Ganglioside GD3 Research Articles

A randomised phase II trial of a trivalent ganglioside vaccine targeting GM2, GD2 and GD3 combined with immunological adjuvant OPT-821 versus OPT-821 alone in metastatic sarcoma patients rendered disease-free by surgery

BackgroundRecurrence after resection of metastatic sarcoma is common. The gangliosides GM2, GD2 and GD3 are strongly expressed across sarcoma subtypes. We hypothesised that generation of anti-ganglioside antibodies would control micrometastases and improve outcomes in sarcoma patients who were disease-free after metastasectomy. MethodsWe conducted a randomised phase II trial of the immunological adjuvant OPT-821 with a KLH-conjugated ganglioside vaccine targeting GM2, GD2 and GD3, versus OPT-821 alone in patients with metastatic sarcoma following complete metastasectomy. Patients received 10 subcutaneous injections at Weeks 1, 2, 3, 8, 16, 28, 40, 52, 68 and 84 and were followed for evidence of recurrent disease. The primary end-point was relapse-free survival. Secondary end-points included overall survival and serologic response. ResultsA total of 136 patients were randomised, 68 to each arm. The mean age was 51.2, 52.2% were male, 90.4% had relapsed disease, 86.8% had high-grade tumours and 14% had ≥4 metastases resected. Histologies included leiomyosarcoma (33%), spindle cell sarcoma (14%), undifferentiated pleomorphic sarcoma (13%), osteosarcoma (10%), synovial sarcoma (9%), liposarcoma (9%) and others (12%). Most adverse events were Grade ≤2 (83.8% and 70.6% in the vaccine and adjuvant arms, respectively). The most common (≥20% of patients) were injection site reaction (89.7%), fatigue (44.1%) and pyrexia (27.9%) on the vaccine arm, and injection site reaction (69.1%) on the adjuvant only arm. The 1-year relapse-free survival rate (34.5% and 34.8% in the vaccine and OPT-821 monotherapy arm, respectively) did not differ between arms (P = 0.725). One-year overall survival rates were 93.1% and 91.5% in the vaccine and OPT-821 monotherapy arm, respectively (P = 0.578). Serologic responses at week 9 were more frequent on the vaccine arm (96.5% of patients) than in the adjuvant arm (32.8%), and the difference between groups was durable. ConclusionsA sustained serologic response to vaccination was induced with the vaccine, but no difference in recurrence-free or overall survival was observed between treatment arms.Clinicaltrials.gov identifier: NCT01141491

T CELLS ENGINEERED TO EXPRESS A CAR ANTI-GD2 AND THE COSTIMULATORY MOLECULE GITRL ERADICATE GLIOBLASTOMA CELLS AND ARE IFNY PRODUCERS

The adoptive transfer of T lymphocytes expressing chimeric antigen receptors (CAR) has resulted in impressive complete remission rates in B-cell malignancies. However, the therapeutic efficacy of CAR-T cells is still low or non-existent against solid tumors, which make up the vast majority of neoplasms. One of the main reasons for this failure is the presence of ligands and immunosuppressive cells in the tumor microenvironment. The complete activation of T cells requires the engagement of costimulatory molecules whose expression is temporally segregated and whose nature of biochemical signals are complementary. This is the case of CD28, expressed constitutively, and GITR, expressed right after the initial activation of T cells. Therefore, our hypothesis is that the combined expression of a CAR containing the costimulatory domain CD28 and the GITR ligand (GITRL) will potentiate the antineoplastic action of CAR-T cells. In addition to providing complementary costimulatory signals for effector CAR-T cells, GITRL can suppress the action of regulatory T cells in the tumor microenvironment, thus possibly reducing local immunosuppression. Then, the aim of this study is to evaluate the therapeutic efficiency of anti-GD2 CAR-T cells coexpressing GITRL in a preclinical model of glioblastoma multiforme. In this project, we used as a model a CAR against the ganglioside GD2, which is highly immunogenic and expressed in tumors of neuroectodermal origin, such as glioblastoma. To test our hypothesis, we generated two lentiviral vectors to express CAR anti-GD2 and another one coexpressing GITRL as a costimulatory domain. After that, we produced lentiviral particles to transduced human T lymphocytes and we detected CAR expression in the T cells transduced with both vectors. To verify the functional activity of these CAR-T cells, we performed a coculture assay against T98G cell line, which express high levels of GD2, and HCT-166 cell, that dont express. These cell lines were previously modified to express luciferase to enable the analysis through bioluminescence imaging. We tested two ratios of effector:target cells (E:T) and we observed that both CAR.GD2 and CAR.GD2-GITRL T cells demonstrated powerful capacity of lysis only against cells that express GD2. Then, we interrogated whether these CAR-T cells could keep their lysis capacity after additional rechallenges. So, another two rounds of coculture were performed and the cells showed persistence of cytotoxicity. In addition, tumor cell killing was associated with secretion of IFNy as evaluated by ELISA quantification of coculture supernatants. These data encouraged us to compare the antineoplastic activity and persistence of CAR-T cells anti-GD2 in an orthotopic pre-clinical model of glioblastoma multiforme, which is under way. We expect that the potential results of this project will foster the development of a new advanced cellular immunotherapy strategy for the treatment of solid GD2+ neoplasms and will pave the way to increase the antineoplastic efficiency of CAR-T cells against other malignancies.

