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Glioblastoma Multiforme Cancer Stem Cells Express Components of the Renin – Angiotensin System

View Article: PubMed Central - PubMed

ABSTRACT

Aim: To investigate the expression of the renin–angiotensin system (RAS) in cancer stem cells (CSCs), we have previously characterized in glioblastoma multiforme (GBM).

Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for the stem cell marker, SOX2, and components of the RAS: angiotensin converting enzyme (ACE), (pro)renin receptor (PRR), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on 4 μm-thick formalin-fixed paraffin-embedded sections of previously characterized GBM samples in six patients was undertaken. Immunofluorescent (IF) IHC staining was performed to demonstrate expression of GFAP, SOX2, PRR, ACE, ATIIR1, and ATIIR2. The protein expression and the transcriptional activities of the genes encoding for ACE, PRR, ATIIR1, and ATIIR2 were studied using Western blotting (WB) and NanoString gene expression analysis, respectively.

Results: DAB and IF IHC staining demonstrated the expression SOX2 on the GFAP+ GBM CSCs. Cytoplasmic expression of PRR by the GFAP+ CSCs and the endothelium of the microvessels was observed. ACE was expressed on the endothelium of the microvessels only, while nuclear and cytoplasmic expression of ATIIR1 and ATIIR2 was observed on the endothelium of the microvessels and the CSCs. ATIIR1 was expressed on the GFAP+ CSCs cells, and ATIIR2 was expressed by the SOX2+ CSCs. The expression of ACE, PRR, and ATIIR1, but not ATIIR2, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of ACE, PRR, and ATIIR1, but not ATIIR2.

Conclusion: This study demonstrated the expression of PRR, ATIIR1, and ATIIR2 by the SOX2 CSC population, and ACE on the endothelium of the microvessels, within GBM. ACE, PRR, and ATIIR1 were expressed at the protein and mRNA levels, with ATIIR2 detectable only by IHC staining. This novel finding suggests that the CSCs may be a novel therapeutic target for GBM by modulation of the RAS.

No MeSH data available.


Representative immunofluorescent immunohistochemical stained images demonstrating the expression of SOX2 [(A), red], PRR [(B), red], and ATIIR2 [(C), red] on GFAP+ CSCs [(A–C), green] and expression of ACE [(D), green] and ATIIR1 [(E), green] on SOX2+ CSCs [(D,E), red]. Negative control was a GBM tissue section with omission of the primary antibody (F). Cell nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole [(A–F), blue]. Scale bars: 20 μm.
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Figure 2: Representative immunofluorescent immunohistochemical stained images demonstrating the expression of SOX2 [(A), red], PRR [(B), red], and ATIIR2 [(C), red] on GFAP+ CSCs [(A–C), green] and expression of ACE [(D), green] and ATIIR1 [(E), green] on SOX2+ CSCs [(D,E), red]. Negative control was a GBM tissue section with omission of the primary antibody (F). Cell nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole [(A–F), blue]. Scale bars: 20 μm.

Mentions: The presence of CSCs within GBM was demonstrated by the relatively abundant expression of the ESC marker SOX2 (Figure 2A, red) on the GFAP+ cells (Figure 2A, green) within GBM, as recently reported (35). We then investigated the expression of PRR (Figure 2B, red) in GBM, by performing IF IHC co-staining with GFAP (Figure 2B, green), which demonstrated that most of the GFAP+ CSCs within GBM expressed PRR. To determine the expression of ACE, we performed dual staining for ACE (Figure 2C, green) and SOX2 (Figure 2C, red) and showed mutually exclusive expression of these markers. Interestingly, ACE was expressed on the endothelial cells with erythrocytes evident within the lumina of the microvessels. We also showed the expression of ATIIR1 (Figure 2D, green) on the SOX2+ (Figure 2D, red) CSC population. ATIIR2 (Figure 2E, red) was expressed on the GFAP+ (Figure 2E, green) CSCs in GBM that were demonstrated to express SOX2 (35). Appropriate negative controls, consisting of omission of the primary antibodies did not reveal any staining (Figure 2F).


