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

AIM To identify and characterize cancer stem cells (CSC) in moderately differentiated buccal mucosa squamous cell carcinoma (MDBMSCC). METHODS Four 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. RESULTS Diaminobenzidine 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. CONCLUSION This 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.