Chemotherapy For Neuroblastoma Research Articles

Early evolutionary branching across spatial domains predisposes to clonal replacement under chemotherapy in neuroblastoma

Neuroblastoma (NB) is one of the most lethal childhood cancers due to its propensity to become treatment resistant. By spatial mapping of subclone geographies before and after chemotherapy across 89 tumor regions from 12 NBs, we find that densely packed territories of closely related subclones present at diagnosis are replaced under effective treatment by islands of distantly related survivor subclones, originating from a different most recent ancestor compared to lineages dominating before treatment. Conversely, in tumors that progressed under treatment, ancestors of subclones dominating later in disease are present already at diagnosis. Chemotherapy treated xenografts and cell culture models replicate these two contrasting scenarios and show branching evolution to be a constant feature of proliferating NB cells. Phylogenies based on whole genome sequencing of 505 individual NB cells indicate that a rich repertoire of parallel subclones emerges already with the first oncogenic mutations and lays the foundation for clonal replacement under treatment.

Atypical hemolytic uremic syndrome during induction chemotherapy in neuroblastoma, a rare phenomenon or common congenital predisposition?

Atypical hemolytic uremic syndrome (aHUS) is a complement-mediated thrombotic microangiopathy sometimes associated with germline variants in genes of the complement system. Clinical findings of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury arise due to aberrant complement protein activation in the circulation. A 13-month-old boy with metastatic neuroblastoma (NB) developed aHUS during his first cycle of induction chemotherapy with germline testing revealing a complement factor H(CFH) gene mutation, currently classified as a variant of uncertain significance (VUS). Now he is in disease remission after successful complement blockade therapy, thus highlighting a unique presentation of aHUS in a patient with newly diagnosed NB.

Self-Assembled Nanocomposite DOX/TPOR4@CB[7]4 for Enhanced Synergistic Photodynamic Therapy and Chemotherapy in Neuroblastoma.

DOX/TPOR4@CB[7]4 was synthesized via self-assembly, and its physicochemical properties and ability to generate reactive oxygen species (ROS) were evaluated. The impact of photodynamic therapy on SH-SY5Y cells was assessed using the MTT assay, while flow cytometry analysis was employed to detect cell apoptosis. Confocal laser scanning microscopy was utilized to observe the intracellular distribution of DOX/TPOR4@CB[7]4 in SH-SY5Y cells. Additionally, fluorescence imaging of DOX/TPOR4@CB[7]4 in nude mice bearing SH-SY5Y tumors and examination of the combined effects of photodynamic and chemical therapies were conducted. The incorporation of CB[7] significantly enhanced the optical properties of DOX/TPOR4@CB[7]4, resulting in increased ROS production and pronounced toxicity towards SH-SY5Y cells. Moreover, both the apoptotic and mortality rates exhibited significant elevation. In vivo experiments demonstrated that tumor growth inhibition was most prominent in the DOX/TPOR4@CB[7]4 group. π-π interactions facilitated the binding between DOX and photosensitizer TPOR, with TPOR's naphthalene hydrophilic groups encapsulated within CB[7]'s cavity through host-guest interactions with CB[7]. Therefore, CB[7] can serve as a nanocarrier to enhance the combined application of chemical therapy and photodynamic therapy, thereby significantly improving treatment efficacy against neuroblastoma tumors.

Neck stiffness and bone osteolytic lesion in a 3-years old child: a case report

BackgroundNeuroblastoma is the most frequent extracranial solid tumor occurring in childhood, representing approximately 28% of all cancers diagnosed in infants. Signs and symptoms of neuroblastoma vary with the site of development of the tumor and can mimic other diseases due to its extreme clinical variability. However, torticollis is not reported in the medical literature as a leading symptom of neuroblastoma.Case presentationHere we report the case of a 3 years-old girl with fever and neck stiffness. Blood tests revealed a mild anemia and a rise in inflammatory markers. CT-scan showed a solid, heterogeneous, predominantly hypodense surrenal mass with eccentric calcification and extensive inhomogeneity of the vertebral metamers. Blood tests revealed raised serum levels of Neuron-Specific Enolase. At the 24-hours urine collection urinary catecholamines were greatly increased. A course of chemotherapy for neuroblastoma was promptly started with immediate clinical improvement.ConclusionsThis case shows that the presence of torticollis could be a chief complaint of neuroblastoma. To our knowledge, neuroblastoma is not mentioned among life-threatening underlying conditions of torticollis in most recent literature reviews.

