Search

Purpose: We have recently shown mesenchymal stem cells (MSCs) isolated from haemangioma stained strongly for OPG. Vimentin immunoreactivity (IR) is an established marker for immature cells of mesenchymal origin. This study characterises haemangioma biopsies at different phases of progression to identify the presence of progenitor cells. Methods: Immunohistochemical staining was carried out on formalin-fixed paraffin sections of haemangioma at different phases of progression for vimentin, osteoprotegerin (OPG), von Willebrand factor and glucose transporter-1 (Glut-1). Results: Positive staining for OPG and vimentin was observed on progenitor cells in the proliferating and early involuting phases of haemangioma. Strong Glut-1 staining of mature capillaries of the involuted phase was inversely related to the degree of OPG and vimentin IR. Positive vimentin staining of cells in the interstitium of involuted heamangioma and weak vimentin IR of blood vessels was found suggesting that these vimentin-positive cells may be MSCs that eventually differentiate into adipocytes that characterise involuted lesions. OPG IR diminished as haemangioma involuted, with little staining seen in involuted lesions. Conclusions: OPG and vimentin IR are early progenitor cell markers that stain immature capillaries of proliferating haemangioma. In involuted haemangioma OPG IR is scant whereas vimentin-positive cells are present in the interstitium. This finding supports our hypothesis that OPG IR is a marker for progenitor cells present in immature capillaries of proliferating haemangioma that is lost as the lesions involute. [ABSTRACT FROM AUTHOR]

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.

Background: This study investigated the expression and localization of cathepsins B, D, and G in relationship to the embryonic stem cell (ESC)-like population we have previously identified in microcystic lymphatic malformation (mLM). Methods and Results: Immunohistochemical staining demonstrated expression of cathepsins B, D, and G in cervicofacial mLM tissue samples from 11 patients. Immunofluorescence staining of two representative mLM samples showed localization of cathepsins B and D to the OCT4+ and the c-MYC+ cells on the endothelium of lesional vessels and the stroma, while cathepsin G was localized to the OCT4+/tryptase+ cells within the stroma. Transcript expression of cathepsins B, D, and G was confirmed using reverse transcription quantitative polymerase chain reaction (RT-qPCR; n  = 5). Western blotting ( n  = 3) performed on the mLM tissue samples revealed protein expression of cathepsins B and D, which were demonstrated to be enzymatically active using enzymatic activity assays. Conclusion: This study demonstrated expression of cathepsins B and D by the ESC-like cells on the endothelium of lesional vessels and the stroma, while cathepsin G was localized to the OCT4+ phenotypic mast cells within the stroma of mLM.

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 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., (Copyright © 2020 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

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., (Copyright © 2020 Kilmister, Patel, van Schaijik, Bockett, Brasch, Paterson, Sim, Davis, Roth, Itinteang and Tan.)

We report an adult case from Kiribati, with a large dermoid cyst, and resultant underlying plagiocephaly, that was managed well with surgical excision. We also discuss the pathogenesis of this condition and the optimum timing for surgical intervention to avoid the deformity.

Background: We have previously shown that the endothelium of the microvessels of infantile hemangioma (IH) exhibits a hemogenic endothelium phenotype and proposed its potential to give rise to mesenchymal stem cells, similar to the development of hematopoietic cells. This endothelial-to-mesenchymal transition (Endo-MT) process involves the acquisition of a migratory phenotype by the endothelial cells, similar to epithelial-to-mesenchymal transition that occurs during neural crest development. We hypothesized that proliferating IH expresses Endo-MT-associated proteins and investigated their expression at the mRNA, protein, and functional levels., Methods: Immunohistochemical staining of paraffin-embedded sections of proliferating IH samples from 10 patients was undertaken to investigate the expression of the Endo-MT proteins Twist1, Twist2, Snail1, and Slug. Transcriptional analysis was performed for the same markers on proliferating IH tissues and CD34 + and CD34 - cells from proliferating IH-derived primary cell lines. Adipogenic and osteogenic differentiation plasticity was determined on the CD34-sorted fractions., Results: The endothelium of the microvessels and the cells within the interstitium of proliferating IH tissues expressed Twist1, Twist2, and Slug proteins. Twist1 was also expressed on the pericyte layer of the microvessels, whereas Snail1 was not expressed. Both CD34 + and CD34 - populations from the IH-derived primary cell lines underwent adipogenic and osteogenic differentiation., Conclusions: The expression of Endo-MT-associated proteins Twist1, Twist2, and Slug by both the endothelium of the microvessels and cells within the interstitium, and Twist1 on the pericyte layer of the microvessels of proliferating IH, suggest the presence of a process similar to Endo-MT. This may enable a tightly controlled primitive endothelium of proliferating IH to acquire a migratory mesenchymal phenotype with the ability to migrate away, providing a plausible explanation for the development of a fibrofatty residuum observed during involution of IH., (Copyright © 2020 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

