Immunohistochemical Analysis of DNA Repair- and Drug-Efflux-Associated Molecules in Tumor and Peritumor Areas of Glioblastoma.

Glioblastoma (GBM), the most commonly occurring primary tumor arising within the central nervous system, is characterized by high invasiveness and poor prognosis. In spite of the improvement in surgical techniques, along with the administration of chemo- and radiation therapy and the incessant investigation in search of prospective therapeutic targets, the local recurrence that frequently occurs within the peritumoral brain tissue makes GBM the most malignant and terminal type of astrocytoma. In the current study, we investigated both GBM and peritumoral tissues obtained from 55 hospitalized patients and the expression of three molecules involved in the onset of resistance/unresponsiveness to chemotherapy: O6-methylguanine methyltransferase (MGMT), breast cancer resistance protein (BCRP1), and A2B5. We propose that the expression of these molecules in the peritumoral tissue might be crucial to promoting the development of early tumorigenic events in the tissue surrounding GBM as well as responsible for the recurrence originating in this apparently normal area and, accordingly, for the resistance to treatment with the standard chemotherapeutic regimen. Notably, the inverse correlation found between MGMT expression in peritumoral tissue and patients' survival suggests a prognostic role for this protein.

Calcium Mobilization in Endothelial Cell Functions.

Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca2+]i). The extremely complex calcium machinery includes plasma membrane bound channels as well as intracellular receptors distributed in distinct cytosolic compartments that act jointly to maintain a physiological [Ca2+]i, which is crucial for triggering many cellular mechanisms. Here, we first survey the overall notions related to intracellular Ca2+ mobilization and later highlight the involvement of this second messenger in crucial ECs functions with the aim at stimulating further investigation that link Ca2+ mobilization to ECs in health and disease.

The caveolar membrane system in endothelium: From cell signaling to vascular pathology.

Caveolae are 50- to 100-nm cholesterol and glycosphingolipid-rich flask-shaped invaginations commonly observed in many terminally differentiated cells. These organelles have been described in many cell types and are particularly abundant in endothelial cells, where they have been involved in the regulation of certain signaling pathways. Specific scaffolding proteins termed caveolins, along with the more recently discovered members of the cavin family, represent the major protein components during caveolae biogenesis. In addition, multiple studies aimed to investigate the expression and the regulation of these proteins significantly contributed to elucidate the role of caveolae and caveolins in endothelial cell physiology and disease. The aim of this review is to survey recent evidence of the involvement of the caveolar network in endothelial cell biology and endothelial cell dysfunction.

VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca2+ signaling.

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.

Knock down of caveolin-1 affects morphological and functional hallmarks of human endothelial cells.

Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA-induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design-based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis.

NAADP links histamine H1 receptors to secretion of von Willebrand factor in human endothelial cells.

A variety of endothelial agonist-induced responses are mediated by rises in intracellular Ca(2+), suggesting that different Ca(2+) signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca(2+) release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca(2+) from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca(2+)-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca(2+) mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca(2+)-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.

Role of Endothelial Cell Metabolism in Normal and Tumor Vasculature.

Endothelial cells (ECs) form a simple squamous epithelium, the endothelium, which lines the lumen of all blood vessels and the heart [...].

Unraveling the Cave: A Seventy-Year Journey into the Caveolar Network, Cellular Signaling, and Human Disease.

In the mid-1950s, a groundbreaking discovery revealed the fascinating presence of caveolae, referred to as flask-shaped invaginations of the plasma membrane, sparking renewed excitement in the field of cell biology. Caveolae are small, flask-shaped invaginations in the cell membrane that play crucial roles in diverse cellular processes, including endocytosis, lipid homeostasis, and signal transduction. The structural stability and functionality of these specialized membrane microdomains are attributed to the coordinated activity of scaffolding proteins, including caveolins and cavins. While caveolae and caveolins have been long appreciated for their integral roles in cellular physiology, the accumulating scientific evidence throughout the years reaffirms their association with a broad spectrum of human disorders. This review article aims to offer a thorough account of the historical advancements in caveolae research, spanning from their initial discovery to the recognition of caveolin family proteins and their intricate contributions to cellular functions. Furthermore, it will examine the consequences of a dysfunctional caveolar network in the development of human diseases.

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells.

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-ß-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.

Autophagy modulators sensitize prostate epithelial cancer cell lines to TNF-alpha-dependent apoptosis.

