title: Interleukin-1β and transforming growth factor-β cooperate to induce neurosphere formation and increase tumorigenicity of adherent LN-229 glioma cells.
abstract: BACKGROUND
  Glioma stem cells (GSCs) have the property of self-renewal and appear to be a driving force for the initiation and recurrence of gliomas. We recently found that the human tumorigenic LN-229 glioma cell line failed to form neurospheres in serum-free conditions and generated mostly small tumors in vivo, suggesting that either LN-229 GSCs are not active in these conditions or GSCs are absent in the LN-229 cell line.
  
  
  METHODS
  Using self-renewal assay, soft-agar colony assay, cell proliferation assay, invasion assay, real time PCR analysis, ELISA and in vivo tumorigenic assay, we investigated the effects of interleukin (IL)-1β and transforming growth factor (TGF)-β on the development of GSCs from LN-229 cells.
  
  
  RESULTS
  Here, we demonstrate that the combination of IL-1β and TGF-β can induce LN-229 cells to form neurospheres in serum-free medium. IL-1β/TGF-β-induced neurospheres display up-regulated expression of stemness factor genes (nestin, Bmi-1, Notch-2 and LIF), and increased invasiveness, drug resistance and tumor growth in vivo: hallmarks of GSCs. These results indicate that IL-1β and TGF-β cooperate to induce a GSC phenotype in the LN-229 cell line. Induction of nestin, LIF and Notch-2 by IL-1β/TGF-β can be reverted after cytokine withdrawal. Remarkably, however, up-regulated Bmi-1 levels remained unchanged after cytokine withdrawal; and the cytokine-withdrawn cells maintained strong clonogenicity, suggesting that Bmi-1 may play a crucial role in tumorigenesis.
  
  
  CONCLUSIONS
  Our finding indicates that glioma cells without self-renewal capability in standard conditions could also contribute to glioma malignancy when cytokines, such as IL-1β and TGF-β, are present in the tumor environment. Targeting GSC-promoting cytokines that are highly expressed in glioblastomas may contribute to the development of more effective glioma therapies.
authors.affiliation: Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA. leiwang@vet.k-state.edu
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Ziyan
authors.initials: Z
authors.lastname: Liu
authors.forename: Sivasai
authors.initials: S
authors.lastname: Balivada
authors.forename: Tej
authors.initials: T
authors.lastname: Shrestha
authors.forename: Stefan
authors.initials: S
authors.lastname: Bossmann
authors.forename: Marla
authors.initials: M
authors.lastname: Pyle
authors.forename: Loretta
authors.initials: L
authors.lastname: Pappan
authors.forename: Jishu
authors.initials: J
authors.lastname: Shi
authors.forename: Deryl
authors.initials: D
authors.lastname: Troyer
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic; D005910:Glioma / Q000378:metabolism / Q000473:pathology; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D053605:Leukemia Inhibitory Factor / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000166:cytology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009687:Nuclear Proteins / Q000378:metabolism; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051883:Receptor, Notch2 / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: C069077:BMI1 protein, human; D053583:Interleukin-1beta; D007381:Intermediate Filament Proteins; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C577484:NES protein, human; C497764:NOTCH2 protein, human; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D051883:Receptor, Notch2; D012097:Repressor Proteins; D016212:Transforming Growth Factor beta; D063150:Polycomb Repressive Complex 1