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Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

Mahller YY, Williams JP, Baird WH, Mitton B, Grossheim J, Saeki Y, Cancelas JA, Ratner N, Cripe TP - PLoS ONE (2009)

Bottom Line: The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse.Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics.These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

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Related in: MedlinePlus

Neuroblastoma tumorspheres express nestin and are efficiently infected by oHSV.(A) Immunohistochemistry on tumorsphere cryosection for nestin (green) and DAPI co-stain (blue). Scale bar = 10 microns. (B, C) A neuroblastoma tumorsphere infected with rQNestin34.5 was imaged at 48 hours post-infection by (B) phase-contrast and (C) fluorescent microscopy for GFP. (D, E) rQNestin34.5-infected neuroblastoma tumorsphere at 48 hours post-infection evaluated by transmission electron microscopy showing viral nucleocapsids in the nucleus (arrows), and mature HSV particles in the cytoplasm in the process of acquiring their envelopes (arrows).
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pone-0004235-g007: Neuroblastoma tumorspheres express nestin and are efficiently infected by oHSV.(A) Immunohistochemistry on tumorsphere cryosection for nestin (green) and DAPI co-stain (blue). Scale bar = 10 microns. (B, C) A neuroblastoma tumorsphere infected with rQNestin34.5 was imaged at 48 hours post-infection by (B) phase-contrast and (C) fluorescent microscopy for GFP. (D, E) rQNestin34.5-infected neuroblastoma tumorsphere at 48 hours post-infection evaluated by transmission electron microscopy showing viral nucleocapsids in the nucleus (arrows), and mature HSV particles in the cytoplasm in the process of acquiring their envelopes (arrows).

Mentions: Based on nestin expression in primary neuroblastoma tumors and tumorspheres, we hypothesized that a nestin-targeted oHSV would be effective in killing both differentiated and tumor initiating neuroblastoma cells. rQNestin34.5 was initially created to target oHSV killing to nestin-positive brain tumor cells [43]. In this recombinant virus, expression of the HSV-1 neurovirulence gene (encoding γ134.5) is driven by the nestin enhancer [43], and the virus remains attenuated in normal cells by a deletion in the virally-encoded large subunit of ribonucleotide reductase (ICP6). LA-N-5 neuroblastoma tumorspheres were positive for nestin by immunostain and all other tested neuroblastoma cell lines showed significant nestin expression (Fig. 7A and Table 1). Neuroblastoma tumorspheres were readily transduced by rQNestin34.5 (Fig. 7B, C). As predicted based on nestin expression, rQNestin34.5 caused significant cell death of both bulk and tumorsphere-derived LA-N-5 cells compared with the control virus, rQLuc (*p<0.05) (Fig. 8A). Virus replication was similar in bulk cultured LA-N-5 cells. In contrast, production of rQNestin34.5 was >10-fold increased compared with rQLuc in tumorsphere-derived cells (*p<0.05) (Fig. 8B). New virus production was confirmed to occur in tumorsphere cells by transmission EM of a virus-infected tumorsphere, demonstrating that virus isn't simply being produced exclusively in non-sphere cells present in the cultures. These cells showed viral nucleocapsids in the nucleus, particles in the cytoplasm acquiring their envelope and fully enveloped HSV particles (Fig. 7D, E).


Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

Mahller YY, Williams JP, Baird WH, Mitton B, Grossheim J, Saeki Y, Cancelas JA, Ratner N, Cripe TP - PLoS ONE (2009)

Neuroblastoma tumorspheres express nestin and are efficiently infected by oHSV.(A) Immunohistochemistry on tumorsphere cryosection for nestin (green) and DAPI co-stain (blue). Scale bar = 10 microns. (B, C) A neuroblastoma tumorsphere infected with rQNestin34.5 was imaged at 48 hours post-infection by (B) phase-contrast and (C) fluorescent microscopy for GFP. (D, E) rQNestin34.5-infected neuroblastoma tumorsphere at 48 hours post-infection evaluated by transmission electron microscopy showing viral nucleocapsids in the nucleus (arrows), and mature HSV particles in the cytoplasm in the process of acquiring their envelopes (arrows).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2626279&req=5

pone-0004235-g007: Neuroblastoma tumorspheres express nestin and are efficiently infected by oHSV.(A) Immunohistochemistry on tumorsphere cryosection for nestin (green) and DAPI co-stain (blue). Scale bar = 10 microns. (B, C) A neuroblastoma tumorsphere infected with rQNestin34.5 was imaged at 48 hours post-infection by (B) phase-contrast and (C) fluorescent microscopy for GFP. (D, E) rQNestin34.5-infected neuroblastoma tumorsphere at 48 hours post-infection evaluated by transmission electron microscopy showing viral nucleocapsids in the nucleus (arrows), and mature HSV particles in the cytoplasm in the process of acquiring their envelopes (arrows).
Mentions: Based on nestin expression in primary neuroblastoma tumors and tumorspheres, we hypothesized that a nestin-targeted oHSV would be effective in killing both differentiated and tumor initiating neuroblastoma cells. rQNestin34.5 was initially created to target oHSV killing to nestin-positive brain tumor cells [43]. In this recombinant virus, expression of the HSV-1 neurovirulence gene (encoding γ134.5) is driven by the nestin enhancer [43], and the virus remains attenuated in normal cells by a deletion in the virally-encoded large subunit of ribonucleotide reductase (ICP6). LA-N-5 neuroblastoma tumorspheres were positive for nestin by immunostain and all other tested neuroblastoma cell lines showed significant nestin expression (Fig. 7A and Table 1). Neuroblastoma tumorspheres were readily transduced by rQNestin34.5 (Fig. 7B, C). As predicted based on nestin expression, rQNestin34.5 caused significant cell death of both bulk and tumorsphere-derived LA-N-5 cells compared with the control virus, rQLuc (*p<0.05) (Fig. 8A). Virus replication was similar in bulk cultured LA-N-5 cells. In contrast, production of rQNestin34.5 was >10-fold increased compared with rQLuc in tumorsphere-derived cells (*p<0.05) (Fig. 8B). New virus production was confirmed to occur in tumorsphere cells by transmission EM of a virus-infected tumorsphere, demonstrating that virus isn't simply being produced exclusively in non-sphere cells present in the cultures. These cells showed viral nucleocapsids in the nucleus, particles in the cytoplasm acquiring their envelope and fully enveloped HSV particles (Fig. 7D, E).

Bottom Line: The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse.Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics.These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

Show MeSH
Related in: MedlinePlus