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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 β-blockers and ACE inhibitors. Recent work on targeting IH stem cells by inhibiting the transcription factor SOX18 using the stereoisomer R(+) propranolol, independent of β-adrenergic blockade, opens up exciting opportunities for novel treatment of IH without the β-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.

Aim To identify and characterize cancer stem cells (CSCs) in moderately differentiated lip squamous cell carcinoma (MDLSCC). Method MDLSCC samples underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for squamous cell carcinoma marker EMA, CSC marker CD44 and embryonic stem cell markers NANOG, octamer-binding transcription factor 4 (OCT4), spalt-like transcription factor 4 (SALL4), sex-determining region Y-box 2 (SOX2), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Immunofluorescent IHC staining was performed on two MDLSCC samples. Western blotting (WB) was used to confirm the expression of the aforementioned proteins and their transcription activation was investigated using NanoString and RT-qPCR. Results IHC staining demonstrated the presence of (1) an EMA+/CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4− CSC subpopulation within the tumor nests (TNs); (2) a CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4− CSC subpopulation; and (3) a CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4+ CSC subpopulation within the stroma, between the TNs. NanoString and RT-qPCR confirmed the presence of mRNA for CD44, SALL4, STAT3, SOX2, and OCT4, and WB confirmed the presence of NANOG, pSTAT3, SOX2, and OCT4. Conclusion This study demonstrates three putative CSC subpopulations within MDLSCC.

Aim To investigate the expression of the renin–angiotensin system (RAS) in cancer stem cells (CSCs), we have previously characterized in glioblastoma multiforme (GBM). Methods 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for the stem cell marker, SOX2, and components of the RAS: angiotensin converting enzyme (ACE), (pro)renin receptor (PRR), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on 4 μm-thick formalin-fixed paraffin-embedded sections of previously characterized GBM samples in six patients was undertaken. Immunofluorescent (IF) IHC staining was performed to demonstrate expression of GFAP, SOX2, PRR, ACE, ATIIR1, and ATIIR2. The protein expression and the transcriptional activities of the genes encoding for ACE, PRR, ATIIR1, and ATIIR2 were studied using Western blotting (WB) and NanoString gene expression analysis, respectively. Results DAB and IF IHC staining demonstrated the expression SOX2 on the GFAP+ GBM CSCs. Cytoplasmic expression of PRR by the GFAP+ CSCs and the endothelium of the microvessels was observed. ACE was expressed on the endothelium of the microvessels only, while nuclear and cytoplasmic expression of ATIIR1 and ATIIR2 was observed on the endothelium of the microvessels and the CSCs. ATIIR1 was expressed on the GFAP+ CSCs cells, and ATIIR2 was expressed by the SOX2+ CSCs. The expression of ACE, PRR, and ATIIR1, but not ATIIR2, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of ACE, PRR, and ATIIR1, but not ATIIR2. Conclusion This study demonstrated the expression of PRR, ATIIR1, and ATIIR2 by the SOX2 CSC population, and ACE on the endothelium of the microvessels, within GBM. ACE, PRR, and ATIIR1 were expressed at the protein and mRNA levels, with ATIIR2 detectable only by IHC staining. This novel finding suggests that the CSCs may be a novel therapeutic target for GBM by modulation of the RAS.

Aim We have recently identified and characterized cancer stem cell (CSC) subpopulations within moderately differentiated buccal mucosal squamous cell carcinoma (MDBMSCC). We hypothesized that these CSCs express components of the renin–angiotensin system (RAS). Methods 3,3′-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on formalin-fixed paraffin-embedded MDBMSCC samples to investigate the expression of the components of the RAS: (pro)renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2). NanoString mRNA gene expression analysis and Western Blotting (WB) were performed on snap-frozen MDBMSCC samples to confirm gene expression and translation of these transcripts, respectively. Double immunofluorescent (IF) IHC staining of these components of the RAS with the embryonic stem cell markers OCT4 or SALL4 was performed to demonstrate their localization in relation to the CSC subpopulations within MDBMSCC. Results DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 in MDBMSCC. IF IHC staining showed that PRR was expressed by the CSC subpopulations within the tumor nests, the peri-tumoral stroma, and the endothelium of the microvessels within the peri-tumoral stroma. ATIIR1 and ATIIR2 were localized to the CSC subpopulations within the tumor nests and the peri-tumoral stroma, while ACE was localized to the endothelium of the microvessels within the peri-tumoral stroma. WB and NanoString analyses confirmed protein expression and transcription activation of PRR, ACE, and ATIIR1, but not of ATIIR2, respectively. Conclusion Our novel findings of the presence and localization of PRR, ACE, ATIIR1, and potentially ATIIR2 to the CSC subpopulations within MDBMSCC suggest CSC as a therapeutic target by modulation of the RAS.

