The heparin-binding growth factor midkine (MK) comprises a family with pleiotrophin/heparin-binding growth-associated molecule. The biological phenomena in which MK is involved can be categorized into five areas: (i) cancer, (ii) inflammation/immunity, (iii) blood pressure, (iv) development and (v) tissue protection. The phenotypes are clear in vivo, but the mechanisms by which MK exerts these actions are not fully understood. Candidate receptors for MK include anaplastic lymphoma kinase, protein tyrosine phosphatase ζ, Notch2, LDL receptor-related protein 1, integrins and proteoglycans. Some physical associations between these candidate receptors are also known. Because of the striking in vivo phenotypes after manipulation of MK, MK could be an important molecular target for the treatment of various diseases. To this end, it will be important to pursue studies to fully understand the mechanisms of MK action.

Neuroblastoma is an embryonal malignancy that affects normal development of the adrenal medulla and paravertebral sympathetic ganglia in early childhood. Extensive studies have revealed the molecular characteristics of human neuroblastomas, including abnormalities at genome, epigenome and transcriptome levels. However, neuroblastoma initiation mechanisms and even its origin are long-standing mysteries. In this review article, we summarize the current knowledge about normal development of putative neuroblastoma sources, namely sympathoadrenal lineage of neural crest cells and Schwann cell precursors that were recently identified as the source of adrenal chromaffin cells. A plausible origin of enigmatic stage 4S neuroblastoma is also discussed. With regard to the initiation mechanisms, we review genetic abnormalities in neuroblastomas and their possible association to initiation mechanisms. We also summarize evidences of neuroblastoma initiation observed in genetically engineered animal models, in which epigenetic alterations were involved, including transcriptomic upregulation by N-Myc and downregulation by polycomb repressive complex 2. Finally, several in vitro experimental methods are proposed that hopefully will accelerate our comprehension of neuroblastoma initiation. Thus, this review summarizes the state-of-the-art knowledge about the mechanisms of neuroblastoma initiation, which is critical for developing new strategies to cure children with neuroblastoma.

Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.

Pediatric cancers such as neuroblastoma are thought to involve a dysregulation of embryonic development. However, it has been difficult to identify the critical events that trigger tumorigenesis and differentiate them from normal development. In this study, we report the establishment of a spheroid culture method that enriches early-stage tumor cells from TH-MYCN mice, a preclinical model of neuroblastoma. Using this method, we found that tumorigenic cells were evident as early as day E13.5 during embryo development, when the MYC and PRC2 transcriptomes were significantly altered. Ezh2, an essential component of PRC2, was expressed in embryonic and postnatal tumor lesions and physically associated with N-MYC and we observed that H3K27me3 was increased at PRC2 target genes. PRC2 inhibition suppressed sphere formation, derepressed its target genes, and suppressed tumor growth. In clinical specimens, expression of MYC and PRC2 target genes correlated strongly and predicted survival outcomes. Together, our findings highlighted PRC2-mediated transcriptional control during embryogenesis as a critical step in the development and clinical outcome of neuroblastoma. .

Tumorsphere culture enriches and expands tumor cells, thus providing important resources for cancer studies. However, as compared with metastatic tissues, primary tumors in the nervous system rarely give rise to long-surviving tumorspheres, thereby seriously limiting studies on these cancers. This might be due to the limited self-renewal capability of tumor cells and/or to inappropriate culture conditions. The growth and maintenance of tumor cells may depend on microenvironments and/or cell origins (e.g., primary or metastatic; stem cell-like or progenitor-like). Here, we attempted to establish a tumorsphere culture condition for primary neuroblastoma (NB). Primary tumors in MYCN transgenic mice, a NB model, could be serially transplanted, suggesting that these tumors contain cells with a high self-renewal potential. However, primary tumors did not give rise to tumorspheres under a serum-free neurosphere culture condition. The newly established culture condition (named PrimNeuS) contained two critical ingredients: fetal bovine serum and β-mercaptoethanol were essential for tumorsphere formation as well as indefinite passages. The spheres could be passaged more than 20 times without exhaustion under this condition, exhibited a property of differentiation and formed tumors in vivo. Unexpectedly, PrimNeuS revealed that the MYCN transgenic mice had bone marrow metastasis. Furthermore, subcutaneous tumors derived from tumorspheres of primary tumors showed bone marrow metastasis. Taken together, PrimNeuS provides resources for the study of NB and can be used as a powerful tool for the detection of minimal residual disease and for in vitro evaluation prior to personalized therapy.

