bluuebunny/crossref_metadata_2025 huggingface dataset:
P09.07 Constitutive activation of the EGFR-STAT1 axis increases proliferation of meningioma tumour cells
Authors: Hanemann C O , Ferluga S , Baiz D , Sharma V , Adams C L , Bassiri K , Dunn J , Ercolano E , Kurian K , Hilton D A
Published: September 2019

BACKGROUND

Meningiomas are the most frequent primary brain tumours of the central nervous system. The standard of treatment is (radio)surgery, but the lack of knowledge of their tumorigenesis contributes to delay the development of additional therapeutic options.

MATERIAL AND METHODS

We used a variety of differen model systems and cell biology techniques

RESULTS

We found STAT1 widely overexpressed in meningioma tumours and in patient-derived meningioma cells but not in the corresponding healthy controls. The protein showed a constitutive phosphorylation on both phosphosites (Y701 and S727), which was not dependent on the JAK/STAT pathway. STAT1 knocked down resulted in a significant reduction of cellular proliferation, showed as a decrease in Ki67-positive cells and Cyclin D1, and deactivation of AKT and ERK 1/2. By studying STAT1 binding partners we isolated a complex composed by STAT1, STAT2, PRMT5 and MEP50. As PRMT5 is known to interact with EGFR, we tested the tyrosine kinase and found that EGFR was constitutively active in meningioma and was responsible for the aberrant phosphorylation of STAT1 on both phosphosites. We tested different drugs inhibiting of EGFR phosphorylation, Canertinib was most effective. It caused a significant reduction in meningioma cells proliferation and a reduction of overall levels of Cyclin D1, phospho- AKT and phospho-ERK 1/2. Hence, STAT1 constitutive phosphorylation, initiated by EGFR activation, is responsible for inducing a positive feedback loop causing its own overexpression and consequently an increased proliferation of the tumour cells.

CONCLUSION

These findings underline a pivotal role of the EGFR and STAT1 axis in meningioma and provides the rationale for further studies aiming to identify novel and effective therapeutic options.
P10.01 Anti-migration and anti-invasion effects of curcumin via suppression of fascin expression in glioblastoma cells
Authors: Ki-Su P , Yoon S , Hwang J , Ahn H
Published: September 2019

BACKGROUND

The natural compound Curcumin was known to inhibit migration and invasion of glioblastoma (GBM) cells. Fascin, a kind of actin-binding proteins, is correlated with migration and invasion of GBM cells. The purpose of this study was to investigate anti-migration and anti-invasion effects of Curcumin via suppression of fascin expression in GBM cells.

MATERIAL AND METHODS

U87 cell line was used as an experimental model of GBM. Fascin was quantified by Western blot analysis. And, the signal transducer and activator of transcription 3 (STAT3), known to play an important role in migration and invasion of tumor cells, were analyzed by sandwich-ELISA. Migration and invasion capacities were assessed by attachment, migration and invasion assays. Cellular morphology was demonstrated by immunofluorescence.

RESULTS

At various concentrations of curcumin and exposure times, fascin expression decreased. After temporarily exposure to 10μM/L Curcumin during 6 hours as less invasive concentration and time, fascin expression temporarily decreased at 12 hours (18.4%, p=0.024), and since then recovered. And, the change of phosphrylated STAT3 level also reflected the temporarily decreased pattern of fascin expression at 12 hours (19.7%, p=0.010). Attachment, migration, and invasion capacities consistently decreased at 6, 12, and 24 hours. And, immunofluorescence showed the change of shape and the reduction of filopodia formation in cells.

CONCLUSION

Curcumin is likely to suppress the fascin expression in GBM cells, and this might be a possible mechanism for anti-migration and anti-invasion effects of Curcumin via inhibition of STAT3 phosphorylation.
P10.06 Stiff limb syndrome associated to anti NMDA-R antibodies
Authors: Birzu C , Jaume L , Rostomashvilli S , Honnorat J , Berzero G , Psimaras D
Published: September 2019

BACKGROUND

Stiff person spectrum disorder is a group of rare conditions clinically characterized by the core symptoms of fluctuating muscular stiffness with superimposed spasms and an exaggerated startle response (hyperkplexia). It includes multiple variants amongst which are focal forms like stiff limb syndrome. Up to date, it was described in association with a spectrum of antibodies interfering with gabaergic transmission, most frequently antibodies targeting GAD, glycine receptor, amphiphysin and more rare dipeptidylpeptidase like protein 6, gephyrin, GABAAR and Glycine transporter 2.