Immunotherapies of retinoblastoma: Effective methods for preserving vision in the future

Retinoblastoma is the most common intraocular tumor in children. Patients can be cured by enucleation, but it can lead to vision loss. Chemotherapy is the main method of treatment for RB currently. Unfortunately, chemoresistant and tumor metastasis often happen, resulting in a relatively poor prognosis. Therefore, immunotherapy becomes one of the optimal choices. Targeting not only tumor cells but also the active tumor microenvironment is a novel strategy for RB treatment. Here, we conclude several potential targets for RB immunotherapy, including gangliosides GD2, PD-1 and PD-L1, B7H3, EpCAM and SYK. We also review the techniques for CART, bispecific antibodies and genetically modified Dendritic cells according to the characteristics of different targets and discuss the feasibility of immunotherapy with different targets.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Dexamethasone Promotes a Stem-Like Phenotype in Human Melanoma Cells via Tryptophan 2,3 Dioxygenase

In addition to its well-established immunosuppressive actions, tryptophan 2,3-dioxygenase (TDO) appears to elicit direct effects on tumor cell function. Although TDO has been associated with cancer stemness, its involvement in melanoma stem cell biology remains largely unknown. Since we showed that by upregulating TDO, dexamethasone (dex) promotes proliferation and migration of SK-Mel-28 human melanoma cells, we sought to investigate dex effects on melanoma spherogenesis and stemness, and whether these events are mediated by TDO. We demonstrate here that dex significantly upregulates TDO in A375, a more aggressive melanoma cell line, confirming that dex effects are not limited to SK-Mel-28 cells. Moreover, dex stimulates spherogenesis of both cell lines, which is mediated by TDO, evident by its suppression with 680C91, a TDO inhibitor. The formed melanospheres appear to be enriched with embryonic stem cell marker mRNAs, the expression of which is potentiated by dex. Expression of cancer stem cell markers (CD133, CD44, ganglioside GD2) was significantly increased in A375 spheres, as detected by flow cytometry. Taken together, our results suggest that TDO could represent a promising target in the management of melanoma and that dex, routinely used as a co-medication also in advanced melanoma, may stimulate melanoma cell function/tumor-supporting properties, a rather debilitating and undesired side effect.

