Glioma Associated Stem Cells (GASCs) Isolation and Culture.

Glioma Associated Stem Cells (GASCs) represent a population of nontumorigenic multipotent stem cells hosted in the microenvironment of human gliomas. In vitro, these cells are able, through the release of exosomes, to increase the biological aggressiveness of glioma-initiating cells. The clinical importance of this finding is supported by the strong prognostic value associated with the GASCs surface immunophenotype thus suggesting that this patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor strom

Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics

Topoisomerase II beta mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs

Background: Glioblastoma stem cells (GSC) have been extensively recognized as a plausible cause of glioblastoma resistance to therapy and recurrence resulting in high glioblastoma mortality. Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the (GSC) subpopulation. Methods: In this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures. MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage. Results: We found several differentially expressed genes and we identified topoisomerase II beta (Top2 beta) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2 beta was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2 beta expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2 beta increased the sensitivity of NCH421k cells to replication stress inducing drugs, such as cisplatin, methyl-methanesulfonate, hydrogen peroxide, and temozolomide. Consistently, we found an increased load of DNA damage and increased Chk1 activation upon Top2 beta depletion in NCH421k cells. Conclusion: We suggest that Top2 beta may represent a new target for gene therapy in glioblastoma. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics

Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression

Nanothermodynamics mediates drug delivery

© Springer International Publishing Switzerland 2015. The efficiency of penetration of nanodrugs through cell membranes imposes further complexity due to nanothermodynamic and entropic potentials at interfaces. Action of nanodrugs is effective after cell membrane penetration. Contrary to diffusion of water diluted common molecular drugs, nanosize imposes an increasing transport complexity at boundaries and interfaces (e.g., cell membrane). Indeed, tiny dimensional systems brought the concept of “nanothermodynamic potential,” which is proportional to the number of nanoentities in a macroscopic system, from either the presence of surface and edge effects at the boundaries of nanoentities or the restriction of the translational and rotational degrees of freedom of molecules within them. The core element of nanothermodynamic theory is based on the assumption that the contribution of a nanosize ensemble to the free energy of a macroscopic system has its origin at the excess interaction energy between the nanostructured entities. As the size of a system is increasing, the contribution of the nanothermodynamic potential to the free energy of the system becomes negligible. Furthermore, concentration gradients at boundaries, morphological distribution of nanoentities, and restriction of the translational motion from trapping sites are the source of strong entropic potentials at the interfaces. It is evident therefore that nanothermodynamic and entropic potentials either prevent or allow enhanced concentration very close to interfaces and thus strongly modulate nanoparticle penetration within the intracellular region. In this work, it is shown that nano-sized polynuclear iron (III)-hydroxide in sucrose nanoparticles have a nonuniform concentration around the cell membrane of macrophages in vivo, compared to uniform concentration at hydrophobic prototype surfaces. The difference is attributed to the presence of entropic and nanothermodynamic potentials at interfaces

Selective aggregation of PAMAM dendrimer nanocarriers and PAMAM/ZnPc nanodrugs on human atheromatous carotid tissues: a photodynamic therapy for atherosclerosis

© 2015, Spyropoulos-Antonakakis et al.; licensee Springer. Photodynamic therapy (PDT) involves the action of photons on photosensitive molecules, where atomic oxygen or OH− molecular species are locally released on pathogenic human cells, which are mainly carcinogenic, thus causing cell necrosis. The efficacy of PDT depends on the local nanothermodynamic conditions near the cell/nanodrug system that control both the level of intracellular translocation of nanoparticles in the pathogenic cell and their agglomeration on the cell membrane. Dendrimers are considered one of the most effective and promising drug carriers because of their relatively low toxicity and negligible activation of complementary reactions. Polyamidoamine (PAMAM) dendrite delivery of PDT agents has been investigated in the last few years for tumour selectivity, retention, pharmacokinetics and water solubility. Nevertheless, their use as drug carriers of photosensitizing molecules in PDT for cardiovascular disease, targeting the selective necrosis of macrophage cells responsible for atheromatous plaque growth, has never been investigated. Furthermore, the level of aggregation, translocation and nanodrug delivery efficacy of PAMAM dendrimers or PAMAM/zinc phthalocyanine (ZnPc) conjugates on human atheromatous tissue and endothelial cells is still unknown. In this work, the aggregation of PAMAM zero generation dendrimers (G0) acting as drug delivery carriers, as well as conjugated G0 PAMAM dendrimers with a ZnPc photosensitizer, to symptomatic and asymptomatic human carotid tissues was investigated by using atomic force microscopy (AFM). For the evaluation of the texture characteristics of the AFM images, statistical surface morphological and fractal analytical methodologies and Minkowski functionals were used. All statistical quantities showed that the deposition of nanodrug carriers on healthy tissue has an inverse impact when comparing to the deposition on atheromatous tissue with different aggregation features between G0 and G0/ZnPc nanoparticles and with considerably larger G0/ZnPc aggregations on the atheromatous plaque. The results highlight the importance of using PAMAM dendrimer carriers as a novel and promising PDT platform for atherosclerosis therapies

Topoisomerase II\u3b2 mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy

Glioma-associated stem cells: A novel class of tumor-supporting cells able to predict prognosis of human low-grade gliomas.

Background: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. Methods and Findings: We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. Conclusions: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma

Diffuse Low-Grade Gliomas in Adults

Numerous diagnostic, prognostic, and therapeutic issues concerning lowgrade gliomas still remain to be clari fi ed. In this regard, in vitro and in vivo models of low-grade gliomas would represent a way to get insight into these crucial aspects. However, many of the in vitro and in vivo models assayed in the literature are focused on high-grade gliomas. For this reason, in this chapter we reviewed the literature on low-grade glioma culture describing the results so far obtained, highlighting limitations and envisioning promising innovative directions that research is undertaking in this critical fi eld. Speci fi cally, fi rst, we critically presented the conventional methods adopted to study low-grade gliomas, such as continuous human tumor cell lines and short-term glioma cultures. Then, we discussed the culture methods utilized to isolate and in vitro expand glioma cancer stem cells from adult and pediatric tumors, underlining the need of optimizing new culture conditions because of the partial failure of the simple transfer to low-grade gliomas of the methods ef fi cient in isolating glioma stem cells from highgrade gliomas. Finally, we presented the innovative, although still in its infancy, possibility to culture nonneoplastic stromal cells as a way to obtain cell lines representative of the biological behavior of the patient tumors. In conclusion, the aim of this chapter was to explain why the culture of patient-derived cancer cells represents a unique opportunity to create in vitro and in vivo models closely mimicking the biological properties of the patient tumor, thus allowing a patient-based approach