bluuebunny/crossref_metadata_2025_split huggingface dataset:
P11.29 Development of ex vivo models for deeper insights into the biology and therapeutic targeting of tumor microtube networks in gliomas
Authors: Jung E , Domínguez Azorín D , Hausmann D , Mall M , Koch P , Wick W , Winkler F 
Published: September 2019

Abstract

BACKGROUND
The formation of multicellular networks via thin cellular protrusions named tumor microtubes (TMs) emerged as a novel mechanism of therapy resistance in malignant glioma. TMs are also involved in tumor cell invasion and growth. Within these tumor cell networks, connected tumor cells communicate via intercellular calcium waves (ICWs). Only few molecular drivers of TMs (Gap43, Ttyh1, Connexin 43) have been identified until now. Furthermore, the molecular mechanisms underlying ICWs as well as their specific biological role in glioma remains to be elucidated. A better understanding of the biology and the identification of molecular key drivers is essential for the development of drugs targeting TM formation and function.

MATERIAL AND METHODS
For this purpose, we have developed novel ex vivo models that not only provide insights into TM biology but further allow medium throughput drug screening. As classical response parameters such as the inhibition of cell growth or cytotoxicity do not necessarily correlate with effects on TM formation or function, a morphometrical approach employing laser scanning microscopy and machine-learning based image analysis tools is used. The application of fluorescent probes and genetic fluorescent reporter systems provides novel longitudinal insights into cytoskeletal dynamics, the role and exchange of organelles such as mitochondria, mechanisms of homeostasis within tumor cell networks (e.g. redox homeostasis) and ICWs in live cells. In addition to 2D glioma cell and co-culture models we have developed a fully human and mature brain organoid model. Here, complex 3D tumor cell networks corresponding to the morphology and exhibiting calcium communication patterns observed in our mouse model can be established and studied ex vivo. Furthermore, with these models not only the role of the brain microenvironment on TM formation but also direct interactions of glioma cells with neurons and glial cells as well as drug effects such as cytotoxicity on these brain cells can be investigated ex vivo.

CONCLUSION
In summary, novel tumor models enable further insights into TM biology and hence provide the basis for development of TM- and network disrupting drugs. First results of this screening opportunity will be presented.
P11.35 Fluorine-18-labeled PET radiotracers for imaging tryptophan uptake and metabolism in brain tumors
Authors: Muzik O , John F , Mittal S , Juhasz C 
Published: September 2019

Abstract

BACKGROUND
Abnormal metabolism of tryptophan via the serotonin and kynurenine pathways plays a key role in multiple disease processes including cancer. Upregulation of key enzymes of the kynurenine pathway (such as indoleamine 2,3-dioxygenase [IDO] and tryptophan 2,3-dioxygenase [TDO]) plays an important role in immune resistance in human brain tumors. IDO inhibitors have recently entered in human clinical trials, and their use can benefit from molecular imaging evaluating IDO activity. Imaging tryptophan uptake and metabolism in vivo can be achieved with tryptophan derivative PET radiotracers. Human studies with such tracers showed promise but have been confined to carbon-11-labeled compounds (such as alpha-[11C]methyl-L-tryptophan). Preclinical development of fluorine-18-labeled tryptophan-based radiotracers has surged only in recent years. We performed a systematic review of studies reporting on such tracers and summarized their biological characteristics and their potential for imaging key enzymes of the kynurenine pathway.

MATERIAL AND METHODS
A PubMed search using the key words “tryptophan” and “PET”/”positron emission tomography” was performed. English language original articles including data on the preparation and/or radiochemical or biological characteristics of fluorine-18-labeled tryptophan derivative radiotracers have been reviewed.

RESULTS
Nineteen original papers identified by the search included data on 15 unique fluorine-18-labeled tryptophan-derived radiotracers. Automated synthesis was reported for 1-(2-[18F]fluoroethyl)-L-tryptophan, the most extensively evaluated tracer among the 15. Biodistribution studies showed high uptake in the pancreas, and the L-type amino acid transporter was the dominant transport mechanism for most of the reported radiotracers. Multiple tracers showed accumulation in various tumor cell lines, including glioma cell lines, in vitro and in xenografts in vivo, with favorable tumor-to-background uptake ratios in comparison to clinically used fluorine-18-labeled radiotracers (such as glucose and non-tryptophan amino acid analogs). Five of the 15 tracers showed promise for imaging IDO activity, including a fluorine-18-labeled analog of alpha-[11C]methyl-L-tryptophan. Two of the 15 radiotracers were metabolized by TDO but showed rapid defluorination in vivo.

