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Establishment and Molecular Characterization of Glioblastoma Primary Cultures

ABSTRACT: Glioblastoma, the most frequent and malignant adult brain tumor has been extensively studied; yet no effective treatment exists. To overcome this dismal scenario, it is essential to improve preclinical biological models. This study aimed to establish and molecularly characterize glioblastoma primary cultures. Additionally, it intended to assess the efficacy of molecular-targeted therapies, in the presence of putative targeting cell lines. Five glioblastoma primary cultures were established exhibiting a diversity of chromosomal alterations by aCGH, with gain of chromosome 7 and loss of chromosome 10, 13 and 17p, the most frequent alterations. Mutation profiling by Ion Torrent and Sanger sequencing showed the present of hotspot mutations in TERT (4/5), TP53 (2/5) and RB, BRAF, PTEN and EGFR (1/5). A similar chromosomal and mutation pattern was observed in the primary cultures and matched frozen tumors. The MTS cell viability assay of the p.Gly598Val EGFR mutated cell line, revealed AST1306 as the most potent EGFR inhibitor, when compared with afatinib, erlotinib, or lapatinib. Herein, glioblastoma primary cultures were successfully established and molecularly characterized. Importantly, we showed that AST1306 inhibits EGFR mutated cells, suggesting that primary cultures are suitable in vitro models for glioblastoma biology and preclinical drugs studies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE59967 | GEO | 2016/07/01

SECONDARY ACCESSION(S): PRJNA257196

REPOSITORIES: GEO

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Establishment and Molecular Characterization of Glioblastoma Primary Cultures

Project description:Glioblastoma, the most frequent and malignant adult brain tumor has been extensively studied; yet no effective treatment exists. To overcome this dismal scenario, it is essential to improve preclinical biological models. This study aimed to establish and molecularly characterize glioblastoma primary cultures. Additionally, it intended to assess the efficacy of molecular-targeted therapies, in the presence of putative targeting cell lines. Five glioblastoma primary cultures were established exhibiting a diversity of chromosomal alterations by aCGH, with gain of chromosome 7 and loss of chromosome 10, 13 and 17p, the most frequent alterations. Mutation profiling by Ion Torrent and Sanger sequencing showed the present of hotspot mutations in TERT (4/5), TP53 (2/5) and RB, BRAF, PTEN and EGFR (1/5). A similar chromosomal and mutation pattern was observed in the primary cultures and matched frozen tumors. The MTS cell viability assay of the p.Gly598Val EGFR mutated cell line, revealed AST1306 as the most potent EGFR inhibitor, when compared with afatinib, erlotinib, or lapatinib. Herein, glioblastoma primary cultures were successfully established and molecularly characterized. Importantly, we showed that AST1306 inhibits EGFR mutated cells, suggesting that primary cultures are suitable in vitro models for glioblastoma biology and preclinical drugs studies. Two-color Agilent 8x60K array CGH (aCGH) was performed in 10 glioblastoma multiforme samples (CY3) and reference DNA (CY5). The 10 glioblastoma multiforme samples were: DNA extracted from 5 primary cultured cells; DNA of 5 primary tumors (matched with the 5 primary cell lines). Gender-matched commercial DNA of each case was used as control.

2016-07-01 | E-GEOD-59967 | biostudies-arrayexpress

Single cell lineage tracing reveals clonal dynamics of anti-EGFR therapy resistance in triple negative breast cancer

Project description:We investigate non-genomic mechanisms determining cellular response to EGFR inhibitors in triple negative breast cancer (TNBC). We integrate methods for cellular barcoding and single-cell transcriptomics to enable cell lineage tracing and explore the subclonal evolution of adaptation in an established preclinical model of TNBC in response to incremental concentrations of Afatinib, a second generation EGFR-TKI that irreversibly inhibits both EGFR and HER2. Retrospective lineage tracing data analysis uncovered a pre-existing subpopulation of rare Afatinib-tolerant cells displaying distinct biological features, such as elevated mRNA levels of the IGFBP2 gene. Furthermore, we investigated temporal coordination of transcriptional programs in drug resistant clones with high replication fitness by reordering cells along a pseudotime trajectory. Interestingly, it revealed the activation of biological processes, such as fatty acid metabolism, which have previously been linked to EGFR-TKIs resistance mechanisms.

