bluuebunny/crossref_metadata_2025 huggingface dataset:
P01.01.A CHARACTERIZATION OF GLIOBLASTOMA’S MIGRATION HETEROGENEITY THROUGH A PATIENT-DERIVED 3D MODEL
Authors: Pujol-Castiblanque A , Savin T , Markaki A E , Mair R
Published: October 2024

BACKGROUND

Glioblastoma (GB) is an aggressive brain cancer with an average survival of 14.6 months. Effective treatment is hindered by GB’s plasticity and invasive phenotype. Little is known regarding the molecular mechanisms driving invasion heterogeneity.

MATERIAL AND METHODS

We developed a bioengineered model of brain-like matrix, which together with patient-derived spheroids, allows study of GB’s migration in vitro. Twenty patient-derived cell lines with different intrinsic (genomic, transcriptomic) characteristics in several extrinsic (extracellular matrix stiffness, hypoxia vs normoxia) conditions were tested.

RESULTS

Time-lapse video analysis identified recurrent migratory phenotypes across the cell lines. GB’s subtype- proneural, neural, classical, and mesenchymal- did not cluster cell lines with similar migratory phenotypes. An increase in matrix stiffness led to a switch in migratory phenotype in certain cell lines.

CONCLUSION

We have identified finite and periodic migration phenotypes of GB in different conditions.
P01.06.B GLIOMA STEM CELLS REMODEL ASTROCYTES TO FACILITATE GBM PROGRESSION VIA EXOSOMAL ZNF16 ACTIVATED TGF-Β SIGNALING AXIS
Authors: Dong J
Published: October 2024

BACKGROUND

Tumor-associated astrocytes (TAAs) play crucial promoting roles during tissue remodeling of glioma stem cells (GSCs), however, molecular mechanisms regulating astrocytes transformation, as well as glioblastoma progression by TAAs have not been fully clarified.

MATERIAL AND METHODS

Normal human astrocytes (NHAs) were cultured in vitro and continuously treated with GSC-derived exosomes (GSC-exo). Cellular phenotypes of TAAs were analyzed, expression of TAAs associated markers TGF-β, CD44, and tenascin-C was assayed by Western blot. Bioinformatic prediction of upstream transcription factor of TGF-β was performed and validated by RT-PCR, dual-luciferase reporter assay. Cell-based assay of glioblastoma proliferation and chemo-resistance were performed through flow cytometric assay, Western blot and colony formation assay, respectively. A xenograft tumor model was applied to verify the role of exosomal ZNF16 activated TGF-β signaling axis in vivo.

RESULTS

Notably, under the influence of GSC-exo, NHAs undergo transformation into tumor-associated astrocytes (TAAs) within three days, expressing hallmark surface markers such as TGF-β, CD44, and Tenascin-C. The transcription factor zinc finger protein 16 (ZNF16) was directly bound to the TGF-β promoter region and enhanced TGF-β expression.This transformation was associated with upregulation of TGF-β in NHAs activated by GSCs exosomal ZNF16. TGF-β is a direct downstream target of ZNF16 in TAAs. Transformed TAAs exhibit morphological changes, enhanced proliferative and migrative capacities, as well as contributing to aggressiveness and chemo-resistance of glioblastoma. In vivo studies disclosed that targeting on GSC-exosomal ZNF16 or TGF-β can suppressive transformation of TAAs and inhibiting their tumor promoting role on glioblastoma progression.

CONCLUSION

Understanding the intricate mutual interactions between GSCs and astrocytes in glioblastoma microenvironment can shed light on new therapeutic strategy on disrupting GSCs-TAAs crosstalk against glioblastoma progression.
P01.12.A PERSISTENT GENETIC AND EPIGENETIC CHANGES ARE INDUCED BY INVASION STIMULI IN GLIOBLASTOMA CELLS
Authors: Müller T , Schreiner S , Cialini C , Salcedo U M , Mittelbronn M , Plate K H , Steinbach J , Hau A
Published: October 2024

BACKGROUND

One key biological challenge in glioblastoma (GB) is the diffuse invasion of GB cells into the brain parenchyma beyond the margins of even supramaximal resection strategies. Various cell-intrinsic and extrinsic factors have been shown to regulate GB invasion, but it is unknown whether invasive GB cells acquire a persistent distinct genetic and epigenetic profile in response to environmental cues.

MATERIAL AND METHODS

Highly invasive isogenic subpopulations of GB cells (GBinv+ cells) were generated by serial invasion through matrix-coated transwell membranes. RNA sequencing and array-based DNA methylation profiling of control and GBinv+ cells were performed. Differentially expressed genes (DEGs) and differentially methylated probes (DMPs) were analysed using DESeq and RnBeads R package, respectively. Functional correlations of DEGs and DMPs were analysed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Candidate genes were validated using TCGA-LGG/GBM datasets and experimentally confirmed by genetic modulation of gene expression changes and applying classical in vitro invasion assays as well as the GLIoma Cerebral Organoid (GLICO) model for complex ex vivo tumor cell invasion analysis.

RESULTS

In classical in vitro invasion assays, the invasion potential of GBinv+ cells were more than 10-fold increased relative to the isogenic control cells baseline (p < 0.0001, students t-test). Increased invasion was also demonstrated using the GLICO model. GBinv+ cells were found to have unique transcriptomic and DNA-methylation profiles that persisted for >2 weeks after removal of the stimulus. Genetic deletion of candidate genes identified by profiling reduced invasive properties.