CONCLUSION
Most fluorine-18-labeled tryptophan derivative PET tracers share common transport mechanisms and biodistribution characteristics. Several of these radiotracers show promise for imaging IDO activity in vivo, and, therefore, could be leading candidates for testing and validation toward human tumor PET imaging applications.
P11.39 Chloroquine as an anti-glioblastoma therapeutic: repurposing of an old drug
Authors: Roy L , Poirier M , Fortin D 
Published: September 2019

Abstract

BACKGROUND
Glioblastoma (GBM) is the most common and aggressive type of primary brain tumour in adults. These tumours depict anarchic proliferation and brain infiltration as well as radio- and chemoresistant profiles. The complete surgical resection is unachievable and responses to standard therapy are transitory. Recurrence is thus inevitable and patient prognosis is generally less than 15 months. Transforming growth factor-beta (TGF-β) holds a substantial role in supporting the GBM phenotype. We showed that TGF-β 1 expression levels correlate with overall and progression-free survival in newly diagnosed GBM patient. We also observed that chloroquine (CQ) can reduce the production of TGF-β together with proliferation, invasion, radioresistance and radio-induced invasion in vitro. Unfortunately, little is known regarding the ability of CQ to penetrate the blood-brain barrier (BBB). Therefore, our objective is to determine whether intravenous (IV) or intra-arterial (IA) infusions of CQ and hydroxychloroquine (HCQ), a pharmacological analog of CQ, can yield therapeutic brain concentrations.

MATERIAL AND METHODS
To assess BBB penetration, the brain, plasma and cerebrospinal fluid (CSF) concentrations of CQ/HCQ were measured by LCMS/MS at different timepoints post-IV or post-IA infusions with 20 mg/kg of CQ/HCQ in tumour-free Wistar rat. For the survival studies, We implanted 10’000 F98 murin glioblastoma cells in the right putamen of Fischer rats. Ten days post-implantation, IA and IV infusion were accomplished through cannulations of the external right carotid and tail vein respectively.

RESULTS
With IV injections, CQ/HCQ brain concentrations 15 minutes post-injection reached 15.76 mg/g (0.18 µM) and 1.67 mg/g (0.078 µM) respectively. However, following IA infusions, we observed a 1.74 and 20.9 fold increase (20 mg/kg HCQ) as well as 7.1 and 84.7-fold-increase (20 mg/kg CQ) in contra- and ipsilateral brain concentrations respectively. Although brain concentrations gradually decreased over time post-IA infusions, the ipsilateral hemisphere CQ concentration was still 82.81 mg/g (34.52 µM) after 6 hours. Whereas plasma concentrations were very similar following IV and IA infusions, both molecules barely accumulated in the CSF and only when using IA infusions. The median survival of the control group (IA phosphate-buffered saline) and the group treated with 20 mg/kg CQ IV were 23.5 days and 24.5 days respectively. However, rats injected with 20 mg/kg CQ IA had a median survival of 28.5 days.

CONCLUSION
These results suggest that IA CQ could be used to abrogate the GBM phenotype. As TGF-β is associated with resistance to both radio- and chemotherapy, we plan to characterize the combination of IA infusions of CQ in combination with radiation or chemotherapy (carboplatin).
P11.41 Comparison of fibroblast activation protein expression and localization in glioblastomas and brain metastases
Authors: Busek P , Zubal M , Chmielova B , Vanickova Z , Hrabal P , Krepela E , Sedo A 
Published: September 2019

Abstract

BACKGROUND
Fibroblast activation protein (FAP) is a transmembrane serine protease that is frequently upregulated in the tumor microenvironment. In several cases, FAP protein itself and/or FAP expressing stromal cells have been shown to contribute to cancer progression and to be associated with more aggressive cancer behaviour and shorter patient survival. The aim of this study was to determine FAP expression in glioblastomas and brain metastases and to identify the cell types that express FAP in the microenvironment of these malignancies.

MATERIAL AND METHODS
FAP enzymatic activity and protein concentration were determined in samples from patients with brain metastases, glioblastomas and pharmacoresistant epilepsy (control non-tumorous brain tissue) by an enzymatic assay using a specific fluorogenic substrate and ELISA, respectively. Immunohistochemical labelling with antibodies against FAP and markers of astroglia, epithelial cancer cells and mesenchymal stromal cells was performed to characterize FAP expressing cells.

RESULTS
FAP was significantly upregulated in the majority of glioblastomas and brain metastases in comparison to non-tumorous brain tissue. In glioblastomas, FAP was localized perivascularly and in mesenchymal cells, and in part of the tumors also in the glioma cells. In brain metastases, FAP positivity was abundantly present in the stroma and predominantly co-localised with markers of mesenchymal stromal cells (TE-7, SMA, PDGFRbeta, NG2), but there was no overlap between FAP and markers of epithelial cancer cells (EpCAM, pancytokeratin).

CONCLUSION
FAP is upregulated in the microenvironment of human glioblastomas and brain metastases compared to non-tumorous brain tissue. In glioblastomas, FAP is expressed in part of the glioma cells, in pericytes and mesenchymal stromal cells, whereas no positivity in cancer cells and more abundant FAP+ stroma was detected in brain metastases. The selective expression of FAP in these brain tumors may be useful for the visualization and possibly therapeutic targeting of their tumor microenvironment.

GRANT SUPPORT
Ministry of Health of the Czech Republic, grant No. 15-31379A, Progres Q28/LF1, 2015064 LM EATRIS and the project,Center for Tumor Ecology - Research of the Cancer Microenvironment Supporting Cancer Growth and Spread” (reg. n. CZ.02.1.01/0.0/0.0/16_019/0000785) supported by the Operational Programme Research, Development and Education.
P11.42 Magnetic targeting of the granzyme B functionalized nanoparticles for therapy of glioblastoma
Authors: Shevtsov M , Stangl S , Marchenko Y , Yakovleva L , Ryzhov V , Nikolaev B , Galibin O , Pitkin E , Multhoff G 
Published: September 2019

Abstract

BACKGROUND
Magnetic drug targeting by employing external magnetic fields represents a promising approach for treatment of glioblastoma. To increase the anti-tumor therapeutic effect magnetic nanocarriers could be functionalized with tumor-targeting bioligands such as granzyme B (GrB). The serine protease GrB that is produced as an effector molecule by activated NK and T cells can induce specific tumor cell apoptosis.

MATERIAL AND METHODS
The targeting potential of the dextran-coated superparamagnetic iron oxide nanoparticles conjugated with GrB (GrB-SPIONs) was assessed in glioblastoma cells (U87, C6, GL261) by flow cytometry, confocal and electron microscopies. Magnetic field strength (320 mT) in the models of U87 glioblastoma in NMRI nu/nu mice and C6 glioma in Wistar rats was achieved by employing NdFeB cylindrical ferromagnet. The irradiation of the implanted tumors was performed using the CT-image guided Small Animal Radiation Research Platform (SARRP). Accumulation of the nanoparticles was assessed in paraffine-embedded specimens employing Prussian blues staining. Sections were additionally analyzed by IHC for apoptosis (caspase 3).

RESULTS
Synthesized GrB-SPIONs incorporated into the cytoplasm of tumor cells via the endolysosomal pathway and induced apoptosis in a dose-dependent manner. Intravenous injection of GrB-SPIONs resulted in the glioma-specific retention of the nanoparticles as was shown by high-field (11 T) magnetic resonance imaging and biodistribution studies (NLR-M2 measurements). Magnetic targeting of the nanoparticles in vivo drastically enhanced the accumulation of nanoparticles to the location of the magnet. Thus the local retention of nanoparticles was 12.3-fold higher as compared to application of GrB-SPIONs without external magnetic field. The presence of granzyme B on SPIONs has been shown to promote tumor cells death (IHC staining for caspase 3) and significantly increased overall survival. A combination of nanoparticle treatment with a single radiation dose (10 Gy) significantly prolonged the survival of rats as compared to animals treated only with magnetic targeting of GrB-SPIONs or radiotherapy alone (P<0.001).

CONCLUSION
Single-agent therapy with GrB-SPIONs demonstrated an impressive increase in overall survival of tumor-bearing animals. Combinatorial regimen employing magnetic targeting and stereotactic radiotherapy further enhanced the therapeutic potential of magnetic conjugates.
P11.45 Synergistic growth inhibition mediated by dual PI3K/mTOR pathway targeting and genetic or direct pharmacological AKT inhibition in human glioblastoma models
Authors: von Achenbach C , Weller M , Kaulich K , Gramatzki D , Zacher A , Fabbro D , Reifenberger G , Szabó E 
Published: September 2019

Abstract

BACKGROUND
Molecular genetic aberrations in the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway are common in human cancers including glioblastoma, yet, novel therapeutic approaches targeting this pathway in glioblastoma have had limited success to date.

