bluuebunny/crossref_metadata_2025_split huggingface dataset:
P11.24 CR13626, an oral tyrosine kinase inhibitor crossing the blood brain barrier, reduces tumour growth and prolongs survival in a mouse model of glioblastoma
Authors: Galimberti C , Montagna M , Piepoli T , Letari O , Caselli G , Artusi R , Colovic M , Persiani S , Rovati L C 
Published: September 2019

Abstract

BACKGROUND
Glioblastoma multiforme (GBM) is the most malignant primary brain cancer. Several mutations and alterations of key cellular pathways including tyrosine kinases (TKs) are involved in GBM etiopathogenesis. Currently there is no cure for GBM. Tumour heterogeneity and the presence of the blood brain barrier (BBB), with efflux transporters, are some of the causes of failure of novel therapeutic agents. Thus, appropriate target selectivity and pharmacokinetics (including brain penetration) are critical issues for the generation of potential drug candidates. The main in-vitro and in-vivo properties and antitumour activity of CR13626, a novel brain penetrant TK inhibitor, are presented.

MATERIAL AND METHODS
CR13626 inhibitory activity against a panel of 173 kinases was assessed. The effect on cellular proliferation was verified in different 2D human glioblastoma cell lines (U87MG, U373, U87MG vIII) and in a U87MG 3D spheroid model by ViaCount assays. The in-vivo antitumour activity was determined in a mouse model of glioblastoma based on the orthotopic injection of U87MG-Luciferase GBM cells in nude mice: oral treatment started on day 9 post-implantation and continued for 10 days (50 mg/kg/daily). Tumour progression was evaluated through the measurement of bioluminescence (BLI) at the end of dosing (day 19) and during follow-up (day 26–33). Survival was also monitored. The pharmacokinetics and brain exposure of CR13626 were assessed by LC/MS/MS in plasma and brain homogenate tissues of CD1 and tumour-bearing nude mice.

RESULTS
CR13626 potently inhibited FYN, YES, KDR and EGFR kinases, relevant for GBM development, with IC50 values of 69 nM, 3.6 nM, 82 nM and 6 nM, respectively. The compound reduced the proliferation of different human glioblastoma cell lines (GI50 1–3 µM). In CD1 mice, CR13626 had a good oral bioavailability (72%) and brain penetration (brain/plasma ratio of 1.4). In-vivo BLI analysis indicated a time-dependent reduction of tumour growth, reaching 60% on the last BLI evaluation 33 days post-implantation (i.e. 15 days after the end of dosing). Tumour growth inhibition translated into an increase of 25% of the median survival time of animals treated with CR13626 compared to the vehicle group (p<0.05). The observed antitumour effects agreed with the exposure of tumour-bearing mice to CR13626, which was above the TKs in-vitro IC50 values.

CONCLUSION
The combined abilities of CR13626 to inhibit the activity of TKs involved in GBM development, to cross the BBB, and to reduce tumour growth in-vivo leading to increased survival, warrant its further development as a drug candidate in GBM.
P11.25 Assessing electrical properties of cells as predictive marker for patient-specific TTFields response and optimal frequency
Authors: Kinzel A , Zeevi E , Gotlib K , Wenger C , Naveh A , Bomzon Z , Kirson E , Weinberg U , Palti Y 
Published: September 2019

Abstract

BACKGROUND
Tumor treating fields (TTFields) are currently approved for the treatment of glioblastoma multiforme (GBM, using 200 kHz), and being tested in other tumor types such as non-small cell lung cancer and brain metastases occurring in this indication (LUNAR and METIS trials, using 150 kHz). Response to TTFields in cancer cells was empirically shown to be frequency-dependent specific for cell type; however, there are no markers available predicting optimal frequency or response in different cancer types or individual patients to date. There is evidence indicating electrical properties determine the optimal anti-mitotic frequency. This study analyzed the correlation of electrical properties of cells with their optimal TTFields frequency and sensitivity using the 3DEP reader (LabTech) to determine the electrical properties with the help of Dielectrophoresis (DEP) force. With this technique, cell movements within electric fields of different frequencies can by analyzed based on the physical effect of DEP, exercising a force on polarizable particles inside a non-homogeneous electric field.

MATERIAL AND METHODS
We used the 3DEP reader to obtain baseline properties (permittivity and conductivity) of 17 different cell lines of several tumor types. The resulting curves were analyzed by a 2-way ANOVA. Additionally, we determined the optimal frequency for maximum cytotoxic effect for each cell line using the inovitroTM system and eventually compared with the detected electrical properties.

