Expression of embryonic stem cell markers in pyogenic granuloma.

BACKGROUND: Recent description of hemangioblastic blood islands within pyogenic granuloma (PG) has led us to investigate the expression of embryonic stem cell (ESC) markers in this tumor. METHODS: In this study we examined the expression of ESC markers, OCT4, SOX2, STAT3 and NANOG in PG samples from 11 patients, by immunohistochemical (IHC) staining, NanoString analysis and in situ hybridization (ISH). RESULTS: IHC staining demonstrated the expression of pSTAT3, OCT4, SOX2 and NANOG by the endothelium of the microvessels in PG whilst pSTAT3, SOX2 and NANOG were also expressed by cells in the interstitium, outside of the microvessels. NanoString and ISH analysis showed mRNA expression for STAT3, OCT4 and NANOG in PG. CONCLUSIONS: The expression of the ESC markers, OCT4, SOX2, pSTAT3 and NANOG, suggests the endothelium of PG displays a primitive phenotype. Cells in the interstitium expressing pSTAT3, SOX2 and NANOG may represent a more downstream derivative of the primitive endothelium, or a separate population. The primitive nature of the endothelium and cells in the interstitium reveals novel insights into the biology of PG. To the best of our knowledge, this is the first demonstration of the expression of ESC markers in PG, implying the presence of a hematopoietic stem cell population.

Differences in methylphenidate dose response between periadolescent and adult rats in the familiar arena-novel alcove task.

Methylphenidate is a psychostimulant widely used in the treatment of attention deficit hyperactivity disorder. In this study, the effects of two nonstereotypy-inducing doses of methylphenidate (2.5 and 5.0 mg/kg s.c.) were examined in periadolescent [postnatal days (P) 35 and 42] and young adult (P70), male Long-Evans rats using a three-period locomotor activity paradigm that affords inferences about exploration, habituation, and attention to a novel stimulus (an "alcove") in a familiar environment in a single test session. In the first test period, P35 and P42 rats were more active than P70 rats, and methylphenidate increased locomotion in a dose-related manner. The introduction of a novel spatial stimulus in the third test period revealed a significant interaction of dose and age such that P70 rats exhibited dose-related increases in distance traveled, but P35 rats did not. Furthermore, methylphenidate dose-relatedly disrupted the rats' tendency to spend increasing amounts of time in the alcove across the test period at P70 but not at P35. Brain and serum methylphenidate concentrations were significantly lower at P35 than at P70, with intermediate levels at P42. Developmental differences in dopaminergic neurochemistry were also observed, including increased dopamine content in the caudate-putamen, nucleus accumbens, and frontal cortex and decreased densities of D(1)-like receptors in the frontal cortex in P70 than in P42 rats. These results raise the possibility that children and adults may respond differently when treated with this drug, particularly in situations involving response to novelty and that these effects involve developmental differences in pharmacokinetics and dopaminergic neurochemistry.

CD73, Tumor Plasticity and Immune Evasion in Solid Cancers.

Regulatory networks controlling cellular plasticity, important during early development, can re-emerge after tissue injury and premalignant transformation. One such regulatory molecule is the cell surface ectoenzyme ecto-5'-nucleotidase that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine (eADO). Ecto-5'-nucleotidase (NT5E) or cluster of differentiation 73 (CD73), is an enzyme that is encoded by NT5E in humans. In normal tissue, CD73-mediated generation of eADO has important pleiotropic functions ranging from the promotion of cell growth and survival, to potent immunosuppression mediated through purinergic G protein-coupled adenosine receptors. Importantly, tumors also utilize several mechanisms mediated by CD73 to resist therapeutics and in particular, evade the host immune system, leading to undesired resistance to targeted therapy and immunotherapy. Tumor cell CD73 upregulation is associated with worse clinical outcomes in a variety of cancers. Emerging evidence indicates a link between tumor cell stemness with a limited host anti-tumor immune response. In this review, we provide an overview of a growing body of evidence supporting the pro-tumorigenic role of CD73 and adenosine signaling. We also discuss data that support a link between CD73 expression and tumor plasticity, contributing to dissemination as well as treatment resistance. Collectively, targeting CD73 may represent a novel treatment approach for solid cancers.

Expression of Cathepsins B, D, and G in Microcystic Lymphatic Malformation.

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.

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 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.

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 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.

The renin-angiotensin system in central nervous system tumors and degenerative diseases.

