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

Abstract

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.

Keywords: buccal; cancer; mucosal; oral cavity; squamous cell carcinoma; stem cells.

Figure 1

Representative DAB IHC-stained sections of MDBMSCC demonstrating nuclear expression of EMA of cells within the tumor nests [(A), brown]. Expression of SOX2 was seen in cells within the tumor nests [(B), brown, thick arrows] and the stroma [(B), brown, arrowheads], and on the endothelium of the microvessels within the stroma [(B), brown, thin arrows]. Expression of SALL4 was limited to cells within the tumor nests [(C), brown, thick arrows] and the endothelium of the microvessels [(C), brown, thin arrows]. OCT4 was also expressed in cells within tumor nests [(D), brown, thick arrows] and the stroma [(D), brown, arrowheads], and the endothelium of the microvessels within the stroma [(D), brown, thin arrows]. Expression of pSTAT3 was detected on cells within the tumor nests [(E), brown, thick arrows] and the stroma [(E), brown, arrowheads], and the endothelium of the microvessels within the stroma [(E), brown, thin arrows]. NANOG was also seen in cells within the tumor nests [(F), brown, thick arrows] and the stroma [(F), brown, arrowheads], and the endothelium of the microvessels within the stroma [(F), brown, thin arrows]. CD44 expression was seen as membranous staining of the tumor nest cells [(G), brown]. Original magnification: 400×.

Figure 2

Representative IF IHC-stained sections of MDBMSCC demonstrating the expression of pSTAT3 [(A), red] and EMA [(A), green] by cells within the tumor nests. There was a CSC subpopulation remonstrating nuclear co-expression of STAT3 [(B), red] and CD34 [(B), green], appearing as orange, on the endothelium of the microvessels within the stroma; and another subpopulation staining only positively for pSTAT3 within the stroma [(B), red]. Nuclear expression of NANOG [(C), red] was demonstrated on the endothelium of the microvessels which expressed CD34 [(C), green] within stroma. The NANOG⁺ cells [(C), red] that do not express CD34 were seen within the tumor nests and the stroma. SOX2 [(D), red] was also expressed by cells within tumor nests and the stroma, and the endothelium of the microvessels expressing CD34 [(D), green]. Nuclear expression of both SOX2 [(E), red] and SALL4 [(E), green], appearing as orange, was seen on the cells within the tumor nests and the stroma. Expression of both SOX2 [(F), red] and OCT4 [(F), green], appearing as orange, was seen on cells within the tumor nests and the stroma, and the endothelium of the microvessels within the stroma. pSTAT3 [(G), red] and membranous staining CD44 [(G), green] were co-expressed by cells within the tumor nests. Scale bars: 20 μm.

Figure 3

Expression of CSC-related mRNA transcripts of NANOG, OCT4, SALL4, SOX2 STAT3, and CD44 in MDBMSCC samples from five patients. Their expression was normalized over GUSB housekeeper.

Figure 4

Representative CISH-stained sections of MDBMSCC demonstrating mRNA expression of SOX2 [(A), pink, arrows], SALL4 [(B), pink, arrows], OCT4 [(C), pink, arrows], STAT3 [(D), pink, arrows], and NANOG [(E), pink, arrows]. Original magnification: 1000×.