2024-03-25 | GSE228154 | GEO

Long non-coding RNA CRNDE Involved in Resistance to EGFR tyrosine kinase inhibitor in EGFR-mutant Lung Cancer via eIF4A3/MUC1/EGFR Signal.

Project description:Introduction: Overcoming of acquired resistance to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) is an intractable obstacle for many clinical oncologists. The mechanisms of resistance to EGFR-TKIs are very complex. Long non-coding RNAs (lncRNAs) may play an important role in cancer development and metastasis. However, the biological process between lncRNAs and drug resistance to EGFR mutated lung cancer largely unknown. Methods: Osimertinib and afatinib-resistant EGFR-mutated lung cancer cells were established using by a stepwise method. Microarray analysis of non-coding and coding RNAs was performed using parental and resistant EGFR-mutant NSCLC cells. Results: Microarray analysis was evaluated by bioinformatics analysis through medical-industrial collaboration. CRNDE and DGCR5 lncRNAs were highly expressed in EGFR-TKIs-resistant cells. CRNDE binds to eIF4A3 protein, down-regulates eIF4A3 and MUC1 expression, and down-regulates p-EGFR expression. CRNDE inhibition activated the eIF4A3/MUC1/EGFR signaling pathway and apoptotic activity and restored sensitivity to EGFR-TKIs. Conclusions: We identified lncRNA CRNDE associated with resistance to EGFR-TKIs in EGFR-mutant NSCLC cells. CRNDE may be a novel therapeutic target for EGFR mutant NSCLC patients.

2021-04-28 | GSE163913 | GEO

Suppression of MicroRNA-9 by Mutant EGFR Signaling Induces FOXP1 to Enhance Glioblastoma Tumorigenicity

Project description:The EGF-receptor (EGFR) is amplified and mutated in glioblastoma (GBM) where its common mutation, (∆EGFR, also called EGFRvIII) has a variety of activities that promote growth and inhibit death, thereby conferring a strong tumor-enhancing effect. This range of activities suggested to us that ∆EGFR might exert its influence through pleiotropic effectors and we hypothesized that microRNAs (miRs) might serve such a function. To test this, we determined the miR profiles of GBM cells with activated wild type EGFR (wtEGFR) and mutant EGFR (∆EGFR) to cells with non-activated EGFR or kinase dead ∆EGFR. To identify miRs regulated by EGFR, RNA from 2 different glioma cell lines (U87 and U373) were hybridized to miR expression arrays and analyzed. Each cell type was engineered to express wild type EGFR (wtEGFR), dead kinase ∆EGFR (DK) or ∆EGFR at elevated levels similar to those observed in primary glioblastomas displaying EGFR overexpression. Parental cells expressing endogenous EGFR and wtEGFR cells stimulated with EGF for 1hr were also included in the analyses.

2013-12-20 | E-GEOD-53504 | biostudies-arrayexpress

Afatinib and Pembrolizumab for Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma (ALPHA Study): A Phase II Study with Biomarker Analysis

Project description:Afatinib may augment pembrolizumab therapy and improve the ORR in HNSCC patients. The paired tissue analysis showed that afatinib, an epidermal growth factor (EGFR) tyrosine kinase inhibitor, may enhance antigen presentation, natural killer cell–mediated cytotoxicity, and immune reactions. We also identified unaltered methyl-thioadenosine phosphorylase (MTAP) levels, EGFR amplification, and high programmed death-ligand 1 expression as possible predictive biomarkers for therapeutic outcomes.

