Patient-derived Glioblastoma Stem-like Cells Research Articles

Abstract 655: Fungal extract screening reveals malformin C to preferentially kill glioblastoma stem-like cells via concerted induction of proteotoxic stress and autophagic flux blockade

Abstract Glioblastoma is a highly aggressive brain tumor for which there is no cure. The dire prognosis of this disease is largely attributable to a high level of heterogeneity, including the presence of a sub-population of tumor initiating glioblastoma stem-like cells (GSCs), which are refractory to chemo- and radiotherapy. Here, in an unbiased marine-derived fungal extract screen, together with bioguided dereplication based on high resolution mass spectrometry, we identified malformin C to preferentially induce cell death in patient-derived GSCs, and explore the potential of this cyclic peptide as a therapeutic agent for glioblastoma. Malformin C significantly reduced tumor growth in an in vivo xenograft model of glioblastoma. Using transcriptomics and chemoproteomics, we found that malformin C binds to many proteins leading to aggregation, and rapidly induces unfolded protein response, including autophagy, in GSCs. Malformin C also appears to accumulate in lysosomes, disrupting autophagic flux and driving cells to death. Supporting this, malformin C synergizes with chloroquine, an inhibitor of autophagy. Strikingly, we observed that autophagic flux is differentially regulated in GSCs compared with normal astrocytes. The sensitivity of GSCs to malformin C highlights the relevance of proteostasis and autophagy as a therapeutic vulnerability in glioblastoma. Citation Format: Emma L. Phillips, Sizèd van Enk, Sara Kildgaard, Silja Schlue, Mona Göttmann, Victoria Jennings, Frederic Bethke, Gabriele Müller, Christel Herold-Mende, Daniel Pastor-Flores, Martin Schneider, Dominic Helm, Thomas Ostenfeld Larsen, Violaine Goidts. Fungal extract screening reveals malformin C to preferentially kill glioblastoma stem-like cells via concerted induction of proteotoxic stress and autophagic flux blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 655.

TRIM25 promotes glioblastoma cell growth and invasion via regulation of the PRMT1/c-MYC pathway by targeting the splicing factor NONO

BackgroundUbiquitination plays an important role in proliferating and invasive characteristic of glioblastoma (GBM), similar to many other cancers. Tripartite motif 25 (TRIM25) is a member of the TRIM family of proteins, which are involved in tumorigenesis through substrate ubiquitination.MethodsDifference in TRIM25 expression levels between nonneoplastic brain tissue samples and primary glioma samples was demonstrated using publicly available glioblastoma database, immunohistochemistry, and western blotting. TRIM25 knockdown GBM cell lines (LN229 and U251) and patient derived GBM stem-like cells (GSCs) GBM#021 were used to investigate the function of TRIM25 in vivo and in vitro. Co-immunoprecipitation (Co-IP) and mass spectrometry analysis were performed to identify NONO as a protein that interacts with TRIM25. The molecular mechanisms underlying the promotion of GBM development by TRIM25 through NONO were investigated by RNA-seq and validated by qRT-PCR and western blotting.ResultsWe observed upregulation of TRIM25 in GBM, correlating with enhanced glioblastoma cell growth and invasion, both in vitro and in vivo. Subsequently, we screened a panel of proteins interacting with TRIM25; mass spectrometry and co-immunoprecipitation revealed that NONO was a potential substrate of TRIM25. TRIM25 knockdown reduced the K63-linked ubiquitination of NONO, thereby suppressing the splicing function of NONO. Dysfunctional NONO resulted in the retention of the second intron in the pre-mRNA of PRMT1, inhibiting the activation of the PRMT1/c-MYC pathway.ConclusionsOur study demonstrates that TRIM25 promotes glioblastoma cell growth and invasion by regulating the PRMT1/c-MYC pathway through mediation of the splicing factor NONO. Targeting the E3 ligase activity of TRIM25 or the complex interactions between TRIM25 and NONO may prove beneficial in the treatment of GBM.

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

CCRG-03. KAT5 REGULATES DEVELOPMENTAL TRANSITIONS AND CELLULAR HETEROGENEITY IN AN IN VIVO MODEL OF GLIOBLASTOMA

Abstract In solid tumors, G0-like states are likely critical for maintaining developmental hierarchies and cellular heterogeneity and promoting tumor growth/recurrence, yet little is known about tumor G0 states or regulation of their ingress/egress. To discover G0-like states and their regulators for glioblastoma (GBM), we previously performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) for genes that trap cells in G0 when inhibited. We identify the protein acetyltransferase KAT5 as a key regulator of GBM G0 ingress/egress. Here, we show in an in vivo GSC-derived orthotopic xenograft model that KAT5 regulates transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states. We show that KAT5 activity suppresses the emergence of non-dividing subpopulations with oligodendrocyte progenitor and radial glial cell characteristics. With regard to chromatin regulation, we show that loss of KAT5 activity alters sites of mixed epigenetic valency (which have both activating and repressive marks in bulk tumor populations) to promote emergence of OPC/RG G0-like populations in tumors. Our data reveal KAT5-associated transcriptional and epigenetic changes in tumors, which indicate that KAT5 activity biases cell state distribution away from OPC and RG-like subpopulations. In addition, loss of KAT5 resulted in a less invasive phenotype and prolonged survival of mice harboring orthotopic xenografts.