We carried out limb lengthening in rabbits and then transplanted osteoblast-like cells derived from the tibial periosteum to the centres of distracted callus immediately after distraction had been terminated. Two weeks later the transaxial area ratio at the centre of the distracted callus and the bone mineral density (BMD) were significantly higher in the transplanted group, by 21% and 42%, respectively, than in the non-injected group or the group injected with physiological saline (p < 0.05). Callus BMD as a percentage of density in uninvolved bone was also significantly higher in the transplanted group (p < 0.05) than in the other two groups, by 27% and 20% in the second and fourth weeks, respectively (p < 0.05). Mechanically, the callus in the transplanted group tended to be stronger as shown by the three-point bending test although the difference in fracture strength was not statistically significant. Our results show that transplantation of osteoblast-like cells promotes maturity of the distracted callus as observed at the second and fourth weeks after lengthening. The method appears promising as a means of shortening the consolidation period of callus distraction and decreasing complications during limb lengthening with an external fixator.

We carried out limb lengthening in rabbits and then transplanted osteoblast-like cells derived from the tibial periosteum to the centres of distracted callus immediately after distraction had been terminated. Two weeks later the transaxial area ratio at the centre of the distracted callus and the bone mineral density (BMD) were significantly higher in the transplanted group, by 21% and 42%, respectively, than in the non-injected group or the group injected with physiological saline (p < 0.05). Callus BMD as a percentage of density in uninvolved bone was also significantly higher in the transplanted group (p < 0.05) than in the other two groups, by 27% and 20% in the second and fourth weeks, respectively (p < 0.05). Mechanically, the callus in the transplanted group tended to be stronger as shown by the three-point bending test although the difference in fracture strength was not statistically significant.Our results show that transplantation of osteoblast-like cells promotes maturity of the distracted callus as observed at the second and fourth weeks after lengthening. The method appears promising as a means of shortening the consolidation period of callus distraction and decreasing complications during limb lengthening with an external fixator. J Bone Joint Surg [Br] 1999;81-B:125-9.

Neurocan (NCAN), a secreted chondroitin sulfate proteoglycan, is one of the major inhibitory molecules for axon regeneration in nervous injury. However, its role in cancer is not clear. Here we observed that high NCAN expression was closely associated with the unfavorable outcome of neuroblastoma (NB). NCAN was also highly and ubiquitously expressed in the early lesions and terminal tumor of TH-MYCN mice, a NB model. Interestingly, exogenous NCAN (i.e., overexpression, recombinant protein and conditioned medium) transformed adherent NB cells into spheres whose malignancies in vitro (anchorage-independent growth and chemoresistance) and in vivo (xenograft tumor growth) were potentiated. Both chondroitin sulfate sugar chains and NCAN's core protein were essential for the sphere formation. The CSG3 domain was essential in the moiety of NCAN. Our comprehensive microarray analysis and RT-qPCR of mRNA expression suggested that NCAN treatment promoted cell division, and urged cells to undifferentiated state. The knockdown of NCAN in tumor sphere cells cultured from TH-MYCN mice resulted in growth suppression in vitro and in vivo. Our findings suggest that NCAN, which stimulates NB cells to promote malignant phenotypes, is an extracellular molecule providing a growth advantage to cancer cells.