Abstract 893: GD2 is a potential biomarker for glioblastoma cancer stem cells

Abstract Introduction: Treatment resistance and tumor recurrence of Glioblastoma (GBM) have been associated with cancer stem cells (CSCs), a subpopulation of cancer cells defined by capabilities of sphere-formation in 3D culture, self-renewal, migration, and survival in serum-deprived conditions. Therefore, therapies specifically targeting CSCs are urgently needed. Previous works have identified Ganglioside GD2 as a biomarker of triple negative breast cancer CSCs and suggested that GD2 is highly expressed in GBM cells. Here, we hypothesize that GD2 is also a potential biomarker of GBM CSCs and is involved with cancer stemness. Methods: GBM cells U-87 MG and U-118 MG were sorted as GD2- and GD2+ cells via FACS. Cells were cultured in 2D and 3D. 3D neurosphere size was measured and GD2 in 2D and 3D cultures was quantified via flow cytometry. Transwell migration assay was used to examine the migration capability of GD2- and GD2+ cells. Unsorted cells were cultured in either complete (10% serum) or deprived (1% serum) media and their GD2 was quantified via flow cytometry. Results: To test our hypothesis, we determined whether GD2+ cells express CSCs capabilities of sphere-formation in 3D culture, migration, and survival in serum-deprived conditions. First, we examined the sphere-forming capability by culturing GD2-, GD2+, and unsorted cells in 3D and found that they all formed neurospheres. However, GD2+ neurosphere size was significantly larger compared with unsorted and GD2- neurospheres for day 1 and 2. Interestingly, the significant difference was no longer seen on day 6. Analysis for GD2 expression in the spheres revealed that GD2- spheres contained GD2+ cells, suggesting that GD2- cells could spontaneously dedifferentiate to generate CSCs. To test this, we sorted GD2- and GD2+ cells into 2D conditions and quantified daily GD2 expression via flow cytometry. We found that GD2- seeded cells spontaneously expressed GD2 over six days, explaining the presence of GD2 in GD2- neurospheres. GD2+ cells also demonstrated the enhanced capability to migrate of CSCs with a significantly higher number of migrated cells compared with GD2- cells in the transwell migration assay. To examine the ability to survive in serum-deprived conditions, we cultured unsorted cells in either complete or deprived media. We found that these cells proliferated in the serum-deprived condition at approximately the same level as complete media. GD2 analysis by flow cytometry also showed a remarkably higher percentage of GD2+ cells in deprived media, suggesting that GD2 is involved in driving cell survival and maintaining cancer stemness in serum-deprived conditions. Conclusion: Our data suggests that GD2+ cells demonstrate characteristics of CSCs and GD2 is a potential biomarker of CSCs in GBM. Therefore, GD2 could be utilized as a differential therapeutic target for GBM. Further mechanistic studies are necessary to elucidate how GD2 is synthesized and maintains the stemness of GBM CSCs. Citation Format: Khoa Nguyen, Minh Tran, Thomas Cheng, Kurtis Willingham, Reza Izadpanah, Matthew Burow. GD2 is a potential biomarker for glioblastoma cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 893.

Ligand‐assisted imprinting‐probe‐labeling strategy reveals Siglec‐7 ‐ glycoprotein interactions

AbstractThe development of a ligand‐assisted imprinting probe (i.e., LIP 1) for the site‐specific labeling of recombinant Siglec‐7 protein (rSiglec‐7) is reported. LIP 1 is a LIP bearing a ganglioside GD3 glycan, a natural ligand for Siglec‐7, that is embedded in an ortho‐hydroxyl cinnamate core, which exhibits UV‐light‐dependent conformational changes, and is appended with a clickable alkyne handle. LIP 1 enables the selective introduction of an alkyne at the threonine residue 91 (Thr91), which is proximal to the carbohydrate‐binding pocket of Siglec‐7, as identified by LC–MS/MS analysis. By using LIP 1, an azide‐containing environment‐sensitive merocyanine dye is conjugated to rSiglec‐7 by a click reaction, which is capable of directly imaging live HeLa cells as a result of highly specific interactions with cell‐surface glycoproteins containing sialic acid. Moreover, the conjugation of doxorubicin (Dox) to alkynylated rSiglec‐7 retains the original activity of protein binding to the interacting natural GD3 glycan ligand, as revealed by microarray analysis; hence, the rSiglec‐7 conjugate can serve as cargo (Dox)‐delivery vehicle to Siglec‐ligand‐expressing HeLa cells. The LIP labeling strategy described herein is expected to be a general strategy for the future development of glycoprobes for investigating lectin‐glycan interactions.

Therapeutic efficacy of antibody-drug conjugates targeting GD2-positive tumors

BackgroundBoth ganglioside GD2-targeted immunotherapy and antibody-drug conjugates (ADCs) have demonstrated clinical success as solid tumor therapies in recent years, yet no research has been carried out to develop anti-GD2 ADCs...

