This may be why a common protocol for tumorsphere cultures cannot be established. Therefore, if appropriate conditions were provided, AbMole Terbuthylazine tumorspheres could be grown in vitro. We addressed this hypothesis in the present study by employing MYCN transgenic mice, a NB model. NB is the most common pediatric extracranial solid tumor and is derived from sympathetic neurons. Despite intensive multimodal therapy, high-risk NB patients with relapse in bone marrow have less than a 10% chance of survival. To overcome this problem, it is particularly important to verify the mechanisms of tumor initiation and the metastasis of NB, which remain elusive. Here, we have established culture condition for long-surviving tumorspheres from primary NBs. This culture condition restores the potential for self-renewal and metastasis of primary NBs. Primary AbMole Aristolochic-acid-A tumors in MYCN transgenic mice could be serially transplanted without exhaustion, suggesting that these primary tumors contain cells with a high self-renewal potential. Nevertheless, primary tumors of MYCN transgenic mice did not give rise to tumorspheres in a serum-free neurosphere culture condition, which is commonly used for tumorsphere cultures for neural tumors. In contrast, allograft tumors gave rise to tumorspheres in this condition. These phenomena are reminiscent of those of clinical samples; tumorspheres are rarely obtained from primary or low-grade neural tumors, in contrast to metastatic or high-grade tumors. In this study, we established the new culture condition PrimNeuS, by which tumorspheres could be formed from a primary tumor and passaged indefinitely. Thus, this study has shown the self-renewal capacity of primary NB cells in vitro. Barrett et al. recently reported that self-renewal does not predict tumor growth potential in mouse models of high-grade glioma. Thus, while Id1low cells generate tumors more rapidly than Id1high cells, Id1low cells show non-self-renewal capacity in a neurosphere culture condition. However, there is still a possibility that Id1low cells might show self-renewal in vitro if an appropriate culture condition were provided. PrimNeuS has enabled us to estimate self-renewal capacity in vitro. Furthermore, PrimNeuS revealed for the first time that tumorspheres from primary tumors as well as primary tumors themselves have the potential for metastasis to the bone marrow. This is unexpected, since bone marrow metastasis has not been reported in the model of MYCN transgenic mice. PrimNeuS could be applicable as a sensitive tool to detect tiny bone marrow metastases or minimal residual disease of NB. Personalized therapy has become a realistic concept for cancer treatment. For example, 
anti-GD2 antibody-treated NB patients lacking HLA class I ligands for their inhibitory killer cell immunoglobulin-like receptors have significantly higher survival rates than those with HLA class I ligands. Unlicensed NK cells are thought to be responsible for this phenomenon. If tumorspheres are obtained from every patient regardless of tumor status, i.e., primary or metastatic, high risk or low risk and high grade or low grade, those are precious resources for evaluating the efficacy of the treatment prior to its clinical use. Although PrimNeuS medium tended to allow a longer culture of tumorspheres from human NB as compared with the classical serum-free medium, a limited number of samples did not allow us to statistically validate the usefulness of PrimNeuS for human NB. Further evaluation or improvement of the culture condition for human NB will facilitate studies of stem cells of NB. To successfully eradicate tumors, it may be necessary to eliminate both the TICs and their more differentiated progeny.