Stationary-to-migratory transition in glioblastoma stem-like cells driven by a fatty acid-binding protein 7-RXRα neurogenic pathway.

Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors

PurposePatient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs).Methods94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively.ResultsExome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S − adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways.ConclusionThe availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Identification of Dysregulated microRNAs in Glioblastoma Stem-like Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite multimodal therapy, median survival is poor at 12-15 months. At the molecular level, radio-/chemoresistance and resulting tumor progression are attributed to a small fraction of tumor cells, termed glioblastoma stem-like cells (GSCs). These CD133-expressing, self-renewing cells display the properties of multi-lineage differentiation, resulting in the heterogenous composition of GBM. MicroRNAs (miRNAs) as regulators of gene expression at the post-transcriptional level can alter many pathways pivotal to cancer stem cell fate. This study explored changes in the miRNA expression profiles in patient-derived GSCs altered on differentiation into glial fiber acid protein (GFAP)-expressing, astrocytic tumor cells using a polymerase chain reaction (PCR) array. Initially, 22 miRNAs showed higher expression in GSCs and 9 miRNAs in differentiated cells. The two most downregulated miRNAs in differentiated GSCs were miR-17-5p and miR-425-5p, whilst the most upregulated miRNAs were miR-223-3p and let-7-5p. Among those, miR-425-5p showed the highest consistency in an upregulation in all three GSCs. By transfection of a 425-5p miRNA mimic, we demonstrated downregulation of the GFAP protein in differentiated patient-derived GBM cells, providing potential evidence for direct regulation of miRNAs in the GSC/GBM cell transition.

Study of Strawberry Notch homolog 1 and 2 expression in human glioblastoma.

In this work, we aimed to comprehensively document the expression of Strawberry Notch homolog (SBNO) 1 and 2 in glioblastoma (GBM) tissue and patient-derived GBM stem-like cell (GSC) cultures. We investigated SBNO1 and SBNO2 expression at the RNA and protein levels in glioma patient tissue and GSCs, respectively by performing immunostainings and qPCR analyses. We also used publicly-available datasets for assessing SBNO1 and SBNO2 gene expression and related copy number alterations. We used lentiviral transduction of SBNO2 to analyze the effect of its expression in patient-derived GSCs. We observed that SBNO2 expression is increased in GBM tissue samples compared to non tumoral brain, or lower-grade gliomas, whereas SBNO1 expression remains unchanged. We hypothesized that such SBNO2 high expression might be linked to copy-number alterations at the level of the 19p13 chromosome section. We located SBNO1 and SBNO2 in different subcellular compartments. Finally, we observed that SBNO2 overexpression induces different phenotypes in different patient-derived GSCs. These results provide the first characterization of SBNO1 and SBNO2 expression in glioma tissue, and indicate SBNO2 as highly expressed in GBM.

A bio-orthogonal linear ubiquitin probe identifies STAT3 as a direct substrate of OTULIN in glioblastoma.

While linear ubiquitin plays critical roles in multiple cell signaling pathways, few substrates have been identified. Global profiling of linear ubiquitin substrates represents a significant challenge because of the low endogenous level of linear ubiquitination and the background interference arising from highly abundant ubiquitin linkages (e.g. K48- and K63-) and from the non-specific attachment of interfering proteins to the linear polyubiquitin chain. We developed a bio-orthogonal linear ubiquitin probe by site-specific encoding of a norbornene amino acid on ubiquitin (NAEK-Ub). This probe facilitates covalent labeling of linear ubiquitin substrates in live cells and enables selective enrichment and identification of linear ubiquitin-modified proteins. Given the fact that the frequent overexpression of the linear linkage-specific deubiquitinase OTULIN correlates with poor prognosis in glioblastoma, we demonstrated the feasibility of the NAEK-Ub strategy by identifying and validating substrates of linear ubiquitination in patient-derived glioblastoma stem-like cells (GSCs). We identified STAT3 as a bona fide substrate of linear ubiquitin, and showed that linear ubiquitination negatively regulates STAT3 activity by recruitment of the phosphatase TC-PTP to STAT3. Furthermore, we demonstrated that preferential expression of OTULIN in GSCs restricts linear ubiquitination on STAT3 and drives persistent STAT3 signaling, and thereby maintains the stemness and self-renewal of GSCs.

Targeting de novo lipid synthesis induces lipotoxicity and impairs DNA damage repair in glioblastoma mouse models.