RESULTS
We found cell lines with an optimal TTFields frequency of 150 kHz (corresponding to cells with a membrane capacitance in the lower range of the observed 3DEP curves, n=9) to possess significantly different (p<0.001) electrical properties from cells with an optimal TTFields frequency of 200 kHz (n=8). According to the curve differences in the lower frequency range, the measure of membrane capacitance served as a good predictor for TTFields response.

CONCLUSION
This study showed a correlation of cell membrane capacitance and optimal TTFields frequency and response. Our results provide a substantial rationale for further studies investigating the predictive potential of electrical properties of tumor cells as a measure for the optimal frequency and sensitivity to TTFields in individual patients.
P11.27 Drug screening of available anti-cancer drugs on patient-derived GBM cultures identifies candidate treatments for a subpopulation of GBM patients
Authors: Ntafoulis I , Kers T , van der Kaaij M , Balvers R , Hayes J , van Rij J , Tching R , Lindner A , Prehn J , Kremer A , Dirven C , Leenstra S , Lamfers M 
Published: September 2019

Abstract

BACKGROUND
The development of new therapeutic agents generally takes many years to translate to clinically-effective new treatments. Among the strategies to reduce this time frame, efforts are now being undertaken to investigate drug repurposing. With this approach, compounds available for a specific disease are evaluated for their therapeutic efficacy in other diseases. We have set up a patient-derived cell culture model to apply this strategy for glioblastoma (GBM).

MATERIAL AND METHODS
Fresh patient-derived tumour tissue was dissociated and cultured in serum-free medium supplemented with EGF and bFGF. MGMT status was determined by methylation-specific PCR.Drug screening was performed using the NIH anti-cancer collection containing 114 approved oncology drugs. Compounds include chemotherapeutic agents as well as small molecule targeted agents. Readout for drug effects is based on ATP-based viability assay. Using systems modelling approaches, integrated analysis of both mutational and expression data of each tumour is applied to identify key pathways involved in response to specific compounds.

RESULTS
Molecular analysis demonstrated that copy number variations are preserved under serum-free culture conditions and that the MGMT methylation status is retained in over 75% of cases. Screening of the NIH anti-cancer collection on 55 GBM cell cultures revealed high intertumoral variation in response to most drugs. This included subsets of GBM revealing exceptionally high sensitivity to specific agents at clinically-feasible concentrations. Further ranking of the compounds was made based on the therapeutic index (IC50 tumour versus normal human astrocytes) and predicted blood-brain-barrier crossing capability. Currently, integrated analysis of molecular profiles of the tumour in relation to the drug response data is ongoing with the aim of identifying response predictors to these clinically-approved anti-cancer agents.

CONCLUSION
Our patient-derived in vitro drug screening assay may offer a tool to identify available anti-cancer agents that are effective in a subpopulation of GBM patients and that may be implemented in future stratified clinical trials for this patient group.
P11.49 An electrophysiological signature of glioma infiltration in the ex vivo human brain
Authors: Kirby A J , Lavrador J P , Brogna C , Vergani F , Chandler C , Zebian B , Bodi I , Bhangoo R , Ashkan K , Finnerty G T 
Published: September 2019

Abstract

BACKGROUND
Invading glioma cells affect the physiological function of the peritumoural cortex. This may manifest clinically as seizures. Here, we investigate the effect the invading glioma cells on the electrophysiological signalling of the peritumoral cortex using living human brain tissue donated by people having a craniotomy for glioma resection (REC approval, 18/SW/002).

MATERIAL AND METHODS
The brain tissue was cut into thin slices, which preserved the architecture of the glioma and the adjacent healthy brain. The brain slices were incubated in 5-aminolevulinic acid to make the glioma cells fluorescent. We observed 5-ALA induced fluorescence in both low-grade and high-grade gliomas. This enabled us to make electrophysiological recordings of brain activity across the boundary between glioma and brain.

RESULTS
We recorded from brain slices of 5 participants with glioblastoma and 4 participants with oligodendroglioma (WHO grade II - III). Spontaneous “seizure-like” discharges were recorded in brain slices from 5/8 participants (3 GBM, 2 oligodendroglioma) who reported seizures and from one participant (GBM) who had not had any clinical seizures. Further analysis of the electrical discharges revealed that they could be subdivided into two distinct types based on the major frequencies in the discharge.

