Validation of Thiosemicarbazone Compounds as P-Glycoprotein Inhibitors in Human Primary Brain-Blood Barrier and Glioblastoma Stem Cells.

P-glycoprotein (Pgp) is highly expressed on blood-brain barrier (BBB) and glioblastoma (GB) cells, particularly on cancer stem cells (SC). Pgp recognizes a broad spectrum of substrates, limiting the therapeutic efficacy of several chemotherapeutic drugs in eradicating GB SC. Finding effective and safe inhibitors of Pgp that improve drug delivery across the BBB and target GB SC is open to investigation. We previously identified a series of thiosemicarbazone compounds that inhibit Pgp with an EC50 in the nanomolar range, and herein, we investigate the efficacy of three of them in bypassing Pgp-mediated drug efflux in primary human BBB and GB cells. At 10 nM, the compounds were not cytotoxic for the brain microvascular endothelial hCMEC/D3 cell line, but they markedly enhanced the permeability of the Pgp-substrate doxorubicin through the BBB. Thiosemicarbazone derivatives increased doxorubicin uptake in GB, with greater effects in the Pgp-rich SC clones than in the differentiated clones derived from the same tumor. All compounds increased intratumor doxorubicin accumulation and consequent toxicity in GB growing under competent BBB, producing significant killing of GB SC. The compounds crossed the BBB monolayer. The most stable derivative, 10a, had a half-life in serum of 4.2 h. The coadministration of doxorubicin plus 10a significantly reduced the growth of orthotopic GB-SC xenografts, without eliciting toxic side effects. Our work suggests that the thiosemicarbazone compounds are able to transform doxorubicin, a prototype BBB-impermeable drug, into a BBB-permeable drug. Bypassing Pgp-mediated drug efflux in both BBB and GB SC, thiosemicarbazones might increase the success of chemotherapy in targeting GB SC, which represent the most aggressive and difficult components to eradicate.

Glioblastoma niches: from the concept to the phenotypical reality.

Recently, the concept of niches as sites of tumor progression, invasion, and angiogenesis in glioblastoma (GB) has been extensively debated. Niches, considered the sites in which glioblastoma stem cells (GSCs) reside, have been classified as perivascular, perinecrotic, and invasive. However, from a neuropathological point of view, it is not easy to establish when a tumor structure can be considered a niche. The relevant literature has been reviewed in the light of our recent experience on the subject. As for perinecrotic niches, the occurrence of GSCs around necrosis is interpreted as triggered by hypoxia through HIF-1α. Our alternative hypothesis is that, together with progenitors, they are the cell constituents of hyper-proliferative areas of GB, where perinecrotic niches have developed, and they would, therefore, represent the remnants of GSCs/progenitors spared by the developing necrosis. Perivascular structures originate from both transport vessels and exchange vessels, i.e., venules, arterioles, or the undefinable neo-formed small vessels, but only those in which a direct contact between GSCs/progenitors and endothelial cells occurs can be called niches. Both pericytes and microglia/macrophages play a role in niche function: Macrophages of blood origin invade GB only after the appearance of "mother vessels" with consequent blood-brain barrier disruption. Not all vessel/tumor cell structures can be considered niches, that is, crucial sites of tumor progression, invasion, and angiogenesis.

The Significance of Chondroitin Sulfate Proteoglycan 4 (CSPG4) in Human Gliomas.

Neuron glial antigen 2 (NG2) is a chondroitin sulphate proteoglycan 4 (CSPG4) that occurs in developing and adult central nervous systems (CNSs) as a marker of oligodendrocyte precursor cells (OPCs) together with platelet-derived growth factor receptor α (PDGFRα). It behaves variably in different pathological conditions, and is possibly involved in the origin and progression of human gliomas. In the latter, NG2/CSPG4 induces cell proliferation and migration, is highly expressed in pericytes, and plays a role in neoangiogenesis. NG2/CSPG4 expression has been demonstrated in oligodendrogliomas, astrocytomas, and glioblastomas (GB), and it correlates with malignancy. In rat tumors transplacentally induced by N-ethyl-N-nitrosourea (ENU), NG2/CSPG4 expression correlates with PDGFRα, Olig2, Sox10, and Nkx2.2, and with new vessel formation. In this review, we attempt to summarize the normal and pathogenic functions of NG2/CSPG4, as well as its potential as a therapeutic target.

Solid Lipid Nanoparticles Carrying Temozolomide for Melanoma Treatment. Preliminary In Vitro and In Vivo Studies.

