In vivo cisplatin-resistant neuroblastoma metastatic model reveals tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as an independent prognostic factor of survival in neuroblastoma.

Neuroblastoma is the most common solid extracranial tumour in children. Despite major advances in available therapies, children with drug-resistant and/or recurrent neuroblastoma have a dismal outlook with 5-year survival rates of less than 20%. Therefore, tackling relapsed tumour biology by developing and characterising clinically relevant models is a priority in finding targetable vulnerability in neuroblastoma. Using matched cisplatin-sensitive KellyLuc and resistant KellyCis83Luc cell lines, we developed a cisplatin-resistant metastatic MYCN-amplified neuroblastoma model. The average number of metastases per mouse was significantly higher in the KellyCis83Luc group than in the KellyLuc group. The vast majority of sites were confirmed as having lymph node metastasis. Their stiffness characteristics of lymph node metastasis values were within the range reported for the patient samples. Targeted transcriptomic profiling of immuno-oncology genes identified tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as a significantly dysregulated MYCN-independent gene. Importantly, differential TNFRSF4 expression was identified in tumour cells rather than lymphocytes. Low TNFRSF4 expression correlated with poor prognostic indicators in neuroblastoma, such as age at diagnosis, stage, and risk stratification and significantly associated with reduced probability of both event-free and overall survival in neuroblastoma. Therefore, TNFRSF4 Low expression is an independent prognostic factor of survival in neuroblastoma.

Assessment of Basic Biological Functions Exerted by miRNAs.

To evaluate basic miRNA function, it is vital to assess various cellular processes, such as cell viability and proliferation, under different miRNA levels. Here we describe the process of overexpression of miRNA in vitro via transfection and subsequent downstream evaluation using the acid phosphatase assay.

Evaluation of miRNA Expression in 3D In Vitro Scaffold-Based Cancer Models.

Accumulating experimental evidence suggests that 3D in vitro cancer models strengthen our understanding of vital processes in the tumor microenvironment (TME) and accelerate the drug discovery pipeline. Previous studies examining the effects of specific miRNAs on cancer cells in vitro have involved ectopic expression of miRNA mimics in 2D in vitro culture. Assessment of cell viability and gene expression ensures that upregulation of the chosen miRNA and repression of its target genes have been achieved. However, this 2D culture is overly simplified and lacks the complex cell to extracellular matrix (ECM) interactions observed in the native TME, yielding results often not reproduced when progressed to in vivo studies. Hence, this chapter describes a novel method of overexpressing the miRNA mimic in cells cultured on 3D collagen-based scaffolds adapted from tissue engineering techniques. Cell growth on scaffolds is sequentially monitored via a DNA quantification assay, and overexpression of the miRNA mimic and repression of its target gene is assessed via reverse transcription quantitative PCR (RT-qPCR).

Methodological advances in the discovery of novel neuroblastoma therapeutics.

INTRODUCTION: Neuroblastoma is a cancer of the sympathetic nervous system that causes up to 15% of cancer-related deaths among children. Among the ~1,000 newly diagnosed cases per year in Europe, more than half are classified as high-risk, with a 5-year survival rate <50%. Current multimodal treatments have improved survival among these patients, but relapsed and refractory tumors remain a major therapeutic challenge. A number of new methodologies are paving the way for the development of more effective and safer therapies to ultimately improve outcomes for high-risk patients. AREAS COVERED: The authors provide a critical review on methodological advances aimed at providing new therapeutic opportunities for neuroblastoma patients, including preclinical models of human disease, generation of omics data to discover new therapeutic targets, and artificial intelligence-based technologies to implement personalized treatments. EXPERT OPINION: While survival of childhood cancer has improved over the past decades, progress has been uneven. Still, survival is dismal for some cancers, including high-risk neuroblastoma. Embracing new technologies (e.g. molecular profiling of tumors, 3D in vitro models, etc.), international collaborative efforts and the incorporation of new therapies (e.g. RNA-based therapies, epigenetic therapies, immunotherapy) will ultimately lead to more effective and safer therapies for these subgroups of neuroblastoma patients.

Three-dimensional In Vitro Biomimetic Model of Neuroblastoma using Collagen-based Scaffolds.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response. This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation. The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining. This model's applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in animal models, and human subjects.

Differentiating Neuroblastoma: A Systematic Review of the Retinoic Acid, Its Derivatives, and Synergistic Interactions.