CONCLUSION
We concluded that human brain slices from people with either a low-grade or a high-grade glioma can generate spontaneous seizure-like discharges. This electrophysiological signature will be compared to infiltration and grade of the glioma cells in the donated sample. The living human brain tissue preparation gives us a platform to study the mechanisms of tumour-associated seizures and how abnormal neural activity affects glioma growth.
P11.50 The dielectric properties of brain tumor tissue
Authors: Proescholdt M A , Lohmeier A , Stoerr E , Eberl P , Brawanski A , Bomzon Z , Hershkovich H S 
Published: September 2019

Abstract

BACKGROUND
Recently, tumor treating fields (TTFields) were established for the treatment of newly diagnosed GBM. One of the most crucial parameters defining the treatment efficacy of TTFields is the electric field intensity. The dielectric properties of the normal intracranial compartments are well established, allowing the prediction of the electric field distribution. In contrast, there is no data available about the dielectric properties of brain tumor tissue. In this study we determined the dielectric properties of brain tumors by analyzing resected tissue following a fast acquisition protocol. To account for the intratumoral heterogeneity, different regions of the tumor were analyzed separately.

MATERIAL AND METHODS
A cohort of 84 patients with tumors of different histology and malignancy grade have been recruited (meningioma: n=26; brain metastases n=18; low grade glioma n=6; glioblastoma n=34). Tissue probes were acquired whenever possible from the vital tumor area, and perinecrotic compartment identified intraoperatively using neuronavigation, intraoperative ultrasound and fluorescence guidance. After acquisition, the tissue was measured immediately to avoid artifacts induced by temperature change, differences of fluid composition as well as post resection ischemia. A fragment was dissected from each tissue sample and was placed into a cylindrical cell with a known diameter. Two parallel electrodes were placed on both sides of the sample and the thickness of the tissue was measured using a micrometer. The impedance was recorded at frequencies 20Hz-1MHz using a software specifically developed for this study, which controls the LCR meter (Keysight Technologies, Santa Rosa, USA). The measured impedance was translated into dielectric properties of the sample (conductivity and relative permittivity) based on the parallel plate model, the recorded complex impedance and the geometry of the samples. Each tissue probe was fixed, H&E stained and histologically analyzed.

RESULTS
We found significant differences between the conductivity of different types of tumors with meningiomas showing the lowest conductivity (mean conductivity [S/m]: 0.193; range: 0.327 - 0.113) and GBM tissue exhibiting the highest conductivity values (mean conductivity [S/m]: 0.402; range: 0.893 - 0.157). Consistently, the perinecrotic areas of tumors displayed lower conductivity values compared to the solid tumor compartments. Also, we found a significant intratumoral heterogeneity in tumors of one specific histological diagnosis.

CONCLUSION
The dielectric properties of intracranial tumors appear to be depending on histological class and malignancy grade and show significant intratumoral heterogeneity. These results may allow a more precise modelling of electric field intensity distribution within the tumor.
P11.59 Integrin a5 heterogeneous expression in glioblastoma is related to glioma stem cell subpopulations
Authors: Dontenwill M , Mercier M , Gillmann G , Reita D , Lelong-Rebel I , Noulet F , Idbaih A , Herold-Mende C , Bagnard D , Spenlé C , Martin S 
Published: September 2019

Abstract

BACKGROUND
Glioblastoma (GBM) is the most aggressive primary brain tumor. Treatment failure and recurrence are explained in part by tumoral heterogeneity. Our previous results showed that the integrin α5β1 is implicated in GBM aggressiveness and represents a relevant therapeutic target. Recently, we observed intra- and inter-tumor heterogeneity of integrin α5β1 expression. Heterogeneity may be linked to different glioma stem cell populations.

MATERIAL AND METHODS
Ten glioma stem cell lines were grown as neurospheres in stem cell medium and their differentiation was induced by serum and/or ATRA. Two cell lines (NCH421k and NCH644) were selected and were modified by depletion (CrisprCas9) or transfection of the α5 integrin gene. Polyclonal lines and individual clones were analyzed phenotypically in vitro, before and after differentiation, and in vivo in orthotopic xenografts of 2x104 cells in nude mice. TCGA datasets were used to validate the heterogeneous expression of α5 integrin in GBM.

