Cell Populations Expressing Stemness-Associated Markers in Lung Adenocarcinoma.

The stemness-associated markers OCT4, NANOG, SOX2, KLF4 and c-MYC are expressed in numerous cancer types suggesting the presence of cancer stem cells (CSCs). Immunohistochemical (IHC) staining performed on 12 lung adenocarcinoma (LA) tissue samples showed protein expression of OCT4, NANOG, SOX2, KLF4 and c-MYC, and the CSC marker CD44. In situ hybridization (ISH) performed on six of the LA tissue samples showed mRNA expression of OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence staining performed on three of the tissue samples showed co-expression of OCT4 and c-MYC with NANOG, SOX2 and KLF4 by tumor gland cells, and expression of OCT4 and c-MYC exclusively by cells within the stroma. RT-qPCR performed on five LA-derived primary cell lines showed mRNA expression of all the markers except SOX2. Western blotting performed on four LA-derived primary cell lines demonstrated protein expression of all the markers except SOX2 and NANOG. Initial tumorsphere assays performed on four LA-derived primary cell lines demonstrated 0-80% of tumorspheres surpassing the 50 µm threshold. The expression of the stemness-associated markers OCT4, SOX2, NANOG, KFL4 and c-MYC by LA at the mRNA and protein level, and the unique expression patterns suggest a putative presence of CSC subpopulations within LA, which may be a novel therapeutic target for this cancer. Further functional studies are required to investigate the possession of stemness traits.

Cathepsins B, D, and G Are Expressed in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma.

AIM: We have previously demonstrated the presence of two cancer stem cell (CSC) subpopulations within metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC) expressing components of the renin-angiotensin system (RAS), which promotes tumorigenesis. Cathepsins B, D and G are enzymes that constitute bypass loops for the RAS. This study investigated the expression and localization of cathepsins B, D, and G in relation to CSC subpopulations within mHNcSCC. METHODS: Immunohistochemical staining was performed on mHNcSCC tissue samples from 20 patients to determine the expression and localization of cathepsins B, D, and G. Immunofluorescence staining was performed on two of these mHNcSCC tissue samples by co-staining of cathepsins B and D with OCT4 and SOX2, and cathepsin G with mast cell markers tryptase and chymase. Western blotting and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were performed on five mHNcSCC samples and four mHNcSCC-derived primary cell lines, to determine protein and transcript expression of these three cathepsins, respectively. Enzyme activity assays were performed on mHNcSCC tissue samples to determine whether these cathepsins were active. RESULTS: Immunohistochemical staining demonstrated the presence of cathepsins B, D and G in in all 20 mHNcSCC tissue samples. Immunofluorescence staining showed that cathepsins B and D were localized to the CSCs both within the tumor nests and peri-tumoral stroma (PTS) and cathepsin G was localized to the phenotypic mast cells within the PTS. Western blotting demonstrated protein expression of cathepsin B and D, and RT-qPCR demonstrated transcript expression of all three cathepsins. Enzyme activity assays showed that cathepsin B and D to be active. CONCLUSION: The presence of cathepsins B and D on the CSCs and cathepsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for mHNcSCC.

Expression of Cathepsins B, D, and G in Extracranial Arterio-Venous Malformation.

