Murine Neuroblastoma Research Articles

Conditional Activation of c-MYC in Distinct Catecholaminergic Cells Drives Development of Neuroblastoma or Somatostatinoma.

c-MYC is an important driver of high-risk neuroblastoma. A lack of c-MYC-driven genetically engineered mouse models (GEMM) has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and to develop effective therapies. Here, we showed that conditional c-MYC induction via Cre recombinase driven by a tyrosine hydroxylase (Th) promoter led to a preponderance of PDX1+ somatostatinoma, a type of pancreatic neuroendocrine tumor (PNET). However, c-MYC activation via an improved Cre recombinase driven by a dopamine β-hydroxylase (Dbh) promoter resulted in neuroblastoma development. The c-MYC murine neuroblastoma tumors recapitulated the pathologic and genetic features of human neuroblastoma and responded to anti-GD2 immunotherapy and DFMO, an FDA-approved inhibitor targeting the MYC transcriptional target ODC1. Thus, c-MYC overexpression results in different but related tumor types depending on the targeted cell. The GEMMs represent valuable tools for testing immunotherapies and targeted therapies for these diseases.

Patterns of corticosterone exposure affect the subcellular localisation of mineralocorticoid and glucocorticoid receptor complexes and gene expression

Mineralocorticoid (MR) and glucocorticoid receptors (GR) act as transcription factors and major mediators of glucocorticoid signalling, with pivotal roles in regulating the stress response and hormonal signalling, mood, cognition and memory. The MR and GR and share many target genes, have a high degree of homology in their DNA binding (DBD) and ligand binding domain (LBD) but differ considerably in the N-terminal domain (NTD). Using Proximity Ligation Assay (PLA) we quantitatively assessed MR-GR complex subcellular localisation and transcriptional regulation in murine neuroblastoma (N2A) cells stimulated by constant or pulsatile corticosterone (CORT) patterns. We observe that continuous receptor activation by CORT caused localisation at the periphery of the cell nucleus. Truncation of the receptor Ligand Binding Domain (LBD) led to a stronger localisation of MR-GR complexes at the periphery of the cell nuclei. This was also observed for GR immunofluorescence (IF), while in cells expressing only MR or GR the mRNA response to pulsatile hormone treatment was substantially attenuated. However, there was no clearcut correlation between the spatial distribution of MR-GR complexes and the mRNA levels of target genes. Overall, our findings suggest that longer presence in the cell nucleus favors more peripheral nuclear localisation.

Evaluation of a Combinatorial Immunotherapy Regimen That Can Cure Mice Bearing MYCN-Driven High-Risk Neuroblastoma That Resists Current Clinical Therapy.

Background: Incorporating GD2-targeting monoclonal antibody into post-consolidation maintenance therapy has improved survival for children with high-risk neuroblastoma. However, ~50% of patients do not respond to, or relapse following, initial treatment. Here, we evaluated additional anti-GD2-based immunotherapy to better treat high-risk neuroblastoma in mice to develop a regimen for patients with therapy-resistant neuroblastoma. Methods: We determined the components of a combined regimen needed to cure mice of established MYCN-amplified, GD2-expressing, murine 9464D-GD2 neuroblastomas. Results: First, we demonstrate that 9464D-GD2 is nonresponsive to a preferred salvage regimen: anti-GD2 with temozolomide and irinotecan. Second, we have previously shown that adding agonist anti-CD40 mAb and CpG to a regimen of radiotherapy, anti-GD2/IL2 immunocytokine and anti-CTLA-4, cured a substantial fraction of mice bearing small 9464D-GD2 tumors; here, we further characterize this regimen by showing that radiotherapy and hu14.18-IL2 are necessary components, while anti-CTLA-4, anti-CD40, or CpG can individually be removed, and CpG and anti-CTLA-4 can be removed together, while maintaining efficacy. Conclusions: We have developed and characterized a regimen that can cure mice of a high-risk neuroblastoma that is refractory to the current clinical regimen for relapsed/refractory disease. Ongoing preclinical work is directed towards ways to potentially translate these findings to a regimen appropriate for clinical testing.