CONCLUSION

Our results indicate that the invasive sub-population of GB cells is characterized by a specific genetic and epigenetic profile and has enduring functional properties. The functional validation of the identified genes associated with invasion underscores their clinical relevance and suggests they may represent interesting new targets for experimental therapies.
P02.04.B CANCER-ASSOCIATED FIBROBLASTS IN LUNG CANCER BRAIN METASTASES: EFFECT ON CANCER CELL MIGRATION AND INVASION, AND MONOCYTE RECRUITMENT AND DIFFERENTIATION
Authors: Vymolova B , Smetana T , Pfeiferova L , Sachova J , Kolar M , Ternerova N , Vymola P , Svablova T , Houdova Megova M , Busek P , Sedo A
Published: October 2024

BACKGROUND

Brain metastases (BrM) are a frequent complication of lung cancer with dismal prognosis. The presence of cancer associated fibroblasts (CAF) was recently described in the BrM microenvironment, but their engagement in BrM biology is largely unknown. In this study, we evaluated the effect of CAF derived from lung cancer brain metastases (BrM-CAF) on cancer cells and the recruitment and differentiation of monocytes.

MATERIAL AND METHODS

Patient-derived BrM-CAF and cancer cells (BrM-CC) were isolated from lung cancer BrM. Normal fibroblasts were isolated from subgalear connective tissue. Monocytes were isolated from healthy donors’ peripheral blood. Patient-derived cell cultures were characterized using immunocytochemistry and RNA expression profiling. A transwell assay and a 3D spheroid-based assay were used to assess migration and invasion, respectively; cell growth was quantified using a kinetic label-free cell counting assay. Monocyte differentiation was evaluated by flow cytometry.

RESULTS

BrM-CAF expressed canonical fibroblast markers and their fibroblast-like phenotype was verified by RNA expression profiling. BrM-CC expressed epithelial markers. Conditioned media from BrM-CAF stimulated the migration of BrM-CC more than conditioned media from normal fibroblasts. In the invasion assay, the cancer cell invasive area was bigger in the presence of BrM-CAF; in addition, we observed a different pattern of invasion in BrM-CAF-containing spheroids, with more protrusions being formed. Direct co-culture with BrM-CAF diminished cancer cell proliferation. BrM-CAF conditioned media induced migration of monocytes. In direct co-culture with BrM-CAF, monocytes acquired a differentiated phenotype with both M1-like and M2-like features similar to that of tumor associated macrophages.

CONCLUSION

Using patient-derived cultures, we show that BrM-CAF promote cancer cell migration and invasion but decrease their proliferation. Moreover, BrM-CAF can contribute to the recruitment of peripheral monocytes and their differentiation into tumor-associated macrophages. Taken together, these results demonstrate the role of BrM-CAF in shaping the brain metastatic microenvironment.

FUNDING

This research was funded by the Charles University grant 342522; project NU22-03-00318 of Ministry of Health of the Czech Republic, Program Cooperatio, research area,,Oncology and Haematology“; Ministry of Education, Youth and Sports of the Czech Republic - LM2023053 of EATRIS-CZ and project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) by the European Union - Next Generation EU.
P02.08.A HERV-W ENVELOPE PROTEIN IS PRESENT IN MICROGLIAL CELLS OF THE HUMAN GLIOMA TUMOR MICROENVIRONMENT AND DIFFERENTIALLY MODULATES NEOPLASTIC CELL BEHAVIOR
Authors: Reiche L , Plaack B , Lehmkuhl M , Weyers V , Gruchot J , Picard D , Perron H , Remke M , Reifenberger G , Küry P , Knobbe-Thomsen C , Kremer D
Published: October 2024

BACKGROUND

Glioma is the most common and aggressive primary tumor of the central nervous system (CNS). With regard to its still unclear etiology, several recent studies focused on human endogenous retroviruses (HERVs) as potential new pathogenic factors in various neurological diseases. Although HERVs account for up to 8 % of the human genome, these retroviral elements are epigenetically silenced but can be reactivated by environmental factors such as infection with modern viruses. The resulting production of viral particles and/or proteins exerts effects on cellular processes such as inflammation, proliferation and migration in neurological diseases such as amyotrophic lateral sclerosis (ALS), schizophrenia, chronic inflammatory polyneuropathy (CIDP) and multiple sclerosis (MS). In previous studies, we demonstrated that in MS, the human endogenous retrovirus type-W envelope protein (HERV-W ENV) interferes with lesion repair through the activation of microglia (MG), the innate myeloid immune cells of the CNS. In this study, we investigated whether HERV-W ENV is expressed in gliomas. In particular, we aimed to understand how and to what extent HERV-W ENV modulates neoplastic cell behavior.

MATERIAL AND METHODS

We used immunohistochemical staining to detect HERV-W ENV in human gliomas. The mRNA expression of HERV-W ENV in tissue samples of human gliomas was quantified via qRT-PCR. Using the human glioblastoma cells T98, A172 and LN229 we investigated the effects of HERV-W ENV on various molecular and cellular markers, including gene and protein expression, proliferation and migration.

RESULTS

We show that HERV-W ENV is present in the microglial cells of the tumor microenvironment (TME) of human glioma. It modulates the behavior of human glioblastoma (GBM) cell lines in GBM/MG cocultures by altering their gene expression, secretome, morphology, proliferation, and migration properties and could thereby contribute to key tumor properties.

