Patient-derived Glioblastoma Samples Research Articles

Glioblastomas within the Subventricular Zone Are Region-Specific Enriched for Mesenchymal Transition Markers: An Intratumoral Gene Expression Analysis.

Simple SummaryInvolvement of the subventricular zone (SVZ) in glioblastoma is associated with poor prognosis and is associated with specific tumor-biological characteristics. In this study, we demonstrate that patient-derived glioblastoma samples from within the SVZ region show increased (epithelial-)mesenchymal transition and angiogenesis/hypoxia signaling as compared to glioblastoma samples from the same patient from outside the SVZ. These results suggest that intratumoral alterations in oncogenic signaling could be mediated by the SVZ microenvironment. Our findings offer rationale for specific targeting of the SVZ in the development of glioblastoma therapy.Background: Involvement of the subventricular zone (SVZ) in glioblastoma is associated with poor prognosis and is associated with specific tumor-biological characteristics. The SVZ microenvironment can influence gene expression in glioblastoma cells in preclinical models. We aimed to investigate whether the SVZ microenvironment has any influence on intratumoral gene expression patterns in glioblastoma patients. Methods: The publicly available Ivy Glioblastoma database contains clinical, radiological and whole exome sequencing data from multiple regions from resected glioblastomas. SVZ involvement of the various tissue samples was evaluated on MRI scans. In tumors that contacted the SVZ, we performed gene expression analyses and gene set enrichment analyses to compare gene (set) expression in tumor regions within the SVZ to tumor regions outside the SVZ. We also compared these samples to glioblastomas that did not contact the SVZ. Results: Within glioblastomas that contacted the SVZ, tissue samples within the SVZ showed enrichment of gene sets involved in (epithelial-)mesenchymal transition, NF-κB and STAT3 signaling, angiogenesis and hypoxia, compared to the samples outside of the SVZ region from the same tumors (p < 0.05, FDR < 0.25). Comparison of glioblastoma samples within the SVZ region to samples from tumors that did not contact the SVZ yielded similar results. In contrast, we observed no differences when comparing the samples outside of the SVZ from SVZ-contacting glioblastomas with samples from glioblastomas that did not contact the SVZ at all. Conclusion: Glioblastoma samples in the SVZ region are enriched for increased (epithelial-)mesenchymal transition and angiogenesis/hypoxia signaling, possibly mediated by the SVZ microenvironment.

CBIO-05. LIPID METABOLIC REPROGRAMMING SENSITIZES A MOLECULARLY-DEFINED SUBSET OF GLIOBLASTOMAS TO FERROPTOSIS

Abstract Cancers, including the universally lethal glioblastoma (GBM), have reprogrammed lipid metabolism to fuel tumor growth. However, the molecular alterations responsible for aberrant lipid metabolism, and the potential for identifying new therapeutic opportunities are not fully understood. To systematically investigate the GBM lipidome, we performed integrated transcriptomic, genomic and shotgun lipidomic analysis of an extensive library of molecularly diverse patient-derived GBM samples. Using this comprehensive approach, we discovered two GBM sub-groups defined by their combined molecular and lipidomic profile. Triacylglycerides (TAGs) enriched in polyunsaturated fatty acids (PUFAs) were among the most significantly altered lipids between the two groups of GBM tumors. TAGs are the main components of lipid droplets, which sequester PUFA-TAGs away from membrane phospholipids where their peroxidation can lead to ferroptosis – a regulated from of PUFA-peroxidation dependent cell death. Accordingly, the GBM subgroup with a depletion of PUFA TAGs showed heightened sensitivity to ferroptosis. Our findings suggest a novel association between specific molecular signatures of GBM, lipid metabolism and ferroptosis. This relationship may present a new therapeutic opportunity to target reprogrammed lipid metabolism in a molecularly-defined subset of GBMs.

Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma.

Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

Identification of novel therapeutic targets in glioblastoma with functional genomic mRNA profiling.

2018 Background: Glioblastoma (GBM), the most common primary brain tumor in adults, universally recurs and has a dismal prognosis. Therefore, there is an unmet need for new and more effective treatment strategies. Here, we aim to discover new therapeutic targets by identifying upregulated genes in GBM with known antineoplastic drug interactions. Methods: Publicly available, raw microarray expression data of patient-derived GBM samples and normal brain tissue were collected from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). Subsequently, we applied functional genomic mRNA profiling (FGmRNA-profiling), a method that is able to correct the gene expression profile of an individual tumor for physiological and experimental factors, which are considered not to be relevant for the observed tumor phenotype. Next, the FGmRNA-profiles of healthy brain tissue and glioblastoma were used to perform a class comparison analysis. Significantly upregulated genes in GBM were prioritized based on: known interaction with antineoplastic agents and the current status of clinical evaluation in humans. Results: After FGmRNA-profiling 66 normal brain tissue samples and 1280 patient-derived GBM samples, class comparison identified 712 significantly upregulated genes. Of all significantly upregulated genes, 27 genes interacted with antineoplastic drugs. 17 out of these 27 druggable genes, including EGFR and VEGFA, have already been clinically evaluated in GBM, and had limited efficacy. Out of the 10 remaining druggable genes, we prioritized RRM2, MAPK9 and XIAP, as these genes are associated with biologic pathways involved in the carcinogenesis of GBM and are therefore considered as novel potential therapeutic targets. Conclusions: Based on data-driven prioritization, we identified three potential therapeutic druggable targets, which have not yet been explored in the context of glioblastoma. Further preclinical and clinical research on the inhibition of these druggable genes is necessary and may lead to an improvement of treatment outcomes for patients with GBM.

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy.

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different sample preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. Each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.

DDIS-13. IDENTIFICATION OF INDIVIDUALIZED THERAPY OPTIONS IN PATIENTS WITH GLIOBLASTOMA BY HIGH-THROUGHPUT DRUG SCREENING

Glioblastoma (GBM) remains rapidly fatal and identification of new treatments has proven difficult due to patient specific characteristics of the tumor. Patient-derived GBM cell cultures enriched for tumor stem cells under serum-free, growth factor-enriched conditions are able to recapitulate the human disease upon xenotransplantation. We hypothesized that patient-derived GBM cultures display patient specific drug sensitivity patterns. We cultured 12 primary GBMs and evaluated viability and cytotoxicity to 470 anticancer drugs in a 5-point dose-response curve using high-throughput screening. The drug collection consisted of both approved and investigational drugs. Screening results were verified in 5 of the screened samples to 4 effective drugs and to temozolomide, the standard of care, across assays measuring viability, cytotoxicity, sphere-forming capability and invasion. We found that inhibition of survivin by an investigational drug was lethal in all samples and effective across all assays. Furthermore, several FDA approved drugs were potent across many samples such as two proteasome inhibitors, a protein synthesis inhibitor and a RNA-/DNA-synthesis inhibitor. Several investigational drugs in clinical phases were potent in many samples, such as a Chk1/Cdk2–, a combined HDAC-/PIK3- and two HSP90 inhibitors. Interestingly, temozolomide was ineffective in all samples except one. In 9 of the screened samples a patient specific drug response was identified. We were able to reproduce the effect of potent drugs and the resistance to temozolomide in our verification set up measuring viability, cytotoxicity and sphere-forming capability. However, the effect on invasion was limited. The high-throughput screening of patient-derived GBM samples creates a drug sensitivity and resistance profile to each patient and leads to identification of possible new effective treatments. By reproducing the screening results in our verification set up we are able to reliably identify preclinical effective therapeutic options on a patient specific level with the possibility to translate preclinical data into a clinical setting.