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).

Background: The authors have previously shown that an embryonic stem cell-like population within keloid-associated lymphoid tissues in keloid lesions expresses components of the renin-angiotensin system that may be dysregulated. The authors hypothesized that cathepsins B, D, and G are present within the embryonic stem cell-like population in keloid lesions and contribute to bypass loops of the renin-angiotensin system., Methods: 3,3'-Diaminobenzidine immunohistochemical staining for cathepsins B, D, and G was performed on formalin-fixed paraffin-embedded sections in keloid tissue samples of 11 patients. Immunofluorescence immunohistochemical staining was performed on three of these keloid tissue samples, by co-staining with CD34, tryptase, and OCT4. Western blotting, reverse transcription quantitative polymerase chain reaction, and enzyme activity assays were performed on five keloid tissue samples and four keloid-derived primary cell lines to investigate protein and mRNA expression, and functional activity, respectively., Results: 3,3'-Diaminobenzidine immunohistochemical staining demonstrated expression of cathepsins B, D, and G in all 15 keloid tissue samples. Immunofluorescence immunohistochemical staining showed localization of cathepsins B and D to the endothelium of microvessels within the keloid-associated lymphoid tissues and localization of cathepsin G to the tryptase-positive perivascular cells. Western blotting confirmed semiquantitative levels of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines. Reverse transcription quantitative polymerase chain reaction showed quantitative transcriptional activation of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines and cathepsin G in keloid tissue samples. Enzyme activity assays demonstrated functional activity of cathepsins B and D., Conclusion: Cathepsins B, D, and G are expressed by the embryonic stem cell-like population within the keloid-associated lymphoid tissues of keloid lesions and may act to bypass the renin-angiotensin system, suggesting a potential therapeutic target using renin-angiotensin system modulators and cathepsin inhibitors.

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.

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.)

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.

Objectives: Vitamin D receptor (VDR) may play a role in keloid disorder. This study investigated the expression of VDR by the embryonic stem cell (ESC)-like population within keloid-associated lymphoid tissues (KALTs) which expresses components of the renin-angiotensin system (RAS). Methods: 11 formalin-fixed paraffin-embedded sections of keloid lesions (KLs) underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for VDR. Immunofluorescence (IF) dual IHC staining of CD34/VDR and OCT4/VDR was performed on two representative KLs. Transcriptional activation of VDR was investigated in four representative snap-frozen KLs using reverse-transcriptase-quantitative polymerase chain reaction (RT-qPCR). Results: DAB IHC staining demonstrated the presence of VDR on the KALTs within the keloid tissue samples. RT-qPCR confirmed transcriptional activation of VDR. IF IHC staining demonstrated expression of VDR on the CD34 + and the OCT4 + endothelium of the microvessels, and the OCT4 + perivascular cells, within the KALTs. Conclusions: This study demonstrated the expression of VDR by the ESC-like population within the KALTs in KLs. Further work is needed to elucidate the precise interaction between VDR and the RAS in regulating the primitive population within the KALTs., Competing Interests: TI, PD and ST are inventors of a PCT application Treatment of Fibrotic Conditions (PCT/NZ2016/050187). The authors are otherwise not aware of any commercial associations or financial relationships that might pose or create a conflict of interest with information presented in any submitted manuscript., (IJCEP Copyright © 2019.)

