Neuroblastoma Cell Proliferation Research Articles

Novel PP2A-Activating Compounds in Neuroblastoma

Background: Neuroblastoma (NB) remains one of the deadliest pediatric solid tumors. Recent advancements aimed at improving outcomes have been insufficient, and patients with high-risk NB continue to have a poor prognosis. Protein phosphatase 2A (PP2A) is a tumor suppressor protein downregulated in many cancers, including NB. PP2A activation has been shown to affect the malignant phenotype in other solid tumors. The present studies aim to investigate the effects of two novel PP2A activators as a NB therapeutic. Methods: Four established NB cell lines and a patient-derived xenoline were utilized to study the effect on cell viability, proliferation, motility, and in vivo tumor growth using two novel tricyclic sulfonamide PP2A activators, ATUX-3364 and ATUX-8385. Results: ATUX-3364 and ATUX-8385 increased PP2A activity. These PP2A activators led to decreased viability, proliferation, and motility of NB cells. Treatment of animals bearing NB tumors with ATUX-3364 or ATUX-8385 resulted in decreased tumor growth in MYCN-amplified SK-N-BE(2) tumors. At the molecular level, PP2A-based reactivation led to dephosphorylation of MYCN-S62 and decreased MYCN protein expression. Conclusions: PP2A activators decreased NB cell viability, proliferation, and motility. In vivo experiments show that PP2A activators have more significant effects on tumorigenesis in MYCN-amplified tumors. Finally, phosphorylation of MYCN protein was decreased following treatment with novel sulfonamide PP2A activators. These data and mechanistic insights may be useful for developing new PP2A-based therapies that target MYCN for the treatment of NB.

Pinus koraiensis essential oil enhances glucose uptake and proliferation in SH-SY5Y neuroblastoma cells

Aromatherapy using essential oils (EOs) is well known for its beneficial effects on mental health and neuroprotection. However, the significant molecular mechanisms have not yet been identified. Recent studies have identified a decrease in glucose uptake as a common feature across various neurodegenerative diseases (NDDs), leading to mitochondrial dysfunction and excessive autophagy. This suggests that glucose may serve not only as an energy source but also as a therapeutic target for NDDs. Using SH-SY5Y neuroblast-like cells and the glucose uptake inhibitor BAY-876, we demonstrated that glucose depletion promoted autophagy. To discover the potential therapeutics that modulate glucose metabolism, we obtained EO from Pinus koraiensis Siebold & Zucc. (PKSZ) using steam distillation. PKSZ-EO upregulated mRNA expression of SLC2A2, SLC2A3, and SLC2A4, leading to increased glucose uptake in SH-SY5Y cells. Furthermore, PKSZ-EO protected SH-SY5Y cells from BAY-876-induced mitochondrial dysfunction, cytostasis, autophagy, and inflammation. Using gas chromatography-mass spectrometry, we confirmed the high levels of α-pinene, an inducer of GLUT4 expression, in PKSZ-EO. These results suggest that PKSZ-EO exerts a protective effect against glucose depletion stress, highlighting its potential as a therapeutic agent for NDDs.

METTL3-induced lncARSR aggravates neuroblastoma tumorigenic properties through stabilizing PHOX2B

Neuroblastoma (NB), the most common extracranial solid tumor in pediatric patients, manifests with considerable variability across multiple primary sites. Despite this, the extent of genetic heterogeneity within these tumor foci and the identification of consistent oncogenic drivers remains largely unexplored. Of particular interest, genetic mutations in PHOX2B have been linked to familial cases of NB, yet the underlying molecular mechanisms are not fully delineated. In our research, we focus on unraveling the role of a novel functional long non-coding RNA (lncRNA) associated with PHOX2B in the context of NB. Using NB cell models with overexpressed PHOX2B, combined with lncRNA microarray analysis, we discovered that lncARSR is significantly upregulated in response to PHOX2B overexpression. Subsequent biological assays demonstrated that lncARSR promotes both the proliferation and metastasis of NB cells. Further molecular investigations revealed that lncARSR plays a crucial role in stabilizing PHOX2B expression within NB cells. Moreover, we identified that the expression of lncARSR is regulated by methylation through methyltransferase-like 3 (METTL3), which itself is positively correlated with PHOX2B expression. Rescue experiments underscored the functional importance of METTL3, lncARSR, and PHOX2B in NB cells. In summary, our findings provide new insights into the molecular functions of PHOX2B in the progression of neuroblastoma and propose a novel therapeutic target for this aggressive malignancy.

