GD2 Expression Research Articles

Associations between glycan signature alterations and the cellular antigenic properties of passaged chondrocytes

BackgroundCartilage repair is a significant clinical challenge because of the limited intrinsic healing capacity. Current therapeutic strategies, such as cell transplantation therapy, aim to overcome this challenge by replacing damaged tissue with healthy cells. However, the long-term survival and functionality of transplanted cells remain major hurdles.ObjectiveThis study investigated the impact of chondrocyte passaging on glycan profiles and their antigenic properties. We hypothesized that alterations in glycan composition due to passaging may contribute to the enhanced ability to activate macrophages, thereby affecting the outcome of cell transplantation therapy.MethodsPeritoneal macrophages and primary articular chondrocytes were isolated from C57BL/6 mice to establish direct and indirect coculture models. Macrophage activation was assessed by measuring the concentrations of IL-6 and nitric oxide in the culture supernatants or their gene expression. Glycome analysis of various glycoconjugates was performed by glycoblotting methods combined with the SALSA procedure for N-glycans and GSLs and the BEP method for O-glycans.ResultsOur results revealed that direct coculture of macrophages with passaged chondrocytes increased the production of proinflammatory cytokines, including IL-6 and NO, as the number of passages increased. With increasing passage number, the expression of GD3 substantially decreased, and the expression of GM3, especially GD1a, significantly increased. Coculturing passaged GM3S knockout chondrocytes with macrophages significantly suppressed IL-6 expression, implying reduced macrophage activation.ConclusionThe observed activation of macrophages due to alterations in the glycan profile of chondrocytes provides a possible explanation for the antigenicity and immune rejection of transplanted cells.

GD2 in Breast Cancer: A Potential Biomarker and Therapeutic Target.

Expression of disialoganglioside GD2 in normal tissues is primarily limited to the central nervous system, peripheral sensory nerve fibers, dermal melanocytes, lymphocytes, and mesenchymal stem cells. Its widespread overexpression in various cancer types allows it to be classified as a tumor-associated antigen with potential diagnostic and therapeutic implications. This article reviews the synthesis pathways of GD2 and its role in cancer cell adhesion, proliferation, and metastasis with a focus on breast cancer. GD2 appears to be overexpressed on the outer membrane of most breast cancer cells and breast cancer stem cells (BCSCs) and is closely linked to epithelial-mesenchymal transition (EMT). GD3 synthase (GD3S) is considered to be the rate-determining step in GD2 synthesis. Clinical studies indicate that GD2 expression is increased in 35-70% of breast cancer samples, with higher levels in triple-negative breast cancer (TNBC). This overexpression correlates with more aggressive tumor features and worse prognosis. Therapeutic targeting of GD2 with monoclonal antibodies (moABs) like dinutuximab and naxitamab has demonstrated anti-cancer activity in preclinical cancer models and human clinical trials against high-risk neuroblastoma reducing tumor growth and enhancing survival. GD2-specific chimeric antigen receptor (CAR) T-cell therapy and GD3S inhibition present other promising therapeutic strategies to improve clinical outcomes. Furthermore, GD2-targeted vaccines are currently being investigated in cancer therapy. This narrative review article underscores the critical role of GD2 in breast cancer pathogenesis and highlights the promising therapeutic opportunities it offers. It advocates for the initiation of clinical trials to further explore the potential of GD2-targeted treatment in combination with standard breast cancer therapies.

Expression of GD2 and GD3 in peripheral neuroblastic tumors.