TNF-alpha levels in prostate cancer correlate with the extent of disease and are significantly elevated in the metastatic stage. TNF receptor superfamily controls two distinct signalling cascades, leading to opposite effects, i.e. apoptosis and survival; in prostate cancer TNF-alpha-mediated signalling induces cell survival and resistance to therapy. The apoptosis of prostate epithelial cancer cells LNCaP and PC3 was investigated upon treatment with the autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin, in combination with TNF-alpha. Cells were exposed to these molecules for 18, 24 and 48 h. Autophagy was assessed via LC3 Western blot analysis; propidium iodide and TUNEL stainings followed by flow cytometry or caspase-8 and caspase-3 activation assays were performed to evaluate apoptosis. TNF-alpha-induced apoptosis was potentiated by 3-methyladenine in the androgen-responsive LNCaP cells, whereas no effect was observed in the androgen-insensitive PC3 cells. Interestingly such pro-apoptosis effect in LNCaP cells was associated with reduced c-Flip levels through proteasomal degradation via increased reactive oxygen species production and p38 activation; such c-Flip reduction was reversed in the presence of either the proteasome inhibitor MG132 or the reactive oxygen species scavenger N-acetyl-cysteine. Conversely in PC3 but not in LNCaP cells, rapamycin stimulated TNF-alpha-dependent apoptosis; such effect was associated with reduced c-Flip promoter activity and FoxO3a activation. We conclude that TNF-alpha-induced apoptosis may be potentiated, in prostate cancer epithelial cells, through autophagy modulators. Increased sensitivity to TNF-alpha-dependent apoptosis correlates with reduced c-Flip levels which are consequent to a post-transcriptional and a transcriptional mechanism in LNCaP and PC3 cells respectively.

Endothelial Cell Metabolism in Vascular Functions.

The endothelium is the innermost layer of all blood and lymphatic vessels composed of a monolayer of specialized endothelial cells (ECs). It is regarded as a dynamic and multifunctional endocrine organ that takes part in essential processes, such as the control of blood fluidity, the modulation of vascular tone, the regulation of immune response and leukocyte trafficking into perivascular tissues, and angiogenesis. The inability of ECs to perform their normal biological functions, known as endothelial dysfunction, is multi-factorial; for instance, it implicates the failure of ECs to support the normal antithrombotic and anti-inflammatory status, resulting in the onset of unfavorable cardiovascular conditions such as atherosclerosis, coronary artery disease, hypertension, heart problems, and other vascular pathologies. Notably, it is emerging that the ability of ECs to adapt their metabolic status to persistent changes of the tissue microenvironment could be vital for the maintenance of vascular functions and to prevent adverse vascular events. The main purpose of the present article is to shed light on the unique metabolic plasticity of ECs as a prospective therapeutic target; this may lead to the development of novel strategies for cardiovascular diseases and cancer.

Message in a Bottle: Endothelial Cell Regulation by Extracellular Vesicles.

Intercellular communication is a key biological mechanism that is fundamental to maintain tissue homeostasis. Extracellular vesicles (EVs) have emerged as critical regulators of cell-cell communication in both physiological and pathological conditions, due to their ability to shuttle a variety of cell constituents, such as DNA, RNA, lipids, active metabolites, cytosolic, and cell surface proteins. In particular, endothelial cells (ECs) are prominently regulated by EVs released by neighboring cell types. The discovery that cancer cell-derived EVs can control the functions of ECs has prompted the investigation of their roles in tumor angiogenesis and cancer progression. In particular, here, we discuss evidence that supports the roles of exosomes in EC regulation within the tumor microenvironment and in vascular dysfunction leading to atherosclerosis. Moreover, we survey the molecular mechanisms and exosomal cargoes that have been implicated in explanations of these regulatory effects.

Targeting of tumor necrosis factor receptor 1 to low density plasma membrane domains in human endothelial cells.

TNFR1 (tumor necrosis factor receptor 1) localizes to caveolae of human endothelial-derived EA.hy926 cells. Transduced TNFR1 molecules lacking amino acid residues 229-244 (spanning the transmembrane/intercellular boundary) are expressed on the cell surface equivalently to full-length TNFR1 molecules but incompletely localize to caveolae. A peptide containing this sequence pulls down CAV-1 (caveolin-1) and TNFR1 from cell lysates but fails to do so following disruption of caveolae with methyl-beta-cyclodextrin. We previously reported that methyl-beta-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-kappaB in EA.hy926 cells. Both CAV-1 and FLOT-2 (flotillin-2), organizing proteins of caveolae and lipid rafts, respectively, associate with caveolae in EA.hy926 cells. Small interfering RNA-mediated knockdown of CAV-1 but not FLOT-2 strikingly reduces caveolae number. Both knockdowns reduce total TNFR1 protein expression, but neither prevents TNFR1 localization to low density membrane domains, TNF-induced internalization of TNFR1, or NF-kappaB activation by TNF. Both CAV-1 and FLOT-2 knockdowns reduce TNF-mediated activation of stress-activated protein kinase (SAPK). However, both knockdowns reduce expression of TRAF2 (TNF receptor-associated factor-2) protein, and small interfering RNA targeting of TRAF2 also selectively inhibits SAPK activation. We conclude that TNFR1 contains a membrane-proximal sequence that targets the receptor to caveolae/lipid rafts. Neither TNFR1 targeting to nor internalization from these low density membrane domains depends upon CAV-1 or FLOT-2. Furthermore, both NF-kappaB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization.