Background: Propranolol is the preferred treatment for problematic proliferating infantile hemangioma (IH) by targeting the renin-angiotensin system (RAS) expressed by IH endothelium. (Pro)renin receptor (PRR) is a major component of the RAS associated with the canonical wnt signaling pathway. We proposed that activation of PRR by renin causes proliferation of IH., Methods: The expression of PRR in IH tissue samples was investigated using immunohistochemical (IHC) staining and NanoString analysis. NanoString analysis was also used to confirm transcriptional expression of PRR in CD34-sorted proliferating IH-derived primary cell lines. MTT assay was utilized to determine the effect of exogenous renin on the number of viable IH cells. RT-qPCR was used to determine the effect of renin on the stem cell gene expression., Results: NanoString analysis and IHC staining confirmed transcriptional and translational expression of PRR, which was localized to the non-endothelial and the endothelial IH cell populations. MTT assay demonstrated an increased number of viable IH cells by administration of renin and the effect was negated by the wnt receptor blocker dickkopf-1., Conclusion: Our results present a model for renin-induced increased proliferation of IH cells through PRR acting via the wnt signaling pathway, which may account for accumulation of cells in IH during the proliferative phase of the tumor.

Background: We investigated expression of prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 by the embryonic stem cell-like population on the endothelium of the microvessels and perivascular cells within keloid-associated lymphoid tissues., Methods: Immunohistochemical staining for prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 was performed on 11 formalin-fixed, paraffin-embedded sections of keloid tissue samples. Immunofluorescence staining was performed on three keloid tissue samples by co-staining with OCT4, CD34, ERG, and tryptase. Real-time quantitative polymerase chain reaction was performed on five keloid tissue samples and four keloid-derived primary cell lines. Western blotting was performed on the four keloid-derived primary cell lines for mRNA and protein expression of these proteins, respectively., Results: Immunohistochemical and immunofluorescence staining showed expression of prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 in all 11 keloid tissue samples. Prorenin receptor and angiotensin II receptor 1 were expressed on the endothelium and the pericyte layer of the microvessels and perivascular cells, angiotensin II receptor 2 was localized to the endothelium of the microvessels and the tryptase-positive perivascular cells, and angiotensin-converting enzyme was localized to the endothelium of the microvessel, within the keloid-associated lymphoid tissues. Real-time quantitative polymerase chain reaction showed transcripts of prorenin receptor, angiotensin-converting enzyme, and angiotensin II receptor 1 in the keloid tissue samples and keloid-derived primary cell lines, whereas angiotensin II receptor 2 was detected in keloid tissue samples only. Western blotting confirmed the presence of prorenin receptor, angiotensin-converting enzyme, and angiotensin II receptor 1 in the keloid-derived primary cell lines., Conclusion: Prorenin receptor, angiotensin-converting enzyme, angiotensin II receptor 1, and angiotensin II receptor 2 were expressed by the embryonic stem cell-like population within the keloid-associated lymphoid tissues, suggesting that this primitive population may be a potential therapeutic target by modulation of the renin-angiotensin system.

Keloid disorder (KD) is a fibroproliferative condition caused by dysregulated wound healing following wounding of the skin. The pathogenesis of KD has not been fully elucidated and current treatment is unsatisfactory. There is increasing evidence of the role of stem cells in KD. This review discusses the role of embryonic stem (ESC)-like cells and mesenchymal stem cells in the pathogenesis of KD. It is proposed that dysfunction of the ESC-like population localized to the endothelium of the microvessels and perivascular cells within the keloid-associated lymphoid tissues may give rise to the aberrant fibroblasts and myofibroblasts via a mesenchymal stem cell intermediate in keloid lesions, by undergoing an endothelial-to-mesenchymal transition. We also discuss the role of the renin-angiotensin system (RAS), the immune system, and the inflammatory response, on stem cell proliferation and differentiation. The understanding of the precise roles of these stem cells and interplay of the associated regulatory pathways could lead to the development of targeted therapy for this enigmatic and challenging condition. The demonstration of the expression of components of the RAS and cathepsins B, D, and G that constitute bypass loops of the RAS, by the ESC-like population, suggests that the primitive population may be a therapeutic target by modulation of the RAS, using existing medications.