The aim of this study was to explore the correlation between the expression of GD2 and GD3 and the histopathological types, risk groups, and chemotherapy in peripheral neuroblastic tumors (pNTs) and provide a theoretical basis for the selection of immunotargeted therapy for pNTs. The expression of GD2 and GD3 in samples of pNTs in all 87 cases, including 39 neuroblastomas (NB), 13 ganglion neuroblastomas nodular (GNBn), 19 ganglion neuroblastomas intermixed (GNBi), 16 ganglioneuroma (GN), and 16 paired NB after chemotherapy, were detected by immunohistochemistry (IHC). SPSS 20.0 statistical software was used for statistical analysis, and P < 0.05 was considered statistically significant. The expression of GD2 was higher than that of GD3 (P < 0.001) in all samples. In NB and GNBn, the expression of GD2 was higher than that of GD3 (P < 0.001 and P = 0.02, respectively). The expression of GD2 in NB was higher than that in GNBn (P = 0.015), and GNBn was higher than GNBi (P < 0.001). The expression of GD2 in the high-risk group was significantly higher than that in the medium-risk group and low-risk group (P = 0.019). The expression of GD2 before chemotherapy was higher than that after chemotherapy (P = 0.022). GD2 was expressed in different degrees in tumor-infiltrating lymphocytes. GD2 may be better than GD3 as a target of immunotherapy for pNTs, especially in the same pathological type. NB and GNBn may be more suitable for anti-GD2 immunotherapy. The expression of GD2 on tumor-infiltrating lymphocytes may be related to the side effects of anti-GD2 immunotherapy.

GD2 and its biosynthetic enzyme GD3 synthase promote tumorigenesis in prostate cancer by regulating cancer stem cell behavior

While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel targetable pathways that contribute to tumor progression in PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC is unexplored. Here, we show that GD2 is expressed in a small subpopulation of PC cells in a subset of patients and a higher proportion of metastatic tumors. Variable levels of cell surface GD2 expression were seen on many PC cell lines, and the expression was highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction was enriched upon growth of PC cells as tumorspheres and GD2high fraction was enriched in tumorsphere-forming ability. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2high CRPC cell models markedly impaired the in vitro oncogenic traits and growth as bone-implanted xenograft tumors and reduced the cancer stem cell and epithelial-mesenchymal transition marker expression. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.

Hu14.18K.322A Causes Direct Cell Cytotoxicity and Synergizes with Induction Chemotherapy in High-Risk Neuroblastoma.

The disialoganglioside, GD2, is a promising therapeutic target due to its overexpression in certain tumors, particularly neuroblastoma (NB), with limited expression in normal tissues. Despite progress, the intricate mechanisms of action and the full spectrum of the direct cellular responses to anti-GD2 antibodies remain incompletely understood. In this study, we examined the direct cytotoxic effects of the humanized anti-GD2 antibody hu14.18K322A (hu14) on NB cell lines, by exploring the associated cell-death pathways. Additionally, we assessed the synergy between hu14 and conventional induction chemotherapy drugs. Our results revealed that hu14 treatment induced direct cytotoxic effects in CHLA15 and SK-N-BE1 cell lines, with a pronounced impact on proliferation and colony formation. Apoptosis emerged as the predominant cell-death pathway triggered by hu14. Furthermore, we saw a reduction in GD2 surface expression in response to hu14 treatment. Hu14 demonstrated synergy with induction chemotherapy drugs with alterations in GD2 expression. Our comprehensive investigation provides valuable insights into the multifaceted effects of hu14 on NB cells, shedding light on its direct cytotoxicity, cell-death pathways, and interactions with induction chemotherapy drugs. This study contributes to the evolving understanding of anti-GD2 antibody therapy and its potential synergies with conventional treatments in the context of NB.

Desensitizing the autonomic nervous system to mitigate anti-GD2 monoclonal antibody side effects.