Evidence of Reelin Signaling in GBM and Its Derived Cancer Stem Cells.

Glioblastoma (GBM) is the most aggressive and malignant form of primary brain cancer, characterized by an overall survival time ranging from 12 to 18 months. Despite the progress in the clinical treatment and the growing number of experimental data aimed at investigating the molecular bases of GBM development, the disease remains characterized by a poor prognosis. Recent studies have proposed the existence of a population of GBM cancer stem cells (CSCs) endowed with self-renewal capability and a high tumorigenic potential that are believed to be responsible for the resistance against common chemotherapy and radiotherapy treatments. Reelin is a large secreted extracellular matrix glycoprotein, which contributes to positioning, migration, and laminar organization of several central nervous system structures during brain development. Mutations of the reelin gene have been linked to disorganization of brain structures during development and behavioral anomalies. In this study, we explored the expression of reelin in GBM and its related peritumoral tissue and performed the same analysis in CSCs isolated from both GBM (GCSCs) and peritumoral tissue (PCSCs) of human patients. Our findings reveal (i) the higher expression of reelin in GBM compared to the peritumoral tissue by immunohistochemical analysis, (ii) the mRNA expression of both reelin and its adaptor molecule Dab1 in either CSC subtypes, although at a different extent; and (iii) the contribution of CSCs-derived reelin in the migration of human primary GBM cell line U87MG. Taken together, our data indicate that the expression of reelin in GBM may represent a potential contribution to the regulation of GBM cancer stem cells behavior, further stimulating the interest on the reelin pathway as a potential target for GBM treatment.

Lipopolysaccharide can trigger a cathepsin B-dependent programmed death response in human endothelial cells.

In this study, we examined the mechanisms that contribute to lipopolysaccharide (LPS)-induced death responses in cultured human umbilical vein endothelial cells (HUVECs). In the presence of the protein synthesis inhibitor cycloheximide, LPS primarily induces caspase-dependent apoptotic cell death of HUVECs, which is blocked by siRNA-mediated knockdown of myeloid differentiation factor 88 adaptor protein but not of Toll-like receptor-associated interferon-inducing factor. Knockdown of Fas-associated death domain protein (FADD) by either siRNA or overexpression of a truncated version of FADD that lacks the N-terminal death effector domain (FADD(DN)) increases the sensitivity of HUVECs to LPS plus cycloheximide-mediated death. However, based on the use of proteinase inhibitors, cell death changes from being principally caspase-dependent to being principally cathepsin B (Cat B)-dependent. Knockdown of cellular FLICE inhibitory protein potentiates the caspase-dependent pathway but does not activate the Cat B-dependent death response. Knockdown of either myeloid differentiation factor 88 or Toll-like receptor-associated interferon-inducing factor expression does not affect the LPS-triggered Cat B death response in FADD-deficient HUVECs. Finally, in the presence of either the phosphatidylinositol 3 kinase inhibitor LY294002 or the inflammatory cytokine interferon-gamma, LPS activates both caspase- and Cat B-dependent death pathways. We conclude that LPS can activate a Cat-B-dependent programmed death response in human endothelial cells that is independent of both myeloid differentiation factor 88 and Toll-like receptor-associated interferon-inducing factor, is blocked by both FADD and phosphatidylinositol 3 kinase, and is potentiated by interferon-gamma.

Caveolae and Lipid Rafts in Endothelium: Valuable Organelles for Multiple Functions.

Caveolae are flask-shaped invaginations of the plasma membrane found in numerous cell types and are particularly abundant in endothelial cells and adipocytes. The lipid composition of caveolae largely matches that of lipid rafts microdomains that are particularly enriched in cholesterol, sphingomyelin, glycosphingolipids, and saturated fatty acids. Unlike lipid rafts, whose existence remains quite elusive in living cells, caveolae can be clearly distinguished by electron microscope. Despite their similar composition and the sharing of some functions, lipid rafts appear more heterogeneous in terms of size and are more dynamic than caveolae. Following the discovery of caveolin-1, the first molecular marker as well as the unique scaffolding protein of caveolae, we have witnessed a remarkable increase in studies aimed at investigating the role of these organelles in cell functions and human disease. The goal of this review is to discuss the most recent studies related to the role of caveolae and caveolins in endothelial cells. We first recapitulate the major embryological processes leading to the formation of the vascular tree. We next discuss the contribution of caveolins and cavins to membrane biogenesis and cell response to extracellular stimuli. We also address how caveolae and caveolins control endothelial cell metabolism, a central mechanism involved in migration proliferation and angiogenesis. Finally, as regards the emergency caused by COVID-19, we propose to study the caveolar platform as a potential target to block virus entry into endothelial cells.