AIM: To develop an innovative delivery system for temozolomide (TMZ) in solid lipid nanoparticles (SLN), which has been preliminarily investigated for the treatment of melanoma. MATERIALS AND METHODS: SLN-TMZ was obtained through fatty acid coacervation. Its pharmacological effects were assessed and compared with free TMZ in in vitro and in vivo models of melanoma and glioblastoma. RESULTS: Compared to the standard free TMZ, SLN-TMZ exerted larger effects, when cell proliferation of melanoma cells, and neoangiogeneis were evaluated. SLN-TMZ also inhibited growth and vascularization of B16-F10 melanoma in C57/BL6 mice, without apparent toxic effects. CONCLUSION: SLN could be a promising strategy for the delivery of TMZ, allowing an increased stability of the drug and thereby its employment in the treatment of aggressive malignacies.

Diagnostic revision of 206 adult gliomas (including 40 oligoastrocytomas) based on ATRX, IDH1/2 and 1p/19q status.

The diagnosis of 206 low and high grade adult gliomas, including 40 oligoastrocytomas, was revised based on the immunohistochemical reactivity for the ATRX protein, IDH1/2 mutation status and 1p/19q chromosomal status. All oligodendrogliomas kept the initial diagnosis. Astrocytomas did not change diagnosis in 30 of 36 cases (83.3 %); four of 36 (11.1 %) cases were reclassified as oligodendroglioma, one (2.8 %) as DNT and the other (2.8 %) as reactive gliosis. Oligoastrocytomas changed diagnosis in 35 of 40 (87.5 %) cases, being reclassified 22 of 40 (55 %) as astrocytoma, 11 of 40 (27.5 %) as oligodendroglioma and two of 40 (5 %) as reactive gliosis. Four (10 %) remained unclassifiable. In one case only (2.5 %), the diagnosis of oligoastrocytoma could not be excluded since tumor astrocytes and tumor oligodendrocytes coexisted in mixed tumor areas. In the GBM tumor subgroup, GBMO disappeared because they were not substantiated by molecular genetics. Pilocytic astrocytomas retained ATRX expression. Loss of nuclear ATRX protein expression was strongly associated to IDH1/2 mutations (p = 0.0001) and mutually exclusive with total 1p/19q co-deletion (p = 0.0001). In astrocytic tumors, loss of immunoreactivity for the ATRX protein was significantly associated to the ALT phenotype (p = 0.0003). The constitutive ATRX expression in microglia/macrophages may be misleading, especially in the identification of an oligodendroglial tumor infiltration. Of paramount importance in the recognition of oligodendroglial and astrocytic tumor cells were the double immunostainings for ATRX/GFAP, ATRX/IDH1R132H, ATRX/Iba-1 and ATRX/CD68.

The neuropathological basis to the functional role of microglia/macrophages in gliomas.

The paper wants to be a tracking shot of the main recent acquisitions on the function and significance of microglia/macrophages in gliomas. The observations have been principally carried out on in vitro cultures and on tumor transplants in animals. Contrary to what is deduced from microglia in non-neoplastic pathologic conditions of central nervous system (CNS), most conclusions indicate that microglia acts favoring tumor proliferation through an immunosuppression induced by glioma cells. By immunohistochemistry, different microglia phenotypes are recognized in gliomas, from ramified microglia to frank macrophagic aspect. One wonders whether the functional conclusions drawn from many microglia studies, but not in conditions of human pathology, apply to all the phenotypes recognizable in them. It is difficult to verify in human pathology a prognostic significance of microglia. Only CD163-positive microglia/macrophages inversely correlate with glioma patients' survival, whereas the total number of microglia does not change with the malignancy grade.

Temozolomide down-regulates P-glycoprotein in human blood-brain barrier cells by disrupting Wnt3 signaling.

Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/ß-catenin signaling, and reduces the binding of ß-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma.

MGMT promoter hypermethylation and its associations with genetic alterations in a series of 350 brain tumors.

