Vitronectin Levels in the Plasma of Neuroblastoma Patients and Culture Media of 3D Models: A Prognostic Circulating Biomarker?

Vitronectin is a glycoprotein present in plasma and the extracellular matrix that is implicated in cell migration. The high amount of vitronectin found in neuroblastoma biopsies has been associated with poor prognosis. Moreover, increased vitronectin levels have been described in the plasma of patients with different cancers. Our aim was to assess vitronectin as a potential circulating biomarker of neuroblastoma prognosis. Vitronectin concentration was quantified using ELISA in culture media of four neuroblastoma cell lines grown in a monolayer and in 3D models, and in the plasma of 114 neuroblastoma patients. Three of the neuroblastoma cell lines secreted vitronectin to culture media when cultured in a monolayer and 3D models. Vitronectin release was higher by neuroblastoma cells cultured in 3D models than in the monolayer and was still elevated when cells were grown in 3D scaffolds with cross-linked vitronectin. Vitronectin secretion occurred independently of cell numbers in cultures. Its concentration in the plasma of neuroblastoma patients ranged between 52.4 and 870 µg/mL (median, 218 µg/mL). A ROC curve was used to establish a cutoff of 361 µg/mL, above which patients over 18 months old had worse prognosis (p = 0.0018). Vitronectin could be considered a new plasma prognostic biomarker in neuroblastoma and warrants confirmation in collaborative studies. Drugs inhibiting vitronectin interactions with cells and/or the extracellular matrix could represent a significant improvement in survival for neuroblastoma patients.

The topology of vitronectin: A complementary feature for neuroblastoma risk classification based on computer-aided detection.

Tumors are complex networks of constantly interacting elements: tumor cells, stromal cells, immune and stem cells, blood/lympathic vessels, nerve fibers and extracellular matrix components. These elements can influence their microenvironment through mechanical and physical signals to promote tumor cell growth. To get a better understanding of tumor biology, cooperation between multidisciplinary fields is needed. Diverse mathematic computations and algorithms have been designed to find prognostic targets and enhance diagnostic assessment. In this work, we use computational digital tools to study the topology of vitronectin, a glycoprotein of the extracellular matrix. Vitronectin is linked to angiogenesis and migration, two processes closely related to tumor cell spread. Here, we investigate whether the distribution of this molecule in the tumor stroma may confer mechanical properties affecting neuroblastoma aggressiveness. Combining image analysis and graph theory, we analyze different topological features that capture the organizational cues of vitronectin in histopathological images taken from human samples. We find that the Euler number and the branching of territorial vitronectin, two topological features, could allow for a more precise pretreatment risk stratification to guide treatment strategies in neuroblastoma patients. A large amount of recently synthesized VN would create migration tracks, pinpointed by both topological features, for malignant neuroblasts, so that dramatic change in the extracellular matrix would increase tumor aggressiveness and worsen patient outcomes.

Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models.

Patient-derived cancer 3D models are a promising tool that will revolutionize personalized cancer therapy but that require previous knowledge of optimal cell growth conditions and the most advantageous parameters to evaluate biomimetic relevance and monitor therapy efficacy. This study aims to establish general guidelines on 3D model characterization phenomena, focusing on neuroblastoma. We generated gelatin-based scaffolds with different stiffness and performed SK-N-BE(2) and SH-SY5Y aggressive neuroblastoma cell cultures, also performing co-cultures with mouse stromal Schwann cell line (SW10). Model characterization by digital image analysis at different time points revealed that cell proliferation, vitronectin production, and migration-related gene expression depend on growing conditions and are specific to the tumor cell line. Morphometric data show that 3D in vitro models can help generate optimal patient-derived cancer models, by creating, identifying, and choosing patterns of clinically relevant artificial microenvironments to predict patient tumor cell behavior and therapeutic responses.

Therapeutic Opportunities in Neuroblastoma Using Nanotechnology.