Pathological and Molecular Features of Glioblastoma and Its Peritumoral Tissue.

Glioblastoma (GBM) is one of the most aggressive and lethal human brain tumors. At present, GBMs are divided in primary and secondary on the basis of the mutational status of the isocitrate dehydrogenase (IDH) genes. In addition, IDH1 and IDH2 mutations are considered crucial to better define the prognosis. Although primary and secondary GBMs are histologically indistinguishable, they retain distinct genetic alterations that account for different evolution of the tumor. The high invasiveness, the propensity to disperse throughout the brain parenchyma, and the elevated vascularity make these tumors extremely recidivist, resulting in a short patient median survival even after surgical resection and chemoradiotherapy. Furthermore, GBM is considered an immunologically cold tumor. Several studies highlight a highly immunosuppressive tumor microenvironment that promotes recurrence and poor prognosis. Deeper insight into the tumor immune microenvironment, together with the recent discovery of a conventional lymphatic system in the central nervous system (CNS), led to new immunotherapeutic strategies. In the last two decades, experimental evidence from different groups proved the existence of cancer stem cells (CSCs), also known as tumor-initiating cells, that may play an active role in tumor development and progression. Recent findings also indicated the presence of highly infiltrative CSCs in the peritumoral region of GBM. This region appears to play a key role in tumor growing and recurrence. However, until recently, few studies investigated the biomolecular characteristics of the peritumoral tissue. The aim of this review is to recapitulate the pathological features of GBM and of the peritumoral region associated with progression and recurrence.

NAADP-induced Ca(2+ signaling in response to endothelin is via the receptor subtype B and requires the integrity of lipid rafts/caveolae.

We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling.

Pivotal role of human stearoyl-CoA desaturases (SCD1 and 5) in breast cancer progression: oleic acid-based effect of SCD1 on cell migration and a novel pro-cell survival role for SCD5.

The influence of cell membrane fluidity on cancer progression has been established in different solid tumors. We previously reported that "cancer-associated fibroblasts" (CAFs) induced epithelial-mesenchymal transition and increased cell membrane fluidity and migration in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. We also found that the membrane fluidity regulating enzyme stearoyl-CoA desaturase 1 (SCD1) was upregulated in tumor cells co-cultured with CAFs and established its essential role for both intrinsic and CAF-driven tumor cell motility. Here, we further explored the mechanisms involved in the SCD1-based modulation of breast cancer cell migration and investigated the role of the other human SCD isoform, SCD5. We showed that the addition of oleic acid, the main SCD1 product, nullified the inhibitory effects produced on MCF-7 and MDA-MB-231 cell migration by SCD1 depletion (pharmacological or siRNA-based). Conversely, SCD5 seemed not involved in the regulation of cancer cell motility. Interestingly, a clear induction of necrosis was observed as a result of the depletion of SCD5 in MCF-7 cells, where the expression of SCD5 was found to be upregulated by CAFs. The necrotic effect was rescued by a 48-h treatment of cells with oleic acid. These results provide further insights in understanding the role of SCD1 in both intrinsic and CAF-stimulated mammary tumor cell migration, unveiling the metabolic basis of this desaturase-triggered effect. Moreover, our data suggest the ability of CAFs to promote the maintenance of tumor cell survival by the induction of SCD5 levels.

Cancer stem cells from peritumoral tissue of glioblastoma multiforme: the possible missing link between tumor development and progression.

In glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for gliomagenesis, resistance to treatment and recurrence. Unfortunately, the prognosis for GBM remains poor and recurrence frequently occurs in the peritumoral tissue within 2 cm from the tumor edge. In this area, a population of CSCs has been demonstrated which may recapitulate the tumor after surgical resection. In the present study, we aimed to characterize CSCs derived from both peritumoral tissue (PCSCs) and GBM (GCSCs) in order to deepen their significance in GBM development and progression. The stemness of PCSC/GCSC pairs obtained from four human GBM surgical specimens was investigated by comparing the expression of specific stem cell markers such as Nestin, Musashi-1 and SOX2. In addition, the growth rate, the ultrastructural features and the expression of other molecules such as c-Met, pMet and MAP kinases, involved in cell migration/invasion, maintenance of tumor stemness and/or resistance to treatments were evaluated. Since it has been recently demonstrated the involvement of the long non-coding RNAs (lncRNAs) in the progression of gliomas, the expression of H19 lncRNA, as well as of one of its two mature products miR-675-5p was evaluated in neurospheres. Our results show significant differences between GCSCs and PCSCs in terms of proliferation, ultrastructural peculiarities and, at a lower extent, stemness profile. These differences might be important in view of their potential role as a therapeutic target.