Anti-GD2 monoclonal antibodies (mAbs) have shown to improve the overall survival of patients with high-risk neuroblastoma (HR-NB). Serious adverse events (AEs), including pain, within hours of antibody infusion, have limited the development of these therapies. In this study, we provide evidence of Autonomic Nervous System (ANS) activation as the mechanism to explain the main side effects of anti-GD2 mAbs. Through confocal microscopy and computational super-resolution microscopy experiments we explored GD2 expression in postnatal nerves of infants. In patients we assessed the ANS using the Sympathetic Skin Response (SSR) test. To exploit tachyphylaxis, a novel infusion protocol (the Step-Up) was mathematically modelled and tested. Through confocal microscopy, GD2 expression is clearly visible in the perineurium surrounding the nuclei of nerve cells. By computational super-resolution microscopy experiments we showed the selective expression of GD2 on the cell membranes of human Schwann cells in peripheral nerves (PNs) significantly lower than on NB. In patients, changes in the SSR were observed 4 minutes into the anti-GD2 mAb naxitamab infusion. SSR latency quickly shortened followed by gradual decrease in the amplitude before disappearance. SSR response did not recover for 24 hours consistent with tachyphylaxis and absence of side effects in the clinic. The Step-Up protocol dissociated on-target off-tumor side effects while maintaining serum drug exposure. We provide first evidence of the ANS as the principal non-tumor target of anti-GD2 mAbs in humans. We describe the development and modeling of the Step-Up protocol exploiting the tachyphylaxis phenomenon we demonstrate in patients using the SSR test.

Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells.

Malignant melanoma represents a form of skin cancer characterized by a bleak prognosis and heightened resistance to traditional therapies. Quercetin has demonstrated notable anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects across various cancer types. However, the intricate relationship between quercetin's anti-cancer properties and ganglioside expression in melanoma remains incompletely understood. In this study, quercetin manifests specific anti-proliferative, anti-migratory, and cell-cycle arrest effects, inducing mitochondrial dysfunction and apoptosis in two melanoma cancer cell lines. This positions quercetin as a promising candidate for treating malignant melanoma. Moreover, our investigation indicates that quercetin significantly reduces the expression levels of ganglioside GD3 and its synthetic enzyme. Notably, this reduction is achieved through the inhibition of the FAK/paxillin/Akt signaling pathway, which plays a crucial role in cancer development. Taken together, our findings suggest that quercetin may be a potent anti-cancer drug candidate for the treatment of malignant melanoma.

Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.

Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. We identified mature (CD105+/CD133-) and undifferentiated (CD133+/CD105-) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.

Metabolic-Glycoengineering-Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans.

In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage-specific embryonic antigen-3 (SSEA-3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA-3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinctive mechanoresponses during the early stages of hPSC differentiation. Taken together, these results demonstrated that MGE-enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs and integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE-based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction.

Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022- 501725-21-00). See related commentary by Locatelli and Quintarelli, p. 3361.

Ganglioside GD3 Regulates Inflammation and Epithelial-to-Mesenchymal Transition in Human Nasal Epithelial Cells.

Chronic sinusitis with nasal polyps (CRSwNP) is one of the most common chronic inflammatory diseases, and involves tissue remodeling. One of the key mechanisms of tissue remodeling is the epithelial-mesenchymal transition (EMT), which also represents one of the pathophysiological processes of CRS observed in CRSwNP tissues. To date, many transcription factors and forms of extracellular stimulation have been found to regulate the EMT process. However, it is not known whether gangliosides, which are the central molecules of plasma membranes, involved in regulating signal transmission pathways, are involved in the EMT process. Therefore, we aimed to determine the role of gangliosides in the EMT process. First, we confirmed that N-cadherin, which is a known mesenchymal marker, and ganglioside GD3 were specifically expressed in CRSwNP_NP tissues. Subsequently, we investigated whether the administration of TNF-α to human nasal epithelial cells (hNECs) resulted in the upregulation of ganglioside GD3 and its synthesizing enzyme, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialytransferase 1 (ST8Sia1), and the consequently promoted inflammatory processes. Additionally, the expression of N-cadherin, Zinc finger protein SNAI2 (SLUG), and matrix metallopeptidase 9 (MMP-9) were elevated, but that of E-cadherin, which is known to be epithelial, was reduced. Moreover, the inhibition of ganglioside GD3 expression by the siRNA or exogenous treatment of neuraminidase 3 (NEU 3) led to the suppression of inflammation and EMT. These results suggest that gangliosides may play an important role in prevention and therapy for inflammation and EMT.