2021-12-10 | GSE190575 | GEO

EGFR pathway expression persists in recurrent glioblastoma independent of amplification status

Project description:Background: Glioblastoma mortality is driven by tumour progression or recurrence despite administering a therapeutic arsenal consisting of surgical resection, radiation, and alkylating chemotherapy. The genetic changes underlying tumour progression and chemotherapy resistance are poorly understood. Methods: In this work, we sought to define the relationship between EGFR amplification status, EGFR mRNA expression, and EGFR pathway activity. We compared RNA-sequencing data from matched primary and recurrent tumour samples (N = 40 patients, 20 with EGFR amplification). Results: In the setting of glioblastoma recurrence, the EGFR pathway was overexpressed regardless of EGFR amplification status, suggesting a common genomic endpoint in recurrent glioblastoma, although EGFR amplification did associate with higher EGFR mRNA expression. Three of forty patients in the study cohort had EGFR-amplified tumours and received targeted EGFR therapy. Their molecular subtypes and clinical outcomes did not significantly differ from patients who received conventional chemotherapy. Conclusion: Our findings suggest that while the EGFR amplification may confer a unique molecular profile in primary glioblastoma, pathway analysis reveals upregulation of the EGFR pathway in recurrence, regardless of amplification status. As such, the EGFR pathway may be a key mediator of glioblastoma progression.

2023-01-25 | GSE222515 | GEO

Suppression of MicroRNA-9 by Mutant EGFR Signaling Induces FOXP1 to Enhance Glioblastoma Tumorigenicity

2013-12-20 | GSE53504 | GEO

Relation between microRNA expression and EGFR amplification status

Project description:Extensive infiltration of the surrounding healthy brain tissue is a critical feature in glioblastoma. Several miRNAs have been related to gliomagenesis, some of them related with the EGFR pathway. We have evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns, studied by fluorescence in situ hybridization in tissue microarrays, of 30 cases of primary glioblastoma multiforme, whose clinicopathological and immunohistochemical features have also been analyzed.

2015-01-16 | E-MTAB-2478 | biostudies-arrayexpress

Acquired resistance to mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling

Project description:To compare gene copy number in cell populations with acquired resistance to specified EGFR inhibitors to parental cell lines. The aim of the experiment was to determine whether gain or loss of copy number of particular genes was associated with resistance to particular EGFR inhibitors. Comparing gene copy number in PC9 parental cells to gene copy number in 5 cell populations resistant to AZD9291, 2 cell populations resistant to WZ4002, 2 cell populations resistant to afatinib and 4 cell populations resistant to gefitinib.

2015-04-14 | E-GEOD-59239 | biostudies-arrayexpress

Clinical Utility of Patient Derived Xenografts to Determine Biomarkers of Prognosis and Map Resistance Pathways in EGFR-Mutant Lung Adenocarcinoma

Project description:PURPOSE: Although epidermal growth factor receptor (EGFR) mutated adenocarcinomas initially have very high response rates to EGFR tyrosine kinase inhibitors (TKIs), most atients eventually develop resistance. Patient derived xenografts (PDXs) are considered preferred preclinical models to study the biology of patient tumors. EGFR-mutant PDX models may be valuable tools to study the biology of these tumors and to elucidate mechanisms of resistance to EGFR-targeted therapies. METHODS: Surgically resected early stage non-small cell lung carcinoma (NSCLC) tumors were implanted into non-obese diabetic severe combined immune deficient (NODSCID) mice. EGFR TKI treatment was initiated at tumor volumes of 150 mm3. Gene expression analysis was performed using microarray platform. RESULTS: Of 33 lung adenocarcinomas with EGFR activating mutations, only 6 engrafted 18%) and could be propagated beyond passage one. Engraftment was associated with upregulation of genes involved in mitotic checkpoint and cell proliferation. A differentially expressed gene set between engrafting and non-engrafting patients could identify EGFRmutant patients with significantly different prognoses in The Cancer Genome Atlas (TCGA) Lung Adenocarcinoma datasets. The PDXs included models with variable sensitivity to first- and second-generation EGFR TKIs and the monoclonal antibody cetuximab. All EGFR-mutant NSCLC PDXs studied closely recapitulated their corresponding patient tumor phenotype and clinical course, including response pattern to EGFR TKIs. CONCLUSIONS: PDX models closely recapitulate primary tumor biology and clinical outcome. They may serve as important laboratory models to investigate mechanisms of resistance to targeted therapies, and for preclinical testing of novel treatment strategies.

2017-03-28 | GSE63685 | GEO

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