Aim: We have recently demonstrated a putative stem cell population within WHO grade I meningioma (MG) that expressed embryonic stem cell (ESC) markers OCT4, NANOG, SOX2, KLF4 and c-MYC, localized to the endothelial and pericyte layers of the microvessels. There is increasing recognition that the renin-angiotensin system (RAS) plays a critical role in stem cell biology and tumorigenesis. This study investigated 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) on the putative stem cell population on the microvessels of WHO grade I MG. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on WHO grade I MG tissue samples from 11 patients for PRR, ACE, ATIIR1, and ATIIR2. Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these components of the RAS in using combinations of CD34 and ESC marker SOX2 or OCT4. NanoString mRNA expression analysis and Western blotting (WB), were performed on six snap-frozen MG tissue samples to confirm mRNA and protein expression of these proteins, respectively. Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 within all 11 MG tissue samples. WB and NanoString mRNA analyses, confirmed protein and mRNA expression of these proteins, respectively. IF IHC staining showed PRR, ATIIR1 and ATIIR2 were localized to the OCT4 + and SOX2 + endothelium and the pericyte layer of MG while ACE was localized to the OCT4 + endothelium of the microvesels. Conclusion: The novel finding of the expression of PRR, ACE, ATIIR1, and ATIIR2 on the putative stem cell population on the microvessels of WHO grade I MG, suggests that these stem cells may be a potential therapeutic target by manipulation of the RAS.

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.

Background: We have recently demonstrated the presence of a NANOG + /pSTAT3 + /OCT4 + /SOX2 + /SALL4 + /CD44 + embryonic stem cell (ESC)-like subpopulation localized to the endothelium and a NANOG + /pSTAT3 + /SOX2 + /CD44 + subpopulation outside of the endothelium, within subcutaneous VM (SCVM) and intramuscular VM (IMVM). We have also shown 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), in both SCVM and IMVM. This study investigated whether the ESC-like subpopulations within SCVM and IMVM expressed the RAS., Methods: Formalin-fixed paraffin-embedded sections of two representative samples from each of the seven SCVM and seven IMVM patients included in our previous studies underwent dual immunofluorescence (IF) immunohistochemical (IHC) staining for ESC marker OCT4 or SOX2 with PRR, ACE, ATIIR1, and ATIIR2., Results: IF IHC staining demonstrated the expression PRR by the OCT4 + cells on the endothelium and outside the endothelium in SCVM and IMVM. ACE was expressed by the SOX2 + cells, predominantly in the endothelium in SCVM and IMVM. ATIIR1 was expressed by the SOX2 + cells on the endothelium and outside the endothelium in SCVM and IMVM. ATIIR2 was expressed by the OCT4 + endothelium and outside the endothelium in SCVM and IMVM., Conclusions: Components of the RAS are expressed by ESC-like subpopulations within both SCVM and IMVM. These primitive subpopulations may be a novel therapeutic target by manipulation of the RAS using existing medications.

Aim: We have recently demonstrated the presence of putative tumor stem cells (TSCs) in World Health Organization (WHO) grade I meningioma (MG) localized to the microvessels, which expresses components of the renin-angiotensin system (RAS). The RAS is known to be dysregulated and promotes tumorigenesis in many cancer types, including glioblastoma. Cathepsins B, D, and G are isoenzymes that catalyze the production of angiotensin peptides, hence providing bypass loops for the RAS. This study investigated the expression of cathepsins B, D, and G in WHO grade I MG in relation to the putative TSC population we have previously demonstrated. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining with antibodies for cathepsins B, D, and G was performed on WHO grade I MG tissue samples from 10 patients. Three of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these cathepsins using combinations of smooth muscle actin (SMA) and embryonic stem cell marker OCT4. NanoString mRNA expression ( n = 6) and Western blotting (WB; n = 5) analyses, and enzyme activity assays (EAAs; n = 3), were performed on snap-frozen WHO grade I MG tissue samples to confirm transcriptional activation, protein expression, and functional activity of these proteins, respectively. Results: DAB IHC staining demonstrated expression of cathepsins B, D, and G in all 10 MG samples. NanoString mRNA expression and WB analyses showed transcriptional activation and protein expression of all three cathepsins, although cathepsin G was expressed at low levels. EAAs demonstrated that cathepsin B and cathepsin D were functionally active. IF IHC staining illustrated localization of cathepsin B and cathepsin D to the endothelium and SMA + pericyte layer of the microvessels, while cathepsin G was localized to cells scattered within the interstitium, away from the microvessels. Conclusion: Cathepsin B and cathepsin D, and to a lesser extent cathepsin G, are expressed in WHO grade I MG. Cathepsin B and cathepsin D are enzymatically active and are localized to the putative TSC population on the microvessels, whereas cathepsin G was localized to cells scattered within the interstitium, These results suggest the presence of bypass loops for the RAS, within WHO grade I MG.