GD2-Targeting CAR T-cell Therapy for Patients with GD2+ Medulloblastoma.

Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Abstract 947: A nanodroplet cell processing platform for identifying synergistic drug combinations for neuroblastoma treatment in TH-MYCN transgenic mice

Abstract Neuroblastoma is the most common pediatric extracranial solid tumor and contributes to more than 15% of child mortality. It is characterized by extreme heterogeneity and high-risk tumors often resulting in poor prognosis. Currently, chemotherapy is a prevailing standard treatment for neuroblastoma. However, it is plagued by multiple challenges such as drug resistance and debilitating side effects. Synergistic drug combination is proposed as an innovative approach to potentially improve clinical outcomes of single-agent chemotherapy. We propose a novel approach to screen for effective chemo-synergistic drug combinations using Bioinspired nanodroplet processing (BioNDP) screening platform. This platform overcomes the limitations of conventional drug screening approaches, including the requirement for large cell numbers and the low translation rate from bench to bedside. In this study, we utilize TH-MYCN transgenic mice, a well-known genetically engineered mouse model of neuroblastoma that recapitulates critical genetic and clinical aspects of high-risk MYCN-amplified neuroblastoma. With its genetically homogeneous background and spontaneous tumor development, TH-MYCN mice offer a valuable comparison to traditional xenograft mouse models. We have also developed an optimal procedure of primary murine tumor isolation using both negative and positive selection approaches. The purity of the isolated cells was verified with GD2 expression, one of well-known marker of neuroblastoma. In addition, we have successfully identified an effective drug combination of cyclophosphamide, doxorubicin, and vincristine, using primary neuroblastoma tumor samples derived from TH-MYCN transgenic mice as a target. The in vitro synergistic effect of primary mouse tumor cell cytotoxicity was confirmed by using luminescent cell viability assay. Further in vivo experiments have demonstrated the therapeutic efficacy of this selected drug combination in suppressing tumor growth and improving survival rate in all TH-MYCN transgenic mice treated. To sum up, we aim to establish a future patient-centric paradigm by which minimal primary cell samples from a personalized biopsy of patients may undergo drug screening, leading to the identification of a tailored drug combination. The BioNDP platform we developed may become a pivotal resource for personalized therapy to ensure a faster and more efficient treatment strategy for patients. Citation Format: Yen-Tzu Liao, Zhi-Kai Yu, Ching-Te Kuo, Wen-Ming Hsu, Yi-Xun Huang, Hsinyu Lee. A nanodroplet cell processing platform for identifying synergistic drug combinations for neuroblastoma treatment in TH-MYCN transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 947.