A neuroblastoma (NB) is a solid paediatric tumour arising from undifferentiated neuronal cells. Despite the recent advances in disease management and treatment, it remains one of the leading causes of childhood cancer deaths, thereby necessitating the development of new therapeutic agents and regimens. Retinoic acid (RA), a vitamin A derivative, is a promising agent that can induce differentiation in NB cells. Its isoform, 13-cis RA or isotretinoin, is used in NB therapy; however, its effectiveness is limited to treating a minimal residual disease as maintenance therapy. As such, research focuses on RA derivatives that might increase the anti-NB action or explores the potential synergy between RA and other classes of drugs, such as cellular processes mediators, epigenetic modifiers, and immune modulators. This review summarises the in vitro, in vivo, and clinical data of RA, its derivatives, and synergising compounds, thereby establishing the most promising RA derivatives and combinations of RA for further investigation.

Neuroblastoma Invasion Strategies Are Regulated by the Extracellular Matrix.

Neuroblastoma is a paediatric malignancy of the developing sympathetic nervous system. About half of the patients have metastatic disease at the time of diagnosis and a survival rate of less than 50%. Our understanding of the cellular processes promoting neuroblastoma metastases will be facilitated by the development of appropriate experimental models. In this study, we aimed to explore the invasion of neuroblastoma cells and organoids from patient-derived xenografts (PDXs) grown embedded in 3D extracellular matrix (ECM) hydrogels by time-lapse microscopy and quantitative image analysis. We found that the ECM composition influenced the growth, viability and local invasion of organoids. The ECM compositions induced distinct cell behaviours, with Matrigel being the preferred substratum for local organoid invasion. Organoid invasion was cell line- and PDX-dependent. We identified six distinct phenotypes in PDX-derived organoids. In contrast, NB cell lines were more phenotypically restricted in their invasion strategies, as organoids isolated from cell line-derived xenografts displayed a broader range of phenotypes compared to clonal cell line clusters. The addition of FBS and bFGF induced more aggressive cell behaviour and a broader range of phenotypes. In contrast, the repression of the prognostic neuroblastoma marker, MYCN, resulted in less aggressive cell behaviour. The combination of PDX organoids, real-time imaging and the novel 3D culture assays developed herein will enable rapid progress in elucidating the molecular mechanisms that control neuroblastoma invasion.

A Context-Dependent Role for MiR-124-3p on Cell Phenotype, Viability and Chemosensitivity in Neuroblastoma in vitro.

Neuroblastoma (NB) is a neural crest-derived tumor, which develops before birth or in early childhood, with metastatic dissemination typically preceding diagnosis. Tumors are characterized by a highly heterogeneous combination of cellular phenotypes demonstrating varying degrees of differentiation along different lineage pathways, and possessing distinct super-enhancers and core regulatory circuits, thereby leading to highly varied malignant potential and divergent clinical outcomes. Cytoskeletal reorganization is fundamental to cellular transformations, including the processes of cellular differentiation and epithelial to mesenchymal transition (EMT), previously reported by our lab and others to coincide with chemotherapy resistance and enhanced metastatic ability of tumor cells. This study set out to investigate the ability of the neuronal miR-124-3p to reverse the cellular transformation associated with drug resistance development and assess the anti-oncogenic role of this miRNA in in vitro models of drug-resistant adrenergic (ADRN) and mesenchymal (MES) neuroblastoma cell lines. Low expression of miR-124-3p in a cohort of neuroblastomas was significantly associated with poor overall and progression-free patient survival. Over-expression of miR-124-3p in vitro inhibited cell viability through the promotion of cell cycle arrest and induction of apoptosis in addition to sensitizing drug-resistant cells to chemotherapeutics in a panel of morphologically distinct neuroblastoma cell lines. Finally, we describe miR-124-3p direct targeting and repression of key up-regulated cytoskeletal genes including MYH9, ACTN4 and PLEC and the reversal of the resistance-associated EMT and enhanced invasive capacity previously reported in our in vitro model (SK-N-ASCis24).

Extracellular Vesicle Dissemination of Epidermal Growth Factor Receptor and Ligands and Its Role in Cancer Progression.

