Characterization of Cancer Stem Cells in Moderately Differentiated Buccal Mucosal Squamous Cell Carcinoma

Abstract

AimTo identify and characterize cancer stem cells (CSC) in moderately differentiated buccal mucosa squamous cell carcinoma (MDBMSCC).MethodsFour micrometer-thick, formalin-fixed, paraffin-embedded MDBMSCC samples from six patients underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers, NANOG, OCT4, SALL4, SOX2, and pSTAT3; cancer stem cell marker, CD44; squamous cell carcinoma (SCC) marker, EMA; and endothelial marker, CD34. The transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, STAT3, and CD44 were studied using NanoString gene expression analysis and colorimetric in situ hybridization (CISH) for NANOG, OCT4, SOX2, SALL4, and STAT3.ResultsDiaminobenzidine and immunofluorescent (IF) IHC staining demonstrated the presence of (1) an EMA+/CD44+/SOX2+/SALL4+/OCT4+/pSTAT3+/NANOG+ CSC subpopulation within the tumor nests; (2) an EMA−/CD44−/CD34−/SOX2+/OCT4+/pSTAT3+/NANOG+ subpopulation within the stroma between the tumor nests; and (3) an EMA−/CD44−/CD34+/SOX2+/SALL4+/OCT4+/pSTAT3+/NANOG+ subpopulation on the endothelium of the microvessels within the stroma. The expression of CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG was confirmed by the presence of mRNA transcripts, using NanoString analysis and NANOG, OCT4, SOX2, SALL4, and STAT3 by CISH staining.ConclusionThis study demonstrated a novel finding of three separate CSC subpopulations within MDBMSCC: (1) within the tumor nests expressing EMA, CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG; (2) within the stroma expressing SOX2, SALL4, OCT4, pSTAT3, and NANOG; and (3) on the endothelium of the microvessels within the stroma expressing CD34, SOX2, SALL4, OCT4, pSTAT3, and NANOG.

The Identification of Three Cancer Stem Cell Subpopulations within Moderately Differentiated Lip Squamous Cell Carcinoma

“…The novel identification of more than one subpopulation of CSCs within MDLSCC parallels our recent findings in oral tongue (21) and buccal mucosa (22) SCC. Determination of which CSC subpopulation within the stroma possess the capacity for epithelial–mesenchymal transition (36) is a subject of further work, and we believe that a new paradigm opens up in the investigation of the biology of this tumor.…”

“…Various methods used for identification of CSCs in OCSCC include Hoechst dye exclusion, sphere forming assays, aldehyde dehydrogenase activity, and CSC marker identification (such as CD44, CD133, E-cadherin, keratins, and integrins) (14–16). More recent studies have utilized the embryonic stem cell (ESC) markers, sex-determining region Y (SRY)-box 2 (SOX2) (17), octamer-binding transcription factor 4 (OCT4) (17, 18), phosphorylated signal transducer and activator of transcription 3 (pSTAT3) (19), spalt-like transcription factor 4 (SALL4) (20), and Homeobox protein NANOG (18) to identify CSCs within OCSCC (21, 22). …”

“…We have recently identified and characterized CSC subpopulations within oral tongue (21) and buccal mucosal (22) SCC. Although there are a number of reports on the presence and role of CSCs in OCSCC (21), there is paucity of data on the presence of CSC in lip SCC.…”

Subcellular localisation of the stem cell markers OCT4, SOX2, NANOG, KLF4 and c-MYC in cancer: a review

“…However, OCT4 mutants containing a nuclear export signal (NES), which were primarily localised to the cytoplasm, have also been shown to maintain the undifferentiated state of human embryonic stem cells (hESCs),11 suggesting that the localisation of OCT4 is insignificant as long as nuclear processing is maintained. Immunohistochemical (IHC) staining was used to identify cytoplasmic OCT4 in CSC subpopulations in two groups of oral cavity squamous cell carcinomas (SCC) (figure 2A),12 13 with a third group showing both nuclear and cytoplasmic localisation,14 consistent with findings in other tumours such as glioma15 and the epithelial cells of CRC 5. A report by Alexander et al ,16 which also used the same antibody as that used by Baillie et al 12 and Ram et al 13 also reported cytoplasmic OCT4, and attributed this to neuroendocrine differentiation; however, the latter remains to be conclusively determined.…”

“…Differential expression patterns of SOX2 have been reported in different cancer types, with oral cavity SCC (OCSCC) CSC subpopulations showing SOX2 localisation to both the nucleus14 and cytoplasm12 14 (figure 2A). Cytoplasmic localisation of SOX2 and OCT4 has also demonstrated CRC CSCs 22.…”