-CSD (19.0%) melanomas. The median survival time for patients with expression of gangliosides GD2 was significantly shorter than patients with absence of gangliosides GD2 expression (21.4 vs 57.6 months, P&amp;lt;0.05). Statistical differences were found between primary melanomas with metastatic melanomas (34.9% vs 53.0%, P&amp;lt;0.05). In addition, the gangliosides GD2 chimeric antigen receptor T cells exhibited specific lysis of melanoma cell with gangliosides GD2 expression in vitro experiment. Conclusion: These data demonstrate the expression of ganglioside GD2 are more frequent in acral and mucosal melanomas than in cutaneous melanomas in Chinese patients. The higher expression of gangliosides GD2 on metastatic melanomas compared to primary melanomas was in conformity with the viewpoint that GD2 expression was related to increased metastatic potential. Gangliosides GD2 chimeric antigen receptor T cells represents a clinically appealing treatment strategy for melanoma patients with ganglioside GD2 expression. Citation Format: Yan Kong, Jiayi Yu, Lu Si, Zhihong Chi, Chuanliang Cui, Xinan Sheng, Jun Guo. The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2303.

Eucaryotic expression of HSV glycoproteins gC2, gD1, gD2 and immunogenicity analysis

Eucaryotic expression of HSV glycoproteins gC2, gD1, gD2 and immunogenicity analysis

Oncotargets GD2 and GD3 are highly expressed in sarcomas of children, adolescents, and young adults.

GD2 and GD3 are the tumor-associated glycolipid antigens found in a broad spectrum of human cancers. GD2-specific antibody is currently a standard of care for high-risk neuroblastoma therapy. In this study, the pattern of GD2 and GD3 expression among pediatric/adolescent or young adult tumors was determined, providing companion diagnostics for targeted therapy. Ninety-two specimens of human osteosarcoma (OS), rhabdomyosarcoma (RMS), Ewing family of tumors, desmoplastic small round cell tumor (DSRCT), and melanoma were analyzed for GD2/GD3 expression by immunohistochemistry. Murine monoclonal antibody 3F8 was used for GD2 staining, and R24 for GD3. Staining was scored according to both intensity and percentage of positive tumor cells from 0 to 4. Both gangliosides were highly prevalent in OS and melanoma. Among other tumors, GD3 expression was higher than GD2 expression. Most OS samples demonstrated strong staining for GD2 and GD3, whereas expression for other tumors was highly variable. Mean intensity of GD2 expression was significantly more heterogeneous (P < 0.001) when compared to GD3 across tumor types. When assessing the difference between GD2 and GD3 expression in all tumor types combined, GD3 expression had a significantly higher score (P = 0.049). When analyzed within each cancer, GD3 expression was significantly higher only in DSRCT (P = 0.002). There was no statistical difference in either GD2 or GD3 expression between primary and recurrent sarcomas. GD2/GD3 expression among pediatric solid tumors is common, albeit with variable level of expression. Especially for patients with sarcoma, these gangliosides can be potential targets for antibody-based therapies.

A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3

We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40μM siRNA, 25μl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000pmol/tumor) was injected into the tumors every 3days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Progenitor/Stem Cell Markers in Brain Adjacent to Glioblastoma: GD3 Ganglioside and NG2 Proteoglycan Expression.

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (a-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

PTPS-20THE GD3 ACETYLATION PATHWAY AS A POTENTIAL THERAPEUTIC TARGET FOR PAEDIATRIC MEDULLOBLASTOMA

Medulloblastoma (MB), the most common malignant paediatric brain tumour, is associated with high mortality rates. Survivors frequently suffer from low quality-of-life as a result of aggressive treatment. Genomic studies have led to novel classification of molecularly and clinically distinct subgroups and are paving the way for novel targeted therapies. GD3, an oncofoetal ganglioside is re-expressed in some cancers of the neural crest. GD3, central to cell movement in developing brain, is internalised during maturation of residual supernumerary cells leading to cell death. When GD3 is re-expressed in cancers, the intracellular form of GD3 is acetylated (GD3A) and is thought to protect cells from mitochondrially-mediated apoptosis. Sialate-O-acetylesterase (SIAE) is responsible for cleaving the acetyl group from GD3A. A genome-wide transcriptional analysis of 103 MB patients revealed that this enzyme is significantly down-regulated in MB tissue compared to non-neoplastic foetal and adult brain (p > 0.02). This suggests that GD3A may be deacetylated at low levels and thus the balance of GD3 to GD3A favours GD3A. Using flow cytometry to examine expression, results revealed that in MB cell lines RES256 and UW402 GD3A expression was higher (84.5% and 74.5%) than GD3 (56.6% and 61.3%) The fluorescent intensity of GD3A expression was 9.9 and 11.4 fold higher and GD3, 4.4 and 6.1 fold higher than negative controls. Immunocytochemical results were in agreement, providing additional evidence that these cells have a balance in favour of GD3A. Previous studies from our group have shown that cell surface expression of GD3 correlates with invasion and in MB cell lines tested surface expression was 31.7% and 65.7% compared to GD3A (29.5% and 11.5%). Inducible SIAE and SIAE catalytically mutated cell lines have been generated and verified in protein and esterase activity assays and will be used to evaluate this pathway as a potential therapeutic target for MB.