Abstract B31: NOX2 generated reactive oxygen species promotes glioblastoma growth through inhibition of PTEN

Abstract The tumor suppressor protein Phosphatase and Tensin Homolog (PTEN) is a negative regulator of the Akt pathway which promotes proliferation, migration, and self-renewal. PTEN is mutated or deleted in ~40% of glioblastoma (GBM) resulting in increased Akt activity; however almost all GBM's have increased Akt activity, regardless of PTEN status. This raises the possibility that GBM may inactivate PTEN through means other than mutation or deletion. Treatment with exogenous reactive oxygen species (ROS), like hydrogen peroxide, inactivates PTEN through oxidation in the phosphatase domain. The low oxygen environment of tumors (1%O2) increases ROS levels as compared to normal body tissue (4% O2), and tissue culture conditions (20% O2). Recent data indicate that low to moderate levels of ROS are required for cell maintenance and stem cell renewal while moderate increases in ROS may promote migration, invasion, and proliferation. Indeed, our lab recently found that normal neural stem cells have higher ROS levels, which is required for stem cell renewal. Moreover, data from our lab and others, have found that growing cells under physiological oxygen levels increases ROS and promotes renewal and proliferation. This suggests that the low-oxygen environment of tumors may promote proliferation and tumorigenesis through post-translational modification of proteins, like PTEN. NADPH oxidase (NOX) is a membrane bound complex of proteins that produces a significant amount of endogenous intracellular ROS. While this protein was originally identified in macrophages as being important for immune surveillance, new data suggests that the family of NOX proteins (1-4) has many other biological functions in a variety of different cell types. New data has found that the constitutively active NOX4 acts as an oncogene in several different cancer types by promoting migration, invasion, and proliferation. Within the brain, NOX2 promotes ROS-induced stem cell renewal in normal neural stem cells. Additionally, increased NOX2 expression in GBM patients correlates with shorter survival, while patients with low NOX2 expression generally live longer. Together these data suggest that, through NOX2, the low-oxygen environment of GBM increases ROS levels that oxidize and inhibit PTEN. This novel post-translational modification may increase proliferation and promote tumorigenicity through activation of Akt in cells with intact PTEN. We examined endogenous ROS levels in patient-derived GBM samples and found that increased ROS correlated with faster proliferating cells. Furthermore, low levels of O2 (1%) increased proliferation and Akt activation in a PTEN dependent manner. Both PTEN null and knockdown cells showed no increase in either proliferation or Akt activation over cells grown at room air (20%). Moreover, pharmacological inhibition of Akt abrogated ROS-induced proliferation, suggesting this mechanism functions through the PTEN/Akt pathway. This response was due directly to ROS production, as treatment with antioxidants decreased both ROS production and proliferation at low oxygen and room air. Additionally, we found that growth at 1% O2 led to the oxidation and inhibition of PTEN as seen by Akt phosphorylation. Treatment with apocynin, a NOX2 inhibitor decreased ROS production, proliferation, and Akt phosphorylation, suggesting that ROS-induced proliferation may be mediated through the NOX2 protein. This data not only show PTEN oxidation and inhibition under physiological conditions for the first time, it suggests that the low-oxygen tumor microenvironment may alter PTEN positive GBM to up-regulate the Akt pathway and promote tumorigenesis. Citation Format: Kirsten Ludwig, Harley Kornblum. NOX2 generated reactive oxygen species promotes glioblastoma growth through inhibition of PTEN. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B31.