Glioblastoma Multiforme Cancer Stem Cells Express Components of the Renin – Angiotensin System
Representative immunofluorescent immunohistochemical stained images demonstrating the expression of SOX2 [(A), red], PRR [(B), red], and ATIIR2 [(C), red] on GFAP+ CSCs [(A–C), green] and expression of ACE [(D), green] and ATIIR1 [(E), green] on SOX2+ CSCs [(D,E), red]. Negative control was a GBM tissue section with omission of the primary antibody (F). Cell nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole [(A–F), blue]. Scale bars: 20 μm.
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Related In: Results  -  Collection

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Figure 2: Representative immunofluorescent immunohistochemical stained images demonstrating the expression of SOX2 [(A), red], PRR [(B), red], and ATIIR2 [(C), red] on GFAP+ CSCs [(A–C), green] and expression of ACE [(D), green] and ATIIR1 [(E), green] on SOX2+ CSCs [(D,E), red]. Negative control was a GBM tissue section with omission of the primary antibody (F). Cell nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole [(A–F), blue]. Scale bars: 20 μm.
Mentions: The presence of CSCs within GBM was demonstrated by the relatively abundant expression of the ESC marker SOX2 (Figure 2A, red) on the GFAP+ cells (Figure 2A, green) within GBM, as recently reported (35). We then investigated the expression of PRR (Figure 2B, red) in GBM, by performing IF IHC co-staining with GFAP (Figure 2B, green), which demonstrated that most of the GFAP+ CSCs within GBM expressed PRR. To determine the expression of ACE, we performed dual staining for ACE (Figure 2C, green) and SOX2 (Figure 2C, red) and showed mutually exclusive expression of these markers. Interestingly, ACE was expressed on the endothelial cells with erythrocytes evident within the lumina of the microvessels. We also showed the expression of ATIIR1 (Figure 2D, green) on the SOX2+ (Figure 2D, red) CSC population. ATIIR2 (Figure 2E, red) was expressed on the GFAP+ (Figure 2E, green) CSCs in GBM that were demonstrated to express SOX2 (35). Appropriate negative controls, consisting of omission of the primary antibodies did not reveal any staining (Figure 2F).

View Article: PubMed Central - PubMed

ABSTRACT

Aim: To investigate the expression of the renin–angiotensin system (RAS) in cancer stem cells (CSCs), we have previously characterized in glioblastoma multiforme (GBM).

Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for the stem cell marker, SOX2, and components of the RAS: angiotensin converting enzyme (ACE), (pro)renin receptor (PRR), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on 4 μm-thick formalin-fixed paraffin-embedded sections of previously characterized GBM samples in six patients was undertaken. Immunofluorescent (IF) IHC staining was performed to demonstrate expression of GFAP, SOX2, PRR, ACE, ATIIR1, and ATIIR2. The protein expression and the transcriptional activities of the genes encoding for ACE, PRR, ATIIR1, and ATIIR2 were studied using Western blotting (WB) and NanoString gene expression analysis, respectively.

Results: DAB and IF IHC staining demonstrated the expression SOX2 on the GFAP+ GBM CSCs. Cytoplasmic expression of PRR by the GFAP+ CSCs and the endothelium of the microvessels was observed. ACE was expressed on the endothelium of the microvessels only, while nuclear and cytoplasmic expression of ATIIR1 and ATIIR2 was observed on the endothelium of the microvessels and the CSCs. ATIIR1 was expressed on the GFAP+ CSCs cells, and ATIIR2 was expressed by the SOX2+ CSCs. The expression of ACE, PRR, and ATIIR1, but not ATIIR2, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of ACE, PRR, and ATIIR1, but not ATIIR2.

Conclusion: This study demonstrated the expression of PRR, ATIIR1, and ATIIR2 by the SOX2 CSC population, and ACE on the endothelium of the microvessels, within GBM. ACE, PRR, and ATIIR1 were expressed at the protein and mRNA levels, with ATIIR2 detectable only by IHC staining. This novel finding suggests that the CSCs may be a novel therapeutic target for GBM by modulation of the RAS.

No MeSH data available.