title: Glioblastoma Multiforme Cancer Stem Cells Express Components of the Renin-Angiotensin System.
abstract: OBJECTIVE
  To investigate the expression of the renin-angiotensin system (RAS) in cancer stem cells (CSCs), we have previously characterized in glioblastoma multiforme (GBM).
  
  
  METHODS
  3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for the stem cell marker, SOX2, and components of the RAS: angiotensin converting enzyme (ACE), (pro)renin receptor (PRR), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on 4 μm-thick formalin-fixed paraffin-embedded sections of previously characterized GBM samples in six patients was undertaken. Immunofluorescent (IF) IHC staining was performed to demonstrate expression of GFAP, SOX2, PRR, ACE, ATIIR1, and ATIIR2. The protein expression and the transcriptional activities of the genes encoding for ACE, PRR, ATIIR1, and ATIIR2 were studied using Western blotting (WB) and NanoString gene expression analysis, respectively.
  
  
  RESULTS
  DAB and IF IHC staining demonstrated the expression SOX2 on the GFAP+ GBM CSCs. Cytoplasmic expression of PRR by the GFAP+ CSCs and the endothelium of the microvessels was observed. ACE was expressed on the endothelium of the microvessels only, while nuclear and cytoplasmic expression of ATIIR1 and ATIIR2 was observed on the endothelium of the microvessels and the CSCs. ATIIR1 was expressed on the GFAP+ CSCs cells, and ATIIR2 was expressed by the SOX2+ CSCs. The expression of ACE, PRR, and ATIIR1, but not ATIIR2, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of ACE, PRR, and ATIIR1, but not ATIIR2.
  
  
  CONCLUSIONS
  This study demonstrated the expression of PRR, ATIIR1, and ATIIR2 by the SOX2 CSC population, and ACE on the endothelium of the microvessels, within GBM. ACE, PRR, and ATIIR1 were expressed at the protein and mRNA levels, with ATIIR2 detectable only by IHC staining. This novel finding suggests that the CSCs may be a novel therapeutic target for GBM by modulation of the RAS.
authors.affiliation: Gillies McIndoe Research Institute , Wellington , New Zealand.
authors.forename: Amy Ruth
authors.initials: AR
authors.lastname: Bradshaw
authors.affiliation: Gillies McIndoe Research Institute, Wellington, New Zealand; Department of Neurosurgery, Wellington Regional Hospital, Wellington, New Zealand.
authors.forename: Agadha Crisantha
authors.initials: AC
authors.lastname: Wickremesekera
authors.affiliation: Gillies McIndoe Research Institute , Wellington , New Zealand.
authors.forename: Helen D
authors.initials: HD
authors.lastname: Brasch
authors.affiliation: Gillies McIndoe Research Institute , Wellington , New Zealand.
authors.forename: Alice M
authors.initials: AM
authors.lastname: Chibnall
authors.affiliation: Gillies McIndoe Research Institute , Wellington , New Zealand.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Davis
authors.affiliation: Gillies McIndoe Research Institute, Wellington, New Zealand; Wellington Regional Plastic, Maxillofacial and Burns Unit, Hutt Hospital, Wellington, New Zealand.
authors.forename: Swee T
authors.initials: ST
authors.lastname: Tan
authors.affiliation: Gillies McIndoe Research Institute , Wellington , New Zealand.
authors.forename: Tinte
authors.initials: T
authors.lastname: Itinteang
keywords: cancer; glioblastoma multiforme; renin–angiotensin system; stem cells