MATERIAL AND METHODS

We report the case of a stiff limb syndrome associated to anti-NMDAR antibodies. The patient, a French 44-year male, with no antecedents presents with a 2-month history of painful spasms in his both legs resulting in a difficulty of walking and multiple falls. The diagnosis was supported by a stiff limb syndrome clinical pattern, a clinical response to treatment with benzodiazepines and baclofen and by the absence of other neurological finding that could have suggested an alternative diagnosis.

RESULTS

Anti NMDA-R antibodies were identified in blood and CSF samples using both immunofluorescence and cell-based assay. Any other intracellular and surface antibodies were negatives, GAD-ab included. Oncological evaluation was negative. The patient didn’t present encephalopathy signs and neuropsychological evaluation was normal. The patient received immunotherapy with one cure of intravenous immunoglobulins after an only partial improvement with symptomatic treatment.

CONCLUSION

This is the first case report of an association between the anti NMDA-R and stiff limb syndrome. Further investigations are needed to provide a better understanding of the pathophysiology of stiff person spectrum disorders.
P11.01 TMZ-LEV- IFN cocktail regimen significantly inhibited the growth of glioma
Authors: Ni X , Qu Y , Wang J , Chen F , Cai H , Yu Y , Yu Y , Yu Z , Cen Z , Chen Z
Published: September 2019

BACKGROUND

TMZ, is the first line chemotherapeutic drug for glioma, and drug resistance is one of the most important reasons for glioma treatment failure. Our previous studies have found that: 1) Type I interferon (IFN) could reverse the resistance of TMZ by inhibiting NF-κB activity, and down-regulating the expression of MGMT in vivo and in vitro; 2) IFN-α could significantly improve chemtherapeautic effect of TMZ for GBM patients. We aim to investigate the therapeutic effect of a cocktail chemotherapy regimen combining temozolomide (TMZ)- Levetiracetam(LEV) - interferon (IFN) on human glioma U138 and U251 subcutaneous xenograft tumor.

MATERIAL AND METHODS

30 xenograft tumors were established by subcutaneously injecting 1×106 glioma cells into the right flank of 4-week-old female BALB/C nude mice and then randomly divided into 5 groups (n=6/group): Control group; TMZ group; TMZ+IFN group; TMZ+LEV group; TMZ+LEV+IFN group. Anti-tumor efficacy of five regimens for tumor-bearing mice was tested after treatment with TMZ (50 mg/kg, i.p.), IFN (2×105 IU, s.c.), LEV (150 mg/kg, i.p.), while TMZ dose were reduced to 12.5 mg/kg for U251 tumors. All drugs are given once a day for five consecutive days. After therapy, the size of tumor was measured every day until the control tumors reached 2000 mm3. Mice bearing U138 tumors were sacrificed at 40 days after therapy, and mice bearing U251 tumors were killed at 26 days after therapy.

RESULTS

We identified that both U138 and U251 tumor growth among TMZ group, TMZ+IFN group, TMZ+LEV group and TMZ+LEV+IFN group were significantly inhibited (P<0.05), compared with the control group. Tumor weight of all treating group was lower than that of the control group (P<0.05). The tumor weight of TMZ+LEV+IFN group was the lowest and significantly lower than that of TMZ+LEV group and TMZ group (P<0.05, respectively). No significant difference was found between TMZ+LEV+IFN group and TMZ+IFN group in U251 subcutaneous xenograft tumors, although the tumor weight was lower in TMZ+LEV+IFN group (P>0.05). In the treatment of mice bearing U138 glioma, TMZ+LEV+IFN regimen was significantly superior to TMZ+IFN regimen.

CONCLUSION

Our data demonstrate that both IFN and LEV can sensitize TMZ effect on glioma. TMZ-LEV-IFN cocktail appears the best regimen.
P11.02 Hypothermia effect on glioblastoma cell viability, proliferation and migration
Authors: Fulbert C , Gaude C , Sulpice E , Chabardès S , Ratel D
Published: September 2019

BACKGROUND

Glioblastoma is the most common and aggressive primary brain tumor in adults. In spite of intensive treatment, patients have a poor prognosis with a median survival of 14–16 months. After surgical resection followed by postoperative chemoradiation (combined temozolomide treatment and radiotherapy), tumor recurs in the resection margin for more than 90% of patients. This recurrence results from the activation of residual glioblastoma cells beyond the resection cavity by therapy-induced injuries. To handle this issue, we propose therapeutic hypothermia as an adjuvant treatment, in order to place the resection margin in a state of hibernation. In fact, hypothermia was introduced as a promising therapeutic approach in various medical applications like cardiac arrest and pharmaco-resistant epilepsy. Only a few in vitro studies explored the effects of hypothermia on cancer cells and showed promising results. The aim of our work is to investigate the effects of hypothermia on glioblastoma cell proliferation and migration, two key cellular processes involved in cancer progression.

