title: Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling.
abstract: BACKGROUND
  High-risk neuroblastoma (HR-NB) presenting with hematogenous metastasis is one of the most difficult cancers to cure. Patient survival is poor. Aggressive tumors contain populations of rapidly proliferating clonogens that exhibit stem cell properties, cancer stem cells (CSCs). Conceptually, CSCs that evade intensive multimodal therapy dictate tumor progression, relapse/recurrence, and poor clinical outcomes. Herein, we investigated the plasticity and stem-cell related molecular response of aggressive metastatic neuroblastoma cells that fit the CSC model.
  
  
  METHODS
  Well-characterized clones of metastatic site-derived aggressive cells (MSDACs) from a manifold of metastatic tumors of clinically translatable HR-NB were characterized for their CSC fit by examining epithelial-to-mesenchymal transition (EMT) (E-cadherin, N-Cadherin), survival (NFκB P65, p50, IκB and pIκB) and drug resistance (ABCG2) by immunoblotting; pluripotency maintenance (Nanog, SOX2) by immunofluorescence; and EMT and stemness related transcription of 93 genes by QPCR profiling. Plasticity of MSDACs under sequential alternation of culture conditions with serum and serum-free stem-cell conditions was assessed by clonal expansion (BrdU incorporation), tumorosphere formation (anchorage independent growth), EMT and stemness related transcriptome (QPCR profiling) and validated with MYC, SOX2, EGFR, NOTCH1 and CXCL2 immunoblotting.
  
  
  RESULTS
  HR-NB MSDACs maintained in alternated culture conditions, serum-free stem cell medium to growth medium with serum and vice versa identified its flexible revocable plasticity characteristics. We observed signatures of stem cell-related molecular responses consistent with phenotypic conversions. Successive reintroduction to the favorable niche not only regained identical EMT, self-renewal capacity, pluripotency maintenance, and other stem cell-related signaling events, but also instigated additional events depicting aggressive adaptive plasticity.
  
  
  CONCLUSIONS
  Together, these results demonstrated the flexible plasticity of HR-NB MSDACs that typically fit the CSC model, and further identified the intrinsic adaptiveness of the successive phenotype switching that clarifies the heterogeneity of HR-NB. Moreover, the continuous ongoing acquisition of stem cell-related molecular rearrangements may hold the key to the switch from favorable disease to HR-NB.
authors.affiliation: Department of Radiation Oncology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 737, Oklahoma City, OK, 73104, USA. vpandian@ouhsc.edu.
authors.forename: Vijayabaskar
authors.initials: V
authors.lastname: Pandian
authors.affiliation: Department of Radiation Oncology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 737, Oklahoma City, OK, 73104, USA. sramraj@ouhsc.edu.
authors.forename: Satishkumar
authors.initials: S
authors.lastname: Ramraj
authors.affiliation: Department of Radiation Oncology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 737, Oklahoma City, OK, 73104, USA. fkhan4@ouhsc.edu.
authors.forename: Faizan H
authors.initials: FH
authors.lastname: Khan
authors.affiliation: Department of Radiation Oncology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 737, Oklahoma City, OK, 73104, USA. Tasfia.Azim@OSSM.EDU.
authors.forename: Tasfia
authors.initials: T
authors.lastname: Azim
authors.affiliation: Department of Radiation Oncology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 737, Oklahoma City, OK, 73104, USA. naravind@ouhsc.edu.
authors.forename: Natarajan
authors.initials: N
authors.lastname: Aravindan
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D000066670:Cell Plasticity / Q000502:physiology*; D049109:Cell Proliferation; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D009362:Neoplasm Metastasis / Q000235:genetics / Q000473:pathology; D009363:Neoplasm Proteins / Q000378:metabolism; D009364:Neoplasm Recurrence, Local; D014411:Neoplastic Stem Cells / Q000473:pathology*; D009447:Neuroblastoma / Q000473:pathology*; D015398:Signal Transduction; D018874:Spheroids, Cellular; D051996:Transcription Factor RelA / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015820:Cadherins; D052002:NF-kappa B p50 Subunit; D009363:Neoplasm Proteins; C498143:RELA protein, human; D051996:Transcription Factor RelA; D051550:I-kappa B Kinase