The Antioxidant Drug Edaravone Binds to the Aryl Hydrocarbon Receptor (AHR) and Promotes the Downstream Signaling Pathway Activation.

A considerable effort has been spent in the past decades to develop targeted therapies for the treatment of demyelinating diseases, such as multiple sclerosis (MS). Among drugs with free radical scavenging activity and oligodendrocyte protecting effects, Edaravone (Radicava) has recently received increasing attention because of being able to enhance remyelination in experimental in vitro and in vivo disease models. While its beneficial effects are greatly supported by experimental evidence, there is a current paucity of information regarding its mechanism of action and main molecular targets. By using high-throughput RNA-seq and biochemical experiments in murine oligodendrocyte progenitors and SH-SY5Y neuroblastoma cells combined with molecular docking and molecular dynamics simulation, we here provide evidence that Edaravone triggers the activation of aryl hydrocarbon receptor (AHR) signaling by eliciting AHR nuclear translocation and the transcriptional-mediated induction of key cytoprotective gene expression. We also show that an Edaravone-dependent AHR signaling transduction occurs in the zebrafish experimental model, associated with a downstream upregulation of the NRF2 signaling pathway. We finally demonstrate that its rapid cytoprotective and antioxidant actions boost increased expression of the promyelinating Olig2 protein as well as of an Olig2:GFP transgene in vivo. We therefore shed light on a still undescribed potential mechanism of action for this drug, providing further support to its therapeutic potential in the context of debilitating demyelinating conditions.

Abstract 2609: Vesicular LGALS3BP is a neuroblastoma biomarker and a therapeutic target for combination therapy with antibody-drug conjugate and checkpoint inhibitors

Abstract We have recently shown that targeting circulating LGALS3BP with a maytansinoid-based non-internalizing antibody-drug conjugate (ADC) resulted in tumor eradication of high-risk NMYC-amplified neuroblastoma patient-derived xenografts. LGALS3BP is a high molecular weight, hyperglycosylated and secreted protein which is enriched at the surface of cancer-derived extracellular vesicles (EVs). Here, using a neuroblastoma pseudometastatic preclinical model, we extended our previous work demonstrating that EVs-associated LGALS3BP strictly correlates with disease progression and dissemination. Moreover, to investigate whether LGALS3BP ADC, named 1959sss/DM4, possesses immune-modulating properties, we analyzed DM4 ability to induce immunogenic cell death (ICD). Interestingly, using NXS2 murine neuroblastoma cells, we found that DM4 induced a time-dependent surface expression of three different ICD marker, i.e. calreticulin, HSP70 and HSP90. As it was recently demonstrated that to limit immune surveillance NXS2 cells increase PD-L1 expression in response to IFNγ production in the tumor immune infiltrate, we evaluated potential synergism between LGALS3BP targeting ADC and an anti-PD1 immune check-point inhibitor. To this aim, we genetically manipulated NXS2 cells which resulted nearly negative for murine LGALS3BP expression. Therefore, using a lentivirus-based gene transduction system, we obtained NXS2 cells producing large amounts of vesicular human LGALS3BP. Of note, the vesicular LGALS3BP could be quantified by ELISA and imaged by confocal microscopy using 1959 antibody. Strikingly, while single agent treatment showed a limited antitumor activity in NXS2-LGALS3BP syngeneic model, the LGALS3BP ADC/anti-PD1 combination resulted in a marked inhibition of tumor growth, which was followed by a prolonged survival. In conclusion, our findings establish that vesicular LGALS3BP is a potential biomarker for liquid biopsy and reveals this protein as a suitable target for therapeutic strategy that combines 1959sss/DM4 ADC with immune checkpoint blockade for the treatment of LGALS3BP expressing neuroblastoma. Citation Format: Gianluca Sala, Emily Capone, Ilaria Cela, Giulio Lovato, Alessia Lamolinara, Manuela Iezzi, Cosmo Rossi, Vincenzo De Laurenzi, Rodolfo Ippoliti, Stefano Iacobelli. Vesicular LGALS3BP is a neuroblastoma biomarker and a therapeutic target for combination therapy with antibody-drug conjugate and checkpoint inhibitors [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 2609.