RESULTS
TCGA data validate that α5 integrin mRNA was only over-expressed in the mesenchymal subclass of GBM. Our results show that α5 integrin protein is not expressed in stem cell culture conditions. However, α5 integrin expression is induced after differentiation in only half of the cell lines supporting the notion of tumoral heterogeneity of glioma stem cells. Interestingly, single cell-derived clone evaluation showed that intra-tumoral stem cell heterogeneity also exists at the level of α5 protein expression. When glioma stem cells are programmed or transduced to express α5 integrin, differentiated cells became more aggressive. Notably, they acquired a fibronectin-dependent motility and a proliferative phenotype. Interestingly, integrin α5 remained expressed in secondary stem cells obtained after dedifferentiation. The in vivo assays suggested that glioma stem cells, programmed to express the integrin, were prone to form larger tumors.

CONCLUSION
Our data support the hypothesis that some glioma stem cells are programmed to express the α5 integrin subunit in their differentiated progeny to form a more aggressive tumor. They add new evidences that both cell populations may be considered for new therapeutic strategies against GBM.
P11.65 Insights into the mechanisms of primary brain tumor invasion
Authors: Bikfalvi A , Daubon T , Billottet C 
Published: September 2019

Abstract
We have made progress in unravelling the mechanisms of tumor cell invasion by focusing the attention on two molecular pathways including chemokines and extracellular matrix molecules. Chemokines are important mediators of cell signaling that operate both on normal cells and tumor cells and in the immune-cell compartment (Billottet et al, 2013). Among the chemokine receptors, CXCR3 mediate diverse biological functions and comes in two major isoforms the A and B isoform. We found that ligand affinities and conformational changes are very different for the A and B form. We have recently elucidated the role and mechanism of CXCR3A in GBM invasion (Boyé et al, 2017b). We demonstrated that agonist stimulation enhances in vitro cell migration and invasion in GBM cells. A major finding was that CXCR3A forms a complex with the trafficking receptor Lipoprotein-related receptor-1 (LRP1). Silencing of LRP1 leads to an increase in the magnitude of ligand-induced conformational change with CXCR3-A focalized at the cell membrane, leading to sustained receptor activity and increase in the migration. This was also clinically validated. Our study defines LRP1 as a new regulator of CXCR3 and indicates that targeting CXCR3-A in GBM may constitute a promising strategy to halt tumor cell invasion. The extracellular matrix (ECM) has morphogenic roles in tumors. Important ECM components are the matricellular proteins, called thrombospondins(THBS1-5) (Adams and Lawler 2011). We recently elucidated the complex role of THSB1 in GBM invasion (Daubon et al.2019). Global expression analysis revealed that THBS1 is up-regulated in GBMs and associated with a poor prognosis. We, furthermore, demonstrated that THBS1 did not activate TGFβ in GBM but that TGFβ1 induced the expression of THBS1 via SMAD3. Furthermore, GBM invasion is compromised when THBS1 is silenced in tumor cells. Thus, our data clearly show that THBS1 is not only involved in the regulation of angiogenesis in GBM, but also impacts the invasive behaviour of glioma cells by interacting with a molecule called CD47 expressed on the surface of GBM cells. RNA-sequencing after microdissection of central and peripheral tumour areas in a human PDX model demonstrated that THBS1 was the gene with the highest connectivity in the peripheral invasive tumour areas. Taken together, these data indicate that THBS1 plays important role in the infiltrative process in GBM.
REFERENCES: Adams JC, Lawler J. Cold Spring Harb Perspect Biol. 2011;3:a009712 Billottet C, Quemener C, Bikfalvi A. Biochim Biophys Acta. 2013;1836:287- Boyé K et al. Sci Rep. 2017;7:10703 Boyé K et al. Nat Commun. 2017;8:1571 Daubon T et al, Nature Communications. Nat Commun. 2019 Mar 8;10(1):1146 Murphy-Ullrich JE, Poczatek M. Cytokine Growth Factor Rev. 2000 11:59
P12.04 Synergistic effects of combination therapy of CAR-NK cells and anti-PD-1 antibody result in high efficacy against advanced stage orthotopic glioblastoma grafts in a syngeneic mouse model and induce protective anti-tumor immunity in vivo
Authors: Strassheimer F , Strecker M I , Zhang C , Mildenberger I C , Harter P N , Tonn T , Steinbach J P , Wels W S , Burger M C 
Published: September 2019

Abstract

BACKGROUND
Checkpoint inhibitors as well as adoptive cell therapy hold great promise for cancer treatment and encouraging treatment responses have already been demonstrated in different cancer indications. Glioblastoma (GB) is the most common and aggressive primary brain tumor. Standard therapy has very limited efficacy in the majority of patients. Analysis of the GB tumor microenvironment (TME) has shown prominent immunosuppressive features including expression of PD-L1 on tumor cells and increased frequency of FOX-P3 positive regulatory T cells. While the surrounding brain is HER2-negative, GB tumors are frequently HER2-positive, suggesting HER2 as a promising target for adoptive immunotherapy.

