bluuebunny/crossref_metadata_2025_split huggingface dataset:
P10.06.A Expression of GDNF and its receptors in glioblastoma
Authors: Ewald J D , Knudsen A M , Kristensen B W 
Published: September 2022

Abstract

Background
Neurotrophic factors are important for brain development, but their role in glioblastoma, the most aggressive primary brain cancer, is not fully understood. We focused on glial cell line-derived neurotrophic factor (GDNF), which has been shown to play a role in growth and migration in glioblastoma. The aim of this study was to investigate the expression of GDNF and its primary receptor GFRA1 as well as GDNF family receptors GFRA2-4 and RET.

Material and Methods
Tissue sections from 10 glioblastoma patients were stained with antibodies against GDNF (frozen tissue) and each of the receptors (formalin-fixed paraffin-embedded tissue). Moreover, the sections were stained with an RNA Scope assay for GDNF mRNA. The area fractions of positive receptor staining were quantified with a software-based classifier. Double immunofluorescence stainings were performed for GFRA1 and the astrocytic tumor cell marker GFAP, the microglia and macrophage marker IBA1, and the tumor stem cell markers OLIG2 and SOX2, respectively, for assessment of co-expression.

Results
GDNF staining in a varying number of cells was seen in 5 of the 10 samples. GDNF mRNA stainings were observed in a varying number of cells in all stained tissues, including in hypoxic areas and in microvascular proliferations. Mean area fractions of the receptors were 31 % (range 7-56) for GFRA1, 3 % (0,2-7) for GFRA2, 0,4 % (0,01-2) for GFRA3, 0,1 % (0,02-0,3) for GFRA4 and 1 % (0,1-2) for RET. Co-expression of GFRA1 and GFAP was widely present. Co-expression of GFRA1 and either OLIG2 or SOX2 was partially present. Co-expression of GFRA1 and IBA1 was limited.

Conclusion
Our results suggest that GDNF and GFRA1 are widely expressed in glioblastoma tissue, but GFRA2-4 and RET are also expressed and may play a role in glioblastoma tumor biology. Future experimental studies are needed to clarify the functional role of GDNF and its receptors in glioblastoma biology.
P10.07.B Differential YAP activity in human glioblastoma tumorspheres
Authors: Kim E , Oh Y , Kang S 
Published: September 2022

Abstract

Background
Although glioblastoma (GBM) is the most common primary brain tumor, the best available treatment options are still associated with poor prognosis. Recently, the Hippo/YAP signaling pathway has been emerged as an important driver of GBM. Nevertheless, extensive studies have not yet been focused on the importance of phosphorylation event in regulating endogenous YAP activity in GBM. Here, we sought to elucidate that the modulation and stabilization of YAP/TAZ in Hippo pathway promotes GBM progression.

Material and Methods
Expression patterns of YAP1, TAZ, CTGF, TEAD4 and LATS1 mRNA were confirmed in human normal and GBM tissue, and patient survival according to YAP1 expression was confirmed in TCGA and Severance cohort. The YAP1 expression patterns in several TSs were classified into SOH and AH according to transcriptome analysis and western blot. For each classified TSs, the characteristics of SOH and AH were comparatively analyzed through immunoprecipitation, nucleus fraction, TEAD4 luciferase assay, and confocal analysis. The effect of YAP1 knockdown/out and overexpression in SOH and AH TSs were measured by WST/ATP analysis, 3D invasion and neurosphere formation, and western blot. In addition, the drug reactivity of SOH and AH TSs was examined by treatment with the YAP1 inhibitors Peptide17 and verteporfin, and the effects of Peptide17, verteporfin and YAP1-shRNA were examined in in-vivo xenograft model.

Results
YAP1 mRNA levels including TAZ, CTGF, TEAD4 and LATS1 were higher in GBM than in normal tissues. Through transcriptome analysis and western blot, TS can be divided into SOH and AH groups according to the expression level of CTGF, but not YAP1 level. In the case of SOH TS, the binding of TEAD4 was strong in immunoprecipitation, the ratio of YAP1 was higher in the nucleus in the nucleus fractionation and confocal analysis, and the luciferase activity of TEAD4 was high in the TEAD4 luciferase assay. On the other hand, in the case of TS of AH, the opposite trend was observed. When YAP1 was knocked down or out, the proliferation, invasion, and stemness of TS tended to be decrease more in SOH TS than in AH TS, and SOH TS showed more sensitive drug response even in Peptide17 and Verteporfin treatment. In the case of YAP1 overexpression, the opposite trend was observed in SOH TS. In the in-vivo xenograft model, injection of Peptide17, verteporfin, and YAP1-shRNA showed a tendency to increase the survival rate of mice.