Abstract 5121: Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies

Abstract The success of CD19-directed chimeric antigen receptor (CAR)-T cell therapy for ALL has yet to be matched in solid pediatric malignancies. Moreover, immune checkpoint blockade has shown limited benefit in pediatric cancer. Despite this unmet need, there is a lack of reliable preclinical models to facilitate novel target discovery, validate therapeutic benefit, anticipate on- or off-target toxicity, and understand mechanisms of resistance. Syngeneic murine model systems are a critical intermediate for preclinical translation, preserving the complex and heterogenous immune landscape and host-immunotherapy interactions. However, model interpretation and validity are limited as expression and function of murine targets and immune effector cells are not always conserved. We have developed a syngeneic immunocompetent C57BL/6 neuroblastoma system to study CAR efficacy, toxicity, and mechanisms of resistance, as well a variety of vaccination strategies. Specifically, we have built two murine CAR T constructs containing a 100% conserved antigen-directed scFv joined to orthologous murine TCR signaling machinery. In parallel, we engineered the murine neuroblastoma cell line 9464D to stably express surface antigen Gpc2 (Bosse, Cancer Cell 2017), or a novel chimeric single chain trimer of a 9mer peptide from intracellular oncoprotein PHOX2B discovered on HLA-A*24:02 (Yarmarkovich, Nature 2023) as Phox2b/A24*02/H2-kb to generate tumor-bearing B6 or B6-A24-Tg mice. We are currently using this system to demonstrate how vaccination approaches enhance the persistence and potency of CART directed to both targets and are extending our syngeneic murine constructs to additional TH-MYCN allograft models. To further study the innate and adaptive immune response to various therapeutics, we have developed peripheral blood mononuclear cell (PBMC) humanized models. Our autologous humanized PDX mouse models of neuroblastoma use immunocompromised NSG mice engrafted with a PDX and humanized with patient-matched PBMCs to evaluate personalized vaccines in vivo. We have shown the ability to stably engraft both lymphoid and myeloid populations in various human cytokine transgenic NSG variants (i.e. SGM3, Flt3L), allowing for T/B cell and APC crosstalk, thus providing a robust means to study targeted interventions. We are currently using this autologously humanized PDX model to test the ability of multivalent, personalized neoantigen vaccines developed specific to the neoantigen landscape of individual PDXs, with a focus on endogenous T cell responses of patient PBMC against their own PDX in vivo. Citation Format: Timothy T. Spear, Elisabeth Posthill, David Groff, Anna M. Giudice, Kristopher R. Bosse, Leyuan Ma, Stephen P. Schoenberger, John M. Maris. Next-generation immunocompetent and humanized neuroblastoma murine models for the discovery and validation of novel immunotherapies [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 5121.

The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

Epidemiological and viral studies of rabies in Bali, Indonesia.

Rabies has been endemic in Bali since 2009, and cases has recently increased. Unfortunately, there is a lack of available vaccines, which hinders the eradication program. This study aimed to investigate the epidemiological and virological aspects of rabies infection in Bali. A total of 24 brain samples were collected from rabid dogs in all districts of Bali. The samples were tested using the direct fluorescent antibody (DFA) test and polymerase chain reaction (PCR) to confirm the presence of rabies virus in the samples. Samples with the highest virus content were propagated in vivo and then inoculated into BALB/c mice. The brains of dead mice were used to prepare an inoculate cultured in murine neuroblastoma cells. Supernatant-positive viruses representing each district were then reinoculated into eight groups of five BALB/c mice. A brain sample from each dead mouse was tested using DFA and PCR and detected under a fluorescence microscope. All rabies virus-positive samples collected from rabid dogs in all districts of Bali were positive. Rabies virus was detected by DFA test and PCR and was consistently confirmed in the in vivo and in vitro studies. BALB/c mice inoculated with the highest viral dilution (105 cells/mL) of culture supernatant showed typical signs of rabies, indicating that the virus could be properly investigated. This study demonstrated a wide epidemiological distribution of rabies in Bali. The obtained virus can be adapted for in vitro and in vivo studies and can be used to develop a homologous vaccine.

