Thonzonium bromide inhibits progression of malignant pleural mesothelioma through regulation of ERK1/2 and p38 pathways and mitochondrial uncoupling.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is a rare malignancy with a poor prognosis. Current therapies are unsatisfactory and novel cures are urgently needed. In a previous drug screening, we identified thonzonium bromide (TB) as one of the most active compounds against MPM cells. Since the biological effects of TB are poorly known, in this work we departed from some hints of previous studies and investigated several hypotheses. Moreover, we evaluated the efficacy of TB in an in vivo xenograft rodent model. METHODS: In vitro assessment was made on five MPM (Mero-14, Mero-25, Ren, NCI-H28, MSTO-211H) and one SV40-immortalized mesothelial cell line (MeT-5A). We evaluated TB ability to affect proliferation, apoptosis, mitochondrial functions and metabolism, and the mevalonate pathway. In vivo assay was carried out on MPM-xenograft NOD-SCID mice (4 mg/kg delivered intraperitoneally, twice a week for 4 weeks) and the overall survival was analysed with Kaplan-Meier curves. RESULTS: After TB treatment, we observed the suppression of ERK 1/2 phosphorylation, the increase of BAX expression and p38 phosphorylation. TB affected Ca2+ homeostasis in both mitochondrial and cytosolic compartments, it regulated the mitochondrial functioning, respiration, and ATP production as well as the mevalonate pathway. The in vivo study showed an increased overall survival for TB treated group vs. vehicle control group (P = 0.0076). CONCLUSIONS: Both in vitro and in vivo results confirmed the effect of TB on MPM and unravelled novel targets with translational potential.

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors.

PURPOSE: Patient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs). METHODS: 94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively. RESULTS: Exome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S - adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways. CONCLUSION: The availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Ultra-hyper-fractionated radiotherapy for high-grade gliomas.

High-grade gliomas (HGGs; WHO grades III and IV) are invariably lethal brain tumors. Low-dose hyper-radiosensitivity (HRS) of HGG is a well-established phenomenon in vitro. However, possibly linked to the unavailability of accurate animal models of the diseases, this therapeutic effect could not be consistently translated to the animal setting, thus impairing its subsequent clinical development. The purpose of this study was to develop radiotherapeutic (RT) schedules permitting to significantly improve the overall survival of faithful animal models of HGG that have been recently made available. We used primary glioma initiating cell (GIC)-driven orthotopic animal models that accurately recapitulate the heterogeneity and growth patterns of the patients' tumors, to investigate the therapeutic effects of low radiation doses toward HGG. With the same total dose, RT fractions ≤0.5 Gy twice per week [ultra-hyper-fractionation (ultra-hyper-FRT)] started at early stages of tumor progression (a condition that in the clinical setting often occurs at the end of the guidelines treatment) improved the effectiveness of RT and the animal survival in comparison to standard fractions. For the same cumulative dose, the use of fractions ≤0.5 Gy may permit to escape one or more tumor resistance mechanisms thus increasing the effectiveness of RT and the overall animal survival. These findings suggest investigating in the clinical setting the therapeutic effect of an ultra-hyper-FRT schedule promptly extending the conventional RT component of the current guideline ("Stupp") therapeutic protocol.

Choline and nicotine increase glioblastoma cell proliferation by binding and activating α7- and α9- containing nicotinic receptors.