Objectives: We have previously identified a population of cells that expressed stemness-associated markers in extracranial arterio-venous malformation (AVM) and demonstrated expression of cathepsins B, D, and G on embryonic stem cell (ESC)-like populations in other vascular anomalies. This study investigated the expression of cathepsins B, D, and G, and their localization in relation to this primitive population in extracranial AVM. Methods: Immunohistochemical staining was performed on AVM tissue samples from 13 patients to demonstrate expression of cathepsins B, D, and G. Western blotting was performed on four AVM tissue samples and three AVM-derived primary cell lines to confirm protein expression of cathepsins B and D proteins. RT-qPCR was performed on three AVM-derived primary cell lines to demonstrate transcript expression of cathepsins B, D, and G. Enzymatic activity assays were performed on three AVM-derived primary cell lines to investigate if cathepsins B and D were active. Localization of the cathepsins was investigated using immunofluorescence dual-staining of the cathepsins with the ESC markers OCT4 and SOX2, and mast cells marker chymase on two of the 13 AVM tissue samples. Results: Immunohistochemical staining demonstrated expression of cathepsins B, D, and G in all 13 AVM tissue samples. Western blotting showed expression of cathepsins B and D proteins in all four AVM tissue samples and all three AVM-derived primary cell lines. RT-qPCR demonstrated transcripts of cathepsins B, D, and G in all three AVM-derived primary cell lines. Enzymatic activity assays showed that cathepsins B and D were active. Immunofluorescence staining showed expression of cathepsins B and D on the OCT4+/SOX2+ endothelium and media of the lesional vessels and cells within the stroma in AVM nidus. Cathepsin G was expressed on the chymase+ phenotypic mast cells. Conclusions: This study demonstrated the novel finding of the expression of cathepsins B, D, and G in AVM. Cathepsins B and D were expressed by the primitive population, and cathepsin G was localized to mast cells, within the AVM nidus.

Expression of cathepsins B and D by cancer stem cells in head and neck metastatic malignant melanoma.

We have previously demonstrated cancer stem cell (CSC) subpopulations in head and neck metastatic malignant melanoma (HNmMM), and the expression of components of the renin-angiotensin system (RAS) by these CSCs. Cathepsins B, D and G are involved in carcinogenesis and constitute bypass loops of the RAS. This study investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. Immunohistochemical staining demonstrated expression of cathepsins B, D and G in HNmMM sections from all 20 patients. Western blotting confirmed the presence of cathepsins B and D proteins in all six HNmMM tissue samples and four HNmMM-derived primary cell lines. RT-qPCR showed transcript expression of cathepsins B, D and G in all six HNmMM tissue samples, and cathepsins B and D but not cathepsin G in all four HNmMM-derived primary cell lines. Enzymatic activity assays demonstrated cathepsins B and D were active in all six HNmMM tissue samples. Immunofluorescence staining performed on two of the HNmMM tissue samples demonstrated expression of cathepsins B and D by the CSCs, and cathepsin G by cells within the peritumoral stroma. Our novel findings suggest the possibility of targeting these CSCs by modulation of paracrine RAS signaling.

Stemness-Associated Markers Are Expressed in Extracranial Arteriovenous Malformation.

Objectives: Arteriovenous malformation (AVM) consists of a nidus with poorly formed low-resistance vessels in place of a functional capillary network. The role of somatic mutations in embryonic stem cells (ESCs) and vascular anomalies and the presence of primitive populations in vascular anomalies led us to investigate the presence of a primitive population in extracranial AVM. Methods: Extracranial AVM tissue samples from 12 patients were stained for stemness-associated markers OCT4, SOX2, NANOG, KLF4, and c-MYC using immunohistochemical staining. In situ hybridization (ISH) was performed on six tissue samples to determine transcript expression. Western blotting and RT-qPCR were performed on two AVM-derived primary cell lines to determine protein and transcript expression of these markers, respectively. Immunofluorescence staining was performed on two tissue samples to investigate marker co-localization. Results: Immunohistochemical staining demonstrated the expression of OCT4, SOX2, KLF4, and c-MYC on the endothelium and media of lesional vessels and cells within the stroma of the nidus in all 12 AVM tissue samples. ISH and RT-qPCR confirmed transcript expression of all five markers. Western blotting showed protein expression of all markers except NANOG. Immunofluorescence staining demonstrated an OCT4+/SOX2+/KLF4+/c-MYC+ population within the endothelium and media of the lesional vessels and cells within the stroma of the AVM nidus. Conclusions: Our findings may suggest the presence of a primitive population within the AVM nidus. Further investigation may lead to novel therapeutic targeting of this population.

Expression of Components of the Renin-Angiotensin System by Cancer Stem Cells in Renal Clear Cell Carcinoma.