Zinc Finger Protein 536 is a Potential Prognostic Biomarker that Promotes Neuroblastoma Progression via VEGFR2-PI3K-AKT Pathway.

Neuroblastoma (NB) is a well-known pediatric malignancy intertwined with neurodevelopment. Previously implicated in neuronal differentiation, Zinc Finger Protein 536 (ZNF536) has emerged as a promising prognostic and immune-related biomarker in our pan-cancer analysis. Single-cell RNA transcriptome sequencing, bulk transcriptome analysis, and immuno-histochemistry were used to assess ZNF536 expression and its association with prognosis. Cell proliferation, migration, invasion, and differentiation in ZNF536-knockdown NB cell lines were detected to evaluate the effect of ZNF536 on tumor cells. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a potential target of ZNF536, and its downstream PI3K/AKT signaling cascade were investigated using transcriptome sequencing, CUT&Tag, quantitative real-time PCR (qRT-PCR), and Western blotting. The role of ZNF536 in tumorigenesis and the potential regulation axis was evaluated in vivo using a BALB/c nude mouse xenograft tumor model. ZNF536 mRNA and protein expression were significantly higher in NB patients with poor prognosis. In vitro, ZNF536 knockdown curtailed proliferation, migration, and invasion of NB cells while fostering differentiation. ZNF536 regulated VEGFR2 expression, thus activating the PI3K-AKT pathway. In vivo, ZNF536 knockdown reduced tumor growth and proliferation via the VEGFR2-PI3K-AKT pathway. ZNF536 resulted as a novel prognostic biomarker in NB, promoting oncogenesis through VEGFR2-PI3K-AKT signaling axis modulation, suggesting its therapeutic potential in managing NB progression.

An Easy-to-Handle Route for Bicomponent Porous Tubes Fabrication as Nerve Guide Conduits

Over the past two decades, the development of nerve guide conduits (NGCs) has gained much attention due to the impellent need to find innovative strategies to take care of damaged or degenerated peripheral nerves in clinical surgery. In this view, significant effort has been spent on the development of high-performance NGCs by different materials and manufacturing approaches. Herein, a highly versatile and easy-to-handle route to process 3D porous tubes made of chitosan and gelatin to be used as a nerve guide conduit were investigated. This allowed us to fabricate highly porous substrates with a porosity that ranged from 94.07 ± 1.04% to 97.23 ± 1.15% and average pore sizes—estimated via X-ray computed tomography (XCT) reconstruction and image analysis—of hundreds of microns and an irregular shape with an aspect ratio that ranged from 0.70 ± 0.19 to 0.80 ± 0.15 as a function of the chitosan/gelatin ratio. More interestingly, the addition of gelatin allowed us to modulate the mechanical properties, which gradually reduced the stiffness—max strength from 0.634 ± 0.015 MPa to 0.367 ± 0.021 MPa—and scaffold toughness—from 46.2 kJ/m3 to 14.0 kJ/m3—as the gelatin content increased. All these data fall into the typical ranges of the morphological and mechanical parameters of currently commercialized NGC products. Preliminary in vitro studies proved the ability of 3D porous tubes to support neuroblastoma cell (SH-SY5Y) adhesion and proliferation. In perspective, the proposed approach could also be easily implemented with the integration of other processing techniques (e.g., electrospinning) for the design of innovative bi-layered systems with an improved cell interface and molecular transport abilities.