The epidermal growth factor receptor (EGFR) pathway functions through the autocrine or paracrine activation of cellular EGFR by a number of transmembrane ligands. Amplified or mutant EGFR can lead to tumour formation due to increased cell proliferation, growth, migration and survival signals. These oncogenic effects were thought to be confined to aberrant cells hosting genetic alterations in EGFR. However, in the past decade, numerous studies identified that tumour cells could harness extracellular vesicles (EVs) to disseminate EGFR, mutant EGFR, phosphorylated EGFR and EGFR ligands to local and distant cells. This functions to impart a pro-tumourigenic phenotype in recipient cells. EVs play an essential role in intracellular communication, through receptor signalling or the release of their intra-vesicular content into recipient cells. This review will discuss the role of EVs delivering EGFR or EGFR ligands either to or from tumour cells and how this can promote metastases, pre-metastatic niche formation, osteoclastogenesis, angiogenesis and immune modulation in cancer. We will examine how circulating EVs positive for EGFR may be exploited as diagnostic, prognostic or therapeutic markers in cancers including breast, lung, glioblastoma, ovarian and prostate. Finally, we will explore recent breakthroughs in bio-engineering EVs with EGFR targeting abilities for targeted drug delivery.

A combined experimental and computational study on peptide nucleic acid (PNA) analogues of tumor suppressive miRNA-34a.

MicroRNAs are a ubiquitous class of non-coding RNAs able to regulate gene expression in diverse biological processes. Widespread miRNAs deregulation was reported in numerous diseases including cancer, with several miRNAs playing oncogenic and/or tumor suppressive role by targeting multiple mRNAs simultaneously. Based on these findings, miRNAs have emerged as promising therapeutic tools for cancer treatment. Herein, for the first time, peptide nucleic acids (PNAs) were studied to develop a new class of molecules able to target 3'UTR on MYCN mRNA without a fully complementary base pairing sequence (as miRNAs). For our proof of concept study we have selected as a model the miRNA-34a, which acts as a tumor suppressor in a number of cancers including neuroblastoma. In particular, miRNA-34a is a direct regulator of MYCN oncogene, whose overexpression is a prominent biomarker for the highly aggressive neuroblastoma phenotype. The design and synthesis of three PNA-based oligomers of different length was described, and their interaction with two binding sites on the target MYCN mRNA was investigated by molecular dynamics simulation, and spectroscopic techniques (CD, UV). Intake assay and confocal microscopy of PNA sequences were also carried out in vitro on neuroblastoma Kelly cells. Despite the presence of multiple mismatches, the PNA/RNA hetero duplexes retain very interesting features in terms of stability, affinity as well as of cellular uptake.

Functional high-throughput screening reveals miR-323a-5p and miR-342-5p as new tumor-suppressive microRNA for neuroblastoma.

Current therapies for most non-infectious diseases are directed at or affect functionality of the human translated genome, barely 2% of all genetic information. By contrast, the therapeutic potential of targeting the transcriptome, ~ 70% of the genome, remains largely unexplored. RNA therapeutics is an emerging field that widens the range of druggable targets and includes elements such as microRNA. Here, we sought to screen for microRNA with tumor-suppressive functions in neuroblastoma, an aggressive pediatric tumor of the sympathetic nervous system that requires the development of new therapies. We found miR-323a-5p and miR-342-5p to be capable of reducing cell proliferation in multiple neuroblastoma cell lines in vitro and in vivo, thereby providing a proof of concept for miRNA-based therapies for neuroblastoma. Furthermore, the combined inhibition of the direct identified targets such as CCND1, CHAF1A, INCENP and BCL-XL could reveal new vulnerabilities of high-risk neuroblastoma.

Endogenous exosome labelling with an amphiphilic NIR-fluorescent probe.

The recognition of the biological, diagnostic and medical importance of exosomes has given rise to an urgent need for efficient labelling of these extracellular vesicles in ways that do not alter their inherent characteristics. We report for the first time an endogenous method to NIR-fluorescent labelled exosomes using an amphiphilic probe without the need for immunolabelling or synthetic or chromatographic manipulation of exosomes. Comparative analyses of labelled and unlabelled exosomes with NTA, AFM, flow cytometry and immunoblot analysis all show a high degree of similarity. Spectroscopic analysis and fluorescence imaging confirmed the ability to visualise purified NIR-exosomes.

MicroRNA-302d targets IRF9 to regulate the IFN-induced gene expression in SLE.

Systemic lupus erythematosus (SLE) is a complex disease targeting multiple organs as a result of overactivation of the type I interferon (IFN) system, a feature currently being targeted by multiple biologic therapies against IFN-α. We have identified an estrogen-regulated microRNA, miR-302d, whose expression is decreased in SLE patient monocytes and identify its target as interferon regulatory factor (IRF)-9, a critical component of the transcriptional complex that regulates expression of interferon-stimulated genes (ISGs). In keeping with the reduced expression of miR-302d in SLE patient monocytes, IRF9 levels were increased, as was expression of a number of ISGs including MX1 and OAS1. In vivo evaluation revealed that miR-302d protects against pristane-induced inflammation in mice by targeting IRF9 and hence ISG expression. Importantly, patients with enhanced disease activity have markedly reduced expression of miR-302d and enhanced IRF9 and ISG expression, with miR-302d negatively correlating with IFN score. Together these findings identify miR-302d as a key regulator of type I IFN driven gene expression via its ability to target IRF9 and regulate ISG expression, underscoring the importance of non-coding RNA in regulating the IFN pathway in SLE.