Conclusion
The association of YAP has been described in several cancers, but there has been no study comparing GBM TS by YAP expression and classifying it at the functional level. Now, for the first time, we show that the characteristics of GBM TS can be predicted according to the expression value of YAP, respectively, and YAP is expected to be applied as an important prognostic factor for GBM treatment in the future.
P10.08.A Lipogenesis inhibition by fatostatin shows effectiveness in glioblastoma models highly expressing fatty acid synthase (FASN)
Authors: Kastler L , Neumayer K , Laaber M , Roschger C , Lenz S , Lötsch-Gojo D , Berger W , Gruber A , Spiegl-Kreinecker S 
Published: September 2022

Abstract

Background
Glioblastoma (GBM) is the most common and most aggressive primary brain tumour in adults. Despite intensive treatment, the medium overall survival of GBM patients remains below 20 months after diagnosis. Therefore, new tumour specific targets allowing successful therapeutic treatment are needed to distinctly enhance GBM patient overall survival. Reprogramming of lipid metabolism leading to highly upregulated anabolic pathways, like phosphatidylinositol-3-kinase (PI3K)/AKT pathway and subsequently lipogenesis, is a hallmark of cancer cells including glioblastomas to meet increased energy demands for proliferation and cell growth. Accordingly, lipogenesis inhibition might be a promising strategy to impede GBM cell growth. Therefore, it was aim of our study to analyse the effect of the lipogenesis inhibitor fatostatin on downstream targets e.g. fatty acid synthase (FASN) and its impact on cell growth in GBM cells in vitro.

Material and Methods
In a cohort of 52 GBM-derived cell lines, FASN mRNA was investigated by qRT-PCR. The impact of fatostatin on cell viability (IC50) was analysed in selected cell lines using cell viability assays (Cell Titer Glo®). To elucidate whether fatostatin exposure affects cell migration, wound healing assays were performed. The effect of fatostatin on cell cycle and apoptosis was analysed by flow cytometry. Additionally, expression of FASN and level of phosphorylated AKT, the active form of AKT serine/threonine kinase, were examined before and after fatostatin treatment with qRT-PCR (mRNA) and Western blot (protein), respectively.

Results
FASN mRNA was found to be expressed at varying levels in our GBM-derived cell line cohort (n=52). Compared to normal astrocytes, FASN mRNA expression was significantly upregulated in GBM cells (p=0.0127). Furthermore, high FASN mRNA expression was significantly associated with higher sensitivity against fatostatin (p=0.0352). Regarding corresponding recurrences, the response to the inhibitor was shown to be persistent. Accordingly, the effectiveness of fatostatin treatment resulted in substantial downregulation of AKT phosphorylation and consequently in the induction of apoptosis and G2/M arrest of the cell cycle. In addition, a significant inhibition of the migration potential by fatostatin treatment was observed.

Conclusion
In summary, FASN expression represents a promising biomarker and therapy target within the lipid metabolism indicated by a significant sensitivity to fatostatin in FASN overexpressing GBM cell lines. Consequently, lipogenesis inhibition by fatostatin might be a promising novel therapeutic option in glioblastoma.
P10.09.B Retroelement expression in glioma tumors exhibits subtype specific patterns
Authors: Rodriguez Almaraz J , Guerra G , Wendt G , Chang S , Francis S S 
Published: September 2022

Abstract

Background
There are ~3 million transposable elements in the human genome constituting about 42% of all basepairs. Retroelements (REs) are ~90% of the transposable elements present in the human genome. Active REs are considered highly mutagenic and have been implicated in multiple steps of cancer development and progression, as well as in neurologic diseases. RE activity has functional effects on the genome, including the maintenance of centromere and telomere integrity, and deleterious gene expression. Previous studies have shown that certain families of RE (HERVK, L1, Alu) are expressed in gliomas, however, their specific role as arbitrators of oncogenesis or promoters of the innate anti-tumor immune response remains uncertain. Moreover, it has been shown that a soluble form of PD-L1 (sPD-L1) that blocks its inhibitory activity is produced by exaptation of an intronic endogenous retroelement (LINE-2A) in the gene encoding PD-L1, highlighting the importance of REs as potential therapeutic targets. In this analysis we aim to identify the unique patterns of RE expression across major subtypes of glioma.