Abstract B110: β3-adrenergic receptor as a new molecular target in neuroblastoma treatment

Abstract Purpose of study: β3-Adrenergic receptor correlation with tumor growth and progression in preclinical and clinical studies in neuroblastoma. Experimental procedures: Knock-out of β3-Adrenergic Receptor (β3-AR) in Neuroblastoma (NB) cell lines using CRISPR-Cas9 technology. In vivo experiment using β3-AR knock-out cells in order to evaluate the tumor growth in mice. RNA Sequencing of tumor masses derived from mice to investigate β3-AR Dependent tumor signaling pathways. Flow Cytometry analysis to observe the expression level of β3- Adrenergic Receptor on circulating tumor cells (CTCs) in peripheral blood and on disseminated tumor cells (DTCs) in bone marrow from NB patients with low-risk and high-risk prognosis. Summary of data: CRISPR/Cas9 technology was performed to obtain ADRB3-/- clones from human and murine NB cell lines. ADRB3-/- knocked-out cells were inoculated in murine models NU-Foxn1nu and A/J mice, showing an absence of tumor growth compared to wild type NB cells. Subsequently, RNA sequencing on tumor masses was performed to investigate β3-AR-dependent tumor signaling pathways. RNA-sequencing analysis showed many pathways were altered. Notably, downregulation of genes involved in epithelial–mesenchymal transition (EMT) pathway and genes involved in cell adhesion were underlined in Knock-out vs control, correlated with the absence of tumor growth. Cytofluorimetric analysis of β3-AR level expression were conducted on circulating tumor cells (CTCs) in peripheral blood and on disseminated tumor cells (DTCs) in bone marrow from NB patients with low-risk and high-risk prognosis to evaluating the expression levels of β3-AR. As expected, most of the high-risk NB patients showed high percentage of β3-AR+ cells among CTCs and DTCs, while the majority of low-risk patients showed lower percentage. Statement of the conclusions: Ours data showed that β3-AR could be a potential target therapy for neuroblastoma treatment. In fact, his deletion results in an absence of tumor growth and progression as demonstrate in mice in vivo experiments. In addition, NB patients with lower β3-AR level, showed a better prognosis . Furter studies should be focused on the pathways related to β3-AR. Moreover, new drugs will be tested in order to normalize the level of β3-AR in NB patients. Citation Format: Rossana Putino, Alessia Boaretto, Gennaro Bruno, Gianluca Mattei, Annalisa Tondo, Claudio Favre, Maura Calvani. β3-adrenergic receptor as a new molecular target in neuroblastoma treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B110.

Venetoclax plus cyclophosphamide and topotecan in heavily pre-treated relapsed metastatic neuroblastoma: a single center case series

The prognosis of relapsed/refractory (R/R) neuroblastoma (NB) is dismal, calling for new therapeutic strategies. Venetoclax (VEN) is a highly selective, potent, orally bioavailable, BCL-2 inhibitor small-molecule that showed a synergistic effect with cyclophosphamide and topotecan (Cy-Topo) in murine NB models. Our aim was to evaluate the feasibility of VEN plus Cy-Topo in children with R/R NB. Four patients, who had previously failed > 3 lines of treatment, were treated with VEN plus Cy-Topo based on a 28-day schedule in an outpatient setting. BCL-2 expression in immunochemistry on tumor samples at relapse and the BCL2 gene status was evaluated in all patients. The main toxicity was hematological, with grade 4 neutropenia and thrombocytopenia occurring in all courses and leading to transient VEN discontinuation. Grade 3 oral mucositis was observed in 1/8 courses. No other grade 2–4 toxicities were observed. BCL-2 was expressed in all tumors, while no molecular abnormalities in the BCL-2 genes were detected. A stable disease was observed in all patients, without any progression during the study period. VEN plus Cy-Topo is well tolerated, with encouraging results that may be improved by testing the schedule in less advanced patients.

Cooperation of acylglycerol hydrolases in neuronal lipolysis

Genetic and biochemical evidence has established DDHD-domain containing 2 (DDHD2) as the principal triacylglycerol (TAG) hydrolase in neuronal lipolysis of cytosolic lipid droplets. In this issue of Journal of Lipid Research, Hofer et al. report that DDHD2 cooperates with adipose triglyceride lipase, the principal TAG hydrolase in adipose lipolysis, contributing to cytosolic hydrolysis of both TAG and diacylglycerols in murine neuroblastoma cells and primary cortical neurons via different configurations of the lipases. This finding highlights the complexity of cytosolic acylglycerol hydrolysis and raises many new questions in the field of lipid metabolism.

