Circulating tumor cells (CTCs) derived from any tumor tissue could contribute to metastasis and resistance to cancer treatments. In this study, we performed single-cell next-generation sequencing of CTCs and evaluated their usefulness for characterizing tumor biology and the mechanisms of metastasis in neuroblastomas (NB). We aimed to isolate CTCs from 10 patients with NB at diagnosis before any treatments and four patients at relapse. GD2(+)CD90(+)CD45(-)CD235a(-)DAPI(-) cells were isolated as neuroblastoma CTCs using fluorescence-activated cell sorting. In five patients with advanced stages (M stage), DNA and RNA sequencing of CTCs at single-cell level were performed. NB CTCs were isolated from eight of the 10 patients at diagnosis and three of the four patients at relapse. More CTCs could be isolated from patients with advanced stages. In one patient, ALK mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with MYCN amplification, this gene was amplified in 12 of 13 CTCs. Using single-cell RNA sequencing, angiogenesis-related and cell cycle-related genes together with CCND1 and TUBA1A genes were found to be upregulated in CTCs. In one patient, CTCs were divided into two subgroups showing different gene expression profiles. In one subgroup, cell cycle-related and proliferation-related genes were differentially upregulated compared with the other group. In conclusion, next-generation sequencing of CTCs at single-cell level might help to characterize the tumor biology and the mechanisms of metastasis in NB.

Outcomes of pediatric hepatoblastoma (HBL) have improved, but refractory cases still occur. More effective and safer drugs are needed that are based on molecular mechanisms. A disintegrin and metalloproteases (ADAMs) are expressed with high frequency in various human carcinomas and play an important role in cancer progression. In this study, we analyzed expression of ADAMs in HBL with a cDNA microarray dataset and found that the expression level of ADAM32 is particularly high. To investigate the role of ADAM32 in cancer, forced expression or knockdown experiments were conducted with HepG2 and HBL primary cells. Colony formation, cell migration and invasion, and cell viability were increased in HepG2 expressing ADAM32, whereas knockdown of ADAM32 induced a decrease in these cellular functions. Quantitative RT-PCR demonstrated an association between ADAM32 expression and the expression of genes related to cancer stem cells and epithelial–mesenchymal transition (EMT), suggesting a role of ADAM32 in cancer stemness and EMT. Furthermore, knockdown of ADAM32 increased cisplatin-induced apoptosis, and this effect was attenuated by a caspase-8 inhibitor, suggesting that ADAM32 plays a role in extrinsic apoptosis signaling. We conclude that ADAM32 plays a crucial role in progression of HBL, so it might be a promising molecular target in anticancer therapy.

Neuroblastoma is the most common extracranial solid tumor in children, and the overall survival rate of patients in the high-risk group is less than 50%. Circulating tumor cells have recently been found to be a potential source of tumor progression, metastasis, and poor prognosis in patients with cancer. There is a dearth of data on the malignant potential of circulating tumor cells in neuroblastoma. Therefore, in this article, we briefly review the methods of quantifying circulating neuroblastoma cells and the relationship between the presence of many circulating tumor cells and prognosis for patients with neuroblastoma. The relationship between higher numbers of circulating neuroblastoma tumor cells and poor prognosis was outlined. However, the data in most studies represented only the tumor burden, and there is no direct evidence that circulating tumor cells are the source of tumor progression and metastasis in patients with neuroblastoma. A comprehensive genetic analysis of circulating tumor cells, including single-cell sequencing and functional assays using ex vivo cell culture and xenografts, may provide insights into the malignant nature of neuroblastoma.

Achaete-scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet-On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, migration, invasion, and sphere-formation assays. Quantitative reverse-transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial-mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal-epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non-induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway-, EMT-, and stemness-related genes. ASCL2 activation may therefore be involved in the progression of HB.

Video-assisted thoracic surgery (VATS) is widely used for thoracic lesions in pediatric patients. VATS is also applied to pediatric mediastinal tumors if there is no adhesion or invasion between the tumor and adjacent neuronal and cardiovascular structures. Here, we present a pediatric case of mediastinal teratoma in which the tumor adhered to the superior vena cava, and resection was safely completed using assisted VATS, an integrated surgical approach comprising mini-thoracotomy with video assistance. A 9 year-old girl presented with right shoulder pain. Chest radiography and computed tomography revealed a 5.4 × 5.1 × 5.8 cm mass in the right upper anterior mediastinum. She was presumed with a mature teratoma, and resection was performed by assisted VATS with muscle sparing axillar skin crease incision (MSASCI) for a mini-thoracotomy. The procedure was safely completed, with the patient discharged on postoperative day 5. At 1 year postoperatively, there was no recurrence with excellent motor and cosmetic results. The combination of MSASCI and VATS would be useful not only for mediastinal teratomas but also for other mediastinal tumors and almost all other thoracic lesions in pediatric patients.