ATPS-92Bmi1 IDENTIFIES TREATMENT-REFRACTORY STEM CELLS IN HUMAN GLIOBLASTOMA

PURPOSE: Glioblastoma (GBM) is a fatal primary adult brain tumor, characterized by extensive cellular heterogeneity. Even with surgery, chemotherapy with temozolomide (TMZ), and radiation, tumor re-growth and patient relapse are inevitable. Brain tumor initiating cells (BTICs), a rare subset of GBM cells with stem cell properties, were shown to be both chemo- and radio-resistant. We hypothesize that these treatment-resistant BTICs cause tumor relapse and a subset of genes regulating BTIC self-renewal drives GBM recurrence. METHODS: Using patient-derived primary GBM samples, we designed an in vitro model of tumor recurrence by treating cells with TMZ and radiation. We then adapted the existing treatment protocol for adults with primary GBM for treatment of immunocompromised mice engrafted with GFP+ human GBM cells. Post-chemoradiotherapy, GFP+ tumor cells were recovered from mouse brains and profiled for self-renewal, proliferation and mRNA expression of important stem cell genes. Using in vitro and in vivo loss-of-function experiments, we investigated the regulatory functions of key BTIC genes in tumor formation. RESULTS: GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy in vitro, suggesting the presence of a treatment-refractory BTIC population. GFP+ cells extracted from chemoradiotherapy treated human tumor xenografts showed increased self-renewal and elevated BTIC marker expression. Although treated mice responded to therapy with decreased tumor size, we observed tumor relapse post-chemoradiotherapy with increased Bmi1 expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis in xenografted mice, highlighting a critical role for Bmi1 in tumor initiation and maintenance. CONCLUSION: Our data confirms the existence of a rare treatment-refractory BTICs population that escapes therapy, and drives tumor relapse and recurrence with enhanced self-renewal capacity. Our human BTIC in vitro assays and human-mouse BTIC xenograft model provide fundamental tools to characterize the functional relevance and of key stem cell self-renewal genes in GBM recurrence.

High-Throughput Screening of Patient-Derived Cultures Reveals Potential for Precision Medicine in Glioblastoma.

Identifying drugs for the treatment of glioblastoma (GBM), a rapidly fatal disease, has been challenging. Most screening efforts have been conducted with immortalized cell lines grown with fetal bovine serum, which have little relevance to the genomic features found in GBM patients. Patient-derived neurosphere cultures, while being more physiologically relevant, are difficult to screen and therefore are only used to test a few drug candidates after initial screening efforts. Laminin has been used to generate two-dimensional cell lines from patient tumors, preserving the genomic signature and alleviating some screening hurdles. We present here the first side-by-side comparison of inhibitor sensitivity of laminin and neurosphere-grown patient-derived GBM cell lines and show that both of these culture methods result in the same pattern of inhibitor sensitivity. We used these screening methods to evaluate the dependencies of seven patient-derived cell models: three grown on laminin and four grown as neurospheres, against 56 agents in 17-point dose-response curves in 384-well format in triplicate. This allowed us to establish differential sensitivity of chemotherapeutic agents across the seven patient-derived models. We found that MEK inhibition caused patient-sample-specific growth inhibition and that bortezomib, an FDA-approved proteasome inhibitor, was potently lethal in all patient-derived models. Furthermore, the screening results led us to test the combination of the Bcl-2 inhibitor ABT-263, and the mTOR inhibitor AZD-8055, which we found to be synergistic in a subset of patient-derived GBM models. Thus, we have identified new candidate therapeutics and developed a high-throughput screening system using patient-derived GBM samples.

Activation of canonical WNT/β-catenin signaling enhances in vitro motility of glioblastoma cells by activation of ZEB1 and other activators of epithelial-to-mesenchymal transition

Here we show that activation of the canonical WNT/β-catenin pathway increases the expression of stem cell genes and promotes the migratory and invasive capacity of glioblastoma. Modulation of WNT signaling alters the expression of epithelial-to-mesenchymal transition activators, suggesting a role of this process in the regulation of glioma motility.Using immunohistochemistry in patient-derived glioblastoma samples we showed higher numbers of cells with intranuclear signal for β-catenin in the infiltrating edge of tumor compared to central tumor parenchyma. These findings suggest that canonical WNT/β-catenin pathway is a critical regulator of GBM invasion and may represent a potential therapeutic target.