Despite their differences, central nervous system (CNS) tumors and degenerative diseases share important molecular mechanisms underlying their pathologies, due to their common anatomy. Here we review the role of the renin-angiotensin system (RAS) in CNS tumors and degenerative diseases, to highlight common molecular features and examine the potential merits in repurposing drugs that inhibit the RAS, its bypass loops, and converging signaling pathways. The RAS consists of key components, including angiotensinogen, (pro)renin receptor (PRR), angiotensin-converting enzyme 1 (ACE1), angiotensin-converting enzyme 2 (ACE2), angiotensin I (ATI), angiotensin II (ATII), ATII receptor 1 (AT1R), ATII receptor 2 (AT2R) and the Mas receptor (MasR). The RAS is integral to systemic and cellular pathways that regulate blood pressure and body fluid equilibrium and cellular homeostasis. The main effector of the RAS is ATII which exerts its effect by binding to AT1R and AT2R through two competitive arms: an ACE1/ATII/AT1R axis, which is involved in regulating oxidative stress and neuroinflammation pathways, and an ATII/AT2R and/or ATII/ACE2/Ang(1-7)/MasR axis that potentiates neuroprotection pathways. Alterations of these axes are associated with cellular dysfunction linked to CNS diseases. The generation of ATII is also influenced by proteases that constitute bypass loops of the RAS. These bypass loops include cathepsins B, D and G and chymase and aminopeptidases. The RAS is also influenced by converging pathways such as the Wnt/ß-catenin pathway which sits upstream of the RAS via PRR, a key component of the RAS. We also discuss the co-expression of components of the RAS and markers of pluripotency, such as OCT4 and SOX2, in Parkinson's disease and glioblastoma, and their potential influences on transduction pathways involving the Wnt/ß-catenin, MAPK/ERK, PI3K/AKT and vacuolar (H+) adenosine triphosphatase (V-ATPase) signaling cascades. Further research investigating modulation of the ACE1/ATII/AT1R and ACE2/Ang(1-7)/MasR axes with RAS inhibitors may lead to novel treatment of CNS tumors and degenerative diseases. The aim of this review article is to discuss and highlight experimental and epidemiological evidence for the role of the RAS, its bypass loops and convergent signaling pathways in the pathogenesis of CNS tumors and degenerative diseases, to direct research that may lead to the development of novel therapy.

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.

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.

Dopamine receptor alterations in female rats with diet-induced decreased brain docosahexaenoic acid (DHA): interactions with reproductive status.

Decreased tissue levels of n-3 (omega-3) fatty acids, particularly docosahexaenoic acid (DHA), are implicated in the etiologies of non-puerperal and postpartum depression. This study examined the effects of a diet-induced loss of brain DHA content and concurrent reproductive status on dopaminergic parameters in adult female Long-Evans rats. An alpha-linolenic acid-deficient diet and breeding protocols were used to produce virgin and parous female rats with cortical phospholipid DHA levels 20-22% lower than those fed a control diet containing adequate alpha-linolenic acid. Decreased brain DHA produced a significant main effect of decreased density of ventral striatal D(2)-like receptors. Virgin females with decreased DHA also exhibited higher density of D(1)-like receptors in the caudate nucleus than virgin females with normal DHA. These receptor alterations are similar to those found in several rodent models of depression, and are consistent with the proposed hypodopaminergic basis for anhedonia and motivational deficits in depression.

Cell Populations Expressing Stemness-Associated Markers in Vascular Anomalies.

Treatment of vascular anomalies (VAs) is mostly empirical and, in many instances unsatisfactory, as the pathogeneses of these heterogeneous conditions remain largely unknown. There is emerging evidence of the presence of cell populations expressing stemness-associated markers within many types of vascular tumors and vascular malformations. The presence of these populations in VAs is supported, in part, by the observed clinical effect of the mTOR inhibitor, sirolimus, that regulates differentiation of embryonic stem cells (ESCs). The discovery of the central role of the renin-angiotensin system (RAS) in regulating stem cells in infantile hemangioma (IH) provides a plausible explanation for its spontaneous and accelerated involution induced by ß-blockers and ACE inhibitors. Recent work on targeting IH stem cells by inhibiting the transcription factor SOX18 using the stereoisomer R(+) propranolol, independent of ß-adrenergic blockade, opens up exciting opportunities for novel treatment of IH without the ß-adrenergic blockade-related side effects. Gene mutations have been identified in several VAs, involving mainly the PI3K/AKT/mTOR and/or the Ras/RAF/MEK/ERK pathways. Existing cancer therapies that target these pathways engenders the exciting possibility of repurposing these agents for challenging VAs, with early results demonstrating clinical efficacy. However, there are several shortcomings with this approach, including the treatment cost, side effects, emergence of treatment resistance and unknown long-term effects in young patients. The presence of populations expressing stemness-associated markers, including transcription factors involved in the generation of induced pluripotent stem cells (iPSCs), in different types of VAs, suggests the possible role of stem cell pathways in their pathogenesis. Components of the RAS are expressed by cell populations expressing stemness-associated markers in different types of VAs. The gene mutations affecting the PI3K/AKT/mTOR and/or the Ras/RAF/MEK/ERK pathways interact with different components of the RAS, which may influence cell populations expressing stemness-associated markers within VAs. The potential of targeting these populations by manipulating the RAS using repurposed, low-cost and commonly available oral medications, warrants further investigation. This review presents the accumulating evidence demonstrating the presence of stemness-associated markers in VAs, their expression of the RAS, and their interaction with gene mutations affecting the PI3K/AKT/mTOR and/or the Ras/RAF/MEK/ERK pathways, in the pathogenesis of VAs.

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.

Proliferating Infantile Hemangioma Tissues and Primary Cell Lines Express Markers Associated with Endothelial-to-Mesenchymal Transition.

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.

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.

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.