Material and Methods
We conducted a differential expression analysis of 49 RE families using RNA-seq data measured in glioma tumors from The Cancer Genome Atlas (TCGA). RE counts were produced using the software REDiscoverTE. Pairwise comparisons between glioma subtypes (defined by WHO2021) were done using in 625 tumor samples adjusting for age, sex and race.

Results
10 of the 49 considered RE families exhibited significantly different (false discovery rate, FDR, &lt;0.05) expression in at least one glioma subtype. Alu(Fold change, FC=1.5), RNA(FC=11.3), PiggyBac(FC=1.6), rRNA(FC=5.23) and Dong-R4(FC=1.8) were overexpressed in IDH-wildtype glioblastoma while Gypsy(FC=0.4) and CRP1(FC=0.26) were decreased in expression. scRNA (FC=2.7) were overexpressed in IDH-mutant oligodendroglioma compared to glioblastoma while Dong-R4 (FC = 0.53) showed decreased expression. LTR (FC=2.02) and tRNA-Deu (FC=1.46), showed increased expression in IDH-wildtype diffuse astrocytomas compared to IDH-mutant, 1p/19q-codeleted oligodendrogliomas while Gypsy (FC =0.41) showed decreased expression.

Conclusion
We have shown that expression of certain RE families within gliomas have subtype-specific patterns. While it is well established that RE expression is dysregulated in cancer, our analysis is the first at exploring a wide range of retroelements in the context of glioma by subtype. Given the important role of REs in transcriptional control, genomic instability, chromosomal rearrangements, and oncogenic activation, the identification of individual families and specific REs in glioma holds an intrinsic value to potential biomarkers and immunotherapy targets.
P10.11.B Re-sensitizing cancer cells to Tumor Treating Fields (TTFields) through PI3K/AKT/mTOR pathway inhibition
Authors: Klein-Goldberg A , Voloshin T , Zemer-Tov E , Paz R , Koren L , Wainer-Katsir K , Wolfbauer C , Haber A , Giladi M , Weinberg U , Palti Y 
Published: September 2022

Abstract

Background
Tumor Treating Fields (TTFields) are alternating electric fields disrupting cellular processes critical for cancer cell replication and tumor progression. TTFields therapy is approved for treatment of newly diagnosed glioblastoma (GBM), recurrent GBM, and unresectable malignant pleural mesothelioma, and is currently under clinical investigation for treating other solid tumors, such as ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The research herein aimed to identify potential treatment escape mechanisms and explore the possibility of targeted inhibition of these pathways for re-sensitizing the cells to TTFields.

Material and Methods
GBM U-87 MG, ovarian A2780, and NSCLC H1299 cells were treated with TTFields (1.7 V/cm RMS, 200 or 150 kHz) for continuous long-term application (7 or 13 days, specific conditions depending on the cell line). Changes in signaling pathways in these cells relative to cells exposed to short term TTFields application (3 or 7 days, depending on cell line) were examined by Luminex multiplex assay. Specific pathway markers were examined by immunohistochemistry of tumor sections from sham or TTFields-treated rats bearing N1S1 HCC tumors. TTFields were then co-applied with relevant pathway inhibitors, followed by cell count measurements and western blot examinations for specific pathway markers. The concomitant application of TTFields with a selected inhibitor was tested in mice inoculated orthotopically with MOSE-L firefly luciferase (FFL) ovarian cancer cells. Tumor volume was measured at study end by luciferin signal detection using the In Vivo Imaging System (IVIS).

Results
Cancer cells exposed to long-term application of TTFields displayed decreased sensitivity to TTFields. The PI3K/AKT/mTOR pathway was activated in these cells, with significant increases in AKT and RPS6 phosphorylation levels also observed in HCC tumors from rats treated with TTFields. PI3K inhibitors re-sensitized the cells to TTFields cytotoxicity, with associated down regulation of AKT phosphorylation. Application of TTFields concomitant with the PI3K inhibitor alpelisib resulted in enhanced efficacy in the ovarian cancer mouse model.