Glioblastomas (GBMs), the most frequent and aggressive human primary brain tumours, have altered cell metabolism, and one of the strongest indicators of malignancy is an increase in choline compounds. Choline is also a selective agonist of some neuronal nicotinic acetylcholine receptor (nAChR) subtypes. As little is known concerning the expression of nAChR in glioblastoma cells, we analysed in U87MG human grade-IV astrocytoma cell line and GBM5 temozolomide-resistant glioblastoma cells selected from a cancer stem cell-enriched culture, molecularly, pharmacologically and functionally which nAChR subtypes are expressed and,whether choline and nicotine can affect GBM cell proliferation. We found that U87MG and GBM5 cells express similar nAChR subtypes, and choline and nicotine increase their proliferation rate and activate the anti-apoptotic AKT and pro-proliferative ERK pathways. These effects are blocked by the presence of non-cell-permeable peptide antagonists selective for α7- and α9-containing nicotinic receptors. siRNA-mediated silencing of α7 or α9 subunit expression also selectively prevents the effects of nicotine and choline on GBM cell proliferation. Our findings indicate that nicotine and choline activate the signalling pathways involved in the proliferation of GBM cells, and that these effects are mediated by α7 and α9-containing nAChRs. This suggests that these nicotinic receptors may contribute to the aggressive behaviour of this tumor and may indicate new therapeutic strategies against high-grade human brain tumours.

Preclinical evaluation of the first intravenous small molecule MDM2 antagonist alone and in combination with temozolomide in neuroblastoma.

High-risk neuroblastoma, a predominantly TP53 wild-type (wt) tumour, is incurable in >50% patients supporting the use of MDM2 antagonists as novel therapeutics. Idasanutlin (RG7388) shows in vitro synergy with chemotherapies used to treat neuroblastoma. This is the first study to evaluate the in vivo efficacy of the intravenous idasanutlin prodrug, RO6839921 (RG7775), both alone and in combination with temozolomide in TP53 wt orthotopic neuroblastoma models. Detection of active idasanutlin using liquid chromatography-mass spectrometry and p53 pathway activation by ELISA assays and Western analysis showed peak plasma levels 1 h post-treatment with maximal p53 pathway activation 3-6 h post-treatment. RO6839921 and temozolomide, alone or in combination in mice implanted with TP53 wt SHSY5Y-Luc and NB1691-Luc cells showed that combined RO6839921 and temozolomide led to greater tumour growth inhibition and increase in survival compared to vehicle control. Overall, RO6839921 had a favourable pharmacokinetic profile consistent with intermittent dosing and was well tolerated alone and in combination. These preclinical studies support the further development of idasanutlin in combination with temozolomide in neuroblastoma in early phase clinical trials.

In vitro and in vivo characterization of stem-like cells from canine osteosarcoma and assessment of drug sensitivity.

Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs.

Down-modulation of SEL1L, an unfolded protein response and endoplasmic reticulum-associated degradation protein, sensitizes glioma stem cells to the cytotoxic effect of valproic acid.

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments.

Pharmacokinetics, pharmacodynamics and efficacy on pediatric tumors of the glioma radiosensitizer KU60019.

We have recently reported that glioblastoma (GB)-initiating cells (GIC) with low expression and/or mutation of TP53 and high expression of PI3K ("responder" genetic profile) can be effectively and safely radiosensitized by the ATM inhibitor KU60019. We report here on drug's diffusion and elimination from the animal body and brain, its effects on orthotopic GB and efficacy toward pediatric GIC. Healthy mice were infused by convection enhanced delivery (CED) with KU60019 and the drug kinetics followed by high performance liquid chromatography-mass spectrometry. Already at the end of CED, KU60019 had diffused from the injection site to the ipsilateral and, to a lower extent, controlateral hemisphere. After 24 hr, no drug could be detected all over the brain or in other organs, indicating rapid draining and excretion. After intraperitoneal injection, traces only of KU60019 could be detected in the brain, indicating inability to cross the brain-blood barrier. Consistent with the induction of cell cycle progression previously observed in vitro, KU60019 stimulated proliferation of orthotopic GB cells with the highest effect observed 96 hr after drug delivery. Adult GIC with high expression of TP53 and low expression of PI3K could be radiosensitized by KU60019, although less promptly than GIC bearing the "responder" profile. Consistent with the kinetics of proliferation induction, the highest radiosensitizing effect was observed 96 hr after delivery of KU60019 to GIC. Pediatric GIC could be similarly radiosensitized after exposure to KU60019. The results indicate that ATM inhibition may allow to radiosensitize a wide range of adult and pediatric high-grade gliomas.