MATERIAL AND METHODS

We performed in vitro experiments on glioblastoma cell lines with different p53 status and various growth rates. For exploring the therapeutic potential of both mild and moderate hypothermia, we studied their impact on cell viability, proliferation and migration. We also performed cell cycle analysis by quantitation of DNA content using flow cytometry.

RESULTS

Results were similar for all glioblastoma cell lines, and demonstrated that cells were extremely sensitive to hypothermia. We showed that both mild and moderate hypothermia induced significant changes on glioblastoma cell lines behavior with a strong inhibition of cell proliferation and migration. Moderate hypothermia also affected glioblastoma cell viability and modified their distribution into the cell cycle phases.

CONCLUSION

Our results were comparable in all glioblastoma cell lines tested, demonstrating a consistent and universal effect of hypothermia. We showed that hypothermia significantly inhibits cell proliferation and migration, which are key processes involved in tumor growth. Proliferation arrest could be explained by the accumulation of cells in the G2/M phase of the cell cycle.

Together, these results support hypothermia as a promising adjuvant therapy for glioblastoma patients. Indeed, combined with current treatments, moderate hypothermia applied at the resection margin could prevent tumor recurrence after surgical resection. There is a crucial need to propose innovative glioblastoma treatments, and hypothermia appears as a promising therapeutic way.

SUPPORT

This work received financial support through grants from the Groupement des Entreprises Françaises de Lutte contre le Cancer (GEFLUC Grenoble - Dauphiné - Savoie) and the Fonds de dotation Clinatec.
P11.16 The stemness andinvasiveness of glioblastoma tumorspheres were suppressed by combined treatment with2’-hydroxycinnamaldehyde andtemozolomide
Authors: Kim W
Published: September 2019

BACKGROUND

Glioblastoma (GBM) is the most common and aggressive human primary brain malignancy. The key properties of GBM, stemness and invasiveness, are known to be associated with a highly unfavorable prognosis. Notably, the process of epithelial-mesenchymal transition (EMT) is closely related to the progression of GBM. On the basis of reports that 2′-hydroxycinnamaldehyde (HCA) and its derivative, 2′-benzoyloxycinnamaldehyde (BCA), suppresses EMT in several human cancer cells, we sought to evaluate the therapeutic efficacy of HCA and BCA, alone and in combination with temozolomide (TMZ), on GBM tumorspheres (TSs).

MATERIAL AND METHODS

Two human GBM TSs were treated with HCA, BCA, or TMZ. Therapeutic effects were evaluated by measuring ATP levels, neurosphere formation, 3D-invasion in collagen matrix, and viability. Protein expression profiles after drug treatment were evaluated by western blotting. In vivo anticancer efficacy of drugs was examined in a mouse orthotopic xenograft model.

RESULTS

Combined treatment of GBM TSs with HCA or BCA and TMZ significantly reduced cell viability, stemness, and invasiveness. Expression levels of stemness-, invasiveness-, and mesenchymal transition-associated markers, Zeb1, N-cadherin, and β-catenin, were also substantially decreased by the combined treatment. The combined treatment also reduced tumor growth in a mouse orthotopic xenograft model.

CONCLUSION

Our findings suggest that HCA and BCA, combined with TMZ, are potential therapeutic agents in the treatment of GBM.
P11.18 Tumor treating fields (TTFields) treatment of spinal cord metastases
Authors: Kinzel A , Yesharim O , Naveh A , Bomzon Z
Published: September 2019

BACKGROUND

Tumor Treating Fields (TTFields) is an anti-mitotic cancer treatment approved for the treatment of Glioblastoma multiforme (GBM) and is currently also investigated in a phase III trial in 1–10 brain metastases from non-small cell lung cancer (METIS). Apart from spread to the brain, some cancer types, such as breast cancer, lung cancer, and melanoma, may lead to metastatic spread to the spinal cord. Previous studies have shown that reported transducer array layouts for the treatment of abdominal/pelvic tumors (e.g. pancreatic cancer), with one pair of arrays positioned on the anterior and posterior of the patient, and the second pair of arrays placed on each side of the thorax, yield therapeutically insufficient field intensities of <1 V/cm in the spinal cord. This finding probably results from the anatomical structure of the spine, consisting of the cerebrospinal fluid as a highly conductive layer, encased by a resistive bone structure that shunts the current delivered across the body by the arrays away from the spine. This simulation-based study aimed at resolving this challenge by identifying novel array layouts on the body that effectively deliver TTFields to the spine.