Abstract 5472: Direct cytotoxicity of hu14.18K.322A in high-risk neuroblastoma

Abstract Background: Neuroblastoma is a childhood cancer of the sympathetic nervous system and is associated with disialoganglioside GD2 overexpression. The main goal of this study is to investigate the involvement of GD2 targeting humanized antidisialoganglioside monoclonal antibody hu14.18K.322A (hu14) in high-risk neuroblastoma direct cell cytotoxicity, assess for synergy with induction chemotherapy agents, and delineate the mechanisms involved in cell death. Methods: For this study, we used a panel of GD2 expressing cell lines [SK-N-BE (2), SK-N-BE (1), LAN5, CHLA15] and a GD2 non-expressing cell line LAN6. We measured surface expression of GD2 and performed cell cycle analysis using flow cytometry. The role of hu14 in neuroblastoma cell death was assessed using cell viability assays, proliferation assays, real time PCR and western blot analysis. We measured synergy between hu14 and induction phase chemotherapy drugs. Further, we employed next generation sequencing (NGS) to analyze the transcriptomic changes in human neuroblastoma cell lines in response to hu14 treatment to understand the pathways involved and the molecular mechanisms of hu14-mediated cellular response. Results: Using a cell viability assay, we found that CHLA15 and SK-N-BE (1) cell lines were sensitive to hu14 treatment whereas SK-N-BE (2), LAN5, LAN6 were resistant. Hu14 treatment resulted in a dose dependent reduction in cell proliferation in these cell lines. We also found that hu14 treatment resulted in increased expression of apoptotic and autophagy pathway proteins, but not ferroptosis or necroptosis pathways proteins. Hu14 induced cell death was partially inhibited by a pan-caspase inhibitor, confirming that caspase-dependent pathways are involved. Western blot analysis also showed decreased levels of p53 phosphorylation in both sensitive cell lines upon hu14 treatment suggesting an important control mechanism centered on the p53 pathway. Hu14 treatment reduced the surface expression of GD2 in the sensitive cells suggesting direct inhibition of the GD2 synthesis pathway. However, PCR analysis of GD2 synthesis genes did not show any significant difference upon treatment suggesting posttranscriptional control of these genes. Furthermore, NGS analysis revealed alterations in several pathways, but these changes were distinct between the cell lines reconfirming the complexity of therapy response in high-risk Neuroblastoma. Conclusion: Taken together, our findings suggest that hu14.18K322A induces direct cell cytotoxicity in a subset of high-risk Neuroblastoma and synergizes well with standard chemotherapy drugs. Furthermore, our research highlights the complex nature of signaling pathways involved in hu14-mediated cytotoxicity that can be harnessed towards targeted therapy. Citation Format: Manu Gnanamony, Maria Thomas, Thu Hien Nguyen, Amber M. D'Souza, Pedro de Alarcon. Direct cytotoxicity of hu14.18K.322A in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5472.

Abstract 2672: GD3 synthase (ST8SIA1) is a key immunomodulator in GD2+ breast cancer cells

Abstract Background: Gangliosides are acidic glycosphingolipids involved in cell adhesion, proliferation, and modulation of signal transduction pathways. It has been reported that tumor-shed gangliosides influence the activity of immune cells, including macrophages, NK cells, and T cells. GD3 synthase (GD3S) is the key enzyme that regulates the biosynthesis of the b and c-series gangliosides, particularly GD3 and GD2, and studies have shown that GD3S is upregulated in most tumors and plays a role in tumor progression. Similarly, we have found GD3S to be significantly upregulated in GD2+ breast cancer stem cells (BCSC) compared to GD2− cells, and the knockdown of this gene prevented tumor formation in mice. Here, we hypothesize that GD3 synthase has an immunomodulatory function in breast cancer (BC) cells through the upregulation of GD2. Methods: We stably overexpressed GD3S in BT549 and MCF-7 breast cancer cell lines by lentiviral transfection and performed CRISPR-Cas9 knockout of GD3S in BT549 and SUM 159 cell lines. We examined the effect of GD3 synthase on macrophage-mediated phagocytosis, NK cell-mediated killing, and T-cell cytotoxicity of BC cells using the live cell imaging system, IncuCyte®. Naxitamab is a US FDA-approved fully humanized anti-GD2 monoclonal antibody for the treatment of neuroblastoma. We evaluated the effects of naxitamab in GD2+ BC cell lines (HCC1395, Hs578T) using the Incucyte, and also examined its effect on in vivo tumor growth using TNBC PDX models. Results: We observed a highly significant decrease in macrophage-mediated phagocytosis (p&amp;lt;0.005; p&amp;lt;0.0001) and NK-mediated killing (p&amp;lt;0.0001; p&amp;lt;0.0001) in BT549 and MCF-7 cell overexpressing GD3S compared to the control cells. Interestingly, we observed a significant increase in phagocytosis in GD3S knockout BT549 and SUM 159 cells (p&amp;lt;0.05; p&amp;lt;0.005) compared to their wild-type counterparts. Moreover, the knockout of GD3S enhanced NK cell-mediated apoptosis in BT549 cells (p&amp;lt;0.001) and T cell killing in BT549 and SUM 159 cells (p&amp;lt;0.0001; p&amp;lt;0.0001). Finally, we found that naxitamab dose-dependently enhances macrophage-mediated phagocytosis and NK cell-mediated apoptosis in HCC1395 and Hs578T BC cells, with 20µg/ml being the most effective concentration in comparison to IgG control (p&amp;lt;0.0001; p&amp;lt;0.0001). In addition, we observed a reduction in tumor volume in naxitamab-treated mice compared to IgG control (p&amp;lt;0.05) Conclusion: GD3 synthase regulates macrophage-mediated phagocytosis, NK cell-induced apoptosis, and T-cell cytotoxicity in BC cells. Naxitamab enhances immune cell killing of breast cancer cells with high GD2 expression. Furthermore, naxitamab inhibited tumor growth in GD2+ TNBC PDX models. Citation Format: Bolutyfe Oderinde, Vivek Anand, Maryam Siddiqui, Anudishi Tyagi, Borgman Jenny, Venkata Lokesh Battula. GD3 synthase (ST8SIA1) is a key immunomodulator in GD2+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2672.