Neuroblastoma (NB) is the most common extracranial solid tumor preferentially occurring in preschoolers. Its characteristic aggressiveness and heterogeneous clinical behavior are especially visible in relapsed or refractory cases and hamper therapeutic success. Although the introduction of novel antitumor agents, such as dinutuximab, isotretinoin, irinotecan, or I-131- metaiodobenzylguanidine, has increased survival rates, the situation in high-risk NB remains dismal. Moreover, treatment is particularly aggressive in these patients, leading to short- and long-term toxicities. The extensive research performed using nanotechnology in recent decades has prompted its application as a therapeutic alternative to overcome some of the common limitations of conventional chemotherapy. Nevertheless, the therapeutic role of nanomedicine in pediatric tumors like NB is not fully elucidated, and to date, only albumin-bound paclitaxel nanoparticles have reached clinic stages. In this review, we summarize the current therapeutic strategies for NB with special attention to the use of nanomedicine. We also highlight the preclinical studies on passive and active targeting nanodelivery of therapeutics in experimental NB models.

Vitronectin as a molecular player of the tumor microenvironment in neuroblastoma.

BACKGROUND: Vitronectin is a multifunctional glycoprotein known in several human tumors for its adhesive role in processes such as cell growth, angiogenesis and metastasis. In this study, we examined vitronectin expression in neuroblastoma to investigate whether this molecule takes part in cell-cell or cell-extracellular matrix interactions that may confer mechanical properties to promote tumor aggressiveness. METHODS: We used immunohistochemistry and image analysis tools to characterize vitronectin expression and to test its prognostic value in 91 neuroblastoma patients. To better understand the effect of vitronectin, we studied its in vitro expression using commercial neuroblastoma cell lines and in vivo using intra-adrenal gland xenograft models by immunohistochemistry. RESULTS: Digital image analysis allowed us to associate vitronectin staining intensity and location discriminating between territorial vitronectin and interterritorial vitronectin expression patterns. High territorial vitronectin expression (strong staining associated with pericellular and intracellular location) was present in tumors from patients with metastatic undifferentiating neuroblastoma, that were MYCN amplified, 11q deleted or with segmental chromosomal profiles, in the high-risk stratification group and with high genetic instability. In vitro studies confirmed that vitronectin is expressed in tumor cells as small cytoplasmic dot drops. In vivo experiments revealed tumor cells with high and dense cytoplasmic vitronectin expression. CONCLUSIONS: These findings highlight the relevance of vitronectin in neuroblastoma tumor biology and suggest its potential as a future therapeutic target in neuroblastoma.

High Oct4 expression: implications in the pathogenesis of neuroblastic tumours.

BACKGROUND: Neuroblastic tumours (NBTs) are paediatric solid tumours derived from embryonic neural crest cells which harbour their own cancer stem cells (CSC). There is evidence indicating that CSC may be responsible for tumour progression, chemotherapy resistance and recurrence in NBTs. Oct4 is a transcription factor which plays a key role in mammal embryonic development and stem cell fate regulation. The aim of the study is to elucidate the clinical significance of Oct4 in NBTs. METHODS: We studied Oct4 expression in 563 primary NBTs using digital image quantification. Chi-square test was applied to analyse the correlation between histopathology and the Oct4+ cell percentage. Survival analysis was carried out with Kaplan-Meier curves and log-rank test. Additionally, a multivariate Cox regression analysis with the stepwise backwards (Wald) method was undertaken to calculate the impact of Oct4 expression level on survival. RESULTS: We found that tumours with a high proportion of cells expressing Oct4 correlated statistically with undifferentiated and poorly differentiated neuroblastoma / nodular ganglioneuroblastoma, and that Oct4 expression was not present in ganglioneuroma (p < 0.05). Statistical analysis also indicated a relationship between high Oct4 expression levels, high-risk patients according to the International Neuroblastoma Risk Group pre-treatment classification parameters, larger blood vessels and low survival rates. CONCLUSIONS: These results suggest that the Oct4 gene may regulate NBT pathogenic differentiation pathways, and should thus be considered as a target for knockdown when developing novel therapies for high-risk NBT patients.