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.

BACKGROUND: Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. METHODS: Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. RESULTS: We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. CONCLUSIONS: Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.

TRIM8 downregulation in glioma affects cell proliferation and it is associated with patients survival.

BACKGROUND: Human gliomas are a heterogeneous group of primary malignant brain tumors whose molecular pathogenesis is not yet solved. In this regard, a major research effort has been directed at identifying novel specific glioma-associated genes. Here, we investigated the effect of TRIM8 gene in glioma. METHODS: TRIM8 transcriptional level was profiled in our own glioma cases collection by qPCR and confirmed in the independent TCGA glioma cohort. The association between TRIM8 expression and Overall Survival and Progression-free Survival in TCGA cohort was determined by using uni-multivariable Cox regression analysis. The effect of TRIM8 on patient glioma cell proliferation was evaluated by performing MTT and clonogenic assays. The mechanisms causing the reduction of TRIM8 expression were explored by using qPCR and in vitro assays. RESULTS: We showed that TRIM8 expression correlates with unfavorable clinical outcome in glioma patients. We found that a restored TRIM8 expression induced a significant reduction of clonogenic potential in U87MG and patient's glioblastoma cells. Finally we provide experimental evidences showing that miR-17 directly targets the 3' UTR of TRIM8 and post-transcriptionally represses the expression of TRIM8. CONCLUSIONS: Our study provides evidences that TRIM8 may participate in the carcinogenesis and progression of glioma and that the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients.

Anti-Tumor Effects of Bak-Proteoliposomes against Glioblastoma.

Despite palliative treatments, glioblastoma (GBM) remains a devastating malignancy with a mean survival of about 15 months after diagnosis. Programmed cell-death is de-regulated in almost all GBM and the re-activation of the mitochondrial apoptotic pathway through exogenous bioactive proteins may represent a powerful therapeutic tool to treat multidrug resistant GBM. We have reported that human Bak protein integrated in Liposomes (LB) was able, in vitro, to activate the mitochondrial apoptotic pathway in colon cancer cells. To evaluate the anti-tumor effects of LB on GBM, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and Western blot analysis were performed on GL26 murine cell line. LB treatment shows a dose-dependent inhibition of cell viability, followed by an up-regulation of Bax and a down-modulation of JNK1 proteins. In GL26-bearing mice, two different routes of administration were tested: intra-tumor and intravenous. Biodistribution, tumor growth and animal survival rates were followed. LB show long-lasting tumor accumulation. Moreover, the intra-tumor administration of LB induces tumor growth delay and total tumor regression in about 40% of treated mice, while the intravenous injection leads to a significant increased life span of mice paralleled by an increased tumor cells apoptosis. Our findings are functional to the design of LB with potentiated therapeutic efficacy for GBM.

Agonist activation of estrogen receptor beta (ERß) sensitizes malignant pleural mesothelioma cells to cisplatin cytotoxicity.

BACKGROUND: Estrogen receptor (ER) ß acts as a tumor suppressor in malignant mesotheliomas. METHODS: Here we explored the anti-proliferative and anti-tumorigenic efficacy of the selective ERß agonist, KB9520, in human mesothelioma cell lines in vitro and in a mesothelioma mouse model in vivo. RESULTS: KB9520 showed significant anti-proliferative effect in ERß positive human malignant pleural mesothelioma cells in vitro. Selective activation of ERß with KB9520 sensitized the cells to treatment with cisplatin, resulting in enhanced growth inhibition and increased apoptosis. Furthermore, in CD1 nude mice mesothelioma tumor growth was significantly inhibited when KB9520 was added on top of the standard of care chemo combination cisplatin/pemetrexed, as compared to the cisplatin/pemetrexed alone group. Importantly, KB9520 exerted a protective effect to cisplatin toxicity in the non-malignant mesothelium derived MET5A cells. CONCLUSIONS: Together, the data presented suggest that selective targeting of ERß may be an efficacious stand-alone treatment option and/or become an important add-on to existing malignant mesothelioma therapy.