MATERIAL AND METHODS

For the simulations of the TTFields delivery to the spine, a human male 34 years old realistic computational model (DUKE v3.1 by ITI’S, Zurich) and the ZMT’s Sim4Life v4.0 electro-quasi-static solver was utilized. TTFields were simulated by imposing an alternating current with a current density of 200 mA/disk and a frequency of 150 kHz on the outer surfaces of the disks of each pair of arrays.

RESULTS

For one of the tested array layouts, a high electric field was shown to be induced within the spinal cord and surrounding CSF: Our calculations of mean field intensity within the spine and nerves from vertebrae T8-T9 at the top to L3-L4 at the bottom added up to 1.77 V/cm. This layout consisted of the placement of a pair of arrays on the back of the patient, with one array positioned above the section in the spine to which treatment would be delivered, and the other array positioned below the target section. Notably, the resulting electric field is directed along the spine in this setting (ie, vertically).

CONCLUSION

Our results demonstrate that treatment of the whole spinal cord and nerves in a single direction can be achieved by placing a pair of transducer arrays on the patient’s back: one array on the neck, and one at the bottom of the spine. For the development of an active treatment in the perpendicular direction, further studies need to be conducted.
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

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

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

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

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

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

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

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

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

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).
P13.08 1H-MRS metabolomics as a predictor of overall survival in glioma patients
Authors: Solivera J , Fuentes Fayos A , Luque R M , Barrios L , Roda J M , Cerdán S , Gandía-González M L
Published: September 2019

BACKGROUND

We assess the efficacy of the metabolomic profile from glioma biopsies, in providing estimates of postsurgical Overall Survival in glioma patients.

MATERIAL AND METHODS

Tumor biopsies from 46 patients bearing gliomas, obtained neurosurgically in the period 1992–1998, were analyzed by high resolution 1H magnetic resonance spectroscopy (HR- 1H MRS), following retrospectively individual postsurgical Overall Survival up to 720 weeks.

RESULTS

The Overall Survival profile could be resolved in three groups; Short (shorter than 52 weeks, n=19), Intermediate (between 53 and 364 weeks, n=19) or Long (longer than 365 weeks, n=8), respectively. Classical histopathological analysis assigned WHO grades II-IV to every biopsy but notably, some patients with low grade glioma depicted unexpectedly Short Overall Survival, while some patients with high grade glioma, presented unpredictably Long Overall Survival. To explore reasons underlying this behavior, we analyzed HR- 1H MRS spectra from acid extracts of these biopsies, to identify the metabolite patterns underlying OS predictions. Poor prognosis was found in biopsies with higher contents of alanine, acetate, glutamate, total choline, phosphorylcholine and glycine, while more favorable prognosis was achieved in biopsies with larger contents of total creatine, glycerol-phosphorylcholine and myo-inositol. We implemented then a multivariate analysis approach to identify hierarchically the influence of these metabolomic biomarkers on OS predictions, using Classification Regression Trees (CRTs). Metabolomic CRTs grew up to 3 branches and split into 8 nodes, predicting correctly the outcome of 94.7% of the patients in the Short Overall Survival group, 78.9 % of the patients in the Intermediate Overall Survival group, and 75% of the patients in the Long Overall Survival group, respectively.

CONCLUSION

Present results suggest that metabolic profiling by HR-1H MRS provides more accurate Overall Survival estimates of glioma patients than classical histopathological grading, thus allowing to implement more accurate therapeutic decisions.
P13.11 Metabolomics and molecular profiling in glioma patients: an interactomic approach
Authors: Fuentes-Fayos A C , Gandía-González M L , Cano-Rojas A , Blanco C J , Negro-Moral E M , Toledano Á , Ramos M J , Cano-Sánchez A , Luque R M , Ortega-Salas R M , Roda J M , Cerdán S , García-Martín M L , Solivera J
Published: September 2019

BACKGROUND

A wider view of the interaction between different omic-domains is needed to identify potential biomarkers of low- and high-grade gliomas. Using an interactomic approach, we analyzed the correlation between radiological data, IDH mutation, gene expression profiling and metabolic signature in glioma samples.