Methods used to achieve different levels of the neuronal differentiation process in SH-SY5Y and Neuro2a cell lines: An integrative review.

To study the process of neuronal differentiation, the human neuroblastoma (SH-SY5Y) and the murine neuroblastoma (Neuro2a) cell lines have proven to be effective models. For this approach, different protocols involving known neurotrophic factors and other molecules, such as retinoic acid (RA), have been assessed to better understand the neuronal differentiation process. Thus, the goal of this manuscript was to provide a brief overview of recent studies that have used protocols to promote neurodifferentiation in SH-SY5Y and Neuro2a cell lines and used acquired morphology and neuronal markers to validate whether differentiation was effective. The published results supply some guidance regarding the relationship between RA and neurotrophins for SH-SY5Y, as well a serum concentrations for both cell lines. Furthermore, they demonstrate the potential application of Neuro2a, which is critical for future research on neuronal differentiation.

Induction of Cholinergic Phenotype in Mouse Neuroblastoma Cells Using Nerve Growth Factor

Nerve growth factor (NGF) is a key factor of neuronal differentiation. NGF plays an important role in growth and differentiation of sensory and sympathetic neurons in the peripheral nervous system. In the mature brain, NGF is important for the maintenance of a cholinergic neuronal phenotype. Here, we studied whether NGF is sufficient to induce cholinergic phenotype in murine neuroblastoma cells, which are often used to model various physiological and pathological conditions. We detected expression of both TrkA and p75NGFR of NGF receptors in NB41A3 and Neuro2a neuroblastoma cells, two the most popular cell lines widely used to study the properties of cholinergic neurons in vitro. Treatment of both types of cell cultures with NGF did not induce in contrast to 8-Br-cAMP, which induced differentiation of Neuro2a cells with formation neuron-like morphology. Furthermore, we did not reveal the markers of cholinergic phenotype, such as ChAT or VAChT mRNA or protein in these cells, after NGF treatment. Thus, NB41A3 and Neuro2a cells cannot be used as an in vitro model of cholinergic cells because they do not differentiate and/or exhibit cholinergic phenotype in response to NGF stimulation.

Soloxolone Methyl Reduces the Stimulatory Effect of Leptin on the Aggressive Phenotype of Murine Neuro2a Neuroblastoma Cells via the MAPK/ERK1/2 Pathway.

Despite the proven tumorigenic effect of leptin on epithelial-derived cancers, its impact on the aggressiveness of neural crest-derived cancers, notably neuroblastoma, remains largely unexplored. In our study, for the first time, transcriptome analysis of neuroblastoma tissue demonstrated that the level of leptin is elevated in neuroblastoma patients along with the severity of the disease and is inversely correlated with patient survival. The treatment of murine Neuro2a neuroblastoma cells with leptin significantly stimulated their proliferation and motility and reduced cell adhesion, thus rendering the phenotype of neuroblastoma cells more aggressive. Given the proven efficacy of cyanoenone-bearing semisynthetic triterpenoids in inhibiting the growth of neuroblastoma and preventing obesity in vivo, the effect of soloxolone methyl (SM) on leptin-stimulated Neuro2a cells was further investigated. We found that SM effectively abolished leptin-induced proliferation of Neuro2a cells by inducing G1/S cell cycle arrest and restored their adhesiveness to extracellular matrix (ECM) proteins to near control levels through the upregulation of vimentin, zonula occludens protein 1 (ZO-1), cell adhesion molecule L1 (L1cam), and neural cell adhesion molecule 1 (Ncam1). Moreover, SM significantly suppressed the leptin-associated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and ribosomal protein S6 kinase A1 (p90RSK), which are key kinases that ensure the survival and proliferation of cancer cells. Further molecular modeling studies demonstrated that the inhibitory effect of SM on the mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathway can be mediated by its direct interaction with ERK2 and its upstream regulators, son of sevenless homolog 1 (SOS) and mitogen-activated protein kinase kinase 1 (MEK1). Taken together, our findings in murine Neuro2a cells provide novel evidence of the stimulatory effect of leptin on the aggressiveness of neuroblastoma, which requires further detailed studies in human neuroblastoma cells and relevant animal models. The obtained results indicate that SM can be considered a promising drug candidate capable of reducing the impact of adipokines on tumor progression.