CONCLUSION

This study shows that HERV-W ENV is a relevant player in the interplay between glioma and microglial cells within the tumor microenvironment. In vitro evidence suggests that HERV-W ENV modulates the drivers of glioma progression via microglia. Whether this can be used therapeutically by blocking HERV-W ENV with the monoclonal antibody temelimab will have to be investigated in future studies.
P02.09.B HYPOXIA MODULATES DIFFUSE ASTROCYTOMA TUMOR MICROENVIRONMENT THROUGH CELL-TYPE-DEPENDENT RESPONSES
Authors: Tiihonen A , Salonen I , Koivisto I , Ritamäki A , Jaatinen S , Hyvärinen T , Kreutzer J , Mohammadlou M , Valkonen M , Ruusuvuori P , Parkkila S , Kallio P , Hagman S , Kesseli J , Ravi V M , Haapasalo H , Jukkola A , Joseph K , Rautajoki K J
Published: October 2024

BACKGROUND

Diffuse astrocytomas, especially glioblastomas (GBs), are aggressive brain tumors with a poor prognosis despite the efforts to improve their treatment. In diffuse astrocytomas, myeloid immune cells, notably monocyte-derived macrophages and brain-resident microglia are pivotal in the immunoregulation of tumor microenvironment (TME). GBs, characterized by higher macrophage frequencies, often exhibit hypoxia in the TME. In this work, we investigated how hypoxia affects the regulation of myeloid immune cells.

MATERIAL AND METHODS

We analyzed 222 diffuse astrocytoma patient samples using multiplex immunohistochemistry, 45 GBs with single-cell RNA sequencing (SCS) data, and 30 diffuse astrocytoma samples using spatial transcriptomics. The resulting data was analyzed with QuPath as well as R and Python-based tools and scripts.

RESULTS

Our findings reveal that hypoxia generates distinct spatial and gene expression responses in macrophages and microglia. Microglia decrease in highly hypoxic areas, while macrophages increase gradually compared to normoxia. In SCS data, macrophages show the highest proportion of cells under hypoxia, and the responding differentially expressed genes in macrophages and microglia are associated with different biological processes. Our results are further validated with spatial transcriptomics and cell culture models.

CONCLUSION

Hypoxia is significantly shaping the TME and immunosuppression in diffuse astrocytomas.
P02.15.A INVESTIGATING THE ROLE OF CELLULAR SENESCENCE IN BRIDGING GLIOBLASTOMA AND ALZHEIMER’S DISEASE
Authors: Krause E , Greutter L , Romanovskaia D , Stepper P , Wöhrer A
Published: October 2024

BACKGROUND

With changing demographics, age-related diseases like neurodegenerative disorders and cancer are becoming increasingly prevalent. We previously demonstrated that over 50% of glioblastoma (GB) patients show Alzheimer’s Disease (AD)-like changes in the tumor-adjacent cortex. However, whether these diseases are biologically linked remains unclear. Here, we investigate cellular senescence, a hallmark of aging, as a shared mechanism.

MATERIAL AND METHODS

We utilized single cell and single nucleus RNA sequencing datasets from 110 GB patients (GBmap core atlas) and 89 individuals with varying stages of AD (SEA-AD MTG atlas). Following data integration, we assessed senescent cells using three established gene lists, performed differential gene expression and conducted gene set enrichment analysis. Senescence scores were compared across different cell types and patients. Co-expression modules with senescence-associated genes were identified using weighted gene co-expression network analysis (WGCNA). Quantitative neuropathological analysis of published measurements (Iba1, AT8, 6E10) complemented the molecular findings.

RESULTS

Senescence was found in both GB and AD patient cells including cell types of the microenvironment. Notably, the variability in scores per patient was significantly higher in GB as compared to AD cell types, indicating greater inter-patient heterogeneity. Microglia were specifically enriched for senescence gene sets among glial cells. Subset analysis of microglia through data harmonization identified a proinflammatory GB microglia subtype that shared high similarity with AD microglia and had highest senescence scores. Notably, the increase in senescent microglia was associated with progression to advanced disease stages in both diseases. WGCNA identified 3 consensus modules of senescence-associated genes that were coexpressed with disease-specific genes, like PTEN implicated phosphorylation in the AD module and SPP1 for extracellular matrix remodeling in the GB module. All senescence associated modules showed increased expression in more advanced disease stages. Quantitative analysis of immunohistochemistry measurements in the AD dataset confirmed that samples with a high senescence score showed a significantly higher density of tau pathology and an altered microglia morphology.

CONCLUSION

Our study identifies cellular senescence as one connection between GB and AD pathology by utilizing atlas-level transcriptomic datasets. Particularly microglia upregulate senescence signatures with disease progression. The co-expression of senescence-associated genes within disease-specific modules underscores the complexity of senescence in this context.
P02.18.A MOLECULAR CHANGES UNDERLYING DECAY OF SENSORY RESPONSES AND ENHANCED SEIZURE PROPENSITY IN PERITUMORAL NEURONS
Authors: De Santis E , Tantillo E , Scalera M , Meneghetti N , Cerri C , Menicagli M , Mazzoni A , Costa M , Mazzanti C , Vannini E , Caleo M
Published: October 2024

BACKGROUND

Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target.

MATERIAL AND METHODS

We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection, we analyzed layer II-III pyramidal neurons molecular profile and with local field potentials recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice.