MATERIALS AND METHODS
The murine glioma cell line GL261 was transfected with HER2. Tumor cells were orthotopically implanted into C57BL/6 mice and treated either with HER2-specific NK-92/5.28.z cells alone or in combination with an anti-PD-1 antibody. Effects on tumor growth and survival were determined, lymphocyte infiltration and immunosuppressive TME were characterized.

RESULTS
Combined treatment with NK-92/5.28.z cells and anti-PD-1 antibody resulted in synergistic tumor regression and long-term survival of advanced-stage tumor bearing mice. Analysis of TME showed enhanced cytotoxic lymphocyte infiltration and altered profiles of exhaustion markers in tumor and immune cells.

CONCLUSION
These data demonstrate that efficacy of NK-92/5.28.z cells can be enhanced by co-therapy with checkpoint inhibitors, resulting in successful treatment of advanced tumors refractory to mono-therapy. Furthermore, this combination therapy induces a cytotoxic rather than immunosuppressive TME, leading to a primed immune system. To address this question in a clinical setting, we are planning a phase I clinical study (CAR2BRAIN-CHECK).
P13.04 Transcriptional profiling differentiates spinal cord ependymal tumours from other glial tumours in children
Authors: Sobocińska A A , Niemira M , Szałkowska A , Wojtaś B , Trubicka J , Grajkowska W , Matyja E , Łastowska M 
Published: September 2019

Abstract

BACKGROUND
Spinal cord tumours in children include ependymal and other tumours of glial origin, all of them displaying GFAP marker. Some tumours may present difficulties in histopathological diagnosis e.g. due to intratumour heterogeneity. Therefore, molecular approach should be examined for its diagnostic usefulness. We investigated a series of originally diagnosed spinal cord ependymal tumours, using transcriptional profiling, to confirm if molecular classification is compatible with histopathological diagnosis.

MATERIAL AND METHODS
Overall, 9 patients diagnosed between 2001 and 2014 in The Children’s Memorial Health Institute (CMHI) in Warsaw, Poland, were included in the analysis. 4 patients were diagnosed with GI myxopapillary ependymomas, 3 patients with GII and 2 patients with GIII grade ependymal tumours. Total RNA was extracted from FFPE tumour samples using RNeasy kits (Qiagen). Transcriptional profiling was performed using NanoString nCounter system analysis. Tumours were analysed according to the manufacturer’s procedures for hybridization, detection and scanning. Raw counts for each gene underwent technical and biological normalization using nSolver software. A custom NanoString CodeSet consisted of 8 marker genes: GFAP as a glial marker, CAPS and FAM81B as the markers for ependymal tumours and OLIG1, OLIG2, PMP2, KLRC3 and C1orf61 as the markers for other gliomas. Marker genes were identified by re-analysis of publically available microarrays data from childhood brain tumour samples. Histopathological reassessment of tumour preparations was performed by two experienced neuropathologists.

RESULTS
Hierarchical clustering of 9 samples revealed that two tumours clustered separately and displayed low expression of ependymal markers but high expression of all other glioma markers. Subsequent histopathological re-analysis of preparation of the later 2 tumours showed that one of them displayed features of pilocytic astrocytoma with loose, microcytic and fibrillary areas accompanied by advanced myxoid degenerative changes mimicking the picture of myxopapillary ependymoma. The second tumour revealed heterogeneous morphology with areas of diffuse low grade astrocytoma, pilocytic astrocytoma and fragments displaying ependymal features, including angiocentric pattern with ependymoma-like perivascular arrangement of neoplastic cells.

CONCLUSION
The results of analysis indicate that RNA expression profiling from FFPE tumour samples using NanoString nCounter system may be a useful approach in differentiation of ependymal tumours from other glial tumours with misleading morphology located in the spinal cord in children. Funded by the National Science Centre, Poland (2016/21/B/NZ2/01785 and 2016/23/B/NZ2/03064).