Conclusions
The current research demonstrated that PI3K/AKT/mTOR signaling pathway activation was involved in the response to long-term application of TTFields, with increased phosphorylation of key proteins observed both in vitro and in vivo. Concomitant treatment with PI3K inhibitors and TTFields re-sensitized the cells to TTFields, as it alleviated TTFields-induced AKT activation.
P10.16.A Repurposing thioridazine as a novel treatment of melanoma brain metastasis
Authors: Moen Taule E , Wikerholmen T , Rigg E , Sarnow K , Wang J , Thorsen F 
Published: September 2022

Abstract

Background
Melanoma shows increasing incidence and has the highest propensity to metastasize to the brain of all primary malignant tumors. Untreated, the median survival time is 2-3 months after diagnosis, while aggressive treatment extends survival to only 4-12 months. Even though targeted therapies and immunotherapy has changed the therapeutic landscape, many of these patient relapse. In this respect, drug repurposing, using old drugs for new purposes, has had several successes for other diseases. The anti-psychotic drug thioridazine (THD) is a dopamine receptor 2 antagonist, with properties to cross an intact blood brain barrier, has shown promising effect on melanoma cells that has escaped prior treatment and on cancer stem cells. However, the therapeutic effects on melanoma brain metastasis (MBM) have not been previously investigated.

Material and Methods
All experiments were done on MBM cell lines (H1, H2, H3 and H10), which were developed in our lab from patient biopsies obtained after surgery. A monolayer viability assay (WST-1) was used to determine IC50 doses. Migration and proliferation after treatment was studied by IncuCyte live cell imaging. Colony forming properties and the ability of cells to grow in an anchorage free environment after THD treatment was investigated using a 2D clonogenic assay and a 3D soft agar model. Potential drug effects were also studied on normal human astrocytes (NHA) and on fetal rat brain organoids (FRBO), using a Live/Dead viability kit and confocal imaging. Apoptosis and necrosis was studied by Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis after 72 hours of treatment.

Results
THD shows a strong cytotoxic effect on MBM cell lines. Cell viability studies showed that THD exerted a dose-dependent inhibition of tumor cell viability (IC50 doses 9-12 µM), while NHA tolerated similar drug concentrations. Tumor cell migration and proliferation were almost completely inhibited with concentrations below the IC50 doses. An inhibition of clonogenic formation and 3D growth was also observed. Bright field microscopy showed changes in MBM morphology in monolayers, but FRBOs were intact after drug treatment. Further, apoptosis in MBM cells was induced after treatment with THD.

Conclusion
Our preliminary data shows that THD has a strong selective cytotoxicity in MBM cells with minimal effects on NHA and FBRO.
P10.18.A Replication of previous GWAS identifies TERT and near EGFR SNVs as risk factors in EPIC glioma patients: a nested case-control study
Authors: Wu W , Dahlin A , Andersson U , Wibom C , Jonsson S , Melin B 
Published: September 2022

Abstract

Background
Gliomas, the most common malignant primary brain tumors in adults, typically have a poor prognosis irrespective of medical care. Previous large genome-wide association studies (GWAS) have identified 27 single-nucleotide variants (SNVs) that are significantly associated with glioma. However, most of the GWAS are conducted by case-control study designs, it is therefore prone to bias when rapidly lethal cases don’t have chance to be included in the study. This study aims to replicate the previous GWAS findings using prospective study design.

Material and Methods
We conducted a nested case-control study within the European Prospective Investigation into Cancer (EPIC) cohort from 7 European countries. GSA-MD Infinium global screening array was used for genotyping. Some subjects were genotyped by other platforms previously. In total, 468 glioma patients and 481 controls were included. The genotypes of 27 SNVs were extracted and for ungenotyped SNVs, datasets were imputed using SHAPEIT v4.1.3 and IMPUTE5 v1.1.5 based on the Haplotype Reference Consortium (Release 1.1) reference panel. Conditional logistic regression model was used to investigate the additive effect of SNVs on the risk of glioma.

Results
21 SNVs showed a consistent direction of effect with previous studies, whereas 6 SNVs did not (ORs between 0.72-0.99 and not significant). After adjusting for multiple testing, two SNVs, rs10069690 (TERT), and rs75061358 (near EGFR) were significantly associated with glioma risk. We observed that prominent OR (2.23, 95%CI=1.49-3.33) of rs75061358 in our study compared to the result from previous GWAS, which implied rs75061358 might be not only a risk factor but also affect survival. Different risk direction was observed for rs77633900 in ETFA gene (OR=0.72, 95%CI=0.51-1.01).