Hybrid Molecules of Benzylguanidine and the Alkylating Group of Melphalan: Synthesis and Effects on Neuroblastoma Cells.

The therapy of neuroblastoma relies, amongst other things, on administering chemotherapeutics and radioactive compounds, e.g., the (meta-iodobenzyl)guanidine [131I]mIBG. For special applications (conditioning before stem cell transplantation), busulfan and melphalan (M) proved to be effective. However, both drugs are not used for normal chemotherapy in neuroblastoma because of their side effects. The alkylating drug melphalan contains a (Cl-CH2-CH2-)2N- group in the para-position of the phenyl moiety of the essential amino acid phenylalanine (Phe) and can, therefore, be taken up by virtually all kinds of cells by amino acid transporters. In contrast, mIBG isotopologs are taken up more selectively by neuroblastoma cells via the noradrenaline transporter (NAT). The present study aimed at synthesising and studying hybrid molecules of benzylguanidine (BG) and the alkylating motif of M. Such hybrids should combine the preferential uptake of BGs into neuroblastoma cells with the cytotoxicity of M. Besides the hybrid of BG with the dialkylating group (Cl-CH2-CH2-)2N- bound in the para-position as in M (pMBG), we also synthesised mMBG, which is BG meta-substituted by a (Cl-CH2-CH2-)2N- group. Furthermore, two monoalkylating hybrid molecules were synthesised: the BG para-substituted by a (Cl-CH2-CH2-)NH- group (pM*BG) and the BG meta-substituted by a (Cl-CH2-CH2-)NH- group (mM*BG). The effects of the four new compounds were studied with human neuroblastoma cell lines (SK-N-SH, Kelly, and LS) with regard to uptake, viability, and proliferation by standard test systems. The dialkylating hybrid molecules pMBG and mMBG were at least as effective as M, whereas the monoalkylating hybrid molecules pM*BG and mM*BG were more effective than M. Considering the preferred uptake via the noradrenaline transporter by neuroblastoma cells, we conclude that they might be well suited for therapy.

Extra-adrenal peripheral neuroblastic tumors: A clinicopathological study of 18 cases

Peripheral neuroblastic tumors arise from the sympathoadrenal lineage of the neural crest. They have been classified according to the International Neuroblastoma Pathology Committee (INPC) into Four categories according to International Neuroblastoma Pathology Committee (INPC): a) Neuroblastoma (NB) b) Ganglioneuroblastoma (GNB), nodular c) Ganglioneuroblastoma, intermixed, and d) Ganglioneuroma (GN). Because of the rarity of extra-adrenal peripheral neuroblastic tumors, limited information is available regarding the chemotherapy of NB and GNB. A few case reports or case series with a small number of patients have been documented in the literature. To describe the clinicopathological characteristics of extra-adrenal peripheral neuroblastic tumors. Materials and. Clinical, histopathological, and immunohistochemistry (IHC) findings of 18 cases were retrieved. Immunohistochemistry at the time of diagnosis was performed using Ventana Benchmark XT. The mean value was calculated using the Microsoft Office Excel 2019 software. The posterior mediastinum was the most commonly affected extra-adrenal site in our study. Neuroblastoma consisted of eight cases (six in children, two in adults), of which four cases were poorly differentiated and the other four cases were differentiating. Two cases had favorable histology. The bone marrow and cervical lymph node metastasis were documented. Of the four GNB cases, one patient developed bone metastasis. All patients of NB and GNB received combination chemotherapy. One out of six GN patients presented with a large retroperitoneal mass encasing the aorta and renal vessels, mimicking a sarcoma. Extra-adrenal peripheral neuroblastic tumors do not pose any diagnostic issue in adequate tissue sampling. In limited material, immunohistochemistry is needed. The chemotherapy regimen has not been standardized due to rarity. Further molecular testing and targeted therapy may be of help in the future.

Clinically relevant treatment of PDX models reveals patterns of neuroblastoma chemoresistance.

Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB). Here, we developed a clinically relevant in vivo treatment protocol mimicking the first-line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN-amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal NB chemoresistance mechanisms. Intrinsic resistance was associated with high genetic diversity and an embryonic phenotype. Relapsed NB with acquired resistance showed a decreased adrenergic phenotype and an enhanced immature mesenchymal-like phenotype, resembling multipotent Schwann cell precursors. NBs with a favorable treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts. Novel integrated phenotypic gene signatures reflected treatment response and patient prognosis. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states. This work sheds light on the mechanisms of NB chemotherapy response and emphasizes the importance of transcriptional cell states in chemoresistance.

Nafamostat mesylate prevents metastasis and dissemination of neuroblastoma through vascular endothelial growth factor inhibition.

Neuroblastoma is a highly malignant disease with a poor prognosis and few treatment options. Despite conventional chemotherapy for neuroblastoma, resistance, invasiveness, and metastatic mobility limit the treatment efficacy. Therefore, it is necessary to develop new strategies for treating neuroblastoma. The present study aimed to evaluate the anticancer effects of nafamostat mesylate, a previously known serine protease inhibitor, on neuroblastoma cells. Effects of nafamostat mesylate on neuroblastoma cell migration and proliferation were analyzed by wound healing assay and WST-8 assay, respectively. To elucidate the mechanisms underlying the effects of nafamostat mesylate on neuroblastoma, the expression levels of NF-κB were measured via western blotting, and the production of the cytokine vascular endothelial growth factor (VEGF) in the cell culture supernatants was determined via ELISA. In addition, a mouse model of hematogenous metastasis was used to investigate the effects of nafamostat mesylate on neuroblastoma. It was determined that nafamostat mesylate significantly inhibited migration and invasion of Neuro-2a cells, but it had no effect on cell proliferation at 24 h after treatment. Exposure of Neuro-2a cells to nafamostat mesylate resulted in decreased vascular endothelial growth factor production, which could be a pivotal mechanism underlying the inhibitory effects of neuroblastoma metastasis. The results of the present study suggest that nafamostat mesylate may be an effective treatment against neuroblastoma invasion and metastasis.

Self-Assembled Nanocomposite DOX/TPOR4@CB[7] for the Enhanced Synergistic Photodynamic Therapy and Chemotherapy in Neuroblastoma

Self-Assembled Nanocomposite DOX/TPOR4@CB[7] for the Enhanced Synergistic Photodynamic Therapy and Chemotherapy in Neuroblastoma

Hemorrhage During Induction Chemotherapy in Neuroblastoma: Additional Risk Factors in High-Risk Patients.

Background: Neuroblastoma is the most common solid extracranial tumor in children. Patients affected by neuroblastoma are stratified into low, intermediate, and high risk in terms of event-free and overall survival. Some high-risk patients have an additional risk of acute hemorrhagic complications during induction chemotherapy.Aim: To find easily and rapidly assessed parameters that help clinicians identify those patients affected by high-risk neuroblastoma who have an additional risk of hemorrhagic complications.Methods: The clinical notes of patients diagnosed with high-risk neuroblastoma from January 2013 until February 2021 were retrospectively reviewed. Clinical, demographic and laboratory data, biological characteristics of the tumor, and information about treatment and hospital stay were identified.Results: In the examined period, 44 patients were diagnosed with high-risk neuroblastoma. Four of these patients had hemorrhagic complications within 2–7 days after the initiation of induction chemotherapy; two patients had hemothorax, one patient had hemoperitoneum and one patient had hemothorax and hemoperitoneum. The patient with isolated hemoperitoneum was treated with blood components transfusions, clotting factors and colloids infusions; the three patients with hemothorax underwent thoracostomy tube placement and respiratory support. At initial presentation, patients who suffered from hemorrhagic complications had a higher degree of hypertension (stage 2, p = 0.0003), higher levels of LDH (median 3,745 U/L, p = 0.009) and lower levels of hemoglobin (mean 7.6 gr/dl, p = 0.0007) compared to other high-risk patients.Conclusions: A subgroup of “additional” high-risk patients can be identified within the high-risk neuroblastoma patients based on mean arterial pressure, LDH levels and hemoglobin levels at presentation. Further studies to define cut-off values and optimal management strategies for these patients are needed.

ALT neuroblastoma chemoresistance due to telomere dysfunction-induced ATM activation is reversible with ATM inhibitor AZD0156.

Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P < 0.05; in vivo mouse event-free survival (EFS), P < 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC50, P < 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P < 0.001) and in four ALT xenografts in vivo (EFS, P < 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma.