Gangliosides in nervous system development, regeneration, and pathologies

Gangliosides, sialic acid-containing sphingolipids, are major constituents of neuronal membranes. According to the number of sialic acids and the structure of the oligosaccharide chain, gangliosides can be classified as simple or complex and grouped in different ganglio-series. Hundreds of gangliosides have been identified in vertebrate cells, with different expression patterns during development and related to several physiological processes, especially in the nervous system. While GD3 and its O-acetylated form, 9acGD3, are highly expressed in early developmental stages, GM1, GD1a, GD1b, and GT1b are the most abundant ganglioside species in the mature nervous system. Mutations in enzymes involved in ganglioside metabolism can lead to the accumulation of specific species, a condition termed gangliosidosis and usually marked by severe neurological impairment. Changes in ganglioside levels have also been described in several neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. In this review, we summarized recent information about the roles of GD3, 9acGD3, GM1, GD1a, GD1b, GT1b, and other ganglioside species in nervous system development and regeneration, as well as clinical trials evaluating possible therapeutic applications of these molecules.

Role of GD3 Synthase ST8Sia I in Cancers.

Simple SummaryThe carbohydrate moiety of cell surface glycolipids is modified in cancers of neuro–ectoderm origin, leading to the expression of more complex structures with two or more sialic acid residues. These alterations result from the upregulation of the ST8SIA1 gene that encodes GD3 synthase, the enzyme controlling the biosynthesis of complex gangliosides, and are usually associated with a more aggressive phenotype and a poor outcome for patients, making GD3 synthase an interesting target for cancer therapy. This review reports our general knowledge of GD3 synthase gene expression and regulation, its role in both epithelial–mesenchymal transition (EMT) and cancer progression, and the different approaches targeting GD3S expression in cancers.GD3 synthase controls the biosynthesis of complex gangliosides, bearing two or more sialic acid residues. Disialylated gangliosides GD3 and GD2 are tumor-associated carbohydrate antigens (TACA) in neuro–ectoderm-derived cancers, and are directly involved in cell malignant properties, i.e., migration, invasion, stemness, and epithelial–mesenchymal transition. Since GD3 and GD2 levels are directly linked to GD3 synthase expression and activity, targeting GD3 synthase appears to be a promising strategy through which to interfere with ganglioside-associated malignant properties. We review here the current knowledge on GD3 synthase expression and regulation in cancers, and the consequences of complex ganglioside expression on cancer cell signaling and properties, highlighting the relationships between GD3 synthase expression and epithelial–mesenchymal transition and stemness. Different strategies were used to modulate GD3 synthase expression in cancer cells in vitro and in animal models, such as inhibitors or siRNA/lncRNA, which efficiently reduced cancer cell malignant properties and the proportion of GD2 positive cancer stem cells, which are associated with high metastatic properties, resistance to therapy, and cancer relapse. These data show the relevance of targeting GD3 synthase in association with conventional therapies, to decrease the number of cancer stem cells in tumors.