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.)

Aim: The presence of cells within meningioma (MG) that express embryonic stem cell (ESC) markers has been previously reported. However, the precise location of these cells has yet to be determined. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on 11 WHO grade I MG tissue samples for the expression of the ESC markers OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence (IF) IHC staining was performed to investigate the localization of each of these ESC markers. NanoString and colorimetric in situ hybridization (CISH) mRNA expression analyses were performed on six snap-frozen MG tissue samples to confirm transcriptional activation of these proteins, respectively. Results: DAB IHC staining demonstrated expression of OCT4, NANOG, SOX2, KLF4, and c-MYC within all 11 MG tissue samples. IF IHC staining demonstrated the expression of the ESC markers OCT4, NANOG, SOX2, KLF4, and c-MYC on both the endothelial and pericyte layers of the microvessels. NanoString and CISH mRNA analyses confirmed transcription activation of these ESC markers. Conclusion: This novel finding of the expression of all aforementioned ESC markers in WHO grade I MG infers the presence of a putative stem cells population which may give rise to MG.

Aim: We have previously identified and characterized cancer stem cell (CSC) subpopulations in liver metastasis from colon adenocarcinoma (LMCA). In this study we investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs., Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D and G was performed on 4μm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients. Immunofluorescence (IF) IHC staining was performed on three representative samples of LMCA from the original cohort of nine patients, to determine the localization of these cathepsins in relation to the CSC subpopulations. NanoString mRNA analysis and Western Blotting (WB) were used to examine the transcript and protein expression of these cathepsins, respectively. Enzyme activity assays were utilized to determine their functional activity. Data acquired from counting of cells staining positively of the cathepsins on the DAB IHC-stained slides and from Nanostring mRNA analysis were subjected to statistical analyses to determine significance., Results: DAB IHC staining demonstrated expression of cathepsins B, D and G within LMCA. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4 - cells within the tumor nests and the OCT4 + CSC subpopulation within the peritumoral stroma. NanoString mRNA analysis showed significantly greater transcript expression of cathepsin B and cathepsin D, compared to cathepsin G. WB confirmed expression of cathepsin B and cathepsin D proteins, while cathepsin G was below detectable levels. Enzyme activity assays showed functional activity of cathepsin B and cathepsin D., Conclusion: Our study demonstrated novel finding of the expression of cathepsin B, cathepsin D, and possibly cathepsin G by the putative CSC subpopulations within LMCA.

The pathogenesis of Dupuytren's disease (DD) remains unclear although there is increasing evidence supporting the role of stem cells in this and other fibrotic conditions. This review examines the role of DD tissue-associated embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), and circulating fibrocytes and circulating MSCs, in the biology of DD. It is exciting to infer that dysfunction of an upstream ESC-like population within the affected tissue leads to the downstream development and proliferation of aberrant myofibroblasts through a putative MSC intermediate. This ESC-like population may be a potential novel therapeutic target through modulation of the renin-angiotensin system. Furthermore, circulating CD34 + fibrocytes and MSCs either derived from the bone marrow, peripheral blood cells, or DD-associated ESC-like population, may serve as potential additional extra-palmar reservoirs that undergo endothelial-to-mesenchymal transition, eventually giving rise to the aberrant myofibroblasts. Further studies examining the relative roles of these stem cells and the precise regulatory pathways that govern them may lead to novel therapy that targets these populations.