MGMT (O6-methylguanine-DNA methyltransferase) promoter hypermethylation is a helpful prognostic marker for chemotherapy of gliomas, although with some controversy for low-grade tumors. The objective of this study was to retrospectively investigate MGMT promoter hypermethylation status for a series of 350 human brain tumors, including 275 gliomas of different malignancy grade, 21 glioblastoma multiforme (GBM) cell lines, and 75 non-glial tumors. The analysis was performed by methylation-specific PCR and capillary electrophoresis. MGMT expression at the protein level was also evaluated by both immunohistochemistry (IHC) and western blotting analysis. Associations of MGMT hypermethylation with IDH1/IDH2 mutations, EGFR amplification, TP53 mutations, and 1p/19q co-deletion, and the prognostic significance of these, were investigated for the gliomas. MGMT promoter hypermethylation was identified in 37.8% of gliomas, but was not present in non-glial tumors, with the exception of one primitive neuroectodermal tumor (PNET). The frequency was similar for all the astrocytic gliomas, with no correlation with histological grade. Significantly higher values were obtained for oligodendrogliomas. MGMT promoter hypermethylation was significantly associated with IDH1/IDH2 mutations (P = 0.0207) in grade II­III tumors, whereas it had a borderline association with 1p deletion (P = 0.0538) in oligodendrogliomas. No other association was found. Significant correlation of MGMT hypermethylation with MGMT protein expression was identified by IHC in GBMs and oligodendrogliomas (P = 0.0001), but not by western blotting. A positive correlation between MGMT protein expression, as detected by either IHC or western blotting, was also observed. The latter was consistent with MGMT promoter hypermethylation status in GBM cell lines. In low-grade gliomas, MGMT hypermethylation, but not MGMT protein expression, was associated with a trend, only, toward better survival, in contrast with GBMs, for which it had favorable prognostic significance.

SEL1L plays a major role in human malignant gliomas.

Suppressor of Lin-12-like (C. elegans) (SEL1L) participates in the endoplasmic reticulum-associated protein degradation pathway, malignant transformation and stem cell biology. We explored the role of SEL1L in 110 adult gliomas, of different molecular subtype and grade, in relation to cell proliferation, stemness, glioma-associated microglia/macrophages (GAMs), prognostic markers and clinical outcome. SEL1L protein expression was assessed by immunohistochemistry and Western blotting. Genetic and epigenetic alterations were detected by molecular genetics techniques. SEL1L was overexpressed in anaplastic gliomas (World Health Organization [WHO] grade III) and in glioblastoma (GB, WHO grade IV) with the highest labelling index (LI) in the latter. Immunoreactivity was significantly associated with histological grade (p = 0.002) and cell proliferation index Ki-67/MIB-1 (p = 0.0001). In GB, SEL1L co-localised with stemness markers Nestin and Sox2. Endothelial cells and vascular pericytes of proliferative tumour blood vessels expressed SEL1L suggesting a role in tumour neo-vasculature. GAMs consistently expressed SEL1L. SEL1L overexpression was significantly associated with TERT promoter mutations (p = 0.0001), EGFR gene amplification (p = 0.0013), LOH on 10q (p = 0.0012) but was mutually exclusive with IDH1/2 mutations (p = 0.0001). SEL1L immunoreactivity correlated with tumour progression and cell proliferation, conditioning poor patient survival and response to therapy. This study emphasises SEL1L as a potential biomarker for the most common subgroup of TERT mutant/EGFR amplified/IDH-WT GBs.

Chondroitin Sulphate Proteoglycan 4 (NG2/CSPG4) Localization in Low- and High-Grade Gliomas.

BACKGROUND: Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. METHODS: NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. RESULTS: NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. CONCLUSION: The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.

Survivin expression in glioblastomas correlates with proliferation, but not with apoptosis.

BACKGROUND: Survivin is expressed in proliferating tissues and in tumors. It is a member of the inhibitory apoptosis protein (IAP) family known to regulate mitosis and to inhibit apoptosis. It has therefore been regarded as a target for therapies. In malignant gliomas it increases with malignancy, even though in glioblastomas it does not seem to correlate with outcome. MATERIALS AND METHODS: Survivin was immunohistochemically studied in 39 selected viable glioblastoma areas belonging to 20 cases which were assayed for apoptosis, using a TUNEL assay, caspase-3, poly(ADP-ribose)polymerase 1 (PARP-1), Bid (BH3-interacting domain death agonist) and with the proliferation index Ki-67/MIB-1 and mitotic index (MI). RESULTS: A positive linear correlation was found between the survivin labelling index (LI) and the Ki-67/MIB-1 LI and MI. No inverse correlation was found with apoptosis. CONCLUSION: This double behavior can be attributed to mechanisms mediating survivin activity, either as a mitosis regulator and apoptosis inhibitor, and should be taken into account in therapeutic strategies using survivin.

Microglia immunophenotyping in gliomas.