Abstract P5-06-10: Tumor microenvironment modulates ganglioside expression leading to immunosuppression in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive human malignancies, characterized by a strikingly poor prognosis linked to high rates of distant metastases and disease relapse. Among several mechanisms that contribute to TNBC aggressiveness is the ability of TNBC cells to evade the anti-tumor immune response. Numerous efforts have been directed to identify and target molecules and signaling pathways that contribute to TNBC immune evasion. A considerable proportion of TNBCs are resistant to immune-checkpoint therapy, implying that additional molecules are involved in immune suppression in these malignancies. We have previously reported that ganglioside GD2 identifies breast cancer stem-like cells (BCSCs) and promotes tumorigenesis. GD3 synthase (GD3S), a key enzyme that regulates GD2 biosynthesis, is significantly upregulated in TNBCs. Gangliosides have been found to promote an immunosuppressive tumor microenvironment. In this study, we hypothesize that tumor-microenvironment influences expression of gangliosides downstream of GD3S, including GD2/GD3, suppressing the anti-tumor immune response while inhibiting GD3S expression enhances immune-mediated killing of TNBC cells. We performed immunohistochemistry (IHC) on primary TNBC patient samples (N=89) and measured GD2 expression using an automated imaging system (Vectra-Polaris). Immunohistochemistry analysis revealed that approximately 60% of samples had detectable GD2 expression of different intensity and was associated with poor overall survival of patients with TNBC (p=0.002). Our IHC data also demonstrated that GD2 is expressed not only in tumors but also in the tumor-associated stroma in ~60% of TNBC samples, suggesting the effect of crosstalk between TME and TNBC cells on GD2 expression. Additionally, to substantiate the role of TME on GD2 expression, we cultured bone marrow-derived mesenchymal stromal cell (BM-MSCs) with MDA-MB-231 cells and found a 2-fold increase in GD2 expression in BM-MSC and MDA-MB-231 cells (p<0.001), suggesting microenvironmental factors contribute to GD2 expression on both tumor and stromal cells. To examine the immunomodulatory role of gangliosides we used the TCGA dataset to analyze the co-expression of GD3S mRNA with T-cell exhaustion markers in all breast cancer patients. We found a significant (p<0.001) positive correlation between ST8SIA1 and CTLA-4 (pearson= 0.4), LAG-3 (pearson= 0.33), and PD1 (pearson=0.3) in breast cancer patients. Furthermore, we overexpressed GD3S in TNBC and non-TNBC cells and observed its effect on T-cell mediated killing of breast cancer (BC) cells by a live-cell imaging system (IncuCyte). Overexpression of GD3S caused upregulation of GD2 in BT549, HIM3 (TNBC), and MCF7 (non-TNBC) cells, and their co-culture with T-cells resulted in a significant reduction (p<0.001) in cell death compared to the controls. In conclusion, GD2 is a tumor-specific marker in patients with TNBC and that GD3S overexpression is associated with T-cell exhaustion and maintenance of an immunosuppressive microenvironment in breast cancer. This immunosuppressive microenvironment in TNBCs fostered by gangliosides downstream of GD3S is also a manifestation of the cross-talk between tumor and tumor-associated stromal cells. Citation Format: Vivek Anand, Fouad El-Dana, Stanley Ly, Michael Andreeff, Venkata Lokesh Battula. Tumor microenvironment modulates ganglioside expression leading to immunosuppression in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-10.

Current and future perspectives of chimeric antigen receptors against glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant form of cancer in the central nervous system; even with treatment, it has a 5-year survival rate of 7.2%. The adoptive cell transfer (ACT) of T cells expressing chimeric antigen receptors (CARs) has shown a remarkable success against hematological malignancies, namely leukemia and multiple myeloma. However, CAR T cell therapy against solid tumors, and more specifically GBM, is still riddled with challenges preventing its widespread adoption. Here, we first establish the obstacles in ACT against GBM, including on-target/off-tumor toxicity, antigen modulation, tumor heterogeneity, and the immunosuppressive tumor microenvironment. We then present recent preclinical and clinical studies targeting well-characterized GBM antigens, which include the interleukin-13 receptor α2 and the epidermal growth factor receptor. Afterward, we turn our attention to alternative targets in GBM, including less-explored antigens such as B7-H3 (CD276), carbonic anhydrase IX, and the GD2 ganglioside. We also discuss additional target ligands, namely CD70, and natural killer group 2 member D ligands. Finally, we present the possibilities afforded by novel CAR architectures. In particular, we examine the use of armored CARs to improve the survival and proliferation of CAR T cells. We conclude by discussing the advantages of tandem and synNotch CARs when targeting multiple GBM antigens.

Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins.

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2−) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin β1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin β1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin β1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin β1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin β1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2− cells. All these results suggest that GD2 and integrin β1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

Pharmacokinetics of the disialoganglioside, GD2, a circulating tumor biomarker for neuroblastoma, in nonhuman primates.