Poly (ADP-ribose) polymerase inhibitor sensitized DNA damage caused by an alkylating pyrrole-imidazole polyamide targeting MYCN in neuroblastoma cells

MYCN amplification (MYCN-amp) is a significant prognostic factor and early genetic marker of high-risk neuroblastoma (NB). MYCN induces the DNA damage response (DDR) and modulates the insensitivity of NB cells to Poly (ADP-ribose) polymerase (PARP) inhibitors. We previously reported that CCC-002, a DNA-alkylating agent conjugated with pyrrole-imidazole polyamide targeting MYCN, inhibits NB cell proliferation and induces DNA damage signaling. In this study, we investigated the synergistic effects of CCC-002 and PARP inhibitors on MYCN-amp NB cells. Combination treatment with PARP inhibitors significantly enhanced the sensitivity of MYCN-amp NB cells to CCC-002. DNA damage signals, such as phosphorylation of H2AX and RPA32 elicited after CCC-002 treatment, were further enhanced by PARP inhibitors, as detected through western blotting and immunofluorescence analyses. The potent cytotoxicity of this combination treatment was confirmed by the significant increase in the subG0-G1 phase. Although MYCN knockdown showed no synergistic effect with PARP inhibitors, fluorescence in situ hybridization and quantitative PCR analyses indicated that PARP inhibitors enhanced the effect of CCC-002 to reduce MYCN copy number and suppress its expression. Overall, our study provides novel insights into a therapeutic approach that combines CCC-002 and PARP inhibition to effectively induce DNA damage and apoptosis in MYCN-amp NB cells.

Repurposing of c-MET Inhibitor Tivantinib Inhibits Pediatric Neuroblastoma Cellular Growth

Background: Dysregulation of receptor tyrosine kinase c-MET is known to promote tumor development by stimulating oncogenic signaling pathways in different cancers, including pediatric neuroblastoma (NB). NB is an extracranial solid pediatric cancer that accounts for almost 15% of all pediatric cancer-related deaths, with less than a 50% long-term survival rate. Results: In this study, we analyzed a large cohort of primary NB patient data and revealed that high MET expression strongly correlates with poor overall survival, disease progression, relapse, and high MYCN levels in NB patients. To determine the effects of c-MET in NB, we repurposed a small molecule inhibitor, tivantinib, and found that c-MET inhibition significantly inhibits NB cellular growth. Tivantinib significantly blocks NB cell proliferation and 3D spheroid tumor formation and growth in different MYCN-amplified and MYCN-non-amplified NB cell lines. Furthermore, tivantinib blocks the cell cycle at the G2/M phase transition and induces apoptosis in different NB cell lines. As expected, c-MET inhibition by tivantinib inhibits the expression of multiple genes in PI3K, STAT, and Ras cell signaling pathways. Conclusions: Overall, our data indicate that c-MET directly regulates NB growth and 3D spheroid growth, and c-MET inhibition by tivantinib may be an effective therapeutic approach for high-risk NB. Further developing c-MET targeted therapeutic approaches and combining them with current therapies may pave the way for effectively translating novel therapies for NB and other c-MET-driven cancers.

Small Molecule Compound DHPA Screened by Computer-Aided Drug Design and Molecular Dynamics Simulation Inhibits Neuroblastoma Cell Proliferation by Targeting TrkB.

Neuroblastoma (NB) is a rare and malignant pediatric solid tumor. Due to its heterogeneity, it poses significant challenges for treatment, resulting in a high mortality rate. This study aimed to identify new therapeutic drugs by modeling the TrkB receptor from PDB 4AT5 and conducting virtual screening of compounds from the YaTCM database (containing 47,696 compounds derived from 6220 Traditional Chinese Medicines). The screening utilized the E-pharmacophore approach to select compounds with potential binding affinity to TrkB. The binding abilities of these compounds were tested through molecular dynamics simulations, stretch dynamics simulations, and US simulations. Among the top 11 optimized hit compounds, DHPA and 3″-demethylhexahydrocurcumin are prominent. Further simulations reveal that they form stable receptor-ligand binary complexes with TrkB. In subsequent in vitro cell experiments, 3″-demethylhexahydrocurcumin is eliminated due to its high IC50 for killing NB cells. Low concentrations of DHPA can significantly kill NB cells. Additionally, DHPA can inhibit the expression of TrkB, the activation of TrkB's downstream signaling pathways, and affect the thermal stability of TrkB protein and its response to streptase protease degradation. DHPA may be a potential TrkB inhibitor.

FTO diversely influences sensitivity of neuroblastoma cells to various chemotherapeutic drugs.