RESULTS

We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (ie, AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signaling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in 2 mouse models of tumor-bearing mice.

CONCLUSION

These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients’ quality of life.
P02.20.A NICHE-DEPENDENT MODES OF IMMUNE CELL TRAFFICKING IN GLIOBLASTOMA OFFER TARGETS FOR NOVEL IMMUNOTHERAPIES
Authors: Sankowski R , Dalmau Gasull A , Prinz M
Published: October 2024

BACKGROUND

Glioblastoma trigger systemic immune responses, resulting in elevated white blood cell counts and elevated temperatures, as the body attempts to combat tumor cells. However, glioblastoma reprograms immune cells to evade immune-mediated destruction and promote tumor growth. While targeted immunotherapies have shown promise in some cancers like melanoma, they have not been efficacious for malignant brain tumors, leading to significant societal costs due to high morbidity and mortality rates.

MATERIAL AND METHODS

Understanding immune cell trafficking is crucial for developing effective immunotherapies. We conducted spatially resolved single-cell analyses to investigate immune cell entry into glioblastoma. Integrating these data with multi-omic sequencing analyses of anatomically dissected specimens from glioblastoma-affected human brain border regions and tumor cores revealed distinct paths of immune cell trafficking into glioblastoma.

RESULTS

One path involves an ordered entry of mononuclear cells through the subarachnoid and perivascular spaces, guided by chemotactic cues. Cells in these regions display markers of immune activation, suggesting immune priming. Another path occurs at sites of vascular leakage due to hypoxia and necrosis, where a mix of mononuclear cells and neutrophils accumulate. These cells exhibit immunosuppressive transcriptomic programs, regulated by the phagocytosis-inducing transcription factor MITF and downstream metabolic changes controlled by PPAR-gamma. Ongoing analyses aim to identify pharmacological agents that can modulate immune cell reprogramming at hypoxic niches.

CONCLUSION

In summary, we have identified distinct modes of immune cell trafficking in glioblastoma. Chemotaxis-mediated trafficking via the brain border regions appears to prime immune cells, while entry at hypoxic niches is associated with immune suppression. Pharmacological agents shielding immune cells from immunosuppressive molecular cues in the tumor microenvironment show promise for glioblastoma immunotherapy.
P02.21.A PROGNOSTIC ROLE OF CD68 AND CD163 IN GLIOMAS
Authors: Petrescu G E D , Dragomir M P , Brehar F M , Gorgan R M
Published: October 2024

BACKGROUND

Tumor-associated macrophages (TAMs) have been demonstrated to play an important role in the growth and progression of multiple types of cancer, including glioblastoma. The aim of this study is to evaluate the distribution of CD68 and CD163 markers for TAMs in glioma samples and to determine their impact on survival.

MATERIAL AND METHODS

A tissue micro-array was performed using glioma samples from patients who underwent surgery at our institution. Diagnosis was established based on the WHO 2021 CNS classification. Thirty-six glioblastoma IDH-wt (GBM) and 15 oligodendroglioma, IDH mutant and astrocytoma, IDH mutant (CNS WHO grade 2 or 3) were included in the study. For comparison CNS WHO grade 2 or 3 samples were grouped. Immunohistochemical staining was performed for the detection of CD68 and CD163. CD68+ and CD163+ stainings were evaluated by counting the number of positive cells using QuPath (ver 0.4.4). For survival analysis the optimal cut-off method was used.

RESULTS

There was a statistically significant difference between the median CD68+ count in GBM vs. gr.2-3 (8.51 vs. 3.27, p =0.0009), and between the median CD163+ count (20.52 vs. 1.38, p<0.0001). By using the Spearman test, we observed a positive correlation (0.595) across all glioma samples between CD68 and CD163 (p < 0.0001). The Log-rank test showed increased survival in samples with low vs. high CD163+ count (median OS: 42 months vs 12 months, p = 0.0004) across all glioma samples. Similarly, there was a difference between the low (median OS: 34 months) and high (median OS: 17 months) CD68+ count, but without statistical significance (p = 0.11). A significant difference was also noted between samples with low count of CD68+ and CD163+ or one of the markers with high count compared to samples where both markers had a high count (median OS: 34 vs. 11.5 months, p = 0.0012).

CONCLUSION

Our findings reinforce the carcinogenic role of TAMs in glioblastoma, as well as their prognostic value, highlighting the possibility of targeted therapy against these targets.
P02.24.B THE IMPACT OF THE IDH1 R132H MUTATION ON MMP2 METHYLATION, EXPRESSION AND INVASION IN EXPERIMENTAL GLIOMAS AND HUMAN GBMS
Authors: Lenkiewicz A M , Ibragimova A , Ghosh M , Szadkowska P , Ellert-Miklaszewska A , Baluszek S , Jacek K , Castro M , Kaminska B
Published: October 2024

BACKGROUND

Glioblastoma (GBM) is a highly diffusive tumor which restricts the efficacy of surgical resection and facilitates tumor recurrence. Matrix metalloproteinase 2 (MMP2) is the extracellular matrix protease that is activated by MMP14 expressed by microglia and plays a central role in tissue remodeling and tumor progression. The R132H mutation in isocitrate dehydrogenase 1 (IDH1R132H) is commonly observed in gliomas, results in hypermethylator phenotype and in GBMs is associated with better survival. The impacts of IDH1R132H on the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored.

