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Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Division of Hematology and Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
PLoS ONE (Impact Factor: 3.53). 02/2009; 4(1):e4235. DOI: 10.1371/journal.pone.0004235
Source: PubMed

ABSTRACT Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.
Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.
These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

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Available from: Yoshinaga Saeki, Aug 21, 2015
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    • "We observed elevated expression of TLX, Oc3/4, Sox2, Nestin, and Nanog in neuroblastoma spheres compared with parental cells (Fig. 1B). Our results are in agreement with reports that SK-N-BE2 cells contain cells with markers of stemness and may form spheres [5] [9]. We observed formation of spheres already after 1 week of culturing. "
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    ABSTRACT: Neuroblastoma develops through processes which include cellular dedifferentiation. Ability of tumors to form spheroids is one of the manifestations of dedifferentiation and carcinogenic transformation. To study mechanisms of dedifferentiation of neuroblastoma cells, we generated spheroids and performed a proteomics study to compare the spheroids with parental SK-N-BE2 cells. We observed that dedifferentiation induced extensive changes in the proteome profiles of the cells, which affected more than 30% of detected cellular proteins. Using mass spectrometry, we identified 239 proteins affected by dedifferentiation into spheroids as compared to the parental cells. These proteins represented such regulatory processes as transcription, cell cycle regulation, apoptosis, cell adhesion, metabolism, intracellular transport, stress response, and angiogenesis. A number of potent regulators of stemness, differentiation and cancer were detected as subnetworks formed by the identified proteins. Our validation tissue microarray study of 30 neuroblastoma cases confirmed that two of the identified proteins, DISC1 and DNA-PKcs, had their expression increased in advanced malignancies. Thus, our report unveiled extensive changes of the cellular proteome upon dedifferentiation of neuroblastoma cells, indicated top subnetworks and clusters of molecular mechanisms involved in dedifferentiation, and provided candidate biomarkers for clinical studies.
    Biochemical and Biophysical Research Communications 10/2014; 454(1). DOI:10.1016/j.bbrc.2014.10.065 · 2.28 Impact Factor
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    • "In NB, CD133 appears to be a marker of TICs as isolated CD133 high cells were able to grow as neurospheres in vitro and to initiate tumors in athymic mice after their subcutaneous (ectopic) injection (Mahller et al., 2009). Recently, it has been demonstrated that CD133 regulates differentiation and proliferation in NB (Takenobu et al., 2011). "
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    ABSTRACT: Neuroblastoma (NB) is the most common and lethal extracranial solid tumor of childhood. Despite aggressive therapy, more than half of the children with advanced NB will die of uncontrolled metastatic disease. After chemotherapy, tumor-initiating cells (TICs) could persist, cause relapses and metastasis. The aim of this study is to demonstrate the tumor-initiating properties of CD133high NB cells and to identify new specific genetic abnormalities. Isolation of the CD133high cell population from NB cell lines was followed by neurosphere formation, soft agar assays, and orthotopic injections in NOD/SCID/IL2Rγc-null mice. A differential genotyping analysis was performed with Affymetrix SNP 6.0 arrays on CD133low and CD133high populations and the frequency of the abnormalities of 36 NB tumors was determined. Our results show that CD133high NB cells possess tumor-initiating properties, as CD133high cells formed significantly more neurospheres and produced significantly more colonies in soft agar than CD133low. Injection of 500 CD133high cells was sufficient to generate primary tumors and frequent metastases in mice. Differential genotyping analysis demonstrated two common regions with gains (16p13.3 and 19p13.3) including the gene EFNA2 in the CD133high population, and two with loss of heterozygosity (16q12.1 and 21q21.3) in the CD133low population. The gain of EFNA2 correlated with increased expression of the corresponding protein. These abnormalities were found in NB samples and some were significantly correlated with CD133 expression. Our results show that CD133high NB cells have TICs properties and present different genotyping characteristics compared to CD133low cells. Our findings reveal insights into new therapeutic targets in NB TICs.
    Genes Chromosomes and Cancer 08/2012; 51(8):792-804. DOI:10.1002/gcc.21964 · 3.84 Impact Factor
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    • "Besides, well-characterized NB cell lines are known to recapitulate the heterogeneity observed in tumors, and some of them were shown to display stem cell–like characteristics. Thus, a highly tumorigenic " side population " of cells capable to specifically efflux a Hoechst dye was isolated from NB cell lines [15] [16] [17]. However, no specific gene expression profiles have been formerly identified for NB-TICs either in primary tumors or in NB cell lines. "
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    ABSTRACT: Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.
    Neoplasia (New York, N.Y.) 10/2011; 13(10):991-1004. DOI:10.1593/neo.11800 · 5.40 Impact Factor
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