Chemotherapy resistance is a significant factor in treatment failure in patients with neuroblastoma (NB), and it directly affects patient prognosis. Therefore, identifying novel therapeutic targets to enhance chemosensitivity is essential to improve the cure rate and prognosis of patients with NB. In this study, we investigated the role of FTO in chemosensitivity of NB cells to various chemotherapeutic drugs. Our results showed that high FTO expression was positively correlated with increased survival probability and favorable prognostic factors in patients with NB. FTO overexpression inhibited cell proliferation, whereas FTO knockdown promoted cell proliferation in NB cells. FTO expression alteration had contrasting effects on NB cells' sensitivity to etoposide but had no significant impact on sensitivity to cisplatin. Downregulation of FTO reduced the sensitivity of NB cells to paclitaxel, whereas upregulation of FTO enhanced its sensitivity. Additionally, the sensitivities between patients with lower and higher FTO expression to various chemotherapeutic drugs or small-molecule inhibitors were different. Thus, FTO affects the sensitivities of NB cells differently depending on the different chemotherapeutic drugs and small-molecule inhibitors. This finding may guide physicians and patients choose the appropriate chemotherapeutic drugs or small-molecule inhibitors for treatment.

L3MBTL2 maintains MYCN-amplified neuroblastoma cell proliferation through silencing NRIP3 and BRME1 genes.

Epigenetic alterations critically affect tumor development. Polycomb-group complexes constitute an evolutionarily conserved epigenetic machinery that regulates stem cell fate and development. They are implicated in tumorigenesis, primarily via histone modification. Polycomb repressive complex 1 (PRC1) complexes 1-6 (PRC1.1-6) mediate the ubiquitination of histone H2A on lysine 119 (H2AK119ub). Here, we studied the functional roles of a PRC1.6 molecule, L3MBTL2, in neuroblastoma (NB) cells. L3MBTL2-knockout and knockdown revealed that L3MBTL2 depletion suppressed NB cell proliferation via cell-cycle arrest and gamma-H2A.X upregulation. L3MBTL2-knockout profoundly suppressed xenograft tumor formation. Transcriptome analysis revealed suppressed cell-cycle-related and activated differentiation-related pathways. Break repair meiotic recombinase recruitment factor 1 (BRME1) and nuclear receptor interacting protein 3 (NRIP3) were notably de-repressed by L3MBTL2-knockout. The deletion of L3MBTL2 reduced enrichment of H2AK119ub and PCGF6 at transcriptional start site proximal regions of the targets. Add-back studies unveiled the importance of L3MBTL2-BRME1 and -NRIP3 axes for NB cell proliferation. We further manifested the association of MYCN with de-repression of NRIP3 in an L3MBTL2-deficient context. Therefore, this study first revealed the significance of L3MBTL2-mediated gene silencing in MYCN-amplified NB cells.

Targeting SWI/SNF ATPases reduces neuroblastoma cell plasticity.

Tumor cell heterogeneity defines therapy responsiveness in neuroblastoma (NB), a cancer derived from neural crest cells. NB consists of two primary subtypes: adrenergic and mesenchymal. Adrenergic traits predominate in NB tumors, while mesenchymal features becomes enriched post-chemotherapy or after relapse. The interconversion between these subtypes contributes to NB lineage plasticity, but the underlying mechanisms driving this phenotypic switching remain unclear. Here, we demonstrate that SWI/SNF chromatin remodeling complex ATPases are essential in establishing an mesenchymal gene-permissive chromatin state in adrenergic-type NB, facilitating lineage plasticity. Targeting SWI/SNF ATPases with SMARCA2/4 dual degraders effectively inhibits NB cell proliferation, invasion, and notably, cellular plasticity, thereby preventing chemotherapy resistance. Mechanistically, depletion of SWI/SNF ATPases compacts cis-regulatory elements, diminishes enhancer activity, and displaces core transcription factors (MYCN, HAND2, PHOX2B, and GATA3) from DNA, thereby suppressing transcriptional programs associated with plasticity. These findings underscore the pivotal role of SWI/SNF ATPases in driving intrinsic plasticity and therapy resistance in neuroblastoma, highlighting an epigenetic target for combinational treatments in this cancer.