We have recently demonstrated the expression of embryonic stem cell markers on the endothelium of infantile hemangioma, a functional hemogenic endothelium with the capacity for primitive erythropoiesis in vitro . Despite recent work characterizing stem cells within proliferating infantile hemangioma, the expression of STAT proteins, well documented for their roles in stem cell signaling, has not been investigated. 3,3-Diaminobenzidine and immunofluorescence immunohistochemical staining revealed expression of pSTAT1, pSTAT3 and pSTAT5 in proliferating infantile hemangioma samples with the strongest expression of pSTAT3. There was reduced expression of these pSTAT proteins in the involuted infantile hemangioma samples. Western blotting confirmed the identification of all these three proteins in proliferating infantile hemangioma. It is therefore not surprising that the phosphorylated/activated forms of these proteins are relatively abundantly expressed in proliferating, in comparison to involuted infantile hemangioma samples. We speculate that the reduced STAT activation, as infantile hemangioma involutes, is a reflection of the depletion of the abundant stem cells within proliferating infantile hemangioma, as the lesion involutes.

Background: The pathogenesis of Dupuytren's disease (DD) remains unclear. An embryonic stem cell (ESC)-like population in the endothelium of the microvessels around tissues that expresses components of the renin-angiotensin system (RAS) has been reported. This study investigated if this primitive population expresses cathepsins B, D, and G, that contribute to RAS bypass loops., Methods: 3,3-Diaminobenzidine immunohistochemical (IHC) staining for cathepsins B, D, and G was performed on sections of formalin-fixed paraffin-embedded DD cords (n = 10) and nodules (n = 10). Immunofluorescence IHC staining was utilized to demonstrate co-expression of these cathepsins with ESC markers. Protein and gene expression of these cathepsins was investigated in snap-frozen DD cords (n = 3) and nodules (n = 3) by Western blotting and NanoString analysis, respectively. Enzymatic activity of these cathepsins was investigated by enzymatic activity assays., Results: 3,3-Diaminobenzidine IHC staining demonstrated expression of cathepsins B, D, and G in DD cords and nodules. Gene expression of cathepsins B, D, and G was confirmed by NanoString analysis. Western blotting confirmed expression of cathepsins B and D, but not cathepsin G. Immunofluorescent IHC staining demonstrated high abundance of cathepsins B and D on the OCT4 + /angiotensin converting enzyme + endothelium and the smooth muscle layer of the microvessels. Cathepsin G was localized to trypase + cells within the stroma in DD cords and nodules with limited expression on the microvessels. Enzyme activity assays demonstrated functional activity of cathepsins B and D., Conclusions: Cathepsins B, D, and G were expressed in the DD tissues, with cathepsins B and D localized to the primitive population in the endothelium of the microvessels, whereas cathepsin G was localized to phenotypic mast cells, suggesting the presence of bypass loops for the RAS.

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.)

Background: Fifty percent of colorectal cancer (CRC) patients develop liver metastasis. This study identified and characterized cancer stem cells (CSCs) within colon adenocarcinoma metastasis to the liver (CAML)., Methods: 3,3-Diaminobenzidine immunohistochemical (IHC) staining was performed on nine CAML samples for embryonic stem cell (ESC) markers OCT4, SOX2, NANOG, c-Myc, and KLF4. Immunofluorescence (IF) IHC staining was performed to investigate coexpression of two markers. NanoString mRNA expression analysis and colorimetric in situ hybridization (CISH) were performed on four snap-frozen CAML tissue samples for transcript expression of these ESC markers. Cells stained positively and negatively for each marker by IHC and CISH staining were counted and analyzed., Results: 3,3-Diaminobenzidine IHC staining, and NanoString and CISH mRNA analyses demonstrated the expression of OCT4, SOX2, NANOG, c-Myc, and KLF4 within in all nine CAML samples, except for SOX2 which was below detectable levels on NanoString mRNA analysis. IF IHC staining showed the presence of a SOX2 + /NANOG + /KLF4 + /c-Myc + /OCT - CSC subpopulation within the tumor nests, and a SOX2 + /NANOG + /KLF4 + /c-Myc + /OCT4 - CSC subpopulation and a SOX2 + /NANOG + /KLF4 + /c-Myc + /OCT4 + CSC subpopulation within the peritumoral stroma., Conclusion: The novel finding of three CSC subpopulations within CAML provides insights into the biology of CRC.

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.)