To identify and characterize cancer stem cells (CSC) in moderately differentiated buccal mucosa squamous cell carcinoma (MDBMSCC).Four micrometer-thick, formalin-fixed, paraffin-embedded MDBMSCC samples from six patients underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers, NANOG, OCT4, SALL4, SOX2, and pSTAT3; cancer stem cell marker, CD44; squamous cell carcinoma (SCC) marker, EMA; and endothelial marker, CD34. The transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, STAT3, and CD44 were studied using NanoString gene expression analysis and colorimetric in situ hybridization (CISH) for NANOG, OCT4, SOX2, SALL4, and STAT3.Diaminobenzidine and immunofluorescent (IF) IHC staining demonstrated the presence of (1) an EMA(+)/CD44(+)/SOX2(+)/SALL4(+)/OCT4(+)/pSTAT3(+)/NANOG(+) CSC subpopulation within the tumor nests; (2) an EMA(-)/CD44(-)/CD34(-)/SOX2(+)/OCT4(+)/pSTAT3(+)/NANOG(+) subpopulation within the stroma between the tumor nests; and (3) an EMA(-)/CD44(-)/CD34(+)/SOX2(+)/SALL4(+)/OCT4(+)/pSTAT3(+)/NANOG(+) subpopulation on the endothelium of the microvessels within the stroma. The expression of CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG was confirmed by the presence of mRNA transcripts, using NanoString analysis and NANOG, OCT4, SOX2, SALL4, and STAT3 by CISH staining.This study demonstrated a novel finding of three separate CSC subpopulations within MDBMSCC: (1) within the tumor nests expressing EMA, CD44, SOX2, SALL4, OCT4, pSTAT3, and NANOG; (2) within the stroma expressing SOX2, SALL4, OCT4, pSTAT3, and NANOG; and (3) on the endothelium of the microvessels within the stroma expressing CD34, SOX2, SALL4, OCT4, pSTAT3, and NANOG.

Aim To identify and characterize cancer stem cells (CSC) in glioblastoma multiforme (GBM). Methods Four-micrometer thick formalin-fixed paraffin-embedded GBM samples from six patients underwent 3,3-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining for the embryonic stem cell (ESC) markers NANOG, OCT4, SALL4, SOX2, and pSTAT3. IF IHC staining was performed to demonstrate co-expression of these markers with GFAP. The protein expression and the transcriptional activities of the genes encoding NANOG, OCT4, SOX2, SALL4, and STAT3 were investigated using Western blotting (WB) and NanoString gene expression analysis, respectively. Results DAB and IF IHC staining demonstrated the presence of a CSC population expressing NANOG, OCT4, SOX2, SALL4, and pSTAT3 with the almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4, within GBM. The expression of NANOG, SOX2 and, pSTAT3 but, not OCT and SALL4, was confirmed by WB. NanoString gene analysis demonstrated transcriptional activation of NANOG, OCT4, SALL4, STAT3, and SOX2 in GBM. Conclusion This study demonstrated a population of CSCs within GBM characterized by the expression of the CSC markers NANOG, SALL4, SOX2, pSTAT3 and OCT4 at the protein and mRNA levels. The almost ubiquitous presence of SOX2 and a relatively low abundance of OCT4 would support the putative existence of a stem cell hierarchy within GBM.