MATERIAL AND METHODS

We employed unique mouse glioma models which mimic human GBM pathology, coupled with high-dimensional technologies (CITEseq and spatial transcriptomics) to analyze and visualize the immune landscape in different gliomas. Additionally, we performed gelatin zymography, qPCR, wound healing assays, and DNA methylation qPCR techniques to validate the obtained results. MMP-2 expression/methylation was studied in the glioma TCGA dataset.

RESULTS

The expression and activity of Mmp2 were different in experimental gliomas with different IDH1 status. Spatial transcriptomics demonstrated that Mmp2 expression is localized within the IDH1 wild-type tumors and absent in IDH1 mutated ones. Gelatin zymography showed the presence of Mmp2 activity in extracts from IDH1 wild-type tumors. Methylation of Mmp2 gene promoter was increased and resulted in decreased Mmp2 expression in IDH1 mutated gliomas. The IDH1 wild-type gliomas showed more diffuse growth patterns (tumors were stained with Nestin antibody) whereas IDH1 mutant gliomas showed more constrained growth patterns. In the TCGA glioma dataset, methylation of the MMP2 gene promoter was increased which was associated with decreased MMP2 expression in IDH1 mutated gliomas.

CONCLUSION

We found that the IDH1 mutation results in hypermethylation of the Mmp2 gene promoter, leading to decreased Mmp2 expression and Mmp2 activity in experimental gliomas and human GBMs. Reduced Mmp2 activity and decreased tumor invasion result in more restricted tumor growth, which may facilitate resection, and radiotherapy, thereby improving patient survival. Studies were supported by National Science Center Poland grant 2020/39/B/NZ4/02683 (BK) and PASIFIC Call 1 PAS (MG).
P02.25.A TUMOR MICROENVIRONMENT (TME) VIS-À-VIS ANTI-VASCULAR TREATMENT IN GLIOBLASTOMA
Authors: Chen Z , Cai H , Ni X
Published: October 2024

BACKGROUND

Glioblastoma (GBM) the most malignant primary central nerves system tumor, usually failed to postoperative treatment since tumor resistance to treatment. Most of Studies for anti-cancer resistance focus on tumor cell its self. Actually, tumor tissue is composed of tumor cells and surrounding matrix components. The components in tumor microenvironment (TME) work in coordination with each other to promote cancer initiation, progression, metastasis, and treatment resistance. Previously, we have found vasculogenic mimicry (VM), a vessel-like structure formed by highly invasive tumor cells, may contribute to anti-vascular resistance. Since TME includes non-cellular components such as extracellular matrix(ECM)and tumor-associated vasculature, as well as various cellular components such as activated tumor-associated fibroblasts, mesenchymal cells, tumor associated macrophages (TAMs), and pericytes. We further invested TME relating to anti-vascular resistance.

MATERIAL AND METHODS

First, we examined tenascin-c (TNC) expression, an extracellular protein, and correlated with VM formation in glioma cell lines as well as clinical samples. Since TNC is composed of 2 domains (EGF-like and fibronectin III), we further examined function of these domains affecting on VM formation in glioma cell lines. Second, we examined association of TAMs with angiogenesis in GBM, and further search possible molecular mechanism.

RESULTS

The expression of TNC increased with the WHO grade of glioma, and the most abundant in GBM, which correspondence to existence of VM structure. Knockdown TNC expression resulted significant reducing the ability of VM formation. Treatment of exogenous TNC protein can enhance the ability of VM formation of glioma cells. The over expression of the EGF-like domain or the full-length TNC in glioma cells revealed significant enhancing VM formation ability. In contrast, over expression of the fibronectin III domain of TNC did not alter VM formation. Furthermore, over expression of the EGF-like domain of TNC or full-length TNC in glioma cells increased the phosphorylation levels of downstream signaling pathways of EGFR. When checked TAMs, we revealed that PTEN deficient GBM may secrete abundant Gal-9, and recruit plenty of TAMs. TAMs may secrete vascular endothelial growth factor A (VEGFA) stimulating angiogenesis and and supporting glioma growth. Moreover, α-lactose through down-regulating VEGFA expression, inhibited glioma angiogenesis.

CONCLUSION

Our findings by searching tumor microenvironment of glioma, may suggest novel antivascularization strategies through TNC (an extracellular protein in ECM) and TAMs.
P04.02.A DISCOVERY OF NEW METABOLIC PATHWAYS IMPLICATED IN THERAPY RESISTANCE IN GLIOBLASTOMA (GBM)
Authors: Cingöz A
Published: October 2024