MATERIAL AND METHODS
Here we analyzed the molecular mechanisms determining sensitivity to PI3K/mTOR inhibition using gene silencing or pharmacological target inhibition, and effects on proliferation, and clonogenicity or spherogenicity as readouts, in an extended panel of human long-term glioma cell (LTC) lines and glioma-initiating cells (GIC).

RESULTS
Glioma cells, including GIC in particular, were universally sensitive to growth inhibition induced by PQR309, a novel, dual pan-PI3K/mTOR antagonist in vitro. Cells exhibited profound growth arrest, but little apoptotic or necrotic cell death as confirmed by electron microscopy; yet, there was evidence of senescence. In vivo studies confirmed the anti-glioma activity of PQR309 in the orthotopic LN-229 glioma model. Cell lines with high basal levels of phosphorylated (active) AKT, low levels of phosphorylated (inactive) protein translation repressor eukaryotic initiation factor (eIF) 4E-binding protein 1 (p4E-BP1), and high levels of Ser9-phosphorylated (inactive) glycogen synthase kinase 3 beta (pGSK3ß) were more sensitive to PQR309. Accordingly, the activity of PQR309 was synergistically enhanced by AKT gene silencing or direct pharmacological AKT inhibition.

CONCLUSION
These data justify to explore combined targeted therapy approaches in glioblastoma that aim at down-regulating AKT function to enhance the therapeutic potential of dual PI3K/mTOR inhibitors.
P11.51 Repurposing the antipsychotic chlorpromazine for the treatment of glioblastoma multiforme
Authors: Paggi M G , Abbruzzese C , Matteoni S , Matarrese P , Giannarelli D , Sperduti I , Villani V , Pace A 
Published: September 2019

Abstract

BACKGROUND
Background Glioblastoma multiforme (GBM) is characterized by short survival rates, thus generating a compelling need for more effective therapeutic schemes. Therapeutic approaches involving novel drugs must travel a long and expensive way in order to reach the bedside; drug repurposing and repositioning are viable strategies to develop therapies, involving shorter timelines and lower costs. Here we analyzed the activity of the antipsychotic drug chlorpromazine (CPZ) in hindering GBM growth in vitro and in vivo. The safety of this drug springs from six decades of clinical use as an antipsychotic. CPZ has been very recently identified as an inhibitor of the mitotic kinesin KSP/Eg5 and the AKT/mTOR pathway in human glioma cells.

MATERIAL AND METHODS
To explore CPZ pharmacodynamic properties, we assayed the effects of CPZ on several GBM cell lines growing in an anchorage-dependent manner or as neurospheres (cancer stem cells). Further experimentation using high-throughput proteomic and metabolomic platforms is underway.

RESULTS
In our hands CPZ, in the low micromolar range, strongly reduced GBM cell viability after 48 h in both anchorage-dependent cells (IC50=10 microM) and neurospheres (IC50=16 microM), cloning efficiency after 15 d (IC 50=4 and 9 microM, respectively), inducing also, at the respective IC50 concentrations for viability, spindle aberrations up to mitotic catastrophe in almost all cells, as highlighted by means of alpha-tubulin immunofluorescent staining. A Phase II clinical trial involving CPZ, in addition to the adjuvant phase of the Stupp protocol, in GBM patients carrying a hypo-methylated MGMT gene is now ongoing.

CONCLUSION
We proved the effectiveness of CPZ in potently hindering GBM cell growth, without sparing cancer stem cells. This last characteristic is important in order to overcome disease relapse and drug resistance. Recently, many drugs have been repositioned as potentially useful in GBM and for many of these, such as metformin, disulfiram, chloroquine, sirolimus and others, there are clinical trials that challenge them individually combined with the Stupp protocol. On the basis on our results, we consider the choice to undertake a Phase II clinical trial adding CPZ to TMZ in the adjuvant phase of the Stupp protocol as a rational and ethical option to be pursued
P11.52 Reduced expression of proteolipid protein 2 increases ER-stress-induced apoptosis and autophagy in glioblastoma
Authors: Feng Z , Zhou W , Wang J , Huang B , Chen A , Zhang D , Bjerkvig R , Wang J , Thorsen F , Li X 
Published: September 2019

Abstract

BACKGROUND
The PLP2 gene encodes for the Proteolipid protein 2 (PLP2) which is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be involved in several human cancers, but the importance of PLP2 in gliomas is poorly understood. In the present study, we therefore investigated the role of PLP2 in human glioma development.

