Single‐cell next‐generation sequencing of circulating tumor cells in patients with neuroblastoma

  • Masato Kojima
    Department of Surgery, Graduate School of Biomedical and Health Sciences Hiroshima University Hiroshima Japan
  • Takanori Harada
    Natural Science Center for Basic Research and Development (N‐BARD) Hiroshima University Hiroshima Japan
  • Takahiro Fukazawa
    Natural Science Center for Basic Research and Development (N‐BARD) Hiroshima University Hiroshima Japan
  • Sho Kurihara
    Department of Surgery, Graduate School of Biomedical and Health Sciences Hiroshima University Hiroshima Japan
  • Ryo Touge
    Department of Pediatric Surgery Hiroshima University Hospital Hiroshima Japan
  • Isamu Saeki
    Department of Pediatric Surgery Hiroshima University Hospital Hiroshima Japan
  • Shinya Takahashi
    Department of Surgery, Graduate School of Biomedical and Health Sciences Hiroshima University Hiroshima Japan
  • Eiso Hiyama
    Department of Pediatric Surgery Hiroshima University Hospital Hiroshima Japan

抄録

<jats:title>Abstract</jats:title><jats:p>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<jats:sup>+</jats:sup>CD90<jats:sup>+</jats:sup>CD45<jats:sup>−</jats:sup>CD235a<jats:sup>−</jats:sup>DAPI<jats:sup>−</jats:sup> 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, <jats:italic>ALK</jats:italic> mutation (p.F1174L), was identified in both tumor tissue and a CTC. In patients with <jats:italic>MYCN</jats:italic> amplification, this gene was amplified in 12 of 13 CTCs. Using single‐cell RNA sequencing, angiogenesis‐related and cell cycle‐related genes together with <jats:italic>CCND1</jats:italic> and <jats:italic>TUBA1A</jats:italic> 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.</jats:p>

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