Metabolic Glycoengineering‐Enabled Molecularly Specific Acoustic Tweezing Cytometry for Targeted Mechanical Stimulation of Cell Surface Sialoglycans

This study developed a new type of dibenzocyclooctyne (DBCO)‐functionalized microbubbles (MBs) and validated their attachment to azide‐labelled sialoglycans on human pluripotent stem cells (hPSCs) generated via metabolic glycoengineering (MGE). This enabled application of mechanical forces to sialoglycans on hPSCs via molecularly specific acoustic tweezing cytometry (mATC), i.e., displacing sialoglycan‐anchored MBs using ultrasound (US). It was shown that subjected to forces of US pulses, sialoglycan‐anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin‐anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage‐specific embryonic antigen‐3 (SSEA‐3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA‐3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinct mechanoresponses during the early stages of hPSC differentiation. Taken together, the results demonstrated that MGE‐enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs than integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE‐based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction.

A novel approach to guide GD2-targeted therapy in pediatric tumors by PET and [64Cu]Cu-NOTA-ch14.18/CHO.

Background: The tumor-associated disialoganglioside GD2 is a bona fide immunotherapy target in neuroblastoma and other childhood tumors, including Ewing sarcoma and osteosarcoma. GD2-targeting antibodies proved to be effective in neuroblastoma and GD2-targeting chimeric antigen receptors (CAR)- expressing T cells as well as natural killer T cells (NKTs) are emerging. However, assessment of intra- and intertumoral heterogeneity has been complicated by ineffective immunohistochemistry as well as sampling bias in disseminated disease. Therefore, a non-invasive approach for the assessment and visualization of GD2 expression in-vivo is of upmost interest and might enable a more appropriate treatment stratification. Methods: Recently, [64Cu]Cu-NOTA-ch14.18/CHO (64Cu-GD2), a radiolabeled GD2-antibody for imaging with Positron-Emission-Tomography (PET) was developed. We here report our first clinical patients' series (n = 11) in different pediatric tumors assessed with 64Cu-GD2 PET/MRI. GD2-expression in tumors and tissue uptake in organs was evaluated by semiquantitative measurements of standardized uptake values (SUV) with PET/MRI on day 1 p.i. (n = 11) as well as on day 2 p.i. (n = 6). Results: In 8 of 9 patients with suspicious tumor lesions on PET/MRI at least one metastasis showed an increased 64Cu-GD2 uptake and a high tracer uptake (SUVmax > 10) was measured in 4 of those 8 patients. Of note, sufficient image quality with high tumor to background contrast was readily achieved on day 1. In case of 64Cu-GD2-positive lesions, an excellent tumor to background ratio (at least 6:1) was observed in bones, muscles or lungs, while lower tumor to background contrast was seen in the spleen, liver and kidneys. Furthermore, we demonstrated extensive tumor heterogeneity between patients as well as among different metastatic sites in individual patients. Dosimetry assessment revealed a whole-body dose of only 0.03 mGy/MBq (range 0.02-0.04). Conclusion: 64Cu-GD2 PET/MRI enables the non-invasive assessment of individual heterogeneity of GD2 expression, which challenges our current clinical practice of patient selection, stratification and immunotherapy application scheme for treatment with anti-GD2 directed therapies.

