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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

Clonally-derived neuroblastoma tumorsphere cells show multi-lineage differentiation.Clonally-derived tumorspheres from three different neuroblastoma cell lines were dissociated and cells were plated on poly-lysine and laminin coated chamber slides. Culture conditions were media containing serum alone or serum with neurotrophic factors (top row), gliogenic factors (middle row) or fibroblastic factors (bottom row) (except negative controls, which were serum alone without factors). Slides were stained with neurofilament-M (NF-M, green, top row), GFAP (red, middle row) S100β (green, middle row), or smooth muscle actin (red, bottom row); each were also co-stained with DAPI (blue). Negative control cultures were incubated without primary antibody and with secondary anti-mouse TRITC or anti-rabbit FITC. Arrows in the top and bottom rows indicate spindle-like cell extensions consistent with either neuronal or fibroblastic differentiation, while arrows in the middle row indicate positively stained cells. On close inspection of the GFAP/S100β stains, IMR-32 cells under serum conditions and CHP-134 cells supplemented with factors show co-staining with a mixture of green/red signals. Scale bars = 65 microns.
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pone-0004235-g003: Clonally-derived neuroblastoma tumorsphere cells show multi-lineage differentiation.Clonally-derived tumorspheres from three different neuroblastoma cell lines were dissociated and cells were plated on poly-lysine and laminin coated chamber slides. Culture conditions were media containing serum alone or serum with neurotrophic factors (top row), gliogenic factors (middle row) or fibroblastic factors (bottom row) (except negative controls, which were serum alone without factors). Slides were stained with neurofilament-M (NF-M, green, top row), GFAP (red, middle row) S100β (green, middle row), or smooth muscle actin (red, bottom row); each were also co-stained with DAPI (blue). Negative control cultures were incubated without primary antibody and with secondary anti-mouse TRITC or anti-rabbit FITC. Arrows in the top and bottom rows indicate spindle-like cell extensions consistent with either neuronal or fibroblastic differentiation, while arrows in the middle row indicate positively stained cells. On close inspection of the GFAP/S100β stains, IMR-32 cells under serum conditions and CHP-134 cells supplemented with factors show co-staining with a mixture of green/red signals. Scale bars = 65 microns.

Mentions: Clonally-derived tumorspheres (LA-N-5, IMR-32, CHP-134) were dissociated, plated on chamber-slides and grown in media containing serum alone or supplemented with neurogenic, gliogenic or smooth muscle fibroblastic growth factors. The cultures were stained with antibodies against various cytoskeletal and membrane proteins that have previously been validated as markers of neural lineage differentiation pathways, including neurofilament-M (NF-M) as a marker of neurogenic cell differentiation, S100β as a marker of Schwannian cell differentiation, glial fibulary acid protein (GFAP) as a marker of glial differentiation, and smooth muscle actin (SMA) as an indicator of fibroblastic differentiation [6], [45], [49], [50] (Fig. 3). All three cell lines grown in both serum and serum supplemented with neurotrophic factors expressed NF-M, though there was more evidence of neuronal-like morphologic changes in the supplemented cultures (Fig. 3, top row, best seen in IMR-32). There was a varied response of the different lines under gliogenic conditions: in LA-N-5 cells, GFAP but not S100β expression was clearly induced, in CHP-134 cells both appeared albeit only faintly, and in IMR-32 cells both were present at baseline but only S100β appeared after exposure to the factors (Fig. 3, middle row). Under fibroblastic conditions, all three cell lines showed marked induction of SMA (Fig. 3, bottom row). IMR-32 cells also showed a more elongated, spindly morphology under these conditions. These data suggest neuroblastoma cell lines harbor a subset of progenitor cells capable of multi-lineage differentiation.


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)

Clonally-derived neuroblastoma tumorsphere cells show multi-lineage differentiation.Clonally-derived tumorspheres from three different neuroblastoma cell lines were dissociated and cells were plated on poly-lysine and laminin coated chamber slides. Culture conditions were media containing serum alone or serum with neurotrophic factors (top row), gliogenic factors (middle row) or fibroblastic factors (bottom row) (except negative controls, which were serum alone without factors). Slides were stained with neurofilament-M (NF-M, green, top row), GFAP (red, middle row) S100β (green, middle row), or smooth muscle actin (red, bottom row); each were also co-stained with DAPI (blue). Negative control cultures were incubated without primary antibody and with secondary anti-mouse TRITC or anti-rabbit FITC. Arrows in the top and bottom rows indicate spindle-like cell extensions consistent with either neuronal or fibroblastic differentiation, while arrows in the middle row indicate positively stained cells. On close inspection of the GFAP/S100β stains, IMR-32 cells under serum conditions and CHP-134 cells supplemented with factors show co-staining with a mixture of green/red signals. Scale bars = 65 microns.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004235-g003: Clonally-derived neuroblastoma tumorsphere cells show multi-lineage differentiation.Clonally-derived tumorspheres from three different neuroblastoma cell lines were dissociated and cells were plated on poly-lysine and laminin coated chamber slides. Culture conditions were media containing serum alone or serum with neurotrophic factors (top row), gliogenic factors (middle row) or fibroblastic factors (bottom row) (except negative controls, which were serum alone without factors). Slides were stained with neurofilament-M (NF-M, green, top row), GFAP (red, middle row) S100β (green, middle row), or smooth muscle actin (red, bottom row); each were also co-stained with DAPI (blue). Negative control cultures were incubated without primary antibody and with secondary anti-mouse TRITC or anti-rabbit FITC. Arrows in the top and bottom rows indicate spindle-like cell extensions consistent with either neuronal or fibroblastic differentiation, while arrows in the middle row indicate positively stained cells. On close inspection of the GFAP/S100β stains, IMR-32 cells under serum conditions and CHP-134 cells supplemented with factors show co-staining with a mixture of green/red signals. Scale bars = 65 microns.
Mentions: Clonally-derived tumorspheres (LA-N-5, IMR-32, CHP-134) were dissociated, plated on chamber-slides and grown in media containing serum alone or supplemented with neurogenic, gliogenic or smooth muscle fibroblastic growth factors. The cultures were stained with antibodies against various cytoskeletal and membrane proteins that have previously been validated as markers of neural lineage differentiation pathways, including neurofilament-M (NF-M) as a marker of neurogenic cell differentiation, S100β as a marker of Schwannian cell differentiation, glial fibulary acid protein (GFAP) as a marker of glial differentiation, and smooth muscle actin (SMA) as an indicator of fibroblastic differentiation [6], [45], [49], [50] (Fig. 3). All three cell lines grown in both serum and serum supplemented with neurotrophic factors expressed NF-M, though there was more evidence of neuronal-like morphologic changes in the supplemented cultures (Fig. 3, top row, best seen in IMR-32). There was a varied response of the different lines under gliogenic conditions: in LA-N-5 cells, GFAP but not S100β expression was clearly induced, in CHP-134 cells both appeared albeit only faintly, and in IMR-32 cells both were present at baseline but only S100β appeared after exposure to the factors (Fig. 3, middle row). Under fibroblastic conditions, all three cell lines showed marked induction of SMA (Fig. 3, bottom row). IMR-32 cells also showed a more elongated, spindly morphology under these conditions. These data suggest neuroblastoma cell lines harbor a subset of progenitor cells capable of multi-lineage differentiation.

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