Comprehensive analysis identifies ubiquitin ligase FBXO42 as a tumor-promoting factor in neuroblastoma

Neuroblastoma, the deadliest solid tumor in children, exhibits alarming mortality rates, particularly among high-risk cases. To enhance survival rates, a more precise risk stratification for patients is imperative. Utilizing proteomic data from 34 cases with or without N-Myc amplification, we identified 28 differentially expressed ubiquitination-related proteins (URGs). From these, a prognostic signature comprising 6 URGs was constructed. A nomogram incorporating clinical-pathological parameters yielded impressive AUC values of 0.88, 0.93, and 0.95 at 1, 3, and 5 years, respectively. Functional experiments targeting the E3 ubiquitin ligase FBXO42, a component of the prognostic signature, revealed its TP53-dependent promotion of neuroblastoma cell proliferation. In conclusion, our ubiquitination-related prognostic model robustly predicts patient outcomes, guiding clinical decisions. Additionally, the newfound pro-proliferative role of FBXO42 offers a novel foundation for understanding the molecular mechanisms of neuroblastoma.

Super-enhancer-driven IRF2BP2 enhances ALK activity and promotes neuroblastoma cell proliferation.

Super-enhancers (SEs) typically govern the expression of critical oncogenes and play a fundamental role in the initiation and progression of cancer. Focusing on genes that are abnormally regulated by SE in cancer may be a new strategy for understanding pathogenesis. In the context of this investigation, we have identified a previously unreported SE-driven gene IRF2BP2 in neuroblastoma (NB). The expression and prognostic value of IRF2BP2 were detected in public databases and clinical samples. The effect of IRF2BP2 on NB cell growth and apoptosis was evaluated through in vivo and in vitro functional loss experiments. The molecular mechanism of IRF2BP2 was investigated by the study of chromatin regulatory regions and transcriptome sequencing. The sustained high expression of IRF2BP2 results from the activation of a novel SE established by NB master transcription factors MYCN, MEIS2, and HAND2, and they form a new complex that regulates the gene network associated with the proliferation of NB cell populations. We also observed a significant enrichment of the AP-1 family at the binding sites of IRF2BP2. Remarkably, within NB cells, AP-1 plays a pivotal role in shaping the chromatin accessibility landscape, thereby exposing the binding site for IRF2BP2. This orchestrated action enables AP-1 and IRF2BP2 to collaboratively stimulate the expression of the NB susceptibility gene ALK, thereby upholding the highly proliferative phenotype characteristic of NB. Our findings indicate that SE-driven IRF2BP2 can bind to AP-1 to maintain the survival of tumor cells via regulating chromatin accessibility of the NB susceptibility gene ALK.

Molecular profiling of core immune-escape genes highlights TNFAIP3 as an immune-related prognostic biomarker in neuroblastoma.

Neuroblastoma (NB) is the most prevalent and deadliest pediatric solid tumor. With of over 50% of high-risk neuroblastoma cases relapse, the imperative for novel drug targets and therapeutic strategies is accentuated. In neuroblastoma, the existence of tumor-associated macrophages (TAMs) correlates with an unfavorable patient prognosis. However, the clinical relevance and prognostic implications of regulatory genes linked to TAMs infiltration in neuroblastoma remain unclear, and further study is required. We conducted a comprehensive analysis utilizing transcriptome expression profiles from three primary datasets associated with neuroblastoma (GSE45547, GSE49710, TARGET) to identify hub genes implicated in immune evasion within neuroblastoma. Subsequently, we utilized single-cell RNA sequencing analysis on 17 clinical neuroblastoma samples to investigate the expression and distribution of these hub genes, leading to the identification of TNFAIP3. The above three public databases were merged to allowed for the validation of TNFAIP3's molecular functions through GO and KEGG analysis. Furthermore, we assessed TNFAIP3's correlation with immune infiltration and its potential immunotherapeutic impact by multiple algorithms. Our single-cell transcriptome data revealed the role of TNFAIP3 in macrophage polarization. Finally, preliminary experimental verifications to confirm the biological functions of TNFAIP3-mediated TAMs in NB. A total of 6 genes related to immune evasion were screened and we found that TNFAIP3 exhibited notably higher expression in macrophages than other immune cell types, based on the scRNA-sequencing data. GO and KEGG analysis showed that low expression of TNFAIP3 significantly correlated with the activation of multiple oncogenic pathways as well as immune-related pathways. Then validation affirmed that individuals within the TNFAIP3 high-expression cohort could potentially derive greater advantages from immunotherapeutic interventions, alongside exhibiting heightened immune responsiveness. Deciphering the pseudotime trajectory of macrophages, we revealed the potential of TNFAIP3 in inducing the polarization of macrophages towards the M1 phenotype. Finally, we confirmed that patients in the TNFAIP3 high expression group might benefit more from immunotherapy or chemotherapy as substantiated by RT-qPCR and immunofluorescence examinations. Moreover, the role of TNFAIP3 in macrophage polarization was validated. Preliminary experiment showed that TNFAIP3-mediated TAMs inhibit the proliferation, migration and invasion capabilities of NB cells. Our results suggest that TNFAIP3 was first identified as a promising biomarker for immunotherapy and potential molecular target in NB. Besides, the presence of TNFAIP3 within TAMs may offer a novel therapeutic strategy for NB.

Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

Preclinical Therapeutic Efficacy of RAF/MEK/ERK and IGF1R/AKT/mTOR Inhibition in Neuroblastoma.

Activating mutations in the RAS/MAPK pathway are observed in relapsed neuroblastoma. Preclinical studies indicate that these tumors have an increased sensitivity to inhibitors of the RAS/MAPK pathway, such as MEK inhibitors. MEK inhibitors do not induce durable responses as single agents, indicating a need to identify synergistic combinations of targeted agents to provide therapeutic benefit. We previously showed preclinical therapeutic synergy between a MEK inhibitor, trametinib, and a monoclonal antibody specific for IGF1R, ganitumab in RAS-mutated rhabdomyosarcoma. Neuroblastoma cells, like rhabdomyosarcoma cells, are sensitive to the inhibition of the RAS/MAPK and IGF1R/AKT/mTOR pathways. We hypothesized that the combination of trametinib and ganitumab would be effective in RAS-mutated neuroblastoma. In this study, trametinib and ganitumab synergistically suppressed neuroblastoma cell proliferation and induced apoptosis in cell culture. We also observed a delay in tumor initiation and prolongation of survival in heterotopic and orthotopic xenograft models treated with trametinib and ganitumab. However, the growth of both primary and metastatic tumors was observed in animals receiving the combination of trametinib and ganitumab. Therefore, more preclinical work is necessary before testing this combination in patients with relapsed or refractory RAS-mutated neuroblastoma.

Proliferation of SH-SY5Y neuroblastoma cells on confined spaces

Background:Microfluidics offers precise drug delivery and continuous monitoring of cell functions, which is crucial for studying the effects of toxins and drugs. Ensuring proper cell growth in these space-constrained systems is essential for obtaining consistent results comparable to standard Petri dishes. New method:We investigated the proliferation of SH-SY5Y cells on circular polycarbonate chambers with varying surface areas. SH-SY5Y cells were chosen for their relevance in neurodegenerative disease research. Results:Our study demonstrates a correlation between the chamber surface area and SH-SY5Y cell growth rates. Cells cultured in chambers larger than 10 mm in diameter exhibited growth comparable to standard 60-mm dishes. In contrast, smaller chambers significantly impeded growth, even at identical seeding densities. Similar patterns were observed for HeLaGFP cells, while 16HBE14σ cells proliferated efficiently regardless of chamber size. Additionally, SH-SY5Y cells were studied in a 12-mm diameter sealed chamber to assess growth under restricted gas exchange conditions. Comparison with existing methods:Our findings underscore the limitations of small chamber sizes in microfluidic systems for SH-SY5Y cells, an issue not typically addressed by conventional methods. Conclusions:SH-SY5Y cell growth is highly sensitive to spatial constraints, with markedly reduced proliferation in chambers smaller than 10 mm. This highlights the need to carefully consider chamber size in microfluidic experiments to achieve cell growth rates comparable to standard culture dishes. The study also shows that while SH-SY5Y and HeLaGFP cells are affected by chamber size, 16HBE14σ cells are not. These insights are vital for designing effective microfluidic systems for bioengineering research.