Abstract A29: Differential expression of the gangliosides GD3 and GD2 in canine and human osteosarcoma cell lines: An immunotherapy target

Abstract Introduction: The disialyl gangliosides GD2/GD3 have been implicated in the enhancement of malignancy in a number human and animal caners (Milner et al Vet Immunology 2006). Moreover, pediatric research trials targeting GD2 osteosarcoma (OSA) surface antigens using humanized mouse antibodies is currently underway. While the coexpression of GD3 and GD2 have been documented in human OSA it has been not reported in canine OSA. Canine OSA is an accepted phenotypic and genotypic large animal model for the study of human OSA. We report on the co-expression of GD3/GD2 in three canine and one human OSA cell lines. Material and Methods: Four canine OSA cell lines (POS, HMPOS, COS31, and Doug) and one human OSA cell line (MG63) were trypsinized from T75 flasks and counted using trypan blue exclusion dye. Live cells (1x106) were spun down at 250 x gravity. Supernatant was removed and cell pellet mixed with 1ml PBS wash solution. Samples were then centrifuged, supernatant removed, and cells were washed two more times. Primary antibodies GD2 antibody (clone 14G2a, Santa Cruz) and GD3 Mel-1 (clone R24, Covance) and isotype controls were added to samples which were then incubated on ice in the dark for 30 minutes. Cells were again washed in PBS wash solution. Samples were blocked using normal goat serum, washed and then incubated with detection antibody goat anti-mouse IgG F(ab')2 conjugated to Alexa Flour 488® or Alexa Fluor 647 Goat anti-mouse IgG2a [Molecular Probes, Eugene, OR USA] ) for 30 minutes on ice in the dark. Cells were then washed in wash buffer and re-suspended in 1ml of PBS wash solution. Samples were read at 10,000 events/tube on FACScan (BD, Franklin Lakes, NJ USA). Immunofluorescence was documented using confocal microscopy was used to establish the co-expression of GD3 and GD2 on the cell surface. Results: All cell lines, expressed varying levels of GD3 and GD2 on the cell surface. The canine OSA cell line appeared to express more GD3 (12.54%) on the surface compared to the human MG63 cell line (0.02%). Remarkably, POS, the primary tumor cell line of HMPOS, expressed very low levels of GD3 (4.65%) and GD2 (0.24%), whereas the metastatic line (HMPOS) expressed higher GD3 (25.4%). All cell lines had cells that expressed either GD2 or GD3 but not both. The human MG63 cell line expressed the highest levels of GD2 alone (68.4%) compared to canine cells (average 0.58%). The average co-expression of GD2/GD3 was 48.1% for all cell lines. Discussion: This is the first report of the expression of both GD3 and GD2 in canine OSA cells. The related cell lines POS and HMPOS showed significant variation in expression, with HMPOS (the metastatic line) showing increased expression of GD3. The differential expression of GD3/GD2 on these two cell lines may indicate a phenotype change associate with metastatic potential. The highest co-expression of GD2/GD3 was found in MG63 and COS31, but was also found in HMPOS and Doug. Current investigation includes expression of GD2 &amp; GD3 in osteosarcoma stem cell populations and invasive potential. Conclusion: Results from this study will provide data to investigate the expression profiles of GD2/GD3 and its role in metastasis as well as targeting OSA tumor antigens. Citation Format: Rowan J. Milner, Noaki Chimura, Kristina D. Bowles, Marc Salute. Differential expression of the gangliosides GD3 and GD2 in canine and human osteosarcoma cell lines: An immunotherapy target. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A29.

Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.

CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigen-presenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaα2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells.

Ligands Binding to Cell Surface Ganglioside GD2 Cause Src-Dependent Activation of N-Methyl-D-Aspartate Receptor Signaling and Changes in Cellular Morphology.

Ganglioside GD2 is a plasma membrane glycosphinogolipid. In healthy adults it is expressed at low levels, but it is over-expressed in many cancers. For cancer therapy, GD2 is targeted with anti-GD2 monoclonal antibodies (mAbs), and one adverse side effect is severe visceral pain. Pain is not neuropathic, cannot be blocked with morphine, and stops on discontinuation of mAb therapy. Here, we provide evidence that ligand binding to cell surface GD2 induces rapid and transient activation of Src-family kinases, followed by Src-dependent phosphorylation of NMDA-receptor NR2B subunits selectively, activation of Ca++ fluxes, production of cAMP, and changes in cellular morphology. These GD2-ligand activated signals differ in kinetics and in pharmacology from activation of the same signals in the same cells by BDNF, the growth factor agonist of the TrkB receptor, suggesting biological specificity. Hence, cell surface GD2 regulates pathways that can be associated with neoplasia and with morphine-intractable pain; and this can explain why expression of GD2 correlates with these two pathologies.