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: 3.23). 02/2009; 4(1):e4235. DOI: 10.1371/journal.pone.0004235
Source: PubMed


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,
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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 · 2.30 Impact Factor
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    • "Since, spheroidal growth under defined conditions is a characteristic of neural cancer stem cells [66]; cancer stem cells express higher levels of SOD2 than non-stem cell counterparts [45], [46]; TrkAIII promotes a more stem cell-like phenotype [1], [4]; and NB staminality associates with increased CD133, CD117, SOX2, Nestin and Nanog expression [66], [68], [69], spheroid growth capacity and the expression of SOD2, CD133, CD117, SOX2, Nestin and Nanog were compared in pcDNA control, TrkA and TrkAIII SH-SY5Y cells. "
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    ABSTRACT: The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.
    PLoS ONE 04/2014; 9(4):e94568. DOI:10.1371/journal.pone.0094568 · 3.23 Impact Factor
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    • "These investigators reported the intratumoral injection of the virus in a child with refractory neuroblastoma, leading to a significant decrease in tumor volume with the child surviving 14 months after therapy [6]. Oncolytic herpes simplex virus (oHSV) mutants have also shown effectiveness in both murine and human neuroblastoma models [7-10]. "
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    ABSTRACT: Despite intensive research efforts and therapeutic advances over the last few decades, the pediatric neural crest tumor, neuroblastoma, continues to be responsible for over 15% of pediatric cancer deaths. Novel therapeutic options are needed for this tumor. Recently, investigators have shown that mice with syngeneic murine gliomas treated with an engineered, neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ 1 34.5 gene, enabling replication in tumor cells but precluding infection of normal neural cells. We hypothesized that M002 would also be effective in the neural crest tumor, neuroblastoma. We showed that M002 infected, replicated, and decreased survival in neuroblastoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly decreased tumor growth, and that this effect was augmented with the addition of ionizing radiation. Importantly, survival could be increased by subsequent doses of radiation without re-dosing of the virus. Finally, these studies showed that the primary entry protein for oHSV, CD111 was expressed by numerous neuroblastoma cell lines and was also present in human neuroblastoma specimens. We concluded that M002 effectively targeted neuroblastoma and that this oHSV may have potential for use in children with unresponsive or relapsed neuroblastoma.
    PLoS ONE 10/2013; 8(10):e77753. DOI:10.1371/journal.pone.0077753 · 3.23 Impact Factor
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