This study investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cells (CSCs) we have recently demonstrated in renal clear cell carcinoma (RCCC). Fifteen RCCC tissue samples underwent immunohistochemical staining for components of the RAS: renin, pro-renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme 2 (ACE2), and angiotensin II receptor 2 (AT2R). Immunofluorescence co-staining or double immunohistochemical staining of these components of the RAS with stemness-associated markers OCT4 or KLF4 was performed on two of the samples. Protein and transcript expression of these components of the RAS in six RCCC tissue samples was investigated using western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, angiotensin II receptor 1 (AT1R) was investigated using RT-qPCR only. Immunohistochemical staining demonstrated expression of renin, PRR, and ACE2 in 11, 13, and 13 out of 15 RCCC samples, respectively, while AT2R was expressed in all 15 samples. ACE was detected in the endothelium of normal vasculature only. Double immunohistochemical staining demonstrated localization of ACE2, but not renin, to the KLF4+ CSCs. Immunofluorescence staining showed localization of PRR and AT2R to the OCT4+ CSCs. Western blotting confirmed protein expression of all components of the RAS except renin. RT-qPCR demonstrated transcript expression of all components of the RAS including AT1R, but not AT2R, in all six RCCC tissue samples. This study demonstrated expression of PRR, ACE2, and AT2R by the CSCs within RCCC. Further studies may lead to novel therapeutic targeting of CSCs by manipulation of the RAS in the treatment of this aggressive cancer.

Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System.

We investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cell (CSC) subpopulations in metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC). Immunohistochemical staining demonstrated expression of prorenin receptor (PRR), angiotensin-converting enzyme (ACE), and angiotensin II receptor 2 (AT2R) in all cases and angiotensinogen in 14 cases; however, renin and ACE2 were not detected in any of the 20 mHNcSCC tissue samples. Western blotting showed protein expression of angiotensinogen in all six mHNcSCC tissue samples, but in none of the four mHNcSCC-derived primary cell lines, while PRR was detected in the four cell lines only. RT-qPCR confirmed transcripts of angiotensinogen, PRR, ACE, and angiotensin II receptor 1 (AT1R), but not renin or AT2R in all four mHNcSCC tissue samples and all four mHNcSCC-derived primary cell lines, while ACE2 was expressed in the tissue samples only. Double immunohistochemical staining on two of the mHNcSCC tissue samples showed expression of angiotensinogen by the SOX2+ CSCs within the tumor nests (TNs), and immunofluorescence showed expression of PRR and AT2R by the SOX2+ CSCs within the TNs and the peritumoral stroma (PTS). ACE was expressed on the endothelium of the tumor microvessels within the PTS. We demonstrated expression of angiotensinogen by CSCs within the TNs, PRR, and AT2R by the CSCs within the TNs and the PTS, in addition to ACE on the endothelium of tumor microvessels in mHNcSCC.

Embryonic stem cell-like subpopulations are present within Schwannoma.

BACKGROUND: There is accumulating evidence of the presence of embryonic stem cell (ESC)-like cells in benign tumors. AIM: This study aimed to identify ESC-like cells in Schwannoma using the induced-pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4 and c-MYC. METHODS: Immunohistochemical (IHC) staining (n = 20) and RT-qPCR (n = 6) were performed on Schwannoma tissue samples (STS) to investigate protein and mRNA expression of these iPSC markers, respectively. Immunofluorescence (IF) staining was performed to investigate co-localization of the iPSC markers with CD34, α-SMA and CD133. RESULTS: IHC staining and RT-qPCR demonstrated protein and mRNA expression of all five iPSC markers, respectively. IF staining showed expression of SOX2, KLF4 and c-MYC on the tumor cells and the endothelium of the tumor microvessels which also expressed OCT4, while NANOG was exclusively expressed on the endothelium of the tumor microvessels. The OCT4+/CD34+ endothelium expressed CD133. CONCLUSION: We have identified a putative OCT4+/SOX2+/NANOG+/KLF4+/c-MYC+/CD133+ ESC-like subpopulation on the endothelium of tumor microvessels and an OCT4-/SOX2+/NANOG-/KLF4+/c-MYC+/CD133+ ESC-like subpopulation, within Schwannoma.

Cancer Stem Cells in Head and Neck Metastatic Malignant Melanoma Express Components of the Renin-Angiotensin System.