Assessment of Basic Biological Functions Exerted by miRNAs.

Assessment of cell viability and proliferation under different miRNA expression levels is an important step in the evaluation of basic miRNA functional effects within the cell. Here, we describe the overexpression of miRNA in question in cells achieved by transfection with subsequent examination of cell viability and proliferation over a period of time using the acid phosphatase assay.

MicroRNA-497 impairs the growth of chemoresistant neuroblastoma cells by targeting cell cycle, survival and vascular permeability genes.

Despite multimodal therapies, a high percentage of high-risk neuroblastoma (NB) become refractory to current treatments, most of which interfere with cell cycle and DNA synthesis or function, activating the DNA damage response (DDR). In cancer, this process is frequently altered by deregulated expression or function of several genes which contribute to multidrug resistance (MDR). MicroRNAs are outstanding candidates for therapy since a single microRNA can modulate the expression of multiple genes of the same or different pathways, thus hindering the development of resistance mechanisms by the tumor. We found several genes implicated in the MDR to be overexpressed in high-risk NB which could be targeted by microRNAs simultaneously. Our functional screening identified several of those microRNAs that reduced proliferation of chemoresistant NB cell lines, the best of which was miR-497. Low expression of miR-497 correlated with poor patient outcome. The overexpression of miR-497 reduced the proliferation of multiple chemoresistant NB cell lines and induced apoptosis in MYCN-amplified cell lines. Moreover, the conditional expression of miR-497 in NB xenografts reduced tumor growth and inhibited vascular permeabilization. MiR-497 targets multiple genes related to the DDR, cell cycle, survival and angiogenesis, which renders this molecule a promising candidate for NB therapy.

The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro.

Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.

Modulation of chemotherapeutic drug resistance in neuroblastoma SK-N-AS cells by the neural apoptosis inhibitory protein and miR-520f.

The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in ∼30% of high-risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high-risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non-MYCN amplified tumour cells, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug-resistance and have implications for future therapeutic research.

The carboxy-terminal segment of the human LINE-1 ORF2 protein is involved in RNA binding.

The human LINE-1/L1 ORF2 protein is a multifunctional enzyme which plays a vital role in the life cycle of the human L1 retrotransposon. The protein consists of an endonuclease domain, followed by a central reverse transcriptase domain and a carboxy-terminal C-domain with unknown function. Here, we explore the nucleic acid binding properties of the 180-amino acid carboxy-terminal segment (CTS) of the human L1 ORF2p in vitro. In a series of experiments involving gel shift assay, we demonstrate that the CTS of L1 ORF2p binds RNA in non-sequence-specific manner. Finally, we report that mutations destroying the putative Zn-knuckle structure of the protein do not significantly affect the level of RNA binding and discuss the possible functional role of the CTS in L1 retrotransposition.

The role of genetic and epigenetic alterations in neuroblastoma disease pathogenesis.

Neuroblastoma is a highly heterogeneous tumor accounting for 15 % of all pediatric cancer deaths. Clinical behavior ranges from the spontaneous regression of localized, asymptomatic tumors, as well as metastasized tumors in infants, to rapid progression and resistance to therapy. Genomic amplification of the MYCN oncogene has been used to predict outcome in neuroblastoma for over 30 years, however, recent methodological advances including miRNA and mRNA profiling, comparative genomic hybridization (array-CGH), and whole-genome sequencing have enabled the detailed analysis of the neuroblastoma genome, leading to the identification of new prognostic markers and better patient stratification. In this review, we will describe the main genetic factors responsible for these diverse clinical phenotypes in neuroblastoma, the chronology of their discovery, and the impact on patient prognosis.

Inhibition of neuroblastoma tumor growth by targeted delivery of microRNA-34a using anti-disialoganglioside GD2 coated nanoparticles.

BACKGROUND: Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD(2)), providing a target for tumor-specific delivery. PRINCIPAL FINDINGS: Nanoparticles encapsulating miR-34a and conjugated to a GD(2) antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors. SIGNIFICANCE: These novel findings highlight the potential of anti-GD(2)-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD(2)-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth.