Metastatic Neuroblastoma Cells Research Articles

The capture of extracellular vesicles endogenously released by xenotransplanted tumours induces an inflammatory reaction in the premetastatic niche.

The capture of tumour-derived extracellular vesicles (TEVs) by cells in the tumour microenvironment (TME) contributes to metastasis and notably to the formation of the pre-metastatic niche (PMN). However, due to the challenges associated with modelling release of small EVs in vivo, the kinetics of PMN formation in response to endogenously released TEVs have not been examined. Here, we have studied the endogenous release of TEVs in mice orthotopically implanted with metastatic human melanoma (MEL) and neuroblastoma (NB) cells releasing GFP-tagged EVs (GFTEVs) and their capture by host cells to demonstrate the active contribution of TEVs to metastasis. Human GFTEVs captured by mouse macrophages in vitro resulted in transfer of GFP vesicles and the human exosomal miR-1246. Mice orthotopically implanted with MEL or NB cells showed the presence of TEVs in the blood between 5 and 28 days after implantation. Moreover, kinetic analysis of TEV capture by resident cells relative to the arrival and outgrowth of TEV-producing tumour cells in metastatic organs demonstrated that the capture of TEVs by lung and liver cells precedes the homing of metastatic tumour cells, consistent with the critical roles of TEVs in PMN formation. Importantly, TEV capture at future sites of metastasis was associated with the transfer of miR-1246 to lung macrophages, liver macrophages, and stellate cells. This is the first demonstration that the capture of endogenously released TEVs is organotropic as demonstrated by the presence of TEV-capturing cells only in metastatic organs and their absence in non-metastatic organs. The capture of TEVs in the PMN induced dynamic changes in inflammatory gene expression which evolved to a pro-tumorigenic reaction as the niche progressed to the metastatic state. Thus, our work describes a novel approach to TEV tracking in vivo that provides additional insights into their role in the earliest stages of metastatic progression.

3D geometry orchestrates the transcriptional landscape of metastatic neuroblastoma cells in a multicellular in vitro bone model.

A key challenge for the discovery of novel molecular targets and therapeutics against pediatric bone metastatic disease is the lack of bona fide in vitro cell models. Here, we show that a beta-tricalcium phosphate (β-TCP) multicellular 3D in vitro bone microtissue model reconstitutes key phenotypic and transcriptional patterns of native metastatic tumor cells while promoting their stemness and proinvasive features. Comparing planar with interconnected channeled scaffolds, we identified geometry as a dominant orchestrator of proangiogenic traits in neuroblastoma tumor cells. On the other hand, the β-TCP-determined gene signature was DNA replication related. Jointly, the geometry and chemical impact of β-TCP revealed a prometastatic landscape of the engineered tumor microenvironment. The proposed 3D multicellular in vitro model of pediatric bone metastatic disease may advance further analysis of the molecular, genetic and metabolic bases of the disease and allow more efficient preclinical target validations.

MIF/CXCR4 signaling axis contributes to survival, invasion, and drug resistance of metastatic neuroblastoma cells in the bone marrow microenvironment

BackgroundThe bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets.MethodsPatient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O2) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo.ResultsWe identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model.ConclusionsOur findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Induction of serine hydroxymethyltransferase 2 promotes tumorigenesis and metastasis in neuroblastoma.

High-risk neuroblastoma (NB) remains an extremely difficult subgroup to cure and is associated with MYCN amplification. Serine hydroxymethyltransferase 2 (SHMT2) regulates serine metabolism in a myc-dependent manner; it is upregulated in several cancers and is associated with tumor aggressiveness. Akt-2, an important regulator of MYCN via the PI3K/Akt pathway, induces metastatic potential in NB. The association between SHMT2 and PI3K/Akt in hepatocyte regeneration has been well established but its mechanistic interaction in cancer has yet to be clearly elucidated. Herein, we evaluated the exact role of SHMT2 on the PI3K/Akt pathway, in addition to NB tumorigenesis and metastatic potential in vitro. SHMT2 gene expression and overall survival (OS) were assessed. Two human NB cell lines were examined. SHMT2 silencing and overexpression were performed. The downstream effects were analyzed with immunoblotting, RT-qPCR and functional assays were performed. We found SHMT2 gene expression is associated with decreased OS and MYCN amplification. SHMT2 protein and mRNA expression are increased in MYCN-amplified cells. SHMT2 expression has a direct interaction with Akt-2 and MYCN. Induction of SHMT2 increased cellular proliferation, colony formation and cellular migration and SHMT2 expression was increased in metastatic NB cells. We conclude that SHMT2 regulates N-Myc via phosphorylation of Akt-2 and plays an important role in NB tumorigenesis by contributing to cell growth, migration, colony formation and metastasis in vitro.

Abstract 1548: Potent activity of CAR T cells targeting the oncofetal protein GPC2 engineered to recognize low antigen density in neuroblastoma

Abstract Background: CAR T-cells targeting solid tumor antigens have not yet demonstrated similar success as seen for CD19, BCMA and CD22 in B-cell malignancies. Truly tumor-specific antigens are rare, yet pediatric solid tumors manifest stalled fetal developmental programs and often overexpress oncofetal antigens, normally restricted to prenatal tissues. Antigen density is a major factor determining CAR potency and whether such nonmutant antigens can be effectively and safely targeted remains unknown. Here, we engineered CAR T-cells targeting GPC2 to recognize clinically relevant antigen thresholds and assess safety. Methods: Expression of GPC2 was assessed during organ development (Cardoso-Moreira et al. 2019), by IHC in prenatal, infant and pediatric brain and in ssRNAseq datasets of fetal (La Manno et. al 2016) and adult brain (Allen Brain Atlas). To measure the molecules/cell of GPC2 and other immunotherapy-relevant targets on bone-marrow infiltrating neuroblastoma (NB) cells of high-risk patients, we developed a Flow Cytometric quantification assay and engineered GPC2-CAR T-cells to recognize relevant antigen thresholds. To assess binding specificity towards GPC2, a membrane proteome array encompassing >5,300 human proteins was utilized. Taking advantage of the murine cross-reactivity of our lead GPC2-CAR, toxicity was assessed in a relevant xenograft model. Results: During organ development, we found GPC2 expression restricted to the developing brain and a gradual decrease until silenced after birth. IHC of prenatal brain revealed an inverse correlation between GPC2 and gestational age (r = -0.775, p = 0.008) and very low staining in infant and pediatric brain (H-scores <100). Analysis of fetal midbrain ssRNAseq data spatializes GPC2 to the neuronal progenitor compartment, a correlation absent in adult brain. We measured a mean of ~4782 GPC2 molecules/cell on patient's metastatic NB cells, a significantly lower number than on NB cell lines (p=0.0496). We found KYMRIAH-based CAR T-cell constructs unable to control tumor due to subthreshold antigen density. Sequential modifications to the CAR architecture, such as CD28-derived hinge-transmembrane and signaling domains domains resulted in a potent lead GPC2-CAR capable of impressive tumor regression in three xenograft models of NB (metastatic, orthotopic and patient-derived xenograft). Potency of our lead GPC2-CAR could be further enhanced by overexpression of c-Jun, mediating long-term complete responses. We found no clinically relevant off-target binding to other membrane proteins and no clinical or histological evidence of toxicity. Conclusion: Our results demonstrate safety and efficacy of a lead GPC2-CAR and illuminate oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors. This work establishes a strong basis for testing GPC2 CAR T-cells in early phase clinical trials. Citation Format: Sabine Heitzeneder, Kristopher R. Bosse, Zhongyu Zhu, Dontcho Jelev, Shaurya Dhingra, Robbie Majzner, Elena Sotillo-Pineiro, Samantha Buongervino, Peng Xu, Jing Huang, Alberto Delaidelli, Martin Hasselblatt, Kevin Parker, Hima Anbunathan, Anya Alag, Jennifer Hwang, Min Huang, Dorota D. Klysz, Johanna L. Theruvath, Jose G. Vilches-Moure, Ansuman T. Satpathy, Poul H. Sorensen, Dimiter S. Dimitrov, John M. Maris, Crystal L. Mackall. Potent activity of CAR T cells targeting the oncofetal protein GPC2 engineered to recognize low antigen density in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1548.

Abstract B72: Dissecting the heterogeneity of metastatic neuroblastoma cells by single-cell RNA-seq

Abstract Introduction: The major cause of death in neuroblastoma (NB) is metastatic relapse, and most therapies do not specifically target metastases. We previously developed a metastatic mouse model using intracardiac injection of tagged human SK-N-AS NB cells, followed by multiple rounds of in vivo selection from bone and brain metastatic sites. Gene expression profiles identified novel genes and pathways dysregulated in metastatic NB cells that, when genetically or pharmacologically manipulated, affected metastasis. A subset of genes up- or downregulated in the enhanced metastatic cells was used to generate a metastatic gene signature that predicted patient survival. We have now asked (1) whether there is a transcriptionally distinct subpopulation of cells in the parental population that selectively metastasizes to bone and brain, and (2) if there are transcriptional differences that can account for the ability of the enhanced metastatic cells to hone to bone/bone marrow vs. brain. Methods: We used 10X Genomics single-cell RNA sequencing (scRNA-seq) to transcriptionally profile parental cells and two in vivo-selected cell lines that showed enhanced metastatic spread to bone/bone marrow (B5) and brain (BR2). Approximately 5,000 genes and 10,000 transcripts per cell were sequenced, with the data analyzed using a unique analysis pipeline incorporating extensive data quality analysis with visualization and clustering methods using evidence-based parameter selection. Genes with high variance were used to compute principal components as inputs to visualize cells in two dimensions (t-SNE plots) in clusters. Results: scRNA-seq data showed that the parental cells clustered separately from B5 and BR2 cells, with clusters in the parental cells that expressed genes enriched in the B5 and BR2 cells that we previously reported drove migration/invasion in vitro and/or metastasis in vivo, including sphingosine kinase 1. These results suggest that subpopulations of the parental cells with enhanced metastatic gene expression are selected in vivo for honing to metastatic sites. We identified genes enriched in B5 and BR2 cells encoding cell surface proteins, chemokines including CCL2, and transcription factors including Msx1 involved in metastasis of other cancers, that we will assess for roles in NB metastasis. While the B2 and BR5 cells were transcriptionally similar to each other, we did identify genes highly expressed in B2 subclusters that are associated with bone cancers including FGF5, and that are highly expressed in BR2 subclusters that are associated with brain cancers including RARRES2. These findings suggest that transcriptional differences between B5 and BR2 cells may influence metastasis to the bone or brain. Conclusion: The identification of genes that are differentially expressed in the more metastatic subpopulations will contribute to our understanding of the molecular mechanisms governing metastases and identify novel targetable pathways that were not detected by profiling bulk tumor populations. Citation Format: Alice R. Shan, Alexander Gont, David R. Kaplan, Meredith S. Irwin. Dissecting the heterogeneity of metastatic neuroblastoma cells by single-cell RNA-seq [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B72.

Abstract 3106: Targeting vitamin D signalling in metastatic neuroblastoma

Abstract Background: Neuroblastoma (NB) is the most common extra-cranial solid tumor and the most frequent cause of cancer-related deaths in children. More than half of patients with NB have metastases and their survival is <50%, and only 5% after relapse. Currently, there are no therapies that specifically target metastatic NB and there is a lack of NB models that recapitulate the sites and burden of metastases observed in patients. Methods: To study metastatic NB, we have developed a mouse model using in vivo selection of SKNAS cells that metastasize following intra-cardiac injection. We isolated subpopulations from bone and brain with enhanced metastatic properties and compared their gene expression profiles to those of the parental SKNAS cells. Using Connectivity Map (CMap) analyses, we identified drugs that were predicted to change the gene expression in the metastatic subpopulations to a profile that is similar to that of parental cell lines. Results: Calcipotriol, a synthetic analogue of vitamin D, was identified from the CMap analysis to be a potentially selective agent for metastatic NB cell lines. In comparison to the parental cells, treatment of the metastatic subpopulations with calcipotriol resulted in reduced proliferation. Furthermore, calcipotriol sensitivity was reduced in metastatic cells with a knockout of vitamin D receptor (VDR) suggesting its effect is on-target. In contrast to the parental cells, following calcipotriol treatment metastatic subpopulations did not exhibit an increase in protein levels of CYP24A1, the enzyme that metabolizes vitamin D, suggesting a potential mechanism for the differential sensitivity of parental and metastatic cells. Calcipotriol treatment also reduced levels of hippo pathway effectors, YAP and TAZ, which we previously reported to play a role in mediating the metastatic phenotype of the isolated subpopulations. Additionally, metastatic cells that were pre-treated with calcipotriol showed reduced migration in a transwell assay whereas the migration potential of parental cells was not affected. Furthermore, RASSF2, an upstream regulator of the hippo pathway, was identified to be upregulated in a VDR-dependent manner after calcipotriol treatment in the metastatic subpopulations indicating a possible mechanistic link for the effects of calcipotriol on the hippo pathway in these metastatic cells. Conclusions: Calcipotriol was identified to be more effective against metastatic NB subpopulations in vitro and this may be in part due to defects in CYP24A1 induction in these metastatic cells. Our data also suggests a novel link between VDR, the hippo pathway and metastasis in NB. Further experiments are required to determine the role of VDR, mechanism of action of calcipotriol in selectively inhibiting growth of metastatic NB cells. Citation Format: Yagnesh Ladumor, Bo Kyung Alex Seong, Robin Hallett, Teresa Adderley, Yingying Wang, Lynn Kee, David Kaplan, Meredith Irwin. Targeting vitamin D signalling in metastatic neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3106.

Neuroblastoma Characteristics and Embryonic Origin of The Primary Lesion Site: A SEER Study

Background: Neuroblastoma, one of the most prevalent infant malignancies, is heterogeneous and easily spreads into other organs causing life-threatening consequences. Different characters of organs (such as the germ layers where the organs derived from) exert different growth microenvironments and potentially influence the behavior of metastatic neuroblastoma cells and the prognosis of patients. However, limited information is been known about this in neuroblastoma. Objectives: To compare characteristics of neuroblastoma, primarily originating from the same germ layer (the seeds), in different tissues (microenvironment) derived from different germ layers. Methods: We performed retrospective analysis of SEER (Surveillance, Epidemiology, and End Results) data (1973 - 2014), patients with malignant neuroblastoma were grouped based on the primary lesion site (mesoderm-, ectoderm- or endoderm-derived tissue). Baseline demographic and clinical characteristics were compared between groups. Due to difficulties of processing incomplete SEER data, therapeutic method and survival rates were analyzed using cases from another SEER database (2000 - 2014). Results: The analysis included 3701 patients: 1970 (53.2%) in the mesoderm group, 1017 (27.5%) in the ectoderm group and 714 (19.3%) in the endoderm group. Tumor histology differed between groups (P < 0.01): the ectoderm group had mostly neuroblastoma (79.2%), as did the mesoderm group (71.1%), whereas the endoderm group contained mainly olfactory neuroblastoma (94.7%). The tumors (mean size: 69.14 ± 58.37 mm) were most commonly poorly differentiated with local extension, although lymph node invasion and distant metastasis occurred in a minority of cases. Compared with the other groups, the endoderm group had smaller (43.89 ± 20.84 mm) and better-differentiated tumors and a lower prevalence of lymph node invasion and metastasis (P < 0.05). Despite this, overall survival was poorest for the endoderm group (P < 0.05). Radiotherapy improved overall survival in the endoderm and ectoderm groups but worsened overall survival in the mesoderm group (P < 0.05). Conclusions: Malignant neuroblastoma characteristics may be influenced by the tumor microenvironment. In the youngest patients, decision-making regarding the best choice of therapy should be delayed until accurate risk stratification is possible.

Calcium phosphate scaffolds with defined interconnecting channel structure provide a mimetic 3D niche for bone marrow metastasized tumor cell growth

Metastasis of tumor cells in the bone marrow (BM) is a multi-step and highly dynamic process during which cells succumb important phenotypic changes. Behavior of disseminated tumor cells in BM is strictly regulated by three-dimensional (3D) cell-cell and cell-matrix interactions. In this study, we explored whether the β-tricalcium-phosphate (β-TCP) scaffolds with a tailored interconnecting channel structure could enable appropriate 3D mimetic BM microenvironment for the growth of metastatic neuroblastoma cells. The scaffolds provided the mechanical support for human mesenchymal stromal cells (hMSC) allowing them to proliferate, differentiate towards osteoblasts, and produce the deposits of extracellular matrix inside the interconnected channels. The in vitro microenvironment shaped by stromal cells was then tailored by neuroblastoma tumor cells. Immunohistological analyses confirmed the organization of tumor cells into the forms of spheres only when co-cultured with hMSC-derived osteoblasts. The growing rate of tumor cells in 3D conditions was less marked comparing to the one of the cells grown as 2D monolayer as confirmed by decreased Ki-67 expression. Instead, the 3D culturing of neuroblastoma cells inside supportive stroma promoted cell quiescence as sustained by increased p27 level. A balance between cell proliferation, survival, and differentiation was more evident for tumor cells grown inside the 3D scaffolds, thus mirroring better the situation that occurs in vivo where the cells do not follow the exponential growth rate. We conclude that the proposed 3D β-TCP scaffold type provides a mimetic 3D in vitro niche suitable for studying behavior of BM metastasized tumor cells. Statement of SignificanceBone marrow (BM) niche is a favorite target of metastatic neuroblastoma cells. To better address the molecular mechanisms that sustain spatiotemporal organization of neuroblastoma cells in the marrow we mimicked the three-dimensional (3D) assembly of stromal and tumor cells inside β-tricalcium-phosphate (β-TCP) scaffolds. β-TCP scaffolds with a tailored interconnecting channel structure provided mechanical support to mesenchymal stromal cells allowing them to differentiate towards osteoblasts and to produce extracellular matrix. A dynamic cell-matrix interplay favored the characteristic rosette-like growth of metastatic neuroblastoma cells and triggered their quiescence. With our study, we confirmed the potential of β-TCP scaffolds with reproduced BM niche as a cost-effective in vitro model for the growth of disseminated tumor cells, and for related biological and pharmacological surveys.

Microvesicles expressing adenosinergic ectoenzymes and their potential role in modulating bone marrow infiltration by neuroblastoma cells

ABSTRACTMetastatic diffusion of Neuroblastoma (NB) cells in the bone marrow (BM) represents the most negative prognostic factors for NB patients. Multiple immune escape mechanisms are postulated as responsible. Our working hypothesis is that adenosine (ADO), an immunosuppressive molecule along with the ectoenzymatic pathways (CD39-CD73 and CD38-CD203a/PC-1-CD73) controlling its production, are involved in the dynamics of NB cells in the BM. The results indicate that ectonucleotidases are expressed by i) NB cell lines, ii) metastatic NB cells isolated from NB patients’ BM, iii) microvesicles (MV) derived from both NB cell types and iv) resident BM cell populations. BM infiltration by NB cells increased CD203a/PC-1 and CD73 expression on lymphoid and myeloid cells, respectively. Expressions of ectoenzymes and GD2 (NB-associated marker) were higher on MV from NB patients’ BM than in controls. Moreover, CD203a/PC-1 expression on BM-derived MV provide a basis for distinguishing NB patients with high or low BM infiltration. ADO production and consumption of related by-products were significantly higher when assessed on NB patients’ MV than those from controls. MV isolated from NB patients’ BM significantly downregulated in vitro T cell proliferation. Lastly, NB patients with worse prognosis are identified by a high percentage of CD38+ or CD73+ MV in the BM. In conclusion, ectonucleotidases are present and functional on NB cells, as well as in NB-infiltrated BM and in MV derived from BM. It is reasonable that MV are involved in BM infiltration by NB cells. Therefore, targeting these molecules may widen the therapeutic armamentarium for metastatic NB patients.

Altered erythropoiesis and decreased number of erythrocytes in children with neuroblastoma.

Neuroblastoma (NB) is a pediatric tumor presenting at diagnosis either as localized or metastatic disease, which mainly involves the bone marrow (BM). The physical occupancy of BM space by metastatic NB cells has been held responsible for impairment of BM function. Here, we investigated whether localized or metastatic NB may alter hematopoietic lineages’ maturation and release of mature cells in the periphery, through gene expression profiling, analysis of BM smears, cell blood count and flow cytometry analysis.Gene ontology and disease-associated analysis of the genes significantly under-expressed in BM resident cells from children with localized and metastatic NB, as compared to healthy children, indicated anemia, blood group antigens, and heme and porphyrin biosynthesis as major functional annotation clusters. Accordingly, in children with NB there was a selective impairment of erythrocyte maturation at the ortho-chromic stage that resulted in reduced erythrocyte count in the periphery, regardless of the presence of metastatic cells in the BM. By considering all NB patients, low erythrocyte count at diagnosis associated with worse survival. Moreover, in the subset of metastatic patients, low erythrocyte count, hemoglobin and hematocrit and high red cell distribution width at follow-up also associated with worse outcome.These observations provide an alternative model to the tenet that infiltrating cells inhibit BM functions due to physical occupancy of space and may open a new area of research in NB to understand the mechanism(s) responsible for such selective impairment.

Molecular galactose-galectin association in neuroblastoma cells: An unconventional tool for qualitative/quantitative screening.

Galectin decorates the cell membrane and forms an extracellular molecular association with galactoside units. Here, galactoside probes have been used to study galectin expression in neuroblastoma cells. The hypothesis behind this investigation has been that the molecular mechanisms by which glycans modulate neural metastatic cells involve a protein-carbohydrate association, galectin-galactose. Preliminary screening to validate the hypothesis has been performed with galactose moieties anchored to beads. The molecular association has been studied by FACS. In vitro experiments reveal the molecular binding preferences of the metastatic neuroblastoma cells. Ex vivo, the galactose probes discriminate healthy tissues. The unconventional assay in microfluidics used in this study displayed results analogous to the above (GI-LI-N cell capture efficiency overcomes IMR-32). At the point of equilibrium of shear and binding forces, the capture yield inside the chamber was measured to 60 ± 4.4% in GI-LI-N versus 40 ± 2.1% in IMR-32. Staining of the fished cells and subsequent conjugation with red beads bearing the galactose also have evidenced that microfluidics can be used to study and quantify the molecular association of galectin-galactose. Most importantly, a crucial insight for obtaining single-cell qualitative/quantitative glycome analysis has been achieved. Finally, the specificity of the assay performed in microfluidics is demonstrated by comparing GI-LI-N fishing efficiency in galactose and fucose environments. The residual adhesion to fucose confirmed the existence of receptors for this glycan and that its eventual unspecific binding (i.e. due to electrostatic interactions) is insignificant compared with the molecular binding. Identification and understanding of this mechanism of discrimination can be relevant for diagnostic monitoring and for producing probes tailored to interfere with galectin activities associated with the malignant phenotype. Besides, the given strategy has implications for the rational design of galectin-specific ligands.

Abstract 5112: Galactose probes hit neuroblastoma cells through a specific galactose-galectin association

Abstract Galectins are a family of proteins that decorate the cell membrane and form extracellular molecular associations with β-galactoside sugars. In order to mimic multivalent molecular recognitions, galactoside moieties have been immobilized on microbeads and used in cytofluorimetric analysis and in microfluidic assay to study the molecular association between carbohydrates and proteins in neuroblastoma (NB) cells. The hypothesis behind this investigation was that the molecular mechanisms by which glycans modulate neural metastatic cells involve protein-carbohydrate association. To evaluate the binding between microbeads and tumor cells, two human NB cell lines were used in cellular association experiments. GI-LI-N cells, derived from the peripheral blood of a bone marrow-infiltrated, high risk, stage IV NB patient, were chosen for metastatic characteristics; IMR-32 cells were chosen because derived from the primary tumor in the abdominal site of an unknown-stage NB patient. In vitro experiments have revealed molecular binding preferences of the metastatic NB cells. This result underscores that the efficiency of the cellular association might be related to the differences in the expression of the galectins between the two cell lines (GI-LI-N cells &amp;gt;&amp;gt; IMR-32 cells) and that the formation of the molecular association galectin-galactose could discriminate the tumor stages. Ex vivo investigations have supported in vitro results; indeed, galactose moieties mostly recognized GI-LI-N cells derived from orthotopic tumor-bearing mice respect to explanted IMR-32 cells. This molecular association was tumor cells specific because it discriminated healthy tissues. The understanding of this mechanism of recognition, and of possible cellular discrimination, can be relevant for the diagnostic monitoring of metastatic NB cells, and for producing probes tailored to interfere with galectin activities associated with the malignant phenotype. Citation Format: Giuseppina Simone, Mirco Ponzoni, Fabio Pastorino. Galactose probes hit neuroblastoma cells through a specific galactose-galectin association. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5112.

Importance of Phox2B Immunohistochemical Stain for Detecting Metastatic Neuroblastoma Cells in Bone Marrow Specimens.

Importance of Phox2B Immunohistochemical Stain for Detecting Metastatic Neuroblastoma Cells in Bone Marrow Specimens.

Hexokinase 2 is a determinant of neuroblastoma metastasis.

Background:Intersecting a genome-wide expression profile of metastatic and nonmetastatic human neuroblastoma xenograft variants with expression profiles of tumours from stage 1 and 4 neuroblastoma patients, we previously characterised hexokinase 2 (HK2) as a gene whose expression was upregulated in both metastatic neuroblastoma variants and tumours from stage 4 neuroblastoma patients.Methods:Local and metastatic neuroblastoma cell variants as well as metastatic neuroblastoma cells genetically manipulated to downregulate the expression of HK2 were utilised for in vitro and in vivo examinations of the involvement of HK2 in neuroblastoma.Results:Hexokinase 2 expression and its activity levels were increased in neuroblastoma metastatic variants as compared with the local variants. The upregulation of HK2 confers upon the metastatic cells high resistance to the antiproliferative effect of the HK2 inhibitor 3-BrPa and to the chemotherapy agent Deferoxamine. The inhibition of HK2 transcript lowered the proliferation and motility of sh-HK2 cells as compared with sh-control cells. Mice that were inoculated with sh-HK2 cells had a lower incidence of local tumours, smaller tumour volumes and a diminished load of lung metastasis compared with mice inoculated with sh-control cells.Conclusions:Hexokinase 2 plays a significant role in shaping the malignant phenotype of neuroblastoma and influences the progression of this disease.

PHOX2B is a suppressor of neuroblastoma metastasis.

Paired like homeobox 2B (PHOX2B) is a minimal residual disease (MRD) marker of neuroblastoma. The presence of MRD, also referred to as micro-metastases, is a powerful marker of poor prognosis in neuroblastoma.Lung metastasis is considered a terminal event in neuroblastoma. Lung micro-metastatic neuroblastoma (MicroNB) cells show high expression levels of PHOX2B and possess a less malignant and metastatic phenotype than lung macro metastatic neuroblastoma (MacroNB) cells, which hardly express PHOX2B.In vitro assays showed that PHOX2B knockdown in MicroNB cells did not affect cell viability; however it decreased the migratory capacity of the MicroNB-shPHOX2B cells. An orthotopic inoculation of MicroNB-shPHOX2B cells into the adrenal gland of nude mice resulted in significantly larger primary tumors and a heavier micro-metastatic load in the lungs and bone-marrow, than when control cells were inoculated.PHOX2B expression was found to be regulated by methylation. The PHOX2B promoter in MacroNB cells is significantly more methylated than in MicroNB cells. Demethylation assays using 5-azacytidine demonstrated that methylation can indeed inhibit PHOX2B transcription in MacroNB cells.These pre-clinical data strongly suggest that PHOX2B functions as a suppressor of neuroblastoma progression.

Abstract 1398: Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling

Abstract High-risk neuroblastoma (HR-NB) presented with hematogenous metastasis is one of the most difficult cancers to cure with poor patient survival. Aggressive tumors contain populations of rapidly proliferating clonogens that exhibit stem cell properties, cancer stem cells (CSCs). Conceptually, CSCs that evade intensive multimodal therapy dictates tumor progression, relapse/recurrence and poor clinical outcomes. Herein, we investigated the plasticity and stem-cell related molecular response of aggressive metastatic neuroblastoma cells that fits CSC model. Well characterized clones of ‘metastatic site derive aggressive cells’ (MSDACs) from the manifold of metastatic tumors of clinically translatable HR-NB were characterized for their CSC fit. Alternated, yet stabilized (three generations) microenvironment simulation experiments with ‘serum-free stem cell medium’ to ‘growth medium with serum’ and vice versa identified flexible revocable plasticity. Signatures stem-cell related molecular responses consistent with phenotypic conversions are observed. Successive reintroduction to the favorable niche not only regained identical EMT, self-renewal capacity, pluripotency maintenance, and other stem-cell related signaling events, but also instigated additional events depicting the aggressive adaptive plasticity. Together, these results demonstrated the flexible plasticity of HR-NB MSDACs that typically fits in CSC model and further identified the intrinsic adaptivness of the successive phenotype switching that clarify the heterogeneity of HR-NB. Moreover, the continuous ongoing acquisition of stem-cell related molecular rearrangements may hold the key for the switch from favorable disease to HR-NB. Citation Format: Satish Kumar Ramraj, Faizan H. Khan, Sheeja Aravindan, Mohan Natarajan, Terence S. Herman, Natarajan Aravindan. Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1398. doi:10.1158/1538-7445.AM2015-1398

Reorganization of metastamiRs in the evolution of metastatic aggressive neuroblastoma cells.

BackgroundMetastamiRs have momentous clinical relevance and have been correlated with disease progression in many tumors. In this study, we identified neuroblastoma metastamiRs exploiting unique mouse models of favorable and high-risk metastatic human neuroblastoma. Further, we related their deregulation to the modulation of target proteins and established their association with clinical outcomes.ResultsWhole genome miRNA microarray analysis identified 74 metastamiRs across the manifold of metastatic tumors. RT-qPCR on select miRNAs validated profile expression. Results from bio-informatics across the ingenuity pathway, miRCancer, and literature data-mining endorsed the expression of these miRNAs in multiple tumor systems and showed their role in metastasis, identifying them as metastamiRs. Immunoblotting and TMA-IHC analyses revealed alterations in the expression/phosphorylation of metastamiRs’ targets, including ADAMTS-1, AKT1/2/3, ASK1, AURKβ, Birc1, Birc2, Bric5, β-CATENIN, CASP8, CD54, CDK4, CREB, CTGF, CXCR4, CYCLIN-D1, EGFR, ELK1, ESR1, CFOS, FOSB, FRA, GRB10, GSK3β, IL1α, JUND, kRAS, KRTAP1, MCP1, MEGF10, MMP2, MMP3, MMP9, MMP10, MTA2, MYB, cMYC, NF2, NOS3, P21, pP38, PTPN3, CLEAVED PARP, PKC, SDF-1β, SEMA3D, SELE, STAT3, TLR3, TNFα, TNFR1, and VEGF in aggressive cells ex vivo and in a manifold of metastatic tumors in vivo. miRNA mimic (hsa-miR-125b, hsa-miR-27b, hsa-miR-93, hsa-miR-20a) and inhibitor (hsa-miR-1224-3p, hsa-miR-1260) approach for select miRNAs revealed the direct influence of the altered metastamiRs in the regulation of identified protein targets. Clinical outcome association analysis with the validated metastamiRs’ targets corresponded strongly with poor overall and relapse-free survival.ConclusionsFor the first time, these results identified a comprehensive list of neuroblastoma metastamiRs, related their deregulation to altered expression of protein targets, and established their association with poor clinical outcomes. The identified set of distinctive neuroblastoma metastamiRs could serve as potential candidates for diagnostic markers for the switch from favorable to high-risk metastatic disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1642-x) contains supplementary material, which is available to authorized users.

Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling.

IntroductionHigh-risk neuroblastoma (HR-NB) presenting with hematogenous metastasis is one of the most difficult cancers to cure. Patient survival is poor. Aggressive tumors contain populations of rapidly proliferating clonogens that exhibit stem cell properties, cancer stem cells (CSCs). Conceptually, CSCs that evade intensive multimodal therapy dictate tumor progression, relapse/recurrence, and poor clinical outcomes. Herein, we investigated the plasticity and stem-cell related molecular response of aggressive metastatic neuroblastoma cells that fit the CSC model.MethodsWell-characterized clones of metastatic site-derived aggressive cells (MSDACs) from a manifold of metastatic tumors of clinically translatable HR-NB were characterized for their CSC fit by examining epithelial-to-mesenchymal transition (EMT) (E-cadherin, N-Cadherin), survival (NFκB P65, p50, IκB and pIκB) and drug resistance (ABCG2) by immunoblotting; pluripotency maintenance (Nanog, SOX2) by immunofluorescence; and EMT and stemness related transcription of 93 genes by QPCR profiling. Plasticity of MSDACs under sequential alternation of culture conditions with serum and serum-free stem-cell conditions was assessed by clonal expansion (BrdU incorporation), tumorosphere formation (anchorage independent growth), EMT and stemness related transcriptome (QPCR profiling) and validated with MYC, SOX2, EGFR, NOTCH1 and CXCL2 immunoblotting.ResultsHR-NB MSDACs maintained in alternated culture conditions, serum-free stem cell medium to growth medium with serum and vice versa identified its flexible revocable plasticity characteristics. We observed signatures of stem cell-related molecular responses consistent with phenotypic conversions. Successive reintroduction to the favorable niche not only regained identical EMT, self-renewal capacity, pluripotency maintenance, and other stem cell-related signaling events, but also instigated additional events depicting aggressive adaptive plasticity.ConclusionsTogether, these results demonstrated the flexible plasticity of HR-NB MSDACs that typically fit the CSC model, and further identified the intrinsic adaptiveness of the successive phenotype switching that clarifies the heterogeneity of HR-NB. Moreover, the continuous ongoing acquisition of stem cell-related molecular rearrangements may hold the key to the switch from favorable disease to HR-NB.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0002-8) contains supplementary material, which is available to authorized users.