Predictability, efficacy and safety of radiosensitization of glioblastoma-initiating cells by the ATM inhibitor KU-60019.

We have previously shown that pharmacological inhibition of ataxia telangiectasia mutated (ATM) protein sensitizes glioblastoma-initiating cells (GICs) to ionizing radiation (IR). Herein, we report the experimental conditions to overcome GIC radioresistance in vitro using the specific ATM inhibitor KU-60019, two major determinants of the tumor response to this drug and the absence of toxicity of this treatment in vitro and in vivo. Repeated treatments with KU-60019 followed by IR substantially delayed GIC proliferation in vitro and even eradicated radioresistant cells, whereas GIC treated with vehicle plus radiation recovered early and expanded. The tumor response to the drug occurred under a cutoff level of expression of TP53 and over a cutoff level of expression of phosphatidylinositol 3-kinase (PI3K). No increased clastogenicity or point mutagenicity was induced by KU-60019 plus radiation when compared to vehicle plus radiation. No significant histological changes to the brain or other organs were observed after prolonged infusion into the brain of KU-60019 at millimolar concentrations. Taken together, these findings suggest that GIC-driven tumors with low expression of TP53 and high expression of PI3K might be effectively and safely radiosensitized by KU-60019.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs.

Guadecitabine increases response to combined anti-CTLA-4 and anti-PD-1 treatment in mouse melanoma in vivo by controlling T-cells, myeloid derived suppressor and NK cells.

BACKGROUND: The combination of Programmed Cell Death 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) blockade has dramatically improved the overall survival rate for malignant melanoma. Immune checkpoint blockers (ICBs) limit the tumor's immune escape yet only for approximately a third of all tumors and, in most cases, for a limited amount of time. Several approaches to overcome resistance to ICBs are being investigated among which the addition of epigenetic drugs that are expected to act on both immune and tumor cells. Guadecitabine, a dinucleotide prodrug of a decitabine linked via phosphodiester bond to a guanosine, showed promising results in the phase-1 clinical trial, NIBIT-M4 (NCT02608437). METHODS: We used the syngeneic B16F10 murine melanoma model to study the effects of immune checkpoint blocking antibodies against CTLA-4 and PD-1 in combination, with and without the addition of Guadecitabine. We comprehensively characterized the tumor's and the host's responses under different treatments by flow cytometry, multiplex immunofluorescence and methylation analysis. RESULTS: In combination with ICBs, Guadecitabine significantly reduced subcutaneous tumor growth as well as metastases formation compared to ICBs and Guadecitabine treatment. In particular, Guadecitabine greatly enhanced the efficacy of combined ICBs by increasing effector memory CD8+ T cells, inducing effector NK cells in the spleen and reducing tumor infiltrating regulatory T cells and myeloid derived suppressor cells (MDSC), in the tumor microenvironment (TME). Guadecitabine in association with ICBs increased serum levels of IFN-γ and IFN-γ-induced chemokines with anti-angiogenic activity. Guadecitabine led to a general DNA-demethylation, in particular of sites of intermediate methylation levels. CONCLUSIONS: These results indicate Guadecitabine as a promising epigenetic drug to be added to ICBs therapy.

Antagonistic modulation of gliomagenesis by Pax6 and Olig2 in PDGF-induced oligodendroglioma.