Microglia, once assimilated to peripheral macrophages, in gliomas has long been discussed and currently it is hypothesized to play a pro-tumor role in tumor progression. Uncertain between M1 and M2 polarization, it exchanges signals with glioma cells to create an immunosuppressive microenvironment and stimulates cell proliferation and migration. Four antibodies are currently used for microglia/macrophage identification in tissues that exhibit different cell forms and cell localization. The aim of the present work was to describe the distribution of the different cell forms and to deduce their significance on the basis of what is known on their function from the literature. Normal resting microglia, reactive microglia, intermediate and bumpy forms and macrophage-like cells can be distinguished by Iba1, CD68, CD16 and CD163 and further categorized by CD11b, CD45, c-MAF and CD98. The number of microglia/macrophages strongly increased from normal cortex and white matter to infiltrating and solid tumors. The ramified microglia accumulated in infiltration areas of both high- and low-grade gliomas, when hypertrophy and hyperplasia occur. In solid tumors, intermediate and bumpy forms prevailed and there is a large increase of macrophage-like cells in glioblastoma. The total number of microglia cells did not vary among the three grades of malignancy, but macrophage-like cells definitely prevailed in high-grade gliomas and frequently expressed CD45 and c-MAF. CD98+ cells were present. Microglia favors tumor progression, but many aspects suggest that the phagocytosing function is maintained. CD98+ cells can be the product of fusion, but also of phagocytosis. Microglia correlated with poorer survival in glioblastoma, when considering CD163+ cells, whereas it did not change prognosis in isocitrate dehydrogenase-mutant low grade gliomas.

Glioblastoma: Microenvironment and Niche Concept.

The niche concept was originally developed to describe the location of normal neural stem cells (NSCs) in the subependymal layer of the sub-ventricular zone. In this paper, its significance has been extended to the location of tumor stem cells in glioblastoma (GB) to discuss the relationship between GB stem cells (GSCs) and endothelial cells (ECs). Their interaction is basically conceived as responsible for tumor growth, invasion and recurrence. Niches are described as the points of utmost expression of the tumor microenvironment (TME), therefore including everything in the tumor except for tumor cells: NSCs, reactive astrocytes, ECs, glioma-associated microglia/macrophages (GAMs), myeloid cells, pericytes, fibroblasts, etc. and all intrinsic and extrinsic signaling pathways. Perivascular (PVNs), perinecrotic (PNNs) and invasive niches were described from the pathological point of view, highlighting the basic significance of the EC/tumor stem cell couple. PNN development was reinterpreted based on the concept that hyperproliferative areas of GB are composed of GSCs/progenitors. TME was depicted in its function as the main regulator of everything that happens in the tumor. A particular emphasis was given to GAMs, pericytes and reactive astrocytes as important elements affecting proliferation, growth, invasion and resistance to therapies of tumor cells.

Carbonic Anhydrase XII Inhibitors Overcome P-Glycoprotein-Mediated Resistance to Temozolomide in Glioblastoma.

The role of carbonic anhydrase XII (CAXII) in the chemoresistance of glioblastoma is unexplored. We found CAXII and P-glycoprotein (Pgp) coexpressed in neurospheres derived from 3 of 3 patients with different genetic backgrounds and low response to temozolomide (time to recurrence: 6-9 months). CAXII was necessary for the Pgp efflux of temozolomide and second-line chemotherapeutic drugs, determining chemoresistance in neurospheres. Psammaplin C, a potent inhibitor of CAXII, resensitized primary neurospheres to temozolomide by reducing temozolomide efflux via Pgp. This effect was independent of other known temozolomide resistance factors present in the patients. The overall survival in orthotopic patient-derived xenografts of temozolomide-resistant neurospheres, codosed with Psammaplin C and temozolomide, was significantly increased over temozolomide-treated (P < 0.05) and untreated animals (P < 0.02), without detectable signs of systemic toxicity. We propose that a CAXII inhibitor in combination with temozolomide may provide a new and effective approach to reverse chemoresistance in glioblastoma stem cells. This novel mechanism of action, via the interaction of CAXII and Pgp, ultimately blocks the efflux function of Pgp to improve glioblastoma patient outcomes.

Correction: SEL1L SNP rs12435998, a predictor of glioblastoma survival and response to radio-chemotherapy.

[This corrects the article DOI: 10.18632/oncotarget.3611.].

Chemotherapeutic Drugs: DNA Damage and Repair in Glioblastoma.

