Cancer Stem Cells in Head and Neck Cutaneous Squamous Cell Carcinoma Express Cathepsins.

Cancer stem cell (CSC) subpopulations within moderately differentiated head and neck cutaneous squamous cell carcinoma (MDHNcSCC) express the components of the renin-angiotensin system (RAS). This study investigated the expression of cathepsins B, D, and G, which constitute bypass loops of the RAS, by CSCs in MDHNcSCC. METHODS: Immunohistochemical staining was performed on MDHNcSCC tissue samples from 15 patients to determine the expression of cathepsins B, D, and G. Co-localization of these cathepsins with the embryonic stem cell markers Octamer-binding transcription factor 4 (OCT4) and c-MYC was investigated with immunofluorescence staining. Reverse transcription quantitative polymerase chain reaction was performed on 5 MDHNcSCC tissue samples to investigate transcript expression of cathepsins B, D and G. Western blotting and enzymatic activity assays were performed on 5 MDHNcSCC tissue samples and 6 MDHNcSCC-derived primary cell lines to confirm protein expression, transcript expression, and functional activity of these cathepsins, respectively. RESULTS: Immunohistochemical staining demonstrated the expression of cathepsins B, D, and G in all MDHNcSCC tissue samples. Immunofluorescence staining showed localization of cathepsins B and D to the c-MYC+ CSC subpopulations and the OCT4+ CSC subpopulations within the tumor nests and the peritumoral stroma. Cathepsin G was expressed on the tryptase+/c-MYC+ cells within the peritumoral stroma. Reverse transcription quantitative polymerase chain reaction demonstrated transcript expression of cathepsins B, D and G in the MDHNcSCC tissue samples. Western blotting and enzymatic activity assays confirmed protein expression and functional activity of cathepsins B and D in the MDHNcSCC tissue samples and MDHNcSCC-derived primary cell lines, respectively. CONCLUSIONS: Cathepsins B, D, and G are expressed in MDHNcSCC with functionally active cathepsins B and D localizing to the CSC subpopulations, and cathepsin G is expressed by mast cells, suggesting the potential use of cathepsin inhibitors in addition to RAS blockade to target CSCs in MDHNcSCC.

Cancer stem cell subpopulations in moderately differentiated head and neck cutaneous squamous cell carcinoma.

Cancer stem cells (CSC), the putative origin of cancer, account for local recurrence and metastasis. We aimed to identify and characterize CSCs within moderately differentiated head and neck cutaneous squamous cell carcinoma (MDHNCSCC). Formalin-fixed paraffin-embedded MDHNCSCC sections of ten patients underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for induced pluripotent stem cell (iPSC) markers OCT4, NANOG, SOX2, KLF4 and c-MYC. Localization of these markers was investigated using immunofluorescence (IF) IHC staining of three of these MDHNCSCC samples. mRNA expression of these iPSC markers in the MDHNCSCC tissue samples was determined by colorimetric in-situ hybridization (CISH, n = 6), and reverse-transcription quantitative polymerase chain reaction (RT-qPCR, n = 4). RT-qPCR was also performed on four MDHNCSCC-derived primary cell lines. DAB IHC staining demonstrated expression of all five iPSC markers within all ten MDHNCSCC tissues samples. CISH and RT-qPCR confirmed mRNA expression of all five iPSC markers within all MDHNCSCC tissues samples examined. RT-PCR demonstrated mRNA transcripts of all five iPSC markers in all four MDHNCSCC-derived primary cell lines. IF IHC staining showed co-expression of OCT4 with SOX2 and KLF4 throughout the tumor nests (TNs) and peri-tumoral stroma (PTS). There was an OCT4+/NANOG+ subpopulation within the TNs, and an OCT4+/NANOG- subpopulation and an OCT4+/NANOG+ subpopulation within the PTS. All iPSC markers were expressed by the endothelium of microvessels within the PTS. Our findings suggest the presence of an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ CSC subpopulation within the TNs, PTS and endothelium of microvessels within the PTS; and an OCT4+/NANOG-/SOX2+/KLF4+/c-MYC+ subpopulation exclusively within the PTS in MDHNCSCC. These CSC subpopulations could be a potential novel therapeutic target for treatment of MDHNCSCC.

Expression of Components of the Renin-Angiotensin System in Pyogenic Granuloma.