Antitumor activity of the investigational B7-H3 antibody-drug conjugate, vobramitamab duocarmazine, in preclinical models of neuroblastoma

IntroductionB7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Vobramitamab duocarmazine (also referred to as MGC018 and herein referred to as vobra duo) is an investigational duocarmycin-based antibody-drug...

Cellulose ether treatment inhibits amyloid beta aggregation, neuroinflammation and cognitive deficits in transgenic mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) is an incurable, progressive and devastating neurodegenerative disease. Pathogenesis of AD is associated with the aggregation and accumulation of amyloid beta (Aβ), a major neurotoxic mediator that triggers neuroinflammation and memory impairment. Recently, we found that cellulose ether compounds (CEs) have beneficial effects against prion diseases by inhibiting protein misfolding and replication of prions, which share their replication mechanism with Aβ. CEs are FDA-approved safe additives in foods and pharmaceuticals. Herein, for the first time we determined the therapeutic effects of the representative CE (TC-5RW) in AD using in vitro and in vivo models. Our in vitro studies showed that TC-5RW inhibits Aβ aggregation, as well as neurotoxicity and immunoreactivity in Aβ-exposed human and murine neuroblastoma cells. In in vivo studies, for the first time we observed that single and weekly TC-5RW administration, respectively, improved memory functions of transgenic 5XFAD mouse model of AD. We further demonstrate that TC-5RW treatment of 5XFAD mice significantly inhibited Aβ oligomer and plaque burden and its associated neuroinflammation via regulating astrogliosis, microgliosis and proinflammatory mediator glial maturation factor beta (GMFβ). Additionally, we determined that TC-5RW reduced lipopolysaccharide-induced activated gliosis and GMFβ in vitro. In conclusion, our results demonstrate that CEs have therapeutic effects against Aβ pathologies and cognitive impairments, and direct, potent anti-inflammatory activity to rescue neuroinflammation. Therefore, these FDA-approved compounds are effective candidates for developing therapeutics for AD and related neurodegenerative diseases associated with protein misfolding.

The effect of the sulfation patterns of dermatan and chondroitin sulfate from vertebrates and ascidians on their neuritogenic and neuroprotective properties

Neurodegeneration is caused by the progressive loss of the structure and function of neurons, leading to cell death, and it is the main cause of many neurodegenerative diseases. Many molecules, such as glycosaminoglycans (GAGs), have been studied for their potential to prevent or treat these diseases. They are widespread in nature and perform an important role in neuritogenesis and neuroprotection. Here we investigated the neuritogenic and neuroprotective role of Phallusia nigra dermatan sulfate (PnD2,6S) and compared it with two distinct structures of chondroitin sulfate (C6S) and dermatan sulfate (D4S). For this study, a neuro 2A murine neuroblastoma cell line was used, and a chemical lesion was induced by the pesticide rotenone (ROT). We observed that PnD2,6S + ROT had a better neuritogenic effect than either C6S + ROT or D4S + ROT at a lower concentration (0.05 μg/mL). When evaluating the mitochondrial membrane potential, PnD2,6S showed a neuroprotective effect at a concentration of 0.4 μg/mL. These data indicate different mechanisms underlying this neuronal potential, in which the sulfation pattern is important for neuritogenic activity, while for neuroprotection all DS/CS structures had similar effects. This finding leads to a better understanding the chemical structures of PnD2,6S, C6S, and D4S and their therapeutic potential.

Iron-Gallic Acid Peptide Nanoparticles as a Versatile Platform for Cellular Delivery with Synergistic ROS Enhancement Effect.