Background: The ganglioside GD2 is a potential circulating tumor biomarker for the childhood cancer, neuroblastoma. Interpreting the levels of a circulating tumor biomarker depends in part on a knowledge of the biomarker’s clinical pharmacology. Background: The ganglioside GD2 is a potential circulating tumor biomarker for the childhood cancer neuroblastoma. Interpreting the levels of a circulating tumor biomarker depends in part on a knowledge of the biomarker’s clinical pharmacology. Methods: We studied the plasma and cerebrospinal fluid (CSF) pharmacokinetics of the C18 lipoform of GD2 in two nonhuman primates with indwelling subcutaneous CSF lateral ventricular reservoir systems. GD2 was quantified with a validated high-performance liquid chromatography (HPLC)/tandem mass spectrometry assay. GD2 was administered as a short intravenous infusion and frequent plasma and CSF samples were drawn over 72 hours. Results: GD2 plasma concentration declined monoexponentially with a half-life of 16 hours. Clearance was 0.0136 and 0.0131 L/h and volume of distribution (Vd) was 0.035 and 0.038 L/kg in the two animals. Vd was equivalent to plasma volume. Greater than 98% of GD2 in plasma is in a bound form consistent with its known association with lipoproteins and accounting for its limited volume of distribution. GD2 did not cross over from plasma into the CSF. Conclusions: The pharmacokinetic profile of GD2 is favorable for a circulating tumor biomarker. This study demonstrates the value of characterizing the clinical pharmacology of circulating biomarkers to better understand their clinical behavior.

The Potential Role of Human NME1 in Neuronal Differentiation of Porcine Mesenchymal Stem Cells: Application of NB-hNME1 as a Human NME1 Suppressor

This study aimed to investigate the effects of the human macrophage (MP) secretome in cellular xenograft rejection. The role of human nucleoside diphosphate kinase A (hNME1), from the secretome of MPs involved in the neuronal differentiation of miniature pig adipose tissue-derived mesenchymal stem cells (mp AD-MSCs), was evaluated by proteomic analysis. Herein, we first demonstrate that hNME1 strongly binds to porcine ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 (pST8SIA1), which is a ganglioside GD3 synthase. When hNME1 binds with pST8SIA1, it induces degradation of pST8SIA1 in mp AD-MSCs, thereby inhibiting the expression of ganglioside GD3 followed by decreased neuronal differentiation of mp AD-MSCs. Therefore, we produced nanobodies (NBs) named NB-hNME1 that bind to hNME1 specifically, and the inhibitory effect of NB-hNME1 was evaluated for blocking the binding between hNME1 and pST8SIA1. Consequently, NB-hNME1 effectively blocked the binding of hNME1 to pST8SIA1, thereby recovering the expression of ganglioside GD3 and neuronal differentiation of mp AD-MSCs. Our findings suggest that mp AD-MSCs could be a potential candidate for use as an additive, such as an immunosuppressant, in stem cell transplantation.

Upregulation of Ganglioside GD2 Synthase (GD2S), as a New Putative Cancer Stem Cell Marker in Breast Carcinomas.

Background: GD2 synthase (GD2S) is the key enzyme required for ganglioside GD2 synthesis. It is commonly expressed in normal tissues and various cancers. Ganglioside GD2 is identified as a breast cancer stem cells (BCSCs) marker that promotes tumorigenesis. As GD2S has been found to be a useful molecular marker in neuroblastoma and retinoblastoma tumors, we suggest that it can be considered as a suitable candidate for the detection of CSCs in breast cancer tissues. Methods: Expression of GD2S was examined in 65 breast tumors compared to adjacent normal tissues, applying quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The association between GD2S expression level and patients’ clinical characteristics was also assessed. Results: Our findings showed that GD2S mRNA expression was significantly higher in breast cancer tissues in comparison to normal adjacent tissue samples (4.92-fold change, p<0.001) in advanced grades (p<0.001) and stages (p<0.001). It was also shown that GD2S protein expression was significantly higher in breast cancer tissues in comparison to normal adjacent tissues (4.86-fold change, p=0.010) in advanced grades (p=0.010), stages (p=0.005) and larger tumor size (p=0.002). Conclusion: The current study showed that increased expression of GD2S in advanced breast cancer potentiates it as a promising tumor marker in these patients.

Chemoenzymatic Synthesis of 9NHAc-GD2 Antigen to Overcome the Hydrolytic Instability of O-Acetylated-GD2 for Anticancer Conjugate Vaccine Development.

