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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

Side population analysis reveals asymmetric cell division of neuroblastoma cells.(A) Side population analysis using Hoechst 33342, +/−verapamil, of bulk LA-N-5 cells (LAN5) and LA-N-5 cells grown in doxorubicin (LAN5-doxR). (B) Analysis of ABCG2 and CD133 expression in LAN5 and LAN5-doxR cells. (C) Sphere-forming efficiency of sorted SP and non-SP (NSP) cells from LAN5 and LAN5-doxR cultures. (D) Sorted SP and NSP cells, from cultures of LAN5 and LAN5-doxR, were plated in serum containing media for 2 weeks and re-evaluated by side population, +/−verapamil. SP-derived cultures show regeneration of SP and NSP cells, while NSP-derived cultures showed only regeneration of NSP cells, not SP cells.
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pone-0004235-g005: Side population analysis reveals asymmetric cell division of neuroblastoma cells.(A) Side population analysis using Hoechst 33342, +/−verapamil, of bulk LA-N-5 cells (LAN5) and LA-N-5 cells grown in doxorubicin (LAN5-doxR). (B) Analysis of ABCG2 and CD133 expression in LAN5 and LAN5-doxR cells. (C) Sphere-forming efficiency of sorted SP and non-SP (NSP) cells from LAN5 and LAN5-doxR cultures. (D) Sorted SP and NSP cells, from cultures of LAN5 and LAN5-doxR, were plated in serum containing media for 2 weeks and re-evaluated by side population, +/−verapamil. SP-derived cultures show regeneration of SP and NSP cells, while NSP-derived cultures showed only regeneration of NSP cells, not SP cells.

Mentions: Expression of ATP-binding cassette transporters such as ABCG2 have been associated on flow cytometric analysis of Hoescht staining with the presence of a so-called “side population” (SP), thought to be enriched for stem cells in some tissues [19]. Thus, the presence of ABCG2 expression suggests that LA-N-5 cultures may contain an efflux channel-dependent SP. Indeed, approximately 1% of bulk LA-N-5 cells appeared in a verapamil-sensitive SP, which was nearly 2-fold enhanced following addition of doxorubicin to culture media (10 ng/ml for 7–12 days) (Fig. 5A). Most other tested neuroblastoma cell lines showed the presence of a verapamil-sensitive SP, ranging from 2.5–40.5% (Table 1). Characterization of cell surface expression of ABCG2 and CD133 in bulk and doxorubicin-treated LA-N-5 cultures revealed a 4.8-fold increase of cells expressing ABCG2 and a 2.2-fold increase in double-positive cells (Fig. 5B). Conversely, doxorubicin exposure significantly reduced the percentage of CD133 expressing cells, indicating that a large number of CD133 positive cells are indeed doxorubicin-sensitive. Further, these studies imply that CD133 expressing cells will reside in both the SP as well as the non-SP.


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)

Side population analysis reveals asymmetric cell division of neuroblastoma cells.(A) Side population analysis using Hoechst 33342, +/−verapamil, of bulk LA-N-5 cells (LAN5) and LA-N-5 cells grown in doxorubicin (LAN5-doxR). (B) Analysis of ABCG2 and CD133 expression in LAN5 and LAN5-doxR cells. (C) Sphere-forming efficiency of sorted SP and non-SP (NSP) cells from LAN5 and LAN5-doxR cultures. (D) Sorted SP and NSP cells, from cultures of LAN5 and LAN5-doxR, were plated in serum containing media for 2 weeks and re-evaluated by side population, +/−verapamil. SP-derived cultures show regeneration of SP and NSP cells, while NSP-derived cultures showed only regeneration of NSP cells, not SP cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004235-g005: Side population analysis reveals asymmetric cell division of neuroblastoma cells.(A) Side population analysis using Hoechst 33342, +/−verapamil, of bulk LA-N-5 cells (LAN5) and LA-N-5 cells grown in doxorubicin (LAN5-doxR). (B) Analysis of ABCG2 and CD133 expression in LAN5 and LAN5-doxR cells. (C) Sphere-forming efficiency of sorted SP and non-SP (NSP) cells from LAN5 and LAN5-doxR cultures. (D) Sorted SP and NSP cells, from cultures of LAN5 and LAN5-doxR, were plated in serum containing media for 2 weeks and re-evaluated by side population, +/−verapamil. SP-derived cultures show regeneration of SP and NSP cells, while NSP-derived cultures showed only regeneration of NSP cells, not SP cells.
Mentions: Expression of ATP-binding cassette transporters such as ABCG2 have been associated on flow cytometric analysis of Hoescht staining with the presence of a so-called “side population” (SP), thought to be enriched for stem cells in some tissues [19]. Thus, the presence of ABCG2 expression suggests that LA-N-5 cultures may contain an efflux channel-dependent SP. Indeed, approximately 1% of bulk LA-N-5 cells appeared in a verapamil-sensitive SP, which was nearly 2-fold enhanced following addition of doxorubicin to culture media (10 ng/ml for 7–12 days) (Fig. 5A). Most other tested neuroblastoma cell lines showed the presence of a verapamil-sensitive SP, ranging from 2.5–40.5% (Table 1). Characterization of cell surface expression of ABCG2 and CD133 in bulk and doxorubicin-treated LA-N-5 cultures revealed a 4.8-fold increase of cells expressing ABCG2 and a 2.2-fold increase in double-positive cells (Fig. 5B). Conversely, doxorubicin exposure significantly reduced the percentage of CD133 expressing cells, indicating that a large number of CD133 positive cells are indeed doxorubicin-sensitive. Further, these studies imply that CD133 expressing cells will reside in both the SP as well as the non-SP.

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