Glioblastoma multiforme (GBM), an aggressive tumor that typically exhibits treatment failure with high mortality rates, is associated with the presence of cancer stem cells (CSCs) within the tumor. CSCs possess the ability for perpetual self-renewal and proliferation, producing downstream progenitor cells that drive tumor growth. Studies of many cancer types 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 GBM and glioblastoma cancer stem cell subtypes, making investigation and establishment of a universal treatment difficult. This review examines the current knowledge on the CSC markers SALL4, OCT-4, SOX2, STAT3, NANOG, c-Myc, KLF4, CD133, CD44, nestin, and glial fibrillary acidic protein, specifically focusing on their use and validity in GBM research and how they may be utilized for investigations into GBM’s cancer biology.

Aims The embryonic-like stem cell origin of infantile hemangioma (IH) and the observed elevated serum levels of alpha-fetoprotein (AFP) in patients with hepatic IH led us to investigate if this tumor was the source of AFP. Materials and methods We measured serial serum levels of AFP in patients with problematic proliferating IH treated with surgical excision or propranolol treatment. We also investigated the expression of AFP in extrahepatic IH samples using immunohistochemical staining, mass spectrometry, NanoString gene expression analysis, and in situ hybridization. Results Serum levels of AFP normalized following surgical excision or propranolol treatment. Multiple regression analysis for curve fittings revealed a different curve compared to reported normal values in the general populations. AFP was not detected in any of the IH samples examined at either the transcriptional or translational levels. Conclusion This study demonstrates the association of proliferating IH with elevated serum levels of AFP, which normalized following surgical excision or propranolol treatment. We have shown that IH is not the direct source of AFP. An interaction between the primitive mesoderm-derived IH and the endogenous endodermal tissues, such as the liver, via an intermediary, may explain the elevated serum levels of AFP in infants with extrahepatic IH.

Aims The role of the renin–angiotensin system (RAS) in the biology of infantile hemangioma (IH) represents an emerging paradigm, particularly the involvement of renin, angiotensin converting enzyme, and angiotensin II. This study investigated the expression of cathepsins B, D, and G, enzymes that may modulate the RAS, in IH. Materials and Methods The expression of cathepsins B, D, and G was examined using immunohistochemistry, enzyme activity assays, mass spectrometry, and NanoString gene expression assay in IH samples at different phases of development. Results Immunohistochemical staining showed the expression of cathepsins B, D, and G in proliferating and involuted IH samples. This was confirmed at the transcriptional level using NanoString gene expression assays. Mass spectrometry confirmed the identification of cathepsins D and G in all three phases of IH development, whereas cathepsin B was detected in 2/2 proliferating and 1/2 involuting lesions. Enzyme activity assays demonstrated the activity of cathepsins B and D, but not G, in all phases of IH development. Conclusion Our data demonstrated the presence of cathepsins B, D, and G in IH. Their role in modulating the RAS and the biology of IH offers potential novel targets for the management of this tumor.

Infantile haemangioma (IH), the most common tumour of infancy, is characterised by an initial proliferation during infancy followed by spontaneous involution over the next 5-10 years, often leaving a fibro-fatty residuum. IH is traditionally considered a tumour of the microvasculature. However, recent data shows the critical role of stem cells in the biology of IH with emerging evidence suggesting an embryonic developmental anomaly due to aberrant proliferation and differentiation of a haemogenic endothelium with a neural crest phenotype that possesses the capacity for endothelial, haematopoietic, mesenchymal and neuronal differentiation. Current evidence suggests a putative placental chorionic mesenchymal core cell embolic origin of IH during the first trimester. This review outlines the emerging role of stem cells and their interplay with the cytokine niche that promotes a post-natal environment conducive for vasculogenesis involving VEGFR-2 and its ligand VEGF-A and the IGF-2 ligand in promoting cellular proliferation, and the TRAIL-OPG anti-apoptotic pathway in preventing cellular apoptosis in IH. The discovery of the role of the renin-angiotensin system in the biology of IH provides a plausible explanation for the programmed biologic behaviour and the β-blocker induced accelerated involution of this enigmatic condition. This crucially involves the vasoactive peptide, angiotensin II, that promotes cellular proliferation in IH predominantly via its action on the ATIIR2 isoform. The role of the RAS in the biology of IH is further supported by the effect of captopril, an ACE inhibitor, in inducing accelerated involution of IH. The discovery of the critical role of RAS in IH represents a novel and fascinating paradigm shift in the understanding of human development, IH and other tumours in general.