Despite improvements in therapeutic strategies, glioblastoma (GB) remains one of the most lethal cancers. The presence of the blood-brain barrier, the infiltrative nature of the tumor and several resistance mechanisms account for the failure of current treatments. Distinct DNA repair pathways can neutralize the cytotoxicity of chemo- and radio-therapeutic agents, driving resistance and tumor relapse. It seems that a subpopulation of stem-like cells, indicated as glioma stem cells (GSCs), is responsible for tumor initiation, maintenance and recurrence and they appear to be more resistant owing to their enhanced DNA repair capacity. Recently, attention has been focused on the pivotal role of the DNA damage response (DDR) in tumorigenesis and in the modulation of therapeutic treatment effects. In this review, we try to summarize the knowledge concerning the main molecular mechanisms involved in the removal of genotoxic lesions caused by alkylating agents, emphasizing the role of GSCs. Beside their increased DNA repair capacity in comparison with non-stem tumor cells, GSCs show a constitutive checkpoint expression that enables them to survive to treatments in a quiescent, non-proliferative state. The targeted inhibition of checkpoint/repair factors of DDR can contribute to eradicate the GSC population and can have a great potential therapeutic impact aiming at sensitizing malignant gliomas to treatments, improving the overall survival of patients.

Comparison among conventional and advanced MRI, 18F-FDG PET/CT, phenotype and genotype in glioblastoma.

Glioblastoma (GB) is a highly heterogeneous tumor. In order to identify in vivo the most malignant tumor areas, the extent of tumor infiltration and the sites giving origin to GB stem cells (GSCs), we combined positron emission tomography/computed tomography (PET/CT) and conventional and advanced magnetic resonance imaging (MRI) with histology, immunohistochemistry and molecular genetics. Prior to dura opening and tumor resection, forty-eight biopsy specimens [23 of contrast-enhancing (CE) and 25 of non-contrast enhancing (NE) regions] from 12 GB patients were obtained by a frameless image-guided stereotactic biopsy technique. The highest values of 2-[18F]-fluoro-2-deoxy-D-glucose maximum standardized uptake value (18F-FDG SUVmax), relative cerebral blood volume (rCBV), Choline/Creatine (Cho/Cr), Choline/N-acetylaspartate (Cho/NAA) and Lipids/Lactate (LL) ratio have been observed in the CE region. They corresponded to the most malignant tumor phenotype, to the greatest molecular spectrum and stem cell potential. On the contrary, apparent diffusion coefficient (ADC) and fractional anisotropy (FA) in the CE region were very variable. 18F-FDG SUVmax, Cho/Cr and Cho/NAA ratio resulted the most suitable parameters to detect tumor infiltration. In edematous areas, reactive astrocytes and microglia/macrophages were influencing variables. Combined MRI and 18F-FDG PET/CT allowed to recognize the specific biological significance of the different identified areas of GB.

Comparison of Allogeneic and Syngeneic Rat Glioma Models by Using MRI and Histopathologic Evaluation.

Research in neurooncology traditionally requires appropriate in vivo animal models, on which therapeutic strategies are tested before human trials are designed and proceed. Several reproducible animal experimental models, in which human physiologic conditions can be mimicked, are available for studying glioblastoma multiforme. In an ideal rat model, the tumor is of glial origin, grows in predictable and reproducible patterns, closely resembles human gliomas histopathologically, and is weakly or nonimmunogenic. In the current study, we used MRI and histopathologic evaluation to compare the most widely used allogeneic rat glioma model, C6-Wistar, with the F98-Fischer syngeneic rat glioma model in terms of percentage tumor growth or regression and growth rate. In vivo MRI demonstrated considerable variation in tumor volume and frequency between the 2 rat models despite the same stereotactic implantation technique. Faster and more reproducible glioma growth occurred in the immunoresponsive environment of the F98-Fischer model, because the immune response is minimized toward syngeneic cells. The marked inability of the C6-Wistar allogeneic system to generate a reproducible model and the episodes of spontaneous tumor regression with this system may have been due to the increased humoral and cellular immune responses after tumor implantation.

Solid lipid nanoparticles by coacervation loaded with a methotrexate prodrug: preliminary study for glioma treatment.

AIM: Methotrexate-loaded biocompatible nanoparticles were tested for preliminary efficacy in glioma treatment. MATERIALS & METHODS: Behenic acid nanoparticles, prepared by the coacervation method, were loaded with the ester prodrug didodecylmethotrexate, which was previously tested in vitro against glioblastoma human primary cultures. Nanoparticle conjugation with an ApoE mimicking chimera peptide was performed to obtain active targeting to the brain. RESULTS & CONCLUSION: Biodistribution studies in healthy rats assessed the superiority of ApoE-conjugated formulation, which was tested on an F98/Fischer glioma model. Differences were observed in tumor growth rate (measured by MRI) between control and treated rats. In vitro tests on F98 cultured cells assessed their susceptibility to treatment, with consequent apoptosis, and allowed us to explain the apoptosis observed in glioma models.