Heterogeneous MYCN amplification in neuroblastoma: a SIOP Europe Neuroblastoma Study.

BACKGROUND: In neuroblastoma (NB), the most powerful prognostic marker, the MYCN amplification (MNA), occasionally shows intratumoural heterogeneity (ITH), i.e. coexistence of MYCN-amplified and non-MYCN-amplified tumour cell clones, called heterogeneous MNA (hetMNA). Prognostication and therapy allocation are still unsolved issues. METHODS: The SIOPEN Biology group analysed 99 hetMNA NBs focussing on the prognostic significance of MYCN ITH. RESULTS: Patients <18 months (18 m) showed a better outcome in all stages as compared to older patients (5-year OS in localised stages: <18 m: 0.95 ± 0.04, >18 m: 0.67 ± 0.14, p = 0.011; metastatic: <18 m: 0.76 ± 0.15, >18 m: 0.28 ± 0.09, p = 0.084). The genomic 'background', but not MNA clone sizes, correlated significantly with relapse frequency and OS. No relapses occurred in cases of only numerical chromosomal aberrations. Infiltrated bone marrows and relapse tumour cells mostly displayed no MNA. However, one stage 4s tumour with segmental chromosomal aberrations showed a homogeneous MNA in the relapse. CONCLUSIONS: This study provides a rationale for the necessary distinction between heterogeneous and homogeneous MNA. HetMNA tumours have to be evaluated individually, taking age, stage and, most importantly, genomic background into account to avoid unnecessary upgrading of risk/overtreatment, especially in infants, as well as in order to identify tumours prone to developing homogeneous MNA.

9-ING-41, a small-molecule glycogen synthase kinase-3 inhibitor, is active in neuroblastoma.

Advanced stage neuroblastoma is a very aggressive pediatric cancer with limited treatment options and a high mortality rate. Glycogen synthase kinase-3ß (GSK-3ß) is a potential therapeutic target in neuroblastoma. Using immunohistochemical staining, we observed positive GSK-3ß expression in 67% of human neuroblastomas (34 of 51 cases). Chemically distinct GSK-3 inhibitors (AR-A014418, TDZD-8, and 9-ING-41) suppressed the growth of neuroblastoma cells, whereas 9-ING-41, a clinically relevant small-molecule GSK-3ß inhibitor with broad-spectrum preclinical antitumor activity, being the most potent. Inhibition of GSK-3 resulted in a decreased expression of the antiapoptotic molecule XIAP and an increase in neuroblastoma cell apoptosis. Mouse xenograft studies showed that the combination of clinically relevant doses of CPT-11 and 9-ING-41 led to greater antitumor effect than was observed with either agent alone. These data support the inclusion of patients with advanced neuroblastoma in clinical studies of 9-ING-41, especially in combination with CPT-11.

A stiff extracellular matrix is associated with malignancy in peripheral neuroblastic tumors.

PURPOSE AND OBJECTIVE: Improved prognosis for patients with peripheral neuroblastic tumors (PNB) depends on enhanced pretreatment risk stratification combined with research into new therapeutic targets. This study investigated the potential contribution of extracellular matrix (ECM) elements toward this endeavor. METHODS: We characterized certain elements such as reticulin fibers, collagen type I fibers, and elastic fibers by digital pathology in almost 400 untreated PNB. RESULTS: A reticular and poorly porous ECM was identified in neuroblastomas (NBs) from patients with clinical and biological features associated with poor prognosis compared with a loose and permeable matrix found in NBs of the favorable cohort. CONCLUSIONS: Aggressiveness patterns of ECM can be accurately determined by morphometric tools and could become candidate elements for novel therapies.

The genetic tumor background is an important determinant for heterogeneous MYCN-amplified neuroblastoma.

Amplification of MYCN is the signature genetic aberration of 20-25% of neuroblastoma and a stratifying marker associated with aggressive tumor behavior. The detection of heterogeneous MYCN amplification (hetMNA) poses a diagnostic dilemma due to the uncertainty of its relevance to tumor behavior. Here, we aimed to shed light on the genomic background which permits hetMNA in neuroblastoma and tied the occurrence to other stratifying markers and disease outcome. We performed SNP analysis using Affymetrix Cytoscan HD arrays on 63 samples including constitutional DNA, tumor, bone marrow and relapse samples of 26 patients with confirmed hetMNA by MYCN-FISH. Tumors of patients ≤18m were mostly aneuploid with numeric chromosomal aberrations (NCAs), presented a prominent MNA subclone and carried none or a few segmental chromosomal aberrations (SCAs). In older patients, tumors were mostly di- or tetraploid, contained a lower number of MNA cells and displayed a multitude of SCAs including concomitant 11q deletions. These patients often suffered disease progression, tumor dissemination and relapse. Restricted to aneuploid tumors, we detected chromosomes with uniparental di- or trisomy (UPD/UPT) in almost every sample. UPD11 was exclusive to tumors of younger patients whereas older patients featured UPD14. In this study, the MNA subclone appears to be constraint by the tumor environment and thus less relevant for tumor behavior in aggressive tumors with a high genomic instability and many segmental aberrations. A more benign tumor background and lower tumor stage may favor an outgrowth of the MNA clone but tumors generally responded better to treatment.

Extracellular matrix composition defines an ultra-high-risk group of neuroblastoma within the high-risk patient cohort.

BACKGROUND: Although survival for neuroblastoma patients has dramatically improved in recent years, a substantial number of children in the high-risk subgroup still die. METHODS: We aimed to define a subgroup of ultra-high-risk patients from within the high-risk cohort. We used advanced morphometric approaches to quantify and characterise blood vessels, reticulin fibre networks, collagen type I bundles, elastic fibres and glycosaminoglycans in 102 high-risk neuroblastomas specimens. The Kaplan-Meier method was used to correlate the analysed elements with survival. RESULTS: The organisation of blood vessels and reticulin fibres in neuroblastic tumours defined an ultra-high-risk patient subgroup with 5-year survival rate <15%. Specifically, tumours with irregularly shaped blood vessels, large sinusoid-like vessels, smaller and tortuous venules and arterioles and with large areas of reticulin fibres forming large, crosslinking, branching and haphazardly arranged networks were linked to the ultra-high-risk phenotype. CONCLUSIONS: We demonstrate that quantification of tumour stroma components by morphometric techniques has the potential to improve risk stratification of neuroblastoma patients.

Intragenic anaplastic lymphoma kinase (ALK) rearrangements: translocations as a novel mechanism of ALK activation in neuroblastoma tumors.

Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK-fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4-11 deletion observed in the CLB-BAR cell line show strong activation of downstream targets STAT3 and extracellular signal-regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis.

Individual patient risk stratification of high-risk neuroblastomas using a two-gene score suited for clinical use.

Several gene expression-based prognostic signatures have been described in neuroblastoma, but none have successfully been applied in the clinic. Here we have developed a clinically applicable prognostic gene signature, both with regards to number of genes and analysis platform. Importantly, it does not require comparison between patients and is applicable amongst high-risk patients. The signature is based on a two-gene score (R-score) with prognostic power in high-stage tumours (stage 4 and/or MYCN-amplified diagnosed after 18 months of age). QPCR-based and array-based analyses of matched cDNAs confirmed cross platform (array-qPCR) transferability. We also defined a fixed cut-off value identifying prognostically differing subsets of high-risk patients on an individual patient basis. This gene expression signature independently contributes to the current neuroblastoma classification system, and if prospectively validated could provide further stratification of high-risk patients, and potential upfront identification of a group of patients that are in need of new/additional treatment regimens.

Neuroblastoma patient-derived orthotopic xenografts retain metastatic patterns and geno- and phenotypes of patient tumours.

Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease often have a poor outcome. Here we describe the establishment of neuroblastoma patient-derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high risk neuroblastoma into immunodeficient mice. In vivo tumour growth was monitored by magnetic resonance imaging and fluorodeoxyglucose-positron emission tomography. Neuroblastoma PDXs retained the undifferentiated histology and proliferative capacity of their corresponding patient tumours. The PDXs expressed neuroblastoma markers neural cell adhesion molecule, chromogranin A, synaptophysin and tyrosine hydroxylase. Whole genome genotyping array analyses demonstrated that PDXs retained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Thus, neuroblastoma PDXs recapitulate the hallmarks of high-risk neuroblastoma in patients. PDX-derived cells were cultured in serum-free medium where they formed free-floating neurospheres, expressed neuroblastoma gene markers MYCN, CHGA, TH, SYP and NPY, and retained tumour-initiating and metastatic capacity in vivo. PDXs showed much higher degree of infiltrative growth and distant metastasis as compared to neuroblastoma SK-N-BE(2)c cell line-derived orthotopic tumours. Importantly, the PDXs presented with bone marrow involvement, a clinical feature of aggressive neuroblastoma. Thus, neuroblastoma PDXs serve as clinically relevant models for studying and targeting high-risk metastatic neuroblastoma.

Quantitative modeling of clinical, cellular, and extracellular matrix variables suggest prognostic indicators in cancer: a model in neuroblastoma.

BACKGROUND: Risk classification and treatment stratification for cancer patients is restricted by our incomplete picture of the complex and unknown interactions between the patient's organism and tumor tissues (transformed cells supported by tumor stroma). Moreover, all clinical factors and laboratory studies used to indicate treatment effectiveness and outcomes are by their nature a simplification of the biological system of cancer, and cannot yet incorporate all possible prognostic indicators. METHODS: A multiparametric analysis on 184 tumor cylinders was performed. To highlight the benefit of integrating digitized medical imaging into this field, we present the results of computational studies carried out on quantitative measurements, taken from stromal and cancer cells and various extracellular matrix fibers interpenetrated by glycosaminoglycans, and eight current approaches to risk stratification systems in patients with primary and nonprimary neuroblastoma. RESULTS: New tumor tissue indicators from both fields, the cellular and the extracellular elements, emerge as reliable prognostic markers for risk stratification and could be used as molecular targets of specific therapies. CONCLUSION: The key to dealing with personalized therapy lies in the mathematical modeling. The use of bioinformatics in patient-tumor-microenvironment data management allows a predictive model in neuroblastoma.

Lack of association between MDM2 promoter SNP309 and clinical outcome in patients with neuroblastoma.

While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T > G), has previously been associated with increased risk and aggressiveness of neuroblastoma and other tumor entities, a protective effect has also been reported in certain other cancers. In this study, we evaluated the association of MDM2 SNP309 with outcome in 496 patients with neuroblastoma and its effect on MDM2 expression. No significant difference in overall or event-free survival was observed among patients with neuroblastoma with or without MDM2 SNP309. The presence of SNP309 does not affect MDM2 expression in neuroblastoma.

Glycosaminoglycan profiling in different cell types using infrared spectroscopy and imaging.