Ganglioside GD2 is an attractive tumor-associated carbohydrate antigen for anti-cancer vaccine development. However, its low immunogenicity and the significant side effects observed with anti-GD2 antibodies present significant obstacles for vaccines. To overcome these, a new GD2 derivative bearing an N-acetamide (NHAc) at its non-reducing end neuraminic acid (9NHAc-GD2) has been designed to mimic the 9-O-acetylated-GD2 (9OAc-GD2), a GD2 based antigen with a restricted expression on tumor cells. 9NHAc-GD2 was synthesized efficiently via a chemoenzymatic method and subsequently conjugated with a powerful carrier bacteriophage Qβ. Mouse immunization with the Qβ-9NHAc-GD2 conjugate elicited strong and long-lasting IgG antibodies, which were highly selective toward 9NHAc-GD2 with little cross-recognition of GD2. Immunization of canines with Qβ-9NHAc-GD2 showed the construct was immunogenic in canines with little adverse effects, paving the way for future clinical translation to humans.

Ganglioside GD2: a novel therapeutic target in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by lack of hormone receptor expression and is known for high rates of recurrence, distant metastases, and poor clinical outcomes. TNBC cells lack targetable receptors; hence, there is an urgent need for targetable markers for the disease. Breast cancer stem-like cells (BCSCs) are a fraction of cells in primary tumors that are associated with tumorigenesis, metastasis, and resistance to chemotherapy. Targeting BCSCs is thus an effective strategy for preventing cancer metastatic spread and sensitizing tumors to chemotherapy. The CD44hi CD24lo phenotype is a well-established phenotype for identification of BCSCs, but CD44 and CD24 are not targetable markers owing to their expression in normal tissues. The ganglioside GD2 has been shown to be upregulated in primary TNBC tumors compared with normal breast tissue and has been shown to identify BCSCs. In this review, we discuss GD2 as a BCSC- and tumor-specific marker in TNBC; epithelial-to-mesenchymal transition and the signaling pathways that are upstream and downstream of GD2 and the role of these pathways in tumorigenesis and metastasis in TNBC; direct and indirect approaches for targeting GD2; and ongoing clinical trials and treatments directed against GD2 as well as future directions for these strategies.

Characterization of Human Medullary Thyroid Carcinoma Glycosphingolipids Identifies Potential Cancer Markers.

Medullary thyroid carcinoma (MTC) accounts for only 1–2% of thyroid cancers; however, metastatic MTC is a mortal disease with no cure. In this study, glycosphingolipids were isolated from human MTCs and characterized by mass spectrometry and binding of carbohydrate recognizing ligands. The tissue distribution of selected compounds was investigated by immunohistochemistry. The amount of acid glycosphingolipids in the MTCs was higher than in the normal thyroid glands. The major acid glycosphingolipid was the GD3 ganglioside. Sulfatide and the gangliosides GM3 and GD1a were also present. The majority of the complex non-acid glycosphingolipids had type 2 (Galβ4GlcNAc) core chains, i.e., the neolactotetraosylceramide, the Lex, H type 2 and x2 pentaosylceramides, the Ley and A type 2 hexaosylceramides, and the A type 2 heptaosylceramide. There were also compounds with globo (GalαGalβ4Glc) core, i.e., globotriaosylceramide, globotetraosylceramide, the Forssman pentaosylceramide, and the Globo H hexaosylceramide. Immunohistochemistry demonstrated an extensive expression av Ley in the MTC cells and also a variable intensity and prevalence of Globo H and Lex. One individual with multiple endocrine neoplasia type 2B expressed the Forssman determinant, which is rarely found in humans. This study of human MTC glycosphingolipids identifies glycans that could serve as potential tumor-specific markers.

Does GD2 synthase (GD2S) detect cancer stem cells in blood samples of breast carcinomas?

Cancer stem cells (CSCs) are a theorized subset of cells within the tumor that is thought to drive disease recurrence and metastatic spread. The aim of this study is to investigate mRNA and protein levels of ganglioside GD2 synthase (GD2S), in breast cancer (BC) patients. 65 PBMCs of preoperative BC patients without chemotherapy were compared to PBMCs after chemotherapy and controls. GD2S were significantly higher in BC patients after chemotherapy compared to pre-chemotherapy at both mRNA and protein. GD2S was higher in pre-chemotherapy blood samples compared to control samples. Higher expression of GD2S in BC samples compared to healthy control indicates the potential utility of GD2S as a marker of malignancy.