Components of the renin-angiotensin system (RAS) are expressed by cancer stem cells (CSCs) in many cancer types. We here investigated expression of the RAS by the CSC subpopulations in human head and neck metastatic malignant melanoma (HNmMM) tissue samples and HNmMM-derived primary cell lines. Immunohistochemical staining demonstrated expression of pro-renin receptor (PRR), angiotensin-converting enzyme (ACE), and angiotensin II receptor 2 (AT2R) in all; renin in one; and ACE2 in none of the 20 HNmMM tissue samples. PRR was localized to cells within the tumor nests (TNs), while AT2R was expressed by cells within the TNs and the peritumoral stroma (PTS). ACE was localized to the endothelium of the tumor microvessels within the PTS. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) detected transcripts for PRR, ACE, ACE2, and AT1R, in all the five HNmMM tissue samples and four HNmMM-derived primary cell lines; renin in one tissue sample and one cell line, and AT2R in none of the five HNmMM tissue samples and cell lines. Western blotting showed variable expression of ACE, PRR, and AT2R, but not ACE2, in six HNmMM tissue samples and two HNmMM-derived primary cell lines. Immunofluorescence staining of two HNmMM tissue samples demonstrated expression of PRR and AT2R by the SOX2+ CSCs within the TNs and the OCT4+ CSCs within the PTS, with ACE localized to the endothelium of the tumor microvessels within the PTS.

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 Are Present Within Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma.

Cancer stem cells (CSCs) have been identified in many cancer types including primary head and neck cutaneous squamous cell carcinoma (HNcSCC). This study aimed to identify and characterize CSCs in metastatic HNcSCC (mHNcSCC). Immunohistochemical staining performed on mHNcSCC samples from 15 patients demonstrated expression of the induced pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4, and c-MYC in all 15 samples. In situ hybridization and RT-qPCR performed on four of these mHNcSCC tissue samples confirmed transcript expression of all five iPSC markers. Immunofluorescence staining performed on three of these mHNcSCC samples demonstrated expression of c-MYC on cells within the tumor nests (TNs) and the peri-tumoral stroma (PTS) that also expressed KLF4. OCT4 was expressed on the SOX2+/NANOG+/KLF4+ cells within the TNs, and the SOX2+/NANOG+/KLF4+ cells within the PTS. RT-qPCR demonstrated transcript expression of all five iPSC markers in all three mHNcSCC-derived primary cell lines, except for SOX2 in one cell line. Western blotting showed the presence of SOX2, KLF4, and c-MYC but not OCT4 and NANOG in the three mHNcSCC-derived primary cell lines. All three cell lines formed tumorspheres, at the first passage. We demonstrated an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ CSC subpopulation and an OCT4+/NANOG-/SOX2+/KLF4+/c-MYC+ subpopulation within the TNs, and an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ subpopulation within the PTS of mHNcSCC.

Identification of Cancer Stem Cell Subpopulations in Head and Neck Metastatic Malignant Melanoma.

Cancer stem cells (CSCs) have been identified in many cancer types. This study identified and characterized CSCs in head and neck metastatic malignant melanoma (HNmMM) to regional lymph nodes using induced pluripotent stem cell (iPSC) markers. Immunohistochemical (IHC) staining performed on 20 HNmMM tissue samples demonstrated expression of iPSC markers OCT4, SOX2, KLF4, and c-MYC in all samples, while NANOG was expressed at low levels in two samples. Immunofluorescence (IF) staining demonstrated an OCT4+/SOX2+/KLF4+/c-MYC+ CSC subpopulation within the tumor nests (TNs) and another within the peritumoral stroma (PTS) of HNmMM tissues. IF also showed expression of NANOG by some OCT4+/SOX2+/KLF4+/c-MYC+ cells within the TNs in an HNmMM tissue sample that expressed NANOG on IHC staining. In situ hybridization (n = 6) and reverse-transcription quantitative polymerase chain reaction (n = 5) on the HNmMM samples confirmed expression of all five iPSC markers. Western blotting of primary cell lines derived from four of the 20 HNmMM tissue samples showed expression of SOX2, KLF4, and c-MYC but not OCT4 and NANOG, and three of these cell lines formed tumorspheres in vitro. We demonstrate the presence of two putative CSC subpopulations within HNmMM, which may be a novel therapeutic target in the treatment of this aggressive cancer.