Background: There is a growing body of research demonstrating expression of the renin-angiotensin system (RAS) by a putative embryonic stem cell (ESC)-like population within vascular anomalies. This study investigated the expression of components of the RAS in relation to the putative ESC-like population within pyogenic granuloma (PG) that we have recently reported. Methods: PG samples from 14 patients were analyzed for the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2), using 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining. Immunofluorescence (IF) IHC staining was performed to localize these proteins on four of the PG samples. RT-qPCR was performed on two snap-frozen PG samples. Western blotting (WB) was performed on one snap-frozen PG sample and two PG-derived primary cell lines. Results: DAB IHC staining demonstrated the expression of ACE, PRR, ATIIR1, and ATIIR2 in all 14 PG tissue samples. RT-qPCR analysis confirmed abundant mRNA transcripts for PRR, ACE, AIITR1 and ATIIR2, relative to the housekeeping gene. WB confirmed the presence of PRR, ATIIR1, and ACE in the PG tissue sample, and PRR and ATIIR1, in the PG-derived primary cell lines. IF IHC staining demonstrated the expression of PRR, ACE, ATIIR1 on the primitive population that expressed NANOG and SOX2 on the ERG+ endothelium of the microvessels within PG. Conclusion: We have demonstrated the expression of PRR, ACE, and ATIIR1 by the putative the ESC-like population within PG.

Expression of Embryonic Stem Cell Markers in Microcystic Lymphatic Malformation.

Aim: To investigate the expression of embryonic stem cell (ESC) markers in microcystic lymphatic malformation (mLM). Methods and Results: Cervicofacial mLM tissue samples from nine patients underwent 3,3'-diaminobenzidine (DAB) immunohistochemical (IHC) staining for ESC markers octamer-binding protein 4 (OCT4), homeobox protein NANOG, sex determining region Y-box 2 (SOX2), Krupple-like factor (KLF4), and proto-oncogene c-MYC. Transcriptional activation of these ESC markers was investigated using real-time polymerase chain reaction (RT-qPCR) and colorimetric in situ hybridization (CISH) on four and five of these mLM tissue samples, respectively. Immunofluorescence (IF) IHC staining was performed on three of these mLM tissue samples to investigate localization of these ESC markers. DAB and IF IHC staining demonstrated the expression of OCT4, SOX2, NANOG, KLF4, and c-MYC on the endothelium of lesional vessels with abundant expression of c-MYC and SOX2, which was also present on the cells within the stroma, in all nine mLM tissue samples. RT-qPCR and CISH confirmed transcriptional activation of all these ESC markers investigated. Conclusions: These findings suggest the presence of a primitive population on the endothelium of lesional vessels and the surrounding stroma in mLM. The abundant expression of the progenitor-associated markers SOX2 and c-MYC suggests that the majority are of progenitor phenotype with a small number of ESC-like cells.

Cancer stem cells within moderately differentiated head and neck cutaneous squamous cell carcinoma express components of the renin-angiotensin system.

PURPOSE: To investigate the expression of components of the renin-angiotensin system (RAS): pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2) by the cancer stem cell (CSC) subpopulations in moderately differentiated head and neck cutaneous squamous cell carcinoma (MDHNCSCC). METHODOLOGY: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for PRR, ACE, ATIIR1 and ATIIR2 was performed on formalin-fixed paraffin-embedded sections of ten MDHNCSCC tissue samples. Immunofluorescence (IF) IHC staining was used to localise components of the RAS. Western blotting (WB) and RT-qPCR were performed on snap-frozen MDHNCSCC tissue samples and MDHNCSCC-derived primary cell lines to investigate protein transcription expression of these proteins, respectively. RESULTS: DAB IHC staining demonstrated the presence of PRR, ACE, ATIIR1 and ATIIR2 in all ten MDHNCSCC tissue samples. IF IHC staining showed expression of PRR and ATIIR2 by the OCT4+ cells, and ACE and ATIIR1 by the SOX2+ cells, within the tumour nests (TNs) and the peritumoural stroma (PTS). PRR, ACE, ATIIR1 and ATIIR2 were expressed by the endothelium of the microvessels within the PTS. WB confirmed protein expression for PRR, ACE and ATIIR1 in MDHNCSCC tissue samples and MDHNCSCC-derived primary cell lines. RT-qPCR showed transcriptional activation of PRR, ACE, ATIIR1 and ATIIR2 in MDHNCSCC tissue samples; and PRR, ACE, ATIIR1 but not ATIIR2, in MDHNCSCC-derived primary cell lines. CONCLUSION: PRR, ACE, ATIIR1 and ATIIR2 are expressed by the CSC subpopulations within the TNs, the PTS, and the endothelium of the microvessels within the PTS, in MDHNCSCC.