Background: Venous malformation (VM) consists of a network of ectatic anomalous thin-walled venous channels. A role for an activating TIE2 mutation in the development of the dilated luminal vessels in VM, and its proposed involvement of embryonic stem cells (ESCs), led us to investigate the expression of ESC markers in subcutaneous VM (SCVM) and intramuscular VM (IMVM)., Methods: Formalin-fixed paraffin-embedded sections of SCVM from seven patients and IMVM samples from seven patients were analyzed for the expression of Nanog, pSTAT3, OCT4, SOX2, SALL4, and CD44, using 3,3'-diaminobenzidine (DAB) immunohistochemical (IHC) staining. All these samples did not express lymphatic marker D2-40. NanoString mRNA analysis and RT-PCR were performed on snap-frozen samples of SCVM ( n  = 3) and IMVM ( n  = 3) from the respective original cohorts of patients included in DAB IHC staining. To confirm co-expression of two proteins, immunofluorescent (IF) IHC staining on two representative samples of IMVM and SCVM samples from the original cohorts of patients included for DAB IHC staining was performed., Results: DAB IHC staining demonstrated expression of all of the above ESC markers in both SCVM and IMVM samples. IF IHC staining showed that these markers were localized to the endothelium within these lesions and that Nanog, pSTAT3, SOX2, and CD44 were also expressed by cells outside of the endothelium. NanoString mRNA analysis confirmed transcription activation of pSTAT3, OCT4, and CD44. RT-qPCR confirmed transcription activation of Nanog, SOX2, and SALL4., Conclusion: Our findings support the presence of two ESC-like subpopulations, one within and one outside of the endothelium, of both SCVM and IMVM. Given that the endothelial ESC-like subpopulation expresses the more primitive marker, OCT4, it is exciting to speculate that they give rise to the non-endothelial subpopulation.

The renin-angiotensin system (RAS) mediates cardiac and renal fibrosis. Dupuytren's disease (DD) is a proliferative fibromatosis affecting the hands. This study investigated the expression of the RAS in DD., Methods: 3,3-Diaminobenzidine (DAB) and immunofluorescent immunohistochemical (IHC) staining for (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) was performed on 4-μm thick formalin-fixed paraffin-embedded sections of DD cords and nodules from 6 patients. Western blotting (WB) and NanoString mRNA analysis were performed to confirm RAS protein expression and transcriptional activation, respectively., Results: IHC staining demonstrated the expression of PRR, ACE, ATIIR1, and ATIIR2 on the ERG + and CD34 + endothelium of the micro vessels surrounding the DD cords and nodules. PRR was also expressed on the pericyte layer of these microvessels. WB confirmed protein expression of PRR, ACE, and ATIIR2 but not ATIIR1. NanoString analysis confirmed transcriptional activation of PRR, ACE, ATIIR1, but ATIIR2 was below detectable levels., Conclusions: We demonstrated expression of PRR, ATIIR1, ATIIR2, and ACE on the embryonic stem cell-like cell population on the microvessels surrounding DD nodules and cords by IHC staining, although the expression of ATIIR1 was not confirmed by WB and that of ATIIR2 was below detectable levels on NanoString analysis. These findings suggest the embryonic stem cell-like cell population as a potential therapeutic target for DD, by using RAS modulators., Competing Interests: Disclosure: Drs. Tan and Itinteang are inventors of a provisional patent application (no. 62/260,953) Treatment of Fibrotic Conditions. The authors are otherwise not aware of any commercial associations or financial relationships that might pose or create a conflict of interest with information presented in any submitted article. The Article Processing Charge was paid for by the Gillies McIndoe Research Institute general fund.

Aim: We have previously demonstrated the putative presence of two cancer stem cell (CSC) subpopulations within moderately differentiated oral tongue squamous cell carcinoma (MDOTSCC), which express components of the renin-angiotensin system (RAS). In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to these CSC subpopulations within MDOTSCC., Methods: 3,3-Diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining was performed on MDOTSCC samples to determine the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations. NanoString mRNA analysis and colorimetric in situ hybridization (CISH) were used to study their transcripts expression. Enzyme activity assays were performed to determine the activity of these cathepsins in MDOTSCC., Results: IHC staining demonstrated expression of cathepsins B, D, and G in MDOTSCC. Cathepsins B and D were localized to CSCs within the tumor nests, while cathepsin B was localized to the CSCs within the peri-tumoral stroma, and cathepsin G was localized to the tryptase + phenotypic mast cells within the peri-tumoral stroma. NanoString and CISH mRNA analyses confirmed transcription activation of cathepsins B, D, and G. Enzyme activity assays confirmed active cathepsins B and D, but not cathepsin G., Conclusion: The presence of cathepsins B and D on the CSCs and cathspsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for MDOTSCC.

