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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 DAB IHC-stained sections of moderately differentiated buccal mucosal squamous cell carcinoma demonstrating cytoplasmic expression of PRR [(A), brown] by cells within the tumor nests (short arrows) and faint staining of endothelium (arrowheads) of the microvessels and peri-tumoral stroma (long arrows). ACE [(B), brown] was expressed by the endothelium of the microvessels within the stroma. Strong cytoplasmic and nuclear expression of ATIIR1 [(C), brown] was demonstrated in cells within the tumor nests (short arrows), with faint staining in cells within the peri-tumoral stroma (long arrows). Strong nuclear and faint cytoplasmic expression of ATIIR2 [(D), brown] was demonstrated in cells within the tumor nests (short arrows) and some cells in the peri-tumoral stroma (long arrows). Nuclei were counterstained with hematoxylin [(A–D), blue]. Original magnification: 400×.
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Figure 1: Representative DAB IHC-stained sections of moderately differentiated buccal mucosal squamous cell carcinoma demonstrating cytoplasmic expression of PRR [(A), brown] by cells within the tumor nests (short arrows) and faint staining of endothelium (arrowheads) of the microvessels and peri-tumoral stroma (long arrows). ACE [(B), brown] was expressed by the endothelium of the microvessels within the stroma. Strong cytoplasmic and nuclear expression of ATIIR1 [(C), brown] was demonstrated in cells within the tumor nests (short arrows), with faint staining in cells within the peri-tumoral stroma (long arrows). Strong nuclear and faint cytoplasmic expression of ATIIR2 [(D), brown] was demonstrated in cells within the tumor nests (short arrows) and some cells in the peri-tumoral stroma (long arrows). Nuclei were counterstained with hematoxylin [(A–D), blue]. Original magnification: 400×.

Mentions: Hematoxylin and eosin staining confirmed the histological grade and the presence of MDBMSCC (n = 6) for each sample. DAB IHC staining showed cytoplasmic expression of PRR (Figure 1A, brown) localized predominantly to cells within the tumor nests (Figure 1A, brown, short arrows). There was also faint immunoreactivity in the endothelium of the microvessels (Figure 1A, brown, arrowheads) and stronger cytoplasmic staining in cells within the peri-tumoral stroma (Figure 1A, brown, long arrows). ACE (Figure 1B, brown) was expressed on the endothelium of the microvessels, which were predominantly located around the periphery of the peri-tumoral stroma. ATIIR1 (Figure 1C, brown) was expressed predominantly in the cytoplasm and nuclei of cells within the tumor nests (Figure 1C, brown, short arrows) with faint cytoplasmic staining in cells within the peri-tumoral stroma (Figure 1C, brown, long arrows). Faint cytoplasmic and stronger nuclear staining of ATIIR2 (Figure 1D, brown) was observed in cells within the tumor nests (Figure 1D, brown, short arrows) and some cells within the peri-tumoral stroma (Figure 1D, brown, long arrows).


Cancer Stem Cells in Moderately Differentiated Buccal Mucosal Squamous Cell Carcinoma Express Components of the Renin – Angiotensin System
Representative DAB IHC-stained sections of moderately differentiated buccal mucosal squamous cell carcinoma demonstrating cytoplasmic expression of PRR [(A), brown] by cells within the tumor nests (short arrows) and faint staining of endothelium (arrowheads) of the microvessels and peri-tumoral stroma (long arrows). ACE [(B), brown] was expressed by the endothelium of the microvessels within the stroma. Strong cytoplasmic and nuclear expression of ATIIR1 [(C), brown] was demonstrated in cells within the tumor nests (short arrows), with faint staining in cells within the peri-tumoral stroma (long arrows). Strong nuclear and faint cytoplasmic expression of ATIIR2 [(D), brown] was demonstrated in cells within the tumor nests (short arrows) and some cells in the peri-tumoral stroma (long arrows). Nuclei were counterstained with hematoxylin [(A–D), blue]. Original magnification: 400×.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037224&req=5

Figure 1: Representative DAB IHC-stained sections of moderately differentiated buccal mucosal squamous cell carcinoma demonstrating cytoplasmic expression of PRR [(A), brown] by cells within the tumor nests (short arrows) and faint staining of endothelium (arrowheads) of the microvessels and peri-tumoral stroma (long arrows). ACE [(B), brown] was expressed by the endothelium of the microvessels within the stroma. Strong cytoplasmic and nuclear expression of ATIIR1 [(C), brown] was demonstrated in cells within the tumor nests (short arrows), with faint staining in cells within the peri-tumoral stroma (long arrows). Strong nuclear and faint cytoplasmic expression of ATIIR2 [(D), brown] was demonstrated in cells within the tumor nests (short arrows) and some cells in the peri-tumoral stroma (long arrows). Nuclei were counterstained with hematoxylin [(A–D), blue]. Original magnification: 400×.
Mentions: Hematoxylin and eosin staining confirmed the histological grade and the presence of MDBMSCC (n = 6) for each sample. DAB IHC staining showed cytoplasmic expression of PRR (Figure 1A, brown) localized predominantly to cells within the tumor nests (Figure 1A, brown, short arrows). There was also faint immunoreactivity in the endothelium of the microvessels (Figure 1A, brown, arrowheads) and stronger cytoplasmic staining in cells within the peri-tumoral stroma (Figure 1A, brown, long arrows). ACE (Figure 1B, brown) was expressed on the endothelium of the microvessels, which were predominantly located around the periphery of the peri-tumoral stroma. ATIIR1 (Figure 1C, brown) was expressed predominantly in the cytoplasm and nuclei of cells within the tumor nests (Figure 1C, brown, short arrows) with faint cytoplasmic staining in cells within the peri-tumoral stroma (Figure 1C, brown, long arrows). Faint cytoplasmic and stronger nuclear staining of ATIIR2 (Figure 1D, brown) was observed in cells within the tumor nests (Figure 1D, brown, short arrows) and some cells within the peri-tumoral stroma (Figure 1D, brown, long arrows).

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