Human neuroblastoma is known for its striking tumor cell heterogeneity and ability to relapse. These features suggest that neuroblastoma may be a stem cell disease. Herein we describe studies using human neuroblastoma cell lines suggesting they contain subpopulations of cells with characteristics shared with normal neural stem cells. All cell lines tested contained cells that express markers previously described on neural stem cells such as CD133, ABCG2, and nestin. Half of the cell lines were capable of growing as “tumorspheres” in neural stem cell media, and three of three tested were capable of multi-lineage differentiation. One line tested further showed that the CD133 positive cells were enriched for tumorigenicity and that the spheres were enriched for a verapamil-sensitive side population, CD133 expression and doxorubicin resistance. Infection with a nestin promoter-directed oncolytc herpes virus prevented tumor formation, suggesting the tumor initiating cells were targetable by virotherapy.
We observed clonal growth in serum-free neurosphere media in four of eight neuroblastoma cells lines tested, which correlated with their MYCN amplification status. It is likely that MYCN expression plays a role in neuroblatoma stem cells given the recently described role of myc-regulated gene networks in generating induced pluripotent stem cells from somatic tissues 
and the role of myc in the maintenance of both normal hematopoietic stem cells 
and glioma cancer stem cells 
. Growth of LA-N-5 neuroblastoma cells as non-adherent, nestin positive, multi-potent tumorspheres was dependent upon γ-secretase and EGFR signaling, similar to neural stem cells 
. Tumorsphere-derived neuroblastoma cultures were enriched for stemness markers CD133 and ABCG2 in comparison to bulk-grown cells. Tumorsphere-derived cells showed relative resistance to doxorubicin.
As predicted, addition of doxorubicin to culture media of neuroblastoma cells increased the percentage of side population cells and correspondingly enriched cultures for ABCG2 expressing cells. Although CD133 positive and doxorubicin-sensitive cells were dramatically reduced in the presence of doxorubicin, those expressing ABCG2 and CD133 were enriched. These results demonstrate that the CD133 positive population is heterogeneous in expression of ABCG2 and that CD133 expression alone is not sufficient to identify neuroblastoma tumor initiating cells. CD133 as been identified as a marker of cancer stem cells in some models but not others, and its utility as a single marker of such cells is controversial 
. Some studies have shown that CD133 expressing human cancer cells show chemoresistance 
. In a tumorsphere formation assay, sorted SP and non-SP cells from bulk cultured LA-N-5 cells showed no difference, suggesting that sphere-forming cells exist in both. In contrast, SP cells from doxorubicin-treated LA-N-5 cultures showed enriched sphere forming ability compared with bulk culture SP cells, likely due to increased cells expressing ABCG2 and CD133. Further, non-SP cells from doxorubicin-treated LA-N-5 cultures lost the ability to form spheres, possibly due to doxorubicin-mediated destruction of ABCG2 null, CD133 expressing cells. These results demonstrate that doxorubicin treatment exerts multiple effects. First, it enriches the culture for cells with drug efflux ability and those able to form tumorspheres. Second, it diminishes the culture of cells lacking drug efflux ability but capable of forming spheres. Thus, side population or drug efflux status and sphere forming efficiency may be related at least partially via CD133 expression.
Asymmetric cell division is a key property of both normal and tumor stem cells. We observed that sorted SP-derived cultures could regenerate their non-SP counterparts, while non-SP-derived cultures could not. CD133 expressing cells showed increased ability to form tumorspheres and xenograft tumors in immunodeficient mice, suggesting increased tumorigenicity. Overall, these studies support the presence of cells with stem cell-like features in neuroblastoma and suggest that multiple criteria be utilized in parallel to further identify, isolate and characterize such cells.
Because cancer stem cells are believed responsible for tumor metastasis, escape from anticancer therapies and ultimately disease relapse, their therapeutic targeting is crucial 
. Small molecule inhibitors designed to target signaling pathways regulating stem cell renewal and maintenance may increase efficacy of traditional therapies. Hypothetical targets for future therapies include signaling via Notch, EGFR, Wnt, Hedgehog and bmi-1 
. Alternatively, it is conceivable that tumor initiating cells may be targeted via tumor cell-specific expression of stem cell surface markers using toxin-coupled antibodies. Combination of current chemotherapeutics with efflux blocking agents, such as calcium channel blockers, may enhance antitumor efficacy.
Another strategy for targeting cancer stem cells could be to utilize biologics. Oncolytic viruses are attractive anticancer therapeutics due to their ability to replicate in vivo
, thereby amplifying the injected dose. These viruses have shown efficacy and safety in clinical trials 
. As drug-resistant tumor initiating cells have been reported to exist in a perivascular niche, intravenous administration of an oncolytic virus may be highly effective to reach this site 
. In addition, because oncolytic viruses circumvent traditional chemotherapy resistance mechanisms, they have been thought to be ideal for targeting cancer stem cells 
. As replication and toxicity of oncolytic viruses may be targeted via tumor specific promoters, we rationalized that such a virus could be targeted against neuroblastoma stem cells. In this study we demonstrated that a nestin-targeted oHSV efficiently infected, replicated and killed neuroblastoma tumorsphere cells. Ex vivo
infection of neuroblastoma cells with a nestin-targeted oHSV resulted in death of tumor initiating cells as it prevented tumor development in animals. Our study used cell lines derived from neuroblastomas, so it will be important to verify our results using primary human samples. Spheres derived from bone marrow metastases of patients with neuroblastoma have been shown to be highly enriched for tumor initiating cells 
, suggesting these cultures may be useful for such validation studies.
Like several other cancers, it is becoming increasingly evident that neuroblastoma is a stem cell disease; thus, therapeutic targeting of cells that cause relapse is vital to improve patient outcome. Further study of neuroblastoma stem cells should reveal their roles in tumor initiation, progression and metastasis. As anticancer therapies incorporate anti-stem cell approaches, it will be important to ensure that these treatments do not adversely affect the function of normal tissue stem cells. Development of such innovative strategies to target cancer stem cell populations in human malignancies is likely to increase treatment success.