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Cancer Stem Cells in Moderately Differentiated Buccal Mucosal Squamous Cell Carcinoma Express Components of the Renin – Angiotensin System

View Article: PubMed Central - PubMed

ABSTRACT

Aim: We have recently identified and characterized cancer stem cell (CSC) subpopulations within moderately differentiated buccal mucosal squamous cell carcinoma (MDBMSCC). We hypothesized that these CSCs express components of the renin–angiotensin system (RAS).

Methods: 3,3′-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on formalin-fixed paraffin-embedded MDBMSCC samples to investigate the expression of the components of the RAS: (pro)renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2). NanoString mRNA gene expression analysis and Western Blotting (WB) were performed on snap-frozen MDBMSCC samples to confirm gene expression and translation of these transcripts, respectively. Double immunofluorescent (IF) IHC staining of these components of the RAS with the embryonic stem cell markers OCT4 or SALL4 was performed to demonstrate their localization in relation to the CSC subpopulations within MDBMSCC.

Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 in MDBMSCC. IF IHC staining showed that PRR was expressed by the CSC subpopulations within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR1 and ATIIR2 were localized to the CSC subpopulations within the tumor nests and the peri-tumoral stroma, while ACE was localized to the endothelium of the microvessels within the peri-tumoral stroma. WB and NanoString analyses confirmed protein expression and transcription activation of PRR, ACE, and ATIIR1, but not of ATIIR2, respectively.

Conclusion: Our novel findings of the presence and localization of PRR, ACE, ATIIR1, and potentially ATIIR2 to the CSC subpopulations within MDBMSCC suggest CSC as a therapeutic target by modulation of the RAS.

No MeSH data available.


Related in: MedlinePlus

Representative immunofluorescent immunohistochemical-stained sections of moderately differentiated buccal mucosal squamous cell carcinoma demonstrating PRR [(A), red] was expressed by cells within the tumor nests that stained positively for EMA [(A), green]. PRR [(B), red] was also expressed by the CD34+ [(B), green] endothelium of the microvessels. In addition, RRR [(C), red] was expressed by cells within the peri-tumoral stroma, which also expressed OCT4 [(C), green]. ACE [(D), green] was expressed on the endothelium of the microvessels, which also expressed SOX2 [(D), red], within the peri-tumoral stroma. Cytoplasmic expression of ATIIR1 [(E), green] and PRR [(E), red] was demonstrated in cells within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR2 [(F,G), red] was expressed by cells within the tumor nests that expressed SALL4 [(F), green] and OCT4 [(G), green]. Cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole [(A–G), blue]. Scale bars: 20 μm.
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Figure 2: Representative immunofluorescent immunohistochemical-stained sections of moderately differentiated buccal mucosal squamous cell carcinoma demonstrating PRR [(A), red] was expressed by cells within the tumor nests that stained positively for EMA [(A), green]. PRR [(B), red] was also expressed by the CD34+ [(B), green] endothelium of the microvessels. In addition, RRR [(C), red] was expressed by cells within the peri-tumoral stroma, which also expressed OCT4 [(C), green]. ACE [(D), green] was expressed on the endothelium of the microvessels, which also expressed SOX2 [(D), red], within the peri-tumoral stroma. Cytoplasmic expression of ATIIR1 [(E), green] and PRR [(E), red] was demonstrated in cells within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR2 [(F,G), red] was expressed by cells within the tumor nests that expressed SALL4 [(F), green] and OCT4 [(G), green]. Cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole [(A–G), blue]. Scale bars: 20 μm.

Mentions: (Pro)renin receptor (Figures 2A–C, red) was expressed by the EMA+ (Figure 2A, green) cells within the tumor nests, as well as the CD34+ endothelium (Figure 2B, green, arrows) and the outer pericyte layer (Figure 2B, red, arrowheads) of the microvessels and OCT4+ (Figure 2C, green) cells within the peri-tumoral stroma (16). ACE (Figure 2D, green, arrows) was expressed on the endothelium of the microvessels within the peri-tumoral stroma, which expressed SOX2 (Figure 2D, red), distinct from the tumor nests. Cytoplasmic expression of ATIIR1 (Figure 2E, green) was demonstrated on cells within the tumor nests and the endothelium of the microvessels within the peri-tumoral stroma expressing PRR (Figure 2E, red). ATIIR2 (Figures 2F,G, red) was expressed by cells within the tumor nests that have been shown to express SALL4 (Figure 2F, green) (16). Interestingly, cells within the peri-tumoral stroma that express OCT4 (16) also expressed ATIIR2 (Figure 2G, green). Separated IF IHC-stained images of Figure 2 are presented in Image S2 in Supplementary Material.