Predicting response to chemotherapy in neuroblastoma using deep learning: A report from the International Neuroblastoma Risk Group.

10039 Background: Metaiodobenzylguanidine (MIBG) scans are a radionucleotide imaging modality used to evaluate neuroblastoma stage at diagnosis and also determine disease response following therapy. Curie scoring is used to semi-quantitatively assess disease burden from an MIBG scan on a scale from none (0) to widespread throughout the body (30). While a Curie score ≤2 after six cycles of induction chemotherapy has been shown to be prognostic of outcome, there is no established correlation between diagnostic Curie score and outcome. Deep learning models, such as convolutional neural networks (CNN), have been shown to learn generalizable patterns within images for successful classification of metastases and detection of multiple adult cancers. We hypothesized a CNN could be developed to predict response to induction chemotherapy, a proxy for outcome, using diagnostic MIBG scans. Methods: DICOM MIBG scans and associated clinical data from a Children’s Oncology Group (COG) pilot study for children diagnosed with high-risk neuroblastoma (ANBL12P1; NCT1798004) were deidentified and linked to clinical data by the Pediatric Cancer Data Commons and obtained from the International Neuroblastoma Risk Group Data Commons. Patients were defined as having a poor response to induction chemotherapy if their Curie score after four cycles of induction chemotherapy was ≥2. An independent external validation cohort was comprised of 29 images from 26 high-risk patients treated at the University of Chicago with clinically-annotated diagnostic and post-cycle six induction DICOM MIBG scans. The CNN was trained using 2D whole body MIBG scans obtained at diagnosis. We developed the CNN using a transfer learning approach using the Xception architecture as the base layer. Hyperparameter optimization was performed using an 80%-20% train-validation strategy. Model performance was evaluated using area under the receiver operating characteristic curve (AUROC). Results: Among 146 patients with high-risk neuroblastoma enrolled on ANBL12P1, 104 had available diagnostic and end-induction MIBG scans. There were no differences in clinical or biological characteristics between included and excluded patients. The base model CNN was able to predict which patients had a poor response to induction chemotherapy with an AUROC of 0.72 in the validation set from the ANBL12P1 cohort. Additionally, the CNN was able to predict patient response to therapy with an AUROC of 0.64 in an independent external dataset from University of Chicago. Conclusions: Our study suggests it is feasible to apply machine learning of diagnostic MIBG scans to predict response to chemotherapy for high-risk neuroblastoma patients. Given these promising results, further work to improve AUROC and performance within larger datasets is ongoing.

Early Decline of Neuron-Specific Enolase during Neuroblastoma Chemotherapy is a Predictive Factor of Clinical Outcome

High risk neuroblastoma (HR-NB) remains one of the most difficult-to-treat pediatric cancers. However, although current risk-stratification is based on multiple pretreatment criteria, HR-NB remains a significant heterogeneity. We examined 60 patients with HR-NB for a median follow-up time of 28 months. We examined the serum neuronspecific enolase (NSE) levels of each chemo cycle, using the survival receiver operating characteristic (survivalROC) method to assess the prognostic power of NSE levels at variant chemo points. We demonstrated that serum NSE was associated with systemic tumor burden. NSE after the third chemo cycle (C3) (C3NSE) was significantly higher in patients who eventually showed cancer relapse or progression. C3NSE had independent prognostic significance for event-free survival (EFS) but not for overall survival (OS) in multivariate cox analysis. SurvivalROC prompted that the C3NSE is a prognostic marker of HR-NB, which had good discrimination for 2- and 3-year EFS with AUC 0.734 and 0.729, respectively. However, its prognositc value for 2- and 3- year OS declined progressively. C3 is the optimal point to predict EFS. Patients whose C3 serum NSE remain at higher level need to undergo more intensive treatment as early as possible to resist recurrence.