Gliomas are aggressive tumors of the central nervous system originating from proliferating neural cells. Regulators of neural stem or progenitor cells biology may thus influence aspects of brain tumorigenesis, such as the maintenance of tumor-propagating potential. We investigated the role of Pax6, a neurogenic transcription factor already suggested as a positive prognostic marker for human gliomas, in a well-characterized in vivo model of PDGF-B-driven oligodendroglioma. In this system, the expression of Pax6 severely impairs tumor propagation by inducing a reduction of cell proliferation and the acquisition of differentiation traits in tumor-initiating cells. The overexpression of Pax6 correlates with a downregulation of Olig2, a bHLH transcription factor that normally antagonizes Pax6 in adult neurogenic niches and that plays a key role in the maintenance of neural stem and progenitor cells. Furthermore, we found that Olig2 is strictly required to maintain the malignancy of oligodendroglioma cells, since its silencing by interfering RNA abrogates tumor propagation. We finally show evidence that this function depends, at least in part, on the silencing of ID4, a dominant negative bHLH protein, whose upregulation follows Olig2 loss. In our model, the upregulation of ID4 mimics the loss of Olig2 in impairing the tumor-propagating potential of glioma cells. Our data, therefore, establish the relevance of physiological regulators of neural stem cell biology in regulating glial tumor malignancy and provide support for their functional interactions in this context.

Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells.

BACKGROUND: Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting.We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type). METHODS: We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis. RESULTS: Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion. CONCLUSIONS: This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting.Massimiliano Monticone and Antonio Daga contributed equally to this work.

Chloride intracellular channel 1 activity is not required for glioblastoma development but its inhibition dictates glioma stem cell responsivity to novel biguanide derivatives.

BACKGROUND: Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings. METHODS: We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos). RESULTS: We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity. CONCLUSIONS: These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach.

NK cells recognize and kill human glioblastoma cells with stem cell-like properties.

In this study, cancer cells were isolated from tumor specimens of nine glioblastoma patients. Glioblastoma cells, cultured under suitable culture conditions, displayed markers typical of neural stem cells, were capable of partial multilineage differentiation in vitro, and gave origin to infiltrating tumors when orthotopically injected in NOD/SCID mice. These cells, although resistant to freshly isolated NK cells, were highly susceptible to lysis mediated by both allogeneic and autologous IL-2 (or IL-15)-activated NK cells. Indeed, all stem cell-cultured glioblastoma cells analyzed did not express protective amounts of HLA class I molecules, while expressing various ligands of activating NK receptors that triggered optimal NK cell cytotoxicity. Importantly, glioblastoma stem cells expressed high levels of PVR and Nectin-2, the ligands of DNAM-1-activating NK receptor.

Demethyl fruticulin A (SCO-1) causes apoptosis by inducing reactive oxygen species in mitochondria.

Demethyl fruticulin A (SCO-1) is a compound found in Salvia corrugata leaves. SCO-1 was reported to induce anoikis in cell lines via the membrane scavenging receptor CD36. However, experiments performed with cells lacking CD36 showed that SCO-1 was able to induce apoptosis also via alternative pathways. To gain some insight into the biological processes elicited by this compound, we undertook an unbiased genomic approach. Upon exposure of glioblastoma tumor initiating cells (GBM TICs) to SCO-1 for 24 h, we observed a deregulation of the genes belonging to the glutathione metabolism pathway and of those belonging to the biological processes related to the response to stress and to chemical stimulus. On this basis, we hypothesized that the SCO-1 killing effect could result from the induction of reactive oxygen species (ROS) in the mitochondria. This hypothesis was confirmed by flow cytometry using MitoSOX, a mitochondria-selective fluorescent reporter of ROS, and by the ability of N-acetyl cysteine (NAC) to inhibit apoptosis when co-administered with SOC-1 to the GBM TICs. We further show that NAC also protects other cell types such as HeLa, MG-63, and COS-7 from apoptosis. We therefore propose that ROS production is the major molecular mechanism responsible for the pro-apoptotic effect induced by SCO-1. Consequently, SCO-1 may have a potential therapeutic value, which deserves further investigation in animal models.