Cancer Stem Cells in Moderately Differentiated Buccal Mucosal Squamous Cell Carcinoma Express Components of the Renin – Angiotensin System
Representative immunofluorescent immunohistochemical-stained sections of moderately differentiated buccal mucosal squamous cell carcinoma demonstrating PRR [(A), red] was expressed by cells within the tumor nests that stained positively for EMA [(A), green]. PRR [(B), red] was also expressed by the CD34+ [(B), green] endothelium of the microvessels. In addition, RRR [(C), red] was expressed by cells within the peri-tumoral stroma, which also expressed OCT4 [(C), green]. ACE [(D), green] was expressed on the endothelium of the microvessels, which also expressed SOX2 [(D), red], within the peri-tumoral stroma. Cytoplasmic expression of ATIIR1 [(E), green] and PRR [(E), red] was demonstrated in cells within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR2 [(F,G), red] was expressed by cells within the tumor nests that expressed SALL4 [(F), green] and OCT4 [(G), green]. Cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole [(A–G), blue]. Scale bars: 20 μm.
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Figure 2: Representative immunofluorescent immunohistochemical-stained sections of moderately differentiated buccal mucosal squamous cell carcinoma demonstrating PRR [(A), red] was expressed by cells within the tumor nests that stained positively for EMA [(A), green]. PRR [(B), red] was also expressed by the CD34+ [(B), green] endothelium of the microvessels. In addition, RRR [(C), red] was expressed by cells within the peri-tumoral stroma, which also expressed OCT4 [(C), green]. ACE [(D), green] was expressed on the endothelium of the microvessels, which also expressed SOX2 [(D), red], within the peri-tumoral stroma. Cytoplasmic expression of ATIIR1 [(E), green] and PRR [(E), red] was demonstrated in cells within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR2 [(F,G), red] was expressed by cells within the tumor nests that expressed SALL4 [(F), green] and OCT4 [(G), green]. Cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole [(A–G), blue]. Scale bars: 20 μm.
Mentions: (Pro)renin receptor (Figures 2A–C, red) was expressed by the EMA+ (Figure 2A, green) cells within the tumor nests, as well as the CD34+ endothelium (Figure 2B, green, arrows) and the outer pericyte layer (Figure 2B, red, arrowheads) of the microvessels and OCT4+ (Figure 2C, green) cells within the peri-tumoral stroma (16). ACE (Figure 2D, green, arrows) was expressed on the endothelium of the microvessels within the peri-tumoral stroma, which expressed SOX2 (Figure 2D, red), distinct from the tumor nests. Cytoplasmic expression of ATIIR1 (Figure 2E, green) was demonstrated on cells within the tumor nests and the endothelium of the microvessels within the peri-tumoral stroma expressing PRR (Figure 2E, red). ATIIR2 (Figures 2F,G, red) was expressed by cells within the tumor nests that have been shown to express SALL4 (Figure 2F, green) (16). Interestingly, cells within the peri-tumoral stroma that express OCT4 (16) also expressed ATIIR2 (Figure 2G, green). Separated IF IHC-stained images of Figure 2 are presented in Image S2 in Supplementary Material.

View Article: PubMed Central - PubMed

ABSTRACT

Aim: We have recently identified and characterized cancer stem cell (CSC) subpopulations within moderately differentiated buccal mucosal squamous cell carcinoma (MDBMSCC). We hypothesized that these CSCs express components of the renin–angiotensin system (RAS).

Methods: 3,3′-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on formalin-fixed paraffin-embedded MDBMSCC samples to investigate the expression of the components of the RAS: (pro)renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2). NanoString mRNA gene expression analysis and Western Blotting (WB) were performed on snap-frozen MDBMSCC samples to confirm gene expression and translation of these transcripts, respectively. Double immunofluorescent (IF) IHC staining of these components of the RAS with the embryonic stem cell markers OCT4 or SALL4 was performed to demonstrate their localization in relation to the CSC subpopulations within MDBMSCC.

Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 in MDBMSCC. IF IHC staining showed that PRR was expressed by the CSC subpopulations within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR1 and ATIIR2 were localized to the CSC subpopulations within the tumor nests and the peri-tumoral stroma, while ACE was localized to the endothelium of the microvessels within the peri-tumoral stroma. WB and NanoString analyses confirmed protein expression and transcription activation of PRR, ACE, and ATIIR1, but not of ATIIR2, respectively.

Conclusion: Our novel findings of the presence and localization of PRR, ACE, ATIIR1, and potentially ATIIR2 to the CSC subpopulations within MDBMSCC suggest CSC as a therapeutic target by modulation of the RAS.

No MeSH data available.


Related in: MedlinePlus