Successful treatment with extracorporeal circulation and chemotherapy for neuroblastoma complicated by cardiac shock and severe renal failure

Successful treatment with extracorporeal circulation and chemotherapy for neuroblastoma complicated by cardiac shock and severe renal failure

Secondary renal neoplasia following chemotherapy or radiation in pediatric patients

Renal neoplasia occurring as a second malignancy following childhood cancer has been most closely associated with neuroblastoma and Wilms tumor. While some cases have been associated with a genetic predisposition, nearly all are thought to result from "late effects" of therapy-related toxicity that involves chemotherapy or radiation. It is unclear if these tumors are enriched for specific molecular or morphologic characteristics. A query of our institutional nephrectomy registry of 8295 patients for renal neoplasia occurring post-treatment for childhood cancer revealed 6 patients with Wilms tumor, 4 with neuroblastoma, and 1 with acute lymphoblastic leukemia (ALL). Three additional cases of MiT family translocation renal cell carcinoma (RCC), from 2 patients, following chemotherapy for neuroblastoma and systemic lupus erythematosus and another of clear cell RCC post-ALL were included. The most common tumor type was clear cell RCC: 9/19 cases (47.4%), followed by metanephric adenoma and MiT family translocation RCC (3/19, 15.8%). There were no characteristic features to indicate a unique renal neoplasia subtype. Potential syndromic renal neoplasia occurred in 2 patients, metanephric adenomas and oncocytoma in a patient with hyperparathyroidism-jaw tumor syndrome post-treatment of Wilms tumor and a fumarate hydratase-deficient RCC in a patient post-treatment for ALL. The mean age at diagnosis of childhood neoplasia or treatment with chemotherapy or radiation was 4.7 years, and the average time to subsequent renal neoplasia was 31 years. Five (of 14) patients developed metastatic RCC, and there were 2 RCC-related deaths. These results indicate the need for extended clinical follow-up of these patients.

LncRNA SNHG7 enhances chemoresistance in neuroblastoma through cisplatin-induced autophagy by regulating miR-329-3p/MYO10 axis.

Neuroblastoma (NB) is a type of extracranial solid tumor that usually occurs in children. Drug resistance has become a major obstacle in NB chemotherapy. Long non-coding RNA small nucleolar RNA host gene 7 (SNHG7) is an oncogene in many cancers, including NB. This study aimed to investigate the role of SNHG7 in cisplatin sensitivity of NB and the underlying mechanism. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability, and the IC50 of cisplatin was calculated. The protein levels of autophagy markers were measured by Western blot assay. The levels of SNHG7, miR-329-3p and myosin X (MYO10) were examined by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot assay. The interaction among SNHG7, miR-329-3p and MYO10 was confirmed by Dual-Luciferase reporter assay. Cisplatin curbed the viability of NB cells in a dose-dependent manner and facilitated autophagy in NB cells. Silencing of SNHG7 reduced cisplatin resistance and suppressed cisplatin-induced autophagy. SNHG7 was a sponge of miR-329-3p and modulated chemosensitivity and autophagy by regulating miR-329-3p. In addition, SNHG7 upregulated MYO10 by sponging miR-329-3p. MYO10 restored the effect of miR-329-3p on cisplatin sensitivity and autophagy. Moreover, suppression of autophagy blocked SNHG7-induced cisplatin resistance. Depletion of SNHG7 potentiated cisplatin sensitivity through inhibition of autophagy by modulating miR-329-3p/MYO10 axis, providing a new therapeutic approach to overcome cisplatin resistance in NB chemotherapy.

Knockdown of galectin-1 facilitated cisplatin sensitivity by inhibiting autophagy in neuroblastoma cells

Neuroblastoma (NB) is a type of solid extracranial tumor that usually occurs in babies and children. Chemotherapy is a common method for NB treatment, however, the drug resistance exerts during the chemotherapy of NB. Galectin-1 is a member of galectin family and plays a potent role in the development of chemotherapy and radiotherapy resistance. However, the effect of galectin-1 on cisplatin resistance in NB remains unknown. The present study aimed to investigate the role of galectin-1 in cisplatin resisitance and the potential mechanism. Human neuroblastoma SH-SY5Y and SK-N-SH cells were treated with cisplatin and/or galectin-1/siRNA targeting galectin-1 (si-Gal-1). The cell viability was measured by MTT assay. The IC50 values for cisplatin of neuroblastoma cells were calculated. The expression levels of autophagy markers including microtubule-associated protein light chain 3 (LC3B), Beclin-1, and p62 were detected by western blot. We found that cisplatin inhibited cell viability of SH-SY5Y and SK-N-SH in a dose-dependent manner. Cisplatin induced the ratio of LC3B-II/LC3B-I and Beclin-1 expression, and inhibited the p62 expression. Knockdown of galectin-1 decreased the IC50 for cisplatin of SH-SY5Y and SK-N-SH cells and inhibited cisplatin-induced autophagy. Moreover, inhibition of autophagy suppressed galectin-1-induced increase in IC50 for cisplatin. In conclusion, galectin-1 knockdown enhanced cisplatin sensitivity of neuroblastoma cells by inhibiting autophagy. The findings might provid a novel therapeutic target to overcome cisplatin resistance in chemotherapy of NB.