We recently identified vibrational spectroscopic markers characteristic of standard glycosaminoglycan (GAG) molecules. The aims of the present work were to further this investigation to more complex biological systems and to characterize, via their spectral profiles, cell types with different capacities for GAG synthesis. After recording spectral information from individual GAG standards (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate) and GAG-GAG mixtures, GAG-defective mutant Chinese hamster ovary (CHO)-745 cells, wild-type CHO cells, and chondrocytes were analyzed as suspensions by high-throughput infrared spectroscopy and as single isolated cells by infrared imaging. Spectral data were processed and interpreted by exploratory unsupervised chemometric methods based on hierarchical cluster analysis and principal component analysis. Our results showed that the spectral information obtained was discriminant enough to clearly delineate between the different cell types both at the cell suspension and single-cell levels. The abilities of the technique are to perform spectral profiling and to identify single cells with different potentials to synthesize GAGs. Infrared microspectroscopy/imaging could therefore be developed for cell screening purposes and further for identifying GAG molecules in normal tissues during physiological conditions (aging, healing process) and numerous pathological states (arthritis, cancer).

Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype.

In neuroblastoma specimens, HIF-2alpha but not HIF-1alpha is strongly expressed in well-vascularized areas. In vitro, HIF-2alpha protein was stabilized at 5% O2 (resembling end capillary oxygen conditions) and, in contrast to the low HIF-1alpha activity at this oxygen level, actively transcribed genes like VEGF. Under hypoxia (1% O2), HIF-1alpha was transiently stabilized and primarily mediated acute responses, whereas HIF-2alpha protein gradually accumulated and governed prolonged hypoxic gene activation. Knockdown of HIF-2alpha reduced growth of neuroblastoma tumors in athymic mice. Furthermore, high HIF-2alpha protein levels were correlated with advanced clinical stage and high VEGF expression and predicted poor prognosis in a clinical neuroblastoma material. Our results demonstrate the relevance of HIF-2alpha in neuroblastoma progression and have general tumor biological implications.

Characterization of Vitronectin Effect in 3D Ewing Sarcoma Models: A Digital Microscopic Analysis of Two Cell Lines.

Ewing sarcoma (ES) is an aggressive bone and soft-tissue pediatric cancer. High vitronectin (VN) expression has been associated with poor prognosis in other cancers, and we aimed to determine the utility of this extracellular matrix glycoprotein as a biomarker of aggressiveness in ES. Silk fibroin plus gelatin-tyramine hydrogels (HGs) were fabricated with and without cross-linked VN and cultivated with A673 and PDX73 ES cell lines for two and three weeks. VN secretion to culture media was assessed using ELISA. Morphometric analysis was applied for phenotypic characterization. VN release to culture media was higher in 3D models than in monolayer cultures, and intracellular, intercellular, and pericluster presence was also observed. A673-HGs showed lower density of clusters but a proportion of larger clusters than PDX73-HGs, which presented low cluster circularity. The cluster density of A673-HGs without added VN was higher than with added VN and slightly lower in the case of PDX73-HGs. Furthermore, a culture time of three weeks provided no benefits in cluster growth compared to two weeks, especially in A673-HGs. These advances in 3D modeling and digital quantification pave the way for future studies in ES and other cancers to deepen understanding about intra- and intercellular heterogeneity and anti-adhesion VN therapies.

MOXD1 is a lineage-specific gene and a tumor suppressor in neuroblastoma.

Neuroblastoma is a childhood developmental cancer; however, its embryonic origins remain poorly understood. Moreover, in-depth studies of early tumor-driving events are limited because of the lack of appropriate models. Herein, we analyzed RNA sequencing data obtained from human neuroblastoma samples and found that loss of expression of trunk neural crest-enriched gene MOXD1 associates with advanced disease and worse outcome. Further, by using single-cell RNA sequencing data of human neuroblastoma cells and fetal adrenal glands and creating in vivo models of zebrafish, chick, and mouse, we show that MOXD1 is a determinate of tumor development. In addition, we found that MOXD1 expression is highly conserved and restricted to mesenchymal neuroblastoma cells and Schwann cell precursors during healthy development. Our findings identify MOXD1 as a lineage-restricted tumor-suppressor gene in neuroblastoma, potentiating further stratification of these tumors and development of novel therapeutic interventions.