MATERIAL AND METHODS

Tumor biopsies from 25 patients with clinical diagnosis of glioma were surgically collected during 2017–2019 at the senior author’s institution. Metabolomic data was obtained by high resolution 31P and 1H magnetic resonance spectroscopy (MRS, 19 metabolites quantified with LCModel). Gene expression profiling was performed using real-time qPCR of 19 genes related to energy metabolism. IDH1/2 common mutation (IDH1R132H/IDH2R172H) was verified by immunohistochemistry and amplicon sanger sequencing. All data was integrated using the R package mixOmics, and we built correlation network plot graphs and correlation maps to identify the most significant interactions, that were analyzed thereafter.

RESULTS

Mean age was 48±10 years and 72% were men. The most frequent clinical presentation was intracranial hypertension and focal deficit. Imaging revealed 88% of single lobar tumors, 96% of contrast enhancement, 52% located near eloquent areas, 48% with augmented perfusion (mean values of 300±130%) and 60% showed restricted diffusion. WHO 2016 diagnosis were glioblastoma IDH mutated (IDHmut, 16%), IDH wildtype (IDHw, 56%); anaplastic astrocytoma IDHmut (4%), IDHw (16%); diffuse astrocytoma IDHmut (4%), SEGA (4%). The genetic and metabolic profiles were normalized per sample using the total sum of all the studied variables per case. This step made the interactomic approach possible. We found no differences between the metabolic or genetic profiles of glioma grade III and IV samples. However, there was a statistical significance or near-threshold correlation between some metabolic patterns and IDH-mutation, where Alanine (4.7±1.3% IDHw vs 2.5±0.7 IDHmut, p=0.046), Glycine (2.7±0.5% vs. 1.6±0.4%, p=0.095), Glycerophosphorylcholine (3.9±0.4% vs. 6.4±0.9%, p=0.013) and Myo-inositol (4.9±1.0% vs 11.9±2.1%, p=0.004) were the most important biomarkers. Overexpression of Lactate Dehydrogenase subunit B (LDHB, 19±3% vs. 31±6%, p=0.039) and Aconitase 1 (ACO1, 0.5±0.1% vs 1.2±0.3%, p=0.08) had also a significant or near-threshold relationship with IDH-mutation. These correlations were shown as hot spots in the correlation graphs and maps.

CONCLUSION

These preliminary results indicate that metabolic patterns by high resolution 31P and 1H MRS could be a useful tool to improve our knowledge about glioma gene expression profiles and to identify potential biomarkers to tackle this pathology.
P13.12 Development and validation of a radiomic model for the preoperative prediction of transsphenoidal surgical response in patients with acromegaly
Authors: Fan Y , Feng M , Wang R
Published: September 2019

BACKGROUND

The preoperative prediction of transsphenoidal surgical (TSS) response is important for determining individual treatment strategies for acromegaly. Therefore, this study aimed to predict TSS response in a non-invasive way based on radiomic analysis.

MATERIAL AND METHODS

273 patients with acromegaly were enrolled and divided into primary (n=180) and validation cohorts (n=93) according to time point. Radiomic features were extracted from the MR images and determined using the ‘Elastic Net’ feature selection algorithm. A radiomic signature was built using a support vector machine. Subsequently, multivariable logistic regression analysis was used to select the most informative clinical features, and a radiomic model, incorporating the radiomic signature and selected clinical features, was constructed and used as the final predictive model. The performance of this radiomic model was validated using receiver operating characteristics analysis, and its calibration, discriminating ability, and clinical usefulness were assessed.

RESULTS

The radiomic signature, which was constructed with six radiomic features selected using the primary cohort, showed a favorable discriminatory ability in the validation cohort. The radiomic model incorporating the radiomic signature and three selected clinical features showed good discrimination abilities and calibration, with an area under the curve (AUC) of 0.93 for the primary cohort and 0.89 for the validation cohort. The radiomic model better estimated the treatment responses of patients with acromegaly than did the clinical features. Decision curve analysis showed the radiomic model was clinically useful.

CONCLUSION

This radiomic model could aid neurosurgeons in the preoperative prediction of TSS response in patients with acromegaly, and could contribute to determining individual treatment strategies.