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Cancer stem cells in moderately differentiated oral tongue squamous cell carcinoma express components of the renin–angiotensin system
  1. Tinte Itinteang1,
  2. Jonathan C Dunne1,
  3. Alice M Chibnall1,
  4. Helen D Brasch1,
  5. Paul F Davis1,
  6. Swee T Tan1,2
  1. 1Gillies McIndoe Research Institute, Wellington, New Zealand
  2. 2Wellington Regional Plastic, Maxillofacial & Burns Unit, Hutt Hospital, Wellington, New Zealand
  1. Correspondence to Dr Swee T Tan ONZM MBBS PhD FRACS, Gillies McIndoe Research Institute, P.O. Box 7184, Newtown, Wellington 6242, New Zealand; swee.tan{at}

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Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cavity cancer.1 We have recently identified two cancer stem cell (CSC) subpopulations within moderately differentiated OTSCC (MDOTSCC): a p63+/NANOG+/SOX2+/SALL4+/pSTAT3+/OCT4− subpopulation within the tumour nests (TNs) and a p63−/NANOG−/SOX2−/SALL4−/pSTAT3−/OCT4+ subpopulation within the stroma.1

The renin–angiotensin system (RAS) is associated with blood pressure regulation,2 but recent publications suggest its role in cancer growth.2 Its components, ACE, angiotensin II receptor 1 (ATIIR1) and ATIIR2 have been demonstrated within different cancers,3 implicating a role for the RAS in carcinogenesis.3 This report demonstrated expression of components of the RAS by the CSC subpopulations within MDOTSCC.

Immunohistochemical staining

Four-micrometre-thick formalin-fixed paraffin-embedded sections from 10 previously characterised MDOTSCC tissue samples1 underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for the primary antibodies SALL4 (1:100; cat#6E3, Cell Marque, Santa Cruz, California, USA), SOX2 (1:500; cat#PA1-094; Thermo Fisher Scientific, Santa Cruz, California, USA), OCT4 (1:30; cat#MRQ-10, Cell Marque), (pro)renin receptor (PRR; 1:2000; cat#ab40790, Abcam, Cambridge, Massachusetts, USA), ACE (1:100; cat#MCA2054, AbD Serotec, Raleigh, North Carolina, USA), ATIIR1 (1:30; cat#ab9391, Abcam) and ATIIR2 (1:2000; cat#NBP1-77368, Novus Biologicals, Littleton, Colorado, USA), as previously described.1

To determine co-expression, two selected MDOTSCC samples from the original cohort were subjected to immunofluorescent (IF) IHC staining using a combination of Vectafluor Excel anti-rabbit 594 (ready-to-use; cat#VEDK-1594, Vector Laboratories, Burlingame, California, USA) and Alexa Fluor anti-mouse 488 (1:500; cat#A21202, Life Technologies, California, USA) for combinations of PRR, and Vectafluor Excel anti-mouse (ready-to-use; cat#VEDK2488, Vector Laboratories) and Alexa Fluor anti-rabbit 594 (1:500; cat#A21207, Life Technologies) for combinations of ACE and ATIIR1.

Positive human control tissues for the primary antibodies were placenta for PRR; liver for ACE and ATIIR1; kidney for ATIIR2; and seminoma for SOX2, OCT4 and SALL4. Omission of the primary antibodies was used as negative controls. Images were captured and …

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