Glioblastoma stands as the most prevalent and aggressive primary brain tumor. The current treatment regimen involves a combination of radiotherapy and chemotherapy post-surgical tumor resection. However, for most patients, tumor recurrence transpires with a more aggressive phenotype. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds promise as it selectively triggers apoptosis in tumor cells, rendering it an attractive therapeutic option. Nonetheless, numerous tumor cells, glioblastoma included, can develop acquired resistance against TRAIL therapy, yet the underlying mechanisms remain poorly elucidated. Apart from resistance to apoptosis, these mechanisms may also encompass non-genetic factors stemming from intracellular dynamics. In our previous research on generating models resistant to TRAIL, we observed differences in the expression of metabolic pathway genes in TRAIL-resistant glioblastoma cells. Our transcriptomic analysis revealed a positive correlation among pathways related to cellular metabolism in these resistant cells. Further examination of this gene family revealed common alterations across various cell lines. Additionally, to delve deeper into the role of cellular metabolism, we conducted in vivo CRISPR/Cas9 screening using a focused library targeting metabolism. We also compared mitochondria densities using MitoTracker dye between sensitive and resistant GBM cells, noting a significant increase in resistant cells. Using the Seahorse Extracellular Flux Analyzer, we measured cellular metabolism activity and observed higher oxygen consumption in resistant cells, indicative of elevated energy levels. Based on these preliminary findings, our study will undergo thorough genetic and metabolomic screening, promising significant insights into cancer metabolism. The identified target genes and metabolites could potentially serve as therapeutic targets for developing novel cancer treatments, particularly glioblastoma.
P04.04.B THE FUNCTIONS AND MECHANISMS OF OSGIN1 INVOLVED IN FERROPTOSIS RESISTANCE OF GLIOBLASTOMA
Authors: Huo L , Luo Z , Liang X , Du X
Published: October 2024

BACKGROUND

Glioblastoma (GBM) is the most prevalent and aggressive primary brain tumor in adults, with an average survival time of less than 15 months despite treatment. Recurrence and chemotherapy resistance are major challenges, with ferroptosis emerging as a potential mechanism contributing to chemotherapy resistance. The role of oxidative stress-induced growth inhibitor 1 (OSGIN1) in GBM, particularly its localization and function, remains poorly understood, though it is associated with poor prognosis.

MATERIAL AND METHODS

We utilized bioinformatics to analyze OSGIN1 expression and GBM patient prognosis from the Cancer Genome Atlas (TCGA). Experimental approaches included C-terminal EGFP tagging of OSGIN1, overexpression, and knockdown experiments in GBM cells. We assessed the effects on cell proliferation, apoptosis, and ferroptosis sensitivity using CCK8 assays, flow cytometry, and lipid peroxidation sensors. The relationship between OSGIN1 and drug sensitivity was explored using data from the Cancer Therapeutics Response Portal (CTRP). Regulatory interactions between OSGIN1 and the cystine transporter SLC7A11 were investigated through polymerase chain reaction, Western blotting, dual luciferase assays, and chromatin immunoprecipitation.

RESULTS

High OSGIN1 expression correlates with poor prognosis in GBM. OSGIN1 does not localize with mitochondria in GBM cells nor induce apoptosis. Instead, OSGIN1-modulated cells exhibited altered ferroptosis sensitivity. Knockdown of OSGIN1 increased ferroptosis sensitivity, an effect reversible in OSGIN1-rescued cells. Moreover, OSGIN1 positively regulates SLC7A11 expression, impacting cystine uptake and ferroptosis resistance. Animal models confirmed that GBM cells overexpressing OSGIN1 are more resistant to ferroptosis.

CONCLUSION

OSGIN1 is a prognostic marker and potential therapeutic target in GBM. It regulates SLC7A11 expression and contributes to ferroptosis resistance, highlighting its role in the chemotherapeutic response. Targeting OSGIN1 could enhance GBM treatment efficacy by modulating ferroptosis sensitivity.
P05.01.A ATR INHIBITORS INCREASE THE ANTI-PROLIFERATIVE EFFECTS OF STANDARD TREATMENT ON PATIENT-DERIVED GLIOBLASTOMA CELLS
Authors: Lozinski M , Bowden N , Graves M , Day B , Stringer B , Fay M , Tooney P
Published: October 2024

BACKGROUND

Glioblastoma is treated with surgery to remove as much as the tumour as possible then radiation therapy (RT) and temozolomide (TMZ) to cause extensive DNA damage in any remaining tumour cells, triggering cell death by apoptosis. However, glioblastoma cells respond by upregulating the DNA damage response (DDR) triggering DNA repair that contributes to treatment resistance. Progression then generally occurs rapidly leaving patients with a poor prognosis and survival. We previously showed that gartisertib, an inhibitor of ATM- and Rad3-Related protein (ATR), a serine/threonine kinase that triggers DNA repair, sensitises glioblastoma cells to TMZ and RT. We extend this investigation to brain-penetrant ATR inhibitors elimusertib (BAY1895344) and ceralasertib (AZD6738).

MATERIAL AND METHODS

Patient-derived glioblastoma cell lines were treated with a dose response of ATRi (elimusertib and ceralasertib) combined with a clinically relevant dose of TMZ (35uM) and/or RT (2Gy). A human astrocyte cell line was also treated with a dose response of single agent elimusertib and ceralasertib. Cells were incubated for 7 days and assessed for cell viability through an MTT assay or cell-cell Incucyte analysis.

RESULTS

Both elimusertib and ceralasertib as single agents reduced glioblastoma cell growth, with elimusertib being more potent compared to ceralasertib. Elimusertib had greater potency in causing cell death of the glioblastoma cell lines than a human astrocyte cell line. However, there was less difference between glioblastoma and astrocyte cell death when treated with ceralasetib. When combined with RT and/or TMZ, elimusertib once again was more potent in reducing glioblastoma cell growth compared to ceralasertib.