Cytosolic delivery of peptides is of great interest owing to their biological functions, which could be utilized for therapeutic applications. However, their susceptibility to enzymatic degradation and multiple cellular barriers generally hinders their clinical application. Integration into nanoparticles, which can enhance the stability and membrane permeability of bioactive peptides, is a promising strategy to overcome extracellular and intracellular obstacles. Herein, we present a versatile platform for the cellular delivery of various cargo peptides by integration into metallo-peptidic coordination nanoparticles. Both termini of cargo peptides were conjugated with gallic acid (GA) to assemble GA-modified peptides into nanostructures upon coordination of Fe(III). Initial pre-complexation of Fe(III) by poly-(vinylpolypyrrolidon) (PVP) as a template favored the formation of nanoparticles, which are able to deliver the peptides into cells efficiently. Iron-gallic acid peptide nanoparticles (IGPNs) are stable in water and are supposed to generate reactive oxygen species (ROS) from endogenous H2O2 in cells via the Fenton reaction. The strategy was successfully applied to an exemplary set of peptide sequences varying in length (1-7 amino acids) and charge (negative, neutral, positive). To confirm the capability of transporting bioactive cargos into cells, pro-apoptotic peptides were integrated into IGPNs, which demonstrated potent killing of human cervix carcinoma HeLa and murine neuroblastoma N2a cells at a 10 µM peptide concentration via the complementary mechanisms of peptide-triggered apoptosis and Fe(III)-mediated ROS generation. This study demonstrates the establishment of IGPNs as a novel and versatile platform for the assembly of peptides into nanoparticles, which can be used for cellular delivery of bioactive peptides combined with intrinsic ROS generation.

Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma.

Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable anti-tumor immune response in an MYCN amplified murine model of NB. Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or in combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated. Herein, we report that Syk is a marker of NB-associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm3) but not larger tumors (250 mm3). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors. Collectively, our findings demonstrate the central role of macrophage Syk in NB progression and demonstrate that Syk blockade can "reeducate" TAMs towards immunostimulatory phenotype, leading to enhanced T cell responses. These findings further support the clinical evaluation of fostamatinib alone or with radiation and ICB, as a novel therapeutic intervention in neuroblastoma.

CD24-Fc suppression of immune related adverse events in a therapeutic cancer vaccine model of murine neuroblastoma.

The combination of Myc-suppressed whole tumor cells with checkpoint inhibitors targeting CTLA-4 and PD-L1 generates a potent therapeutic cancer vaccine in a mouse neuroblastoma model. As immunotherapies translate from pre-clinical to clinical trials, the potential immune-related adverse events (irAEs) associated with induction of potent immunity must be addressed. The CD24-Siglec 10/G interaction is an innate checkpoint that abrogates inflammatory responses to molecules released by damaged cells, but its role in cancer immunology is not well defined. We investigate irAEs of an effective whole cell neuroblastoma vaccine and subsequently the effect of CD24-Fc, a CD24 and Fc fusion protein, on both the vaccine efficacy and induced irAEs in a mouse neuroblastoma model. To test whether the whole tumor cell vaccination leads to autoimmune responses in other organ systems we harvested lung, heart, kidney and colon from naïve mice (n=3), unvaccinated tumor only mice (n=3), and vaccinated mice with CD24 Fc (n=12) or human IgG-Fc control (n=12) after tumor inoculation and vaccination therapy at day 30. The Immune cell infiltrates and immunogenic pathway signatures in different organ systems were investigated using NanoString Autoimmune Profiling arrays. Nanostring RNA transcript results were validated with immunohistochemistry staining. The whole tumor cell vaccine combined with immune checkpoint therapy triggers occult organ specific immune cell infiltrates, primarily in cardiac tissue and to a lesser extent in the renal and lung tissue, but not in the colon. CD24-Fc administration with vaccination partially impedes anti-tumor immunity but delaying CD24-Fc administration after initial vaccination reverses this effect. CD24-Fc treatment also ameliorates the autoimmune response induced by effective tumor vaccination in the heart. This study illustrates that the combination of Myc suppressed whole tumor cell vaccination with checkpoint inhibitors is an effective therapy, but occult immune infiltrates are induced in several organ systems in a mouse neuroblastoma model. The systemic administration of CD24-Fc suppresses autoimmune tissue responses, but appropriate timing of administration is critical for maintaining efficacy of the therapeutic vaccine.