Immunomodulatory properties of extracellular vesicles isolated from bone marrow of patients with neuroblastoma: role of PD-L1 and HLA-G.

Extracellular vesicles (EVs) can be released by any cell and are crucial for cell-to-cell communications. EVs have been characterized in patients with solid and hematological tumors, where they play an important role in tumor progression and metastasis. EVs may express different surface proteins derived from the parental cells, including immunomodulatory molecules, such as HLA-G and PDL1. We isolated EV from bone marrow (BM) samples of patients with Neuroblastoma (NB) and healthy controls and we analyzed the expression of CD56, GD2 and immune checkpoints on EV by flow cytometry. Next, we analyzed the function of T cells in vitro in the presence or absence of NB patients' BM-derived EV, in terms of proliferation and cytokine production. Finally, we analyzed the correlation between the expression of immune checkpoints on EV and the clinical outcome of patients. We found a higher expression of CD56 on EVs derived from BM of patients with NB than in those from healthy donors (HD). However, CD56 expression was not dependent on BM infiltration of NB cells. Moreover, the analysis of GD2 expression revealed that only a small fraction of EVs was released by infiltrating NB cells, whereas the majority may derive from BM-resident cells. BM-derived EVs from NB patients display a higher expression of HLA-G and PD-L1 than those derived from HD. Nonetheless, such EVs are able to modulate T cell immune responses. We measured a robust response, in vitro, towards a common bacterial antigen, including the release of GM-CSF and proinflammatory cytokines, like IFN-a and IL-6, from mononuclear cells. Some of these immunomodulatory features are dependent on the expression of HLA-G and PD-L1, whereas others may rely on other mechanism(s). Finally, a high expression of CD56, HLA-G and PD-L1 on BM-derived EVs may represent a good prognostic factor. We described the presence of HLA-G and PDL1-bearing EVs in the BM of NB patients, which may represent a mechanism performed by resident BM cells to counteract the inflammation occurring in the BM microenvironment of NB patients.

Upregulation of human GD3 synthase (hST8Sia I) gene expression during serum starvation-induced osteoblastic differentiation of MG-63 cells.

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase (hST8Sia I) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway.

A Multi-Color Flow Cytometric Assay for Quantifying Dinutuximab Binding to Neuroblastoma Cells in Tumor, Bone Marrow, and Blood.

GD2, a disialoganglioside, is present on the surface of most neuroblastomas, as well as on some other cancers, such as melanoma and osteogenic sarcoma. The anti-GD2 antibody ch14.18 (dinutuximab) has an FDA-registered indication for use as maintenance therapy for high-risk neuroblastoma with cytokines and 13-cis-retinoic acid after myeloablative therapy. Recent studies using immunohistochemistry of tumor or tumor cells in marrow have shown that some neuroblastomas are negative for GD2. Dinutuximab and other anti-GD2 antibodies are increasingly used in combination with cytotoxic chemotherapy for treating relapsed neuroblastoma, so it is important to be able to identify patients with tumor cells with low GD2 expression, as such patients may experience toxicity but not benefit from the antibody therapy. As the most common clinical samples available for relapsed neuroblastoma are bone marrow aspirates, we developed a method to quantify dinutuximab binding density and the frequency of neuroblastoma cells positive for the antibody in bone marrow aspirates. Here, we describe a multi-color flow cytometry assay that employs non-GD2 antibodies to identify neuroblastoma cells in a mixed population (tumor, bone marrow, or blood) and an anti-GD2 antibody to quantify both the frequency and density of GD2 expression on neuroblastoma cells.