Massive Intra-Tumor Hemorrhage during Induction Chemotherapy in Neuroblastoma: A Case Series Report

Massive Intra-Tumor Hemorrhage during Induction Chemotherapy in Neuroblastoma: A Case Series Report

TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas.

The objective of this study was to examine the combined effect of Interferon-gamma (IFN-γ) and Tumor Necrosis factor-alpha (TNF-α) on cytotoxicity and expression of prostate apoptosis response-4 (Par-4) and Par-4 interacting proteins B-cell lymphoma (Bcl-2), nuclear factor kappa-light-chain-enhancer of activated B cells/p65 subunit (NF-κB/p65), Ak mouse strain thymoma (Akt) in human neuroblastoma (NB) cells. Materials and methods included human neuroblastoma cell lines-SK-N-MC, SK-N-SH, and SH-SY5Y, which were treated with IFN-γ and TNF-α individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flow cytometry), DNA fragmentation, Poly (ADP-ribose) polymerase (PARP) cleavage, and caspase-8 activity. Transcript level of Par-4 was measured by RT-PCR. Protein levels of Par-4 and suppressor of cytokine signaling 3 (SOCS-3) were assessed by immunoblotting. Cellular localization of Par-4 and p65 was examined by immunofluorescence. Unbiased transcript analysis for IFN-γ, TNF-α, and Par-4 were analyzed from three independent clinical datasets from neuroblastoma patients. In terms of results, SK-N-MC cells treated with a combination of, but not individually with, IFN-γ and TNF-α induced apoptosis characterized by hypodiploidy, DNA fragmentation, PARP cleavage, and increased caspase-8 activity. Apoptosis was associated with up-regulation of Par-4 mRNA and protein expression. Immunofluorescence studies revealed that Par-4 was localized exclusively in cytoplasm in SK-N-MC cells cultured for 24 h. but showed nuclear localization at 48 h. Treatment with IFN-γ and TNF-α together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-γ, and TNF-α have strong synergy with Par-4 expression and provide good survival advantage. The findings also demonstrated that apoptosis was associated with reduced level of pro-survival proteins–Bcl-2 and Akt and NF-κB/p65. Furthermore, the apoptotic effect induced by IFN-γ-induced Signal Transducer and Activator of Transcription-1(STAT-1), and could be due to down-regulation of suppressor of cytokine signaling-3 (SOCS3). The study concludes that a combinatorial approach using IFN-γ and TNF-α can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process.

Neuroblastoma chemotherapy can be augmented by immunotargeting O-acetyl-GD2 tumor-associated ganglioside

ABSTRACTDespite recent advances in high-risk neuroblastoma therapy, the prognosis for patients remains poor. In addition, many patients suffer from complications related to available therapies that are highly detrimental to their quality of life. New treatment modalities are, thus, urgently needed to further improve the efficacy and reduce the toxicity of existing therapies. Since antibodies specific for O-acetyl GD2 ganglioside display pro-apoptotic activity against neuroblastoma cells, we hypothesized that combination of immunotherapy could enhance tumor efficacy of neuroblastoma chemotherapy.We demonstrate here that combination of anti-O-acetyl GD2 monoclonal antibody 8B6 with topotecan synergistically inhibited neuroblastoma cell proliferation, as shown by the combination index values. Mechanistically, we evidence that mAb 8B6 induced plasma cell membrane lesions, consistent with oncosis. Neuroblastoma tumour cells treated with mAb 8B6 indeed showed an increased uptake of topotecan by the tumor cells and a more profound tumor cell death evidenced by increased caspase-3 activation. We also found that the combination with topotecan plus monoclonal antibody 8B6 showed a more potent anti-tumor efficacy in vivo than either agent alone. Importantly, we used low-doses of topotecan with no noticeable side effect. Our data suggest that chemo-immunotherapy combinations may improve the clinical efficacy and safety profile of current chemotherapeutic modalities of neuroblastoma.