CONCLUSION

We have expanded our in vitro data showing that various ATR inhibitors (elimusertib, ceralasertib and previously gartisertib) are able to reduce the growth of glioblastoma cells in combination with RT and TMZ treatment. This provides further support for initiating a clinical trial of ATR inhibition in combination with standard treatment for patients with glioblastoma.
P06.05.B FLYDENTIFICATION - CHARACTERIZATION OF NOVEL MOLECULAR DRIVERS IN GLIOBLASTOMA BASED ON FUNCTIONAL GENOMICS SCREENING IN DROSOPHILA MELANOGASTER
Authors: Lewandowska O , Picard D , Hariri B , Klein T , Reifenberger G , Reiff T , Leprivier G
Published: October 2024

Glioblastoma is an extremely aggressive form of brain tumor. The median overall survival of patients is only 15 to 18 months, and the five-year survival rate is less than 10% despite aggressive multimodal treatment that includes total resection of the tumor followed by a combination of radio-, chemo- and Tumor Treating Fields (TTF)-therapy. Therefore, new therapeutic interventions are urgently needed. For this reason, it is essential to define novel therapeutic targets for this tumor entity. To this aim, we developed a combinatorial approach, termed “

FLY dentification”, to identify and characterize novel driver genes in glioblastoma. This pipeline combines bioinformatics analysis of patients proteomics data together with functional screening in Drosophila models of glioblastoma, and validation with human glioblastoma cell models. This approach allowed us to initially uncover the top 300 most overexpressed proteins in glioblastoma tissues using in silico preselection algorithms. By taking advantage of a Drosophila model of glioblastoma ‒ driven by overexpression of the oncogenic forms of EGFR and PI3K ‒ and using an RNAi approach we tested the functional contribution of 192 of the gene candidates to glioblastoma lethality in vivo. This targeted screening allowed us to identify 41 clinically and functionally relevant gene candidates. Focusing on the top candidates with no reported function in glioblastoma, we demonstrated that the DNA binding and chromatin remodeling DEK protein contributes to human glioblastoma tumorigenesis in vitro. Some of these findings will be validated using in vivo models of human glioblastoma. Together, the established “FLY dentification” pipeline represents a combinatorial approach designed to uncover previously unrecognized glioblastoma driver genes, which has the power to reveal novel biomarkers and therapeutic targets for this tumor type.
P06.08.B OF TRAVELING HOMEOPROTEINS AND MEDULLOBLASTOMAS
Authors: Vigneul E , Clotman F
Published: October 2024

BACKGROUND

Medulloblastomas (MB) are the most common pediatric malignancies. These WHO grade IV neoplasms are divided into four main subgroups called SHH-activated, WNT-activated, group 3 and group 4. These subgroups are governed by different molecular patterns involving transcription factors (TFs), in particular (TF) Orthodenticle homeobox 2 (OTX2) which, when overexpressed, is a key factor in group 3 and 4 medulloblastoma formations. In addition to being a transcription factor crucial for the proper cerebral and cerebellar embryogenesis, OTX2 is a homeoprotein with intercellular travelling properties. Indeed, via secretion and penetration sequences, OTX2 can travel between cells and influence the tumor neighborhood in a paracrine manner. The objective of this study is to establish the list of traveling homeoproteins associated with the development of MB.

MATERIALS AND METHODS

We found 57 homeoproteins with demonstrated intercellular transfer capabilities in vivo. We then systematically reviewed the literature and found 89 articles eligible for full-text analysis.

RESULTS

Ten traveling homeoproteins (CUX1, EN1, ISL1, LHX1, NKX2.2, OTX2, PAX5, PAX6, PAX8 and POU5F1) are involved in medulloblastoma formation. Apart from PAX8, which is underexpressed, all other homeoproteins are overexpressed in medulloblastoma.

CONCLUSION

This study highlights the role of several traveling homeoproteins in the formation of medulloblastoma. Most of these proteins are overexpressed in MB and have the ability to enter and modify the transcriptional and non-transcriptional mechanisms of surrounding cells. Antibodies targeting these transcription factors, as used in animal models, could become a promising tool to circumscribe the potential chain reaction of deregulation associated with these roaming homeoproteins.
P06.09.B RATIONAL DRUG COMBINATIONS TARGETING FAK AND MEK OVERCOMES TUMOUR HETEROGENEITY IN GLIOBLASTOMA
Authors: Furqan M , Elliott R , Nagle P , Loftus A , Masalmeh R , Munro A , Drake C , Dawson J , Brunton V , Frame M , Carragher N
Published: October 2024

BACKGROUND

Glioblastoma (GBM) presents a formidable clinical challenge with limited treatment options and poor outcomes. Despite efforts, the standard therapy has not improved for almost two decades. The heterogeneity of GBM, driven by multiple intracellular signalling pathways, complicates treatment response and contributes to drug resistance. Recent advancements in targeted combination therapy, such as dabrafenib and trametinib, offer promise for specific GBM subgroups, but identifying effective drug combinations remains challenging. Integrin mediated signalling, particularly through Focal Adhesion Kinase (FAK), plays a crucial role in GBM pathogenesis and invasion. While targeting FAK shows promise, identifying synergistic combinations is essential. Our study employs a chemogenomic screening approach to identify FAK targeted drug combinations in glioblastoma.

MATERIAL AND METHODS

Glioblastoma cells were cultured and subjected to chemogenomic screening to identify synergistic drug combinations with FAK depletion. Cell viability was assessed using the Alamar Blue assay, while morphological changes were observed using high content imaging. Synergies in drug combinations were determined using the SynergyFinder online tool. Molecular mechanism was elucidated through Reverse Phase Protein Array and western blots, with statistical analysis determining significance. Finally, in vivo efficacy was evaluated in an orthotopic transplantation model.

