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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 (AT 2 R). 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 (AT 1 R) 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 AT 2 R 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 AT 2 R 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 AT 1 R, but not AT 2 R, in all six RCCC tissue samples. This study demonstrated expression of PRR, ACE2, and AT 2 R 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.

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 (AT 2 R) 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 (AT 1 R), but not renin or AT 2 R 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 AT 2 R 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 AT 2 R by the CSCs within the TNs and the PTS, in addition to ACE on the endothelium of tumor microvessels in mHNcSCC.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. T.I., P.F.D., and S.T.T. are inventors of the patent Cancer Diagnosis and Therapy (United States Patent No. 10281472), provisional patents Cancer Diagnosis and Therapy (PCT/NZ2015/050108), and Cancer Therapeutic (PCT/NZ2018/050006), provisional patent application Novel Pharmaceutical Compositions for Cancer Therapy (US/62/711709).

Aims: The cancer stem cell concept proposes that tumor growth and recurrence is driven by a small population of cancer stem cells (CSCs). In this study we investigated the expression of induced-pluripotent stem cell (iPSC) markers and their localization in primary low-grade adenocarcinoma (LGCA) and high-grade adenocarcinoma (HGCA) and their patient-matched normal colon samples., Materials and Methods: Transcription and translation of iPSC markers OCT4, SOX2, NANOG, KLF4 and c-MYC were investigated using immunohistochemical (IHC) staining, RT-qPCR and in-situ hybridization (ISH)., Results: All five iPSC markers were detected at the transcriptional and translational levels. Protein abundance was found to be correlated with tumor grade. Based on their protein expression within the tumors, two sub-populations of cells were identified: a NANOG+/OCT4- epithelial subpopulation and an OCT4+/NANOG- stromal subpopulation. All cases were accurately graded based on four pieces of iPSC marker-related data., Conclusions: This study suggests the presence of two putative sub-populations of CSCs: a NANOG+/OCT4- epithelial subpopulation and an OCT4+/NANOG- stromal subpopulation. Normal colon, LGCA and HGCA could be accurately distinguished from one another using iPSC marker expression. Once validated, novel combinations of iPSC markers may provide diagnostic and prognostic value to help guide patient management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. TI and STT are inventors of the provisional patents Cancer Diagnosis and Therapy (PCT/NZ2015/050108) and Cancer Therapeutic (PCT/NZ2018/050006), and provisional patent application Novel Pharmaceutical Compositions for Cancer Therapy (US/62/711709).

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., (Copyright © 2018 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Glioblastoma (GB) is the most aggressive primary brain tumor in adults. The aggressive nature of GB has been attributed to the presence of cancer stem cells (CSCs) which drive tumorigenesis and are thought to be the root cause of the disease. Circulating tumor stem cells (CTSCs), which can be derived from CSCs, have been identified in numerous types of cancer including GB, have been proposed to contribute to local and distant recurrence. There are many technical difficulties in studying CTSCs, therefore there is a significant gap in the literature pertaining to how they arise and function, and how the understanding of the biology of CTSCs could elucidate the underlying cause of local recurrence and metastasis. An initial epithelial-to-mesenchymal transition (EMT) followed by mesenchymal-to-epithelial transition involving these primitive cells appear to be the critical processes underpinning metastasis. This review focuses on the association between CSCs undergoing EMT to become CTSCs, and how this could arise from the CSC subpopulation in GB, and contribute to the understanding of the pathogenesis and treatment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We have recently characterized cancer stem cell (CSC) subpopulations in different types of cancer. This study aimed to identify and characterize CSCs within metastatic melanoma (MM) to the brain. 3, 3-diaminobenzidine (DAB) immunohistochemical (IHC) staining of ten samples of MM to the brain demonstrated the expression of the embryonic stem cell (ESC) markers OCT4, NANOG, SALL4, SOX2 and pSTAT3. Protein expression of all five ESC markers except SALL4 were confirmed by Western blotting on five samples and transcriptional activation of all five markers was demonstrated using NanoString mRNA analysis on four samples. Immunofluorescence IHC staining suggested the presence of CSCs that stained for OCT4, SALL4, SOX2 or NANOG. Some of these cells also stained for Melan-A. Also, a pSTAT3 + /CD34 + primitive subpopulation was detected on the endothelium of microvessels. These CSCs may be a novel therapeutic target for MM to the brain., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Colorectal cancer (CRC) is the second most common cancer in women and the third most common in men. Adenocarcinoma accounts for 90% of CRC cases. There has been accumulating evidence in support of the cancer stem cell (CSC) concept of cancer which proposes that CSCs are central in the initiation of cancer. CSCs have been the focus of study in a range of cancers, including CRC. This has led to the identification and understanding of genes involved in the induction and maintenance of pluripotency of stem cells, and markers for CSCs, including those investigated specifically in CRC. Knowledge of the expression pattern of CSCs in CRC has been increasing in recent years, revealing a heterogeneous population of cells within CRC ranging from pluripotent to differentiated cells, with overlapping and sometimes unique combinations of markers. This review summarises current literature on the understanding of CSCs in CRC, including evidence of the presence of CSC subpopulations, and the stem cell markers currently used to identify and localise these CSC subpopulations. Future research into this field may lead to improved methods for early detection of CRC, novel therapy and monitoring of treatment for CRC and other cancer types., Competing Interests: Competing interests: TI and STT are inventors of the PCT patent application (No. PCT/NZ2015/050108) Cancer Diagnosis and Therapy, and Cancer Therapeutic (US62/452479). The authors declare no other conflicts of interest., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The stem cell markers octamer-binding transcription factor 4, sex-determining region Y-box 2, NANOG, Kruppel-like factor 4 and c-MYC are key factors in inducing pluripotency in somatic cells, and they have been used to detect cancer stem cell subpopulations in a range of cancer types. Recent literature has described the subcellular localisation of these markers and their potential implications on cellular function. This is a relatively complex and unexplored area of research, and the extent of the effect that subcellular localisation has on cancer development and growth is largely unknown. This review analyses this area of research in the context of the biology of stem cells and cancer and explores the potential modulating effect of subcellular localisation of these proteins as supported by the literature., Competing Interests: Competing interests: TI, STT and PFD are inventors of the PCT patent applications (NZ2015/050108) Cancer Diagnosis and Therapy, and Cancer Therapeutic (US62/452479). The authors are otherwise not aware of any commercial associations or financial relationships that might create a conflict of interest with any information presented in this manuscript., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Meningioma is a common intracranial and intraspinal neoplasm accounting for 25-30% of all primary neurological tumours. It is associated with high rates of recurrence especially in higher-grade tumours and lesions located at the skull base. Cancer stem cells are increasingly recognised as the origin of cancer and are attributed to loco-regional recurrence, metastasis and treatment resistance. This review presents the accumulating evidence of the presence of tumour stem cells within meningioma and the stem cell markers being used to characterise this putative primitive population within this common tumour., (Copyright © 2017 Elsevier Ltd. All rights reserved.)