MATERIAL AND METHODS
Immunohistochemistry was carried out for paraffin-embedded glioma samples, and changes in protein level were detected by western blot analysis. Small interfering RNA transfections were used for knockdown of specific genes. Cell viability and proliferation was then assessed by CCK-8 assay and EdU assay. Transmission electron microscopy was used to observe the ultrastructure of the cells and flow cytometry for the evaluation of apoptosis. Finally, U87 and U251 cells were treated with lentiviral transduction to obtain stably PLP2-knockdown cell lines, which were used for an in vivo study.

RESULTS
Data from publicly available datasets (Rembrandt, TCGA and CGGA) showed a correlation between up-regulation of PLP2 levels and increased malignancy. This was confirmed by IHC staining of sections from our own clinical glioma samples. Mechanistically, down-regulation of PLP2 in U87 and U251 glioma cell lines decreased the proliferation and increased apoptosis and autophagy, mediated by ER-stress. In PLP2 knockdown U87 and U251 cells, autophagy inhibition by chloroquine (CQ) augmented apoptotic cell death. Finally, orthotopic U87-shPLP2 and U251-shPLP2 intracranial xenograft models revealed that down-regulating of PLP2 inhibited glioma development in vivo.

CONCLUSION
In conclusion, the results indicate that PLP2 expression is related to glioma progression, and could be a potential target for future treatment strategies.
P11.53 Genotype trumps phenotype: Oligodendroglioma and oligodendroglioma like tumors- A 1p19q fluorescence in situ hybridization based study at a tertiary referral centre in north India
Authors: Dewan A , Kini L , Sharma S , Bhardwaj M 
Published: September 2019

Abstract

BACKGROUND
The 2016 WHO update of CNS tumours defines oligodendroglioma as a diffusely infiltrating glioma with IDH 1 or IDH 2 mutation and co-deletion of chromosomal arms 1p and 19q. We evaluated the role of 1p19q testing by Fluorescence in situ hybridization (FISH) in the diagnosis and correct classification of CNS tumours in accordance with WHO 2016 classification and exclusion of morphological mimickers of oligodendrogliomas.

MATERIAL AND METHODS
A retrospective analysis of CNS tumours was carried out that were tested for 1p19q co-deletion by FISH over a period of fourteen months at our institute. Correlation with tumour morphology as well as other molecular tests (IDH mutation and MGMT) where available was then done. A tumour was considered to have 1p or 19q deletion when the 1p probe to 1q probe ratio (1p/1q) or the 19q probe to 19p probe ratio (19q/19p) was <0.80.

RESULTS
A total of 125 cases underwent FISH testing for 1p19q co-deletion over a period of last fourteen months at our institute with 74 male patients and 51 women. Thirty-eight of the 125 evaluated patients demonstrated 1p19q co-deletion on FISH. These included 25 oligodendrogliomas, 9 astrocytomas (Grade II/III) and 4 cases of oligoastrocytomas by morphology. Out of these cases, 21 cases had a co-existent IDH mutation, while two cases were IDH wild type and for 15 cases the status was unknown. Rest of the 87 patients were negative for 1p19q co-deletion and included cases of oligodendroglioma, other gliomas (pilocytic astrocytoma, oligoastrocytoma, glioblastoma) and few glioneuronal tumours (ganglioglioma, dysembryoplastic neuroepithelial tumour and central neurocytoma).

CONCLUSION
1. Analysis of 1p19q co-deletion by FISH plays an important role in correctly identifying cases of oligodendroglioma as shown in our study. This is important as it is both a predictive as well as prognostic biomarker 2. In cases with co-deletion but IDH wild type status, the 1p19q status should be confirmed by other methods such as comparative genomic hybridization to confirm the presence of whole chromosome loss.3. FISH analysis also helps in excluding morphologic mimics of oligodendroglioma such as glioneuronal tumours and pilocytic astrocytomas in young patients. 4. However, in a resource limited country like ours, a good morphological diagnosis should still form the basis for FISH and other molecular tests to avoid unnecessary testing.