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.

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.

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.

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.

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.

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 four 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.