RESULTS

Using a CRISPR engineered model, we identified synergistic drug combinations with FAK loss, notably MEK inhibitors. Combining FAK inhibitors with MEK inhibitors showed potent synergy, validated by target engagement assays. Across a diverse panel of patient derived GBM stem cells, this combination inhibited spheroid growth and invasion. RPPA profiling elucidated their molecular effects. In vivo, the combination significantly reduced tumour volume. These results highlight a promising therapeutic strategy for glioblastoma.

CONCLUSION

In conclusion, our study reveals promising synergies with MEK inhibitors, notably trametinib, across diverse GBM models. This approach also highlights the potential of combination therapy to address the complex challenges of GBM treatment.
P06.10.A THE MIRNA 216B-5P ACTS AS A PROMISING TUMOR-SUPPRESSOR BY MANIPULATING CELL CYCLE PROGRESSION IN HIGH-GRADE GLIOMA CELLS
Authors: Lang A , Bürger S , Mager L , Kiesel B , Madlener S , Ricken G , Lämmerer A , Spiegl-Kreinecker S , Hackl M , Pultar M , Reisecker J , Kirchhofer D , Mischkulnig M , Berghoff A S , Woehrer A , Rössler K , Roetzer-Pejrimovsky T , Berger W , Jaunecker C N , Hainfellner J A , Hoeftberger R , Widhalm G , Erhart F , Loetsch-Gojo D
Published: October 2024

BACKGROUND

Glioblastoma is the most common brain tumor in adults and with an approximate survival rate of 15 months, new treatment strategies are of utmost urgency. In our previous phase II clinical trial, applying immunotherapy with dendritic cells to glioblastoma patients, no differences in overall survival were observed. However, distinct miRNAs and in particular miR-216b, harbored a predictive power and were associated with therapy susceptibility. Thus, we hypothesize that amongst others, miR-216b is involved in glioblastoma aggressiveness and thereby impacting therapeutic success.

MATERIAL AND METHODS

First, we screened glioblastoma tissue samples (n=39) and several cell models for the expression of five different miRNAs. As miR-216b was generally low expressed compared to others, we investigated whether transfection with miR-216b “mimic” (upregulation) or “inhibitor” (blocking) had an impact on clonogenic survival, sphere-forming and migratory capacity in three patient-derived and two immortalized glioblastoma cell models. To gain deeper knowledge about miR-216b targets, miRNA sequencing was performed. Subsequently, predicted targets were confirmed by Western Blot, FACS analysis and drug sensitivity assays.

RESULTS

MiR-216b upregulation resulted in reduced cell proliferation and migration as well as in decreased stemness characteristics in the majority of the investigated high-grad glioma (HGG) models. RNA sequencing predicted target genes of miR-216b that are involved in cell division (e.g. CDK4). Accordingly, upon transfection with “miR-216b mimics”, protein levels of CDK4 and subsequently phosphorylation of RB were reduced, accompanied by an increase of cells in G0/G1 phase. When miR-216b was blocked, the sensitivity towards CDK4/6 inhibitors was distinctly increased, pointing towards a drug-targeting engagement.

CONCLUSION

Our data indicates that miR-216b acts as a tumor suppressor via the inhibition of CDK4 in glioblastoma cells. The prognostic and predictive value of this miRNA in glioblastoma is currently under investigation and harbors the potential to manipulate glioma cells into cell cycle arrest.
P06.12.B Microbiota-gut-brain axis in glioblastoma development and therapeutic resistance
Authors: Lavielle S , Delrot G , Lemaitre M , Pateras I S , Nikolski M , Daubon T , Martin O C B
Published: October 2024

BACKGROUND

Glioblastoma (GB) is the most common subtype of glioma in adults. Despite treatment through tumor resection associated with chemo and radiotherapies, this cancer still has a very poor prognosis. Factors contributing to etiology, pathogenesis, or treatment resistance are not well known. The significance of understanding the microbiome-gut-brain axis (MGBA) in GB, a topic that has been recently shown to be crucial in several neurodegenerative diseases, cannot be overstated. Therefore, our project aims to understand how modulation of gut physiopathology (i.e., gut inflammation or microbiota dysbiosis) affects GB development and therapeutic resistance.

MATERIAL AND METHODS

We have examined the relationship between gut inflammation, microbiome modulation, GB development, and therapeutic resistance. Mice received dextran-sulfate sodium (DSS), a gut pro-inflammatory agent, and were orthotopically injected with mGB2 GB cells. Some mice were then resected and treated by radio and chemotherapy using Temozolomide (TMZ). To understand the role of the gut microbiota in GB, we performed gut bacterial microbiota depletion using antibiotics.

RESULTS

Our results showed that DSS-treated mice had a higher GB growth than non-treated mice. Moreover, the recurrence after treatment was higher in mice bearing gut inflammation. Interestingly, we also observed on DSS-treated mice that the GB-bearing mice had lower intestinal inflammation than the control. GB growth was also associated with microbiota modifications, which were restored by the treatment. Gut microbiota alteration leads to increased tumoral growth, suggesting an implication of the microbiota in GB development.

CONCLUSION

Altogether, our results support the bidirectional communication between the gut and the brain in the context of GB. Alteration of gut physiopathology strongly impacts GB development and therapeutic resistance. This connection suggests that targeting the gut microbiome could slow down GB progression and/or improve treatment efficacy.