Aim: We investigated the expression of the renin-angiotensin system (RAS) by cancer stem cell (CSC) subpopulations we have identified in moderately differentiated lip squamous cell carcinoma (MDLSCC)., Method: Ten MDLSCC samples underwent 3,3-diaminobenzidine (DAB) and immunofluorescent immunohistochemical (IHC) staining for (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and receptor 2 (ATIIR2). NanoString analysis and Western blotting (WB) were performed on six MDLSCC samples for gene and protein expression, respectively., Results: IHC staining showed expression of PRR, ATIIR1, and ATIIR2 on cells within the tumor nests (TNs) and the stroma. ACE was localized to the microvessels within the stroma. WB detected PRR, ACE, and ATIIR2. NanoString analysis confirmed gene expression of PRR, ACE, and ATIIR1., Conclusion: Components of the RAS: PRR, ATIIR1, and ATIIR2 are expressed on two CSC subpopulations in MDLSCC, one within the TNs and the other within the stroma. The endothelium of the microvessels within the stroma expresses ACE.

Cancer stem cells (CSCs) have been identified in oral cavity squamous cell carcinoma (OCSCC). CSCs possess the ability for perpetual self-renewal and proliferation, producing downstream progenitor cells and cancer cells that drive tumor growth. Studies of many cancer types including OCSCC have identified CSCs using specific markers, but it is still unclear as to where in the stem cell hierarchy these markers fall. This is compounded further by the presence of multiple CSC subtypes within OCSCC, making investigation reliant on the use of multiple markers. This review examines the current knowledge in CSC markers OCT4, SOX2, NANOG, ALDH1, phosphorylated STAT3, CD44, CD24, CD133, and Musashi-1, specifically focusing on their use and validity in OCSCC CSC research and how they may be organized into the CSC hierarchy. OCSCC CSCs also express components of the renin-angiotensin system (RAS), which suggests CSCs may be novel therapeutic targets by modulation of the RAS using existing medications.

Aim: To investigate the expression of cathepsins B, D, and G, in relation to the cancer stem cell (CSC) subpopulations, we have previously characterized within isocitrate dehydogenase (IDH)-wildtype glioblastoma (IDHWGB)., Methods: 3,3-Diaminobezidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D, and G, was performed on 4μm-thick formalin-fixed paraffin-embedded IDHWGB samples obtained from six patients. Two representative DHWGB samples from the original cohort of patients were selected for immunofluorescent (IF) IHC staining, to identify the localization of the cathepsins in relation to the CSC subpopulations. NanoString gene expression analysis and colorimetric in situ hybridization (CISH) were conducted to investigate the transcriptional activation of genes encoding for cathepsins B, D, and G. Data obtained from cell counting of DAB IHC-stained slides and from NanoString analysis were subjected to statistical analyses to determine significance., Results: Cathepsin B and cathepsin D were detected in IDHWGB by DAB IHC staining. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4 + and SALL4 + CSC subpopulations. NanoString gene analysis and CISH confirmed the abundant transcript expression of these cathepsins. The transcriptional and translational expressions of cathepsin G were minimal and were confined to cells within the microvasculature., Conclusion: This study demonstrated the expression of cathepsin B and cathepsin D but not cathepsin G within the CSC subpopulations of IDHWGB at both the transcriptional and translational level. Cathepsin G was expressed at low levels and was not localized to the CSC population of IDHWGB. The novel finding of cathepsin B and cathepsin D in IDHWGB suggests the presence of bypass loops for the renin-angiotensin system, which may facilitate the production of angiotensin peptides. Elucidating the precise role of these cathepsins may lead to better understanding and more effective treatment of this aggressive tumor.