Conclusion
Our findings further confirmed the genetic role on the etiology of glioma in the European population. The potential biases from the previous GWAS are required to be elucidated.
P10.20.A Mechanisms of synergistic glioma growth suppression by radiotherapy and MET inhibition
Authors: Silginer M , Papa E , Szabo E , Vasella F , Pruschy M , Stroh C , Roth P , Weiss T , Weller M 
Published: September 2022

Abstract

Background
Glioblastoma remains to be one of the most lethal solid cancers. Despite multi-modal therapy including surgery as safely feasible, radiotherapy and chemotherapy with the alkylating agent temozolomide, the median survival of affected patients is still limited to approximately one year on a population level. Thus, novel therapies are urgently needed. There is increasing interest in the role of the HGF/MET pathway in the response of glioblastoma to radiotherapy since MET may be involved in radioresistance via proinvasive and DNA damage response pathways.

Material and Methods
Here we assessed the role of the MET pathway in the response to radiotherapy in vitro and in vivo in syngeneic mouse glioma models and explored potential modes of action responsible for the synergistic effects of MET pathway inhibition and irradiation on tumor growth in vivo.

Results
Murine glioma cells express HGF and MET and show increased MET phosphorylation upon exposure to exogenous HGF. In vitro, glioma cell viability and proliferation are not affected by pharmacological MET inhibition using tepotinib or genetic MET inhibition using CRISPR/Cas9-engineered Met gene knockout and sensitization to irradiation by MET inhibition is not seen. In vivo, the combination of MET inhibition with focal radiotherapy mediates prolonged survival of syngeneic orthotopic glioma-bearing mice compared with either treatment alone. Complementary studies demonstrate that synergy is lost when gliomas are established and treated in immunodeficient mice, but also if MET gene expression is disrupted in the tumor of wildtype mice. Combination therapy suppresses a set of pro-inflammatory mediators that are upregulated by radiotherapy alone and which are positively regulated by transforming growth factor (TGF)-β. In line with this data, ex vivo analysis of mouse brains reveal increased TGF-β pathway activity upon irradiation alone that is counteracted by concomitant MET inhibition.

Conclusion
In summary, we demonstrate synergistic suppression of syngeneic glioma growth by irradiation and MET inhibition that requires MET expression in the tumor as well as an intact immune system. Clinical evaluation of this combined treatment approach in newly diagnosed glioblastoma is warranted.
P11.02.A Gliotarget: a Danish nationwide phase I/II platform trial focusing on individualized targeted treatment for newly diagnosed glioblastoma patients based on genomic profiling
Authors: Fougner V N , Urup T H , Hasselbalch B , Lassen U , Poulsen H S 
Published: September 2022

Abstract

INTRODUCTION
The survival in glioblastoma has been unchanged since 2005. The introduction of targeted treatments, which have improved survival in several cancers has yet to influence the treatment of glioblastoma. In Gliotarget, we will individualize the targeted treatment, and give it in the primary setting alongside standard therapy with the intention to improve the likelihood of effect.

METHODS
Gliotarget is a biomarker enriched phase I/II platform trial with initially 4 predefined biomarker selected arms and one control arm. All patients receive standard therapy with concomitant radiochemotherapy and adjuvant temozolomide. The experimental treatment is given alongside the adjuvant temozolomide. Molecular analysis, including Whole Genome Sequencing, is performed on all patients, to identify actionable biomarkers. Gliotarget includes newly diagnosed IDH-wt glioblastoma patients. In addition, to exclude the patients where the inherent poor prognosis might conceal the drug efficacy, the patients must have a 50% probability of being alive 12 months after initial surgery, according to a prognostic model developed in our institution. The sample size is calculated with Simon’s two-stage design using treatment effect at 9-months progression free survival (PFS9). We anticipate 9 patients for stage one and 24 in total, for each arm. Treatment effect is defined as 65% of the patients in an experimental arm reaching PFS9. For the survival analysis, supplementing the control arm data with leveraged external controls taken from our prospectively registered database will decrease the probability of false positive results.

CONCLUSION
Gliotarget complements the field of ongoing platform trials with its distinctive trial design. The chosen biomarkers and treatments will be presented at the 2022 EANO meeting. Enrollment is set to open in Q4 2022.