title: Induction of stem-like cells with malignant properties by chronic exposure of human lung epithelial cells to single-walled carbon nanotubes.
abstract: BACKGROUND
  Carbon nanotubes (CNT) hold great promise to create new and better products for commercial and biomedical applications, but their long-term adverse health effects are a major concern. The objective of this study was to address human lung cancer risks associated with chronic pulmonary exposure to single-walled (SW) CNT through the fundamental understanding of cellular and molecular processes leading to carcinogenesis. We hypothesized that the acquisition of cancer stem cells (CSC), a subpopulation that drive tumor initiation and progression, may contribute to CNT carcinogenesis.
  
  
  METHODS
  Non-tumorigenic human lung epithelial cells were chronically exposed to well-dispersed SWCNT for a period of 6 months at the physiologically relevant concentration of 0.02 μg/cm2 surface area dose. Chronic SWCNT-exposed cells were evaluated for the presence of CSC-like cells under CSC-selective conditions of tumor spheres and side population (SP). CSC-like cells were isolated using fluorescence-activated cell sorting and were assessed for aggressive behaviors, including acquired apoptosis resistance and increased cell migration and invasion in vitro, and tumor-initiating capability in vivo. Non-small cell lung cancer cells served as a positive control.
  
  
  RESULTS
  We demonstrated for the first time the existence of CSC-like cells in all clones of chronic SWCNT-exposed lung epithelial cells. These CSC-like cells, in contrary to their non-CSC counterpart, possessed all biological features of lung CSC that are central to irreversible malignant transformation, self-renewal, aggressive cancer behaviors, and in vivo tumorigenesis. These cells also displayed aberrant stem cell markers, notably Nanog, SOX-2, SOX-17 and E-cadherin. Restored expression of tumor suppressor p53 abrogated CSC properties of CSC-like cells. Furthermore, we identified specific stem cell surface markers CD24low and CD133high that are associated with SWCNT-induced CSC formation and tumorigenesis.
  
  
  CONCLUSIONS
  Our findings provide new and compelling evidence for the acquisition of CSC-like cells induced by chronic SWCNT exposure, which are likely to be a major driving force for SWCNT tumorigenesis. Thus, our study supports prudent adoption of prevention strategies and implementation of exposure control for SWCNT. We also suggest that the detection of CSC and associated surface markers may provide an effective screening tool for prediction of the carcinogenic potential of SWCNT and related nanoparticles.
authors.forename: Sudjit
authors.initials: S
authors.lastname: Luanpitpong
authors.forename: Liying
authors.initials: L
authors.lastname: Wang
authors.forename: Vincent
authors.initials: V
authors.lastname: Castranova
authors.affiliation: Pharmaceutical and Pharmacological Sciences Program, West Virginia University, Morgantown, WV 26506, USA. yrojan@hsc.wvu.edu.
authors.forename: Yon
authors.initials: Y
authors.lastname: Rojanasakul
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology*; D005434:Flow Cytometry; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000473:pathology*; D008175:Lung Neoplasms / Q000139:chemically induced / Q000473:pathology; D051379:Mice; D037742:Nanotubes, Carbon / Q000633:toxicity*; D009361:Neoplasm Invasiveness / Q000473:pathology; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D010957:Plasmids / Q000235:genetics; D013234:Stem Cells; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
chemical_list: D015415:Biomarkers; D014408:Biomarkers, Tumor; D037742:Nanotubes, Carbon