A mitochondria-related genes associated neuroblastoma signature - based on bulk and single-cell transcriptome sequencing data analysis, and experimental validation.

Neuroblastoma (NB), characterized by its marked heterogeneity, is the most common extracranial solid tumor in children. The status and functionality of mitochondria are crucial in regulating NB cell behavior. While the significance of mitochondria-related genes (MRGs) in NB is still missing in key knowledge. This study leverages consensus clustering and machine learning algorithms to construct and validate an MRGs-related signature in NB. Single-cell data analysis and experimental validation were employed to characterize the pivotal role of FEN1 within NB cells. MRGs facilitated the classification of NB patients into 2 distinct clusters with considerable differences. The constructed MRGs-related signature and its quantitative indicators, mtScore and mtRisk, effectively characterize the MRGs-related patient clusters. Notably, the MRGs-related signature outperformed MYCN in predicting NB patient prognosis and was adept at representing the tumor microenvironment (TME), tumor cell stemness, and sensitivity to the chemotherapeutic agents Cisplatin, Topotecan, and Irinotecan. FEN1, identified as the most contributory gene within the MRGs-related signature, was found to play a crucial role in the communication between NB cells and the TME, and in the developmental trajectory of NB cells. Experimental validations confirmed FEN1's significant influence on NB cell proliferation, apoptosis, cell cycle, and invasiveness. The MRGs-related signature developed in this study offers a novel predictive tool for assessing NB patient prognosis, immune infiltration, stemness, and chemotherapeutic sensitivity. Our findings unveil the critical function of FEN1 in NB, suggesting its potential as a therapeutic target.

Regulatory roles of insulin growth factor binding protein family in neuroblastoma cell proliferation and differentiation: Potential prognostic biomarkers and therapeutic targets for neuroblastoma

AbstractNeuroblastoma (NB), as a representative of tumors of embryonic origin in children, has specific clinical features. On the one hand, a very small number of NBs may appear to regress on their own. On the other hand, highly malignant NBs can invade the surrounding blood vessels and organs and metastasize to distant bone, bone marrow, and lymph nodes in the early stages of the disease. Based on differential affinities to insulin growth factors (IGFs), insulin growth factor binding proteins (IGFBPs) are classified into two groups: IGF binding proteins (IGFBP1‐6) with high‐affinity and IGF low‐affinity binding proteins, such as IGFBP‐related proteins (IGFBP rP1‐10). IGFBP are crucial regulators of the bioavailability and function of IGF in metabolic signaling and as modulators of IGF signaling, and their role in NB is gaining increasing attention. In this study, we investigate the involvement of IGFBP family members in the growth and differentiation of NB cells, as well as the potential of IGFBPs as prognostic biomarkers and therapeutic targets for human NB.

USP44 Overexpression Drives a MYC-Like Gene Expression Program in Neuroblastoma through Epigenetic Reprogramming.

Neuroblastoma is an embryonic cancer that contributes disproportionately to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer, although epigenetic pathways have been implicated in disease pathogenesis. We used an expression-based computational screen that examined the impact of deubiquitinating enzymes on patient survival to identify potential new targets. We identified the histone H2B deubiquitinating enzyme USP44 as the enzyme with the greatest impact on survival in patients with neuroblastoma. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN amplification. The subset of patients with tumors expressing high levels of USP44 had significantly worse survival, including those with tumors lacking MYCN amplification. We showed experimentally that USP44 regulates neuroblastoma cell proliferation, migration, invasion, and neuronal development. Depletion of the histone H2B ubiquitin ligase subunit RNF20 resulted in similar findings, strongly implicating this histone mark as the target of USP44 activity in this disease. Integration of transcriptome and epigenome in analyses demonstrates a distinct set of genes that are regulated by USP44, including those in Hallmark MYC target genes in both murine embryonic fibroblasts and the SH-SY5Y neuroblastoma cell line. We conclude that USP44 is a novel epigenetic regulator that promotes aggressive features and may be a novel target in neuroblastoma. Implications: This study identifies a new genetic marker of aggressive neuroblastoma and identifies the mechanisms by which its overactivity contributes to the pathophysiology of this disease.