Deregulated de novo lipid synthesis (DNLS) is a potential druggable vulnerability in glioblastoma (GBM), a highly lethal and incurable cancer. Yet the molecular mechanisms that determine susceptibility to DNLS-targeted therapies remain unknown, and the lack of brain-penetrant inhibitors of DNLS has prevented their clinical evaluation as GBM therapeutics. Here, we report that YTX-7739, a clinical-stage inhibitor of stearoyl CoA desaturase (SCD), triggers lipotoxicity in patient-derived GBM stem-like cells (GSCs) and inhibits fatty acid desaturation in GSCs orthotopically implanted in mice. When administered as a single agent, or in combination with temozolomide (TMZ), YTX-7739 showed therapeutic efficacy in orthotopic GSC mouse models owing to its lipotoxicity and ability to impair DNA damage repair. Leveraging genetic, pharmacological, and physiological manipulation of key signaling nodes in gliomagenesis complemented with shotgun lipidomics, we show that aberrant MEK/ERK signaling and its repression of the energy sensor AMP-activated protein kinase (AMPK) primarily drive therapeutic vulnerability to SCD and other DNLS inhibitors. Conversely, AMPK activation mitigates lipotoxicity and renders GSCs resistant to the loss of DNLS, both in culture and in vivo, by decreasing the saturation state of phospholipids and diverting toxic lipids into lipid droplets. Together, our findings reveal mechanisms of metabolic plasticity in GSCs and provide a framework for the rational integration of DNLS-targeted GBM therapies.

Effectiveness of Flattening-Filter-Free versus Flattened Beams in V79 and Glioblastoma Patient-Derived Stem-like Cells

Literature data on the administration of conventional high-dose beams with (FF) or without flattening filters (FFF) show conflicting results on biological effects at the cellular level. To contribute to this field, we irradiated V79 Chinese hamster lung fibroblasts and two patient-derived glioblastoma stem-like cell lines (GSCs-named #1 and #83) using a clinical 10 MV accelerator with FF (at 4 Gy/min) and FFF (at two dose rates 4 and 24 Gy/min). Cell killing and DNA damage induction, determined using the γ-H2AX assay, and gene expression were studied. No significant differences in the early survival of V79 cells were observed as a function of dose rates and FF or FFF beams, while a trend of reduction in late survival was observed at the highest dose rate with the FFF beam. GSCs showed similar survival levels as a function of dose rates, both delivered in the FFF regimen. The amount of DNA damage measured for both dose rates after 2 h was much higher in line #1 than in line #83, with statistically significant differences between the two dose rates only in line #83. The gene expression analysis of the two GSC lines indicates gene signatures mimicking the prognosis of glioblastoma (GBM) patients derived from a public database. Overall, the results support the current use of FFF and highlight the possibility of identifying patients with candidate gene signatures that could benefit from irradiation with FFF beams at a high dose rate.

Nanobody-based retargeting of an oncolytic herpesvirus for eliminating CXCR4+ GBM cells: A proof of principle

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, which remains difficult to cure. The very high recurrence rate has been partly attributed to the presence of GBM stem-like cells (GSCs) within the tumors, which have been associated with elevated chemokine receptor 4 (CXCR4) expression. CXCR4 is frequently overexpressed in cancer tissues, including GBM, and usually correlates with a poor prognosis. We have created a CXCR4-retargeted oncolytic herpesvirus (oHSV) by insertion of an anti-human CXCR4 nanobody in glycoprotein D of an attenuated HSV-1 (ΔICP34.5, ΔICP6, and ΔICP47), thereby describing a proof of principle for the use of nanobodies to target oHSVs toward specific cellular entities. Moreover, this virus has been armed with a transgene expressing a soluble form of TRAIL to trigger apoptosis. In vitro, this oHSV infects U87MG CXCR4+ and patient-derived GSCs in a CXCR4-dependent manner and, when armed, triggers apoptosis. In a U87MG CXCR4+ orthotopic xenograft mouse model, this oHSV slows down tumor growth and significantly improves mice survival. Customizing oHSVs with diverse nanobodies for targeting multiple proteins appears as an interesting approach for tackling the heterogeneity of GBM, especially GSCs. Altogether, our study must be considered as a proof of principle and a first step toward personalized GBM virotherapies to complement current treatments.

Infiltrating natural killer cells bind, lyse and increase chemotherapy efficacy in glioblastoma stem-like tumorospheres

Glioblastomas remain the most lethal primary brain tumors. Natural killer (NK) cell-based therapy is a promising immunotherapeutic strategy in the treatment of glioblastomas, since these cells can select and lyse therapy-resistant glioblastoma stem-like cells (GSLCs). Immunotherapy with super-charged NK cells has a potential as antitumor approach since we found their efficiency to kill patient-derived GSLCs in 2D and 3D models, potentially reversing the immunosuppression also seen in the patients. In addition to their potent cytotoxicity, NK cells secrete IFN-γ, upregulate GSLC surface expression of CD54 and MHC class I and increase sensitivity of GSLCs to chemotherapeutic drugs. Moreover, NK cell localization in peri-vascular regions in glioblastoma tissues and their close contact with GSLCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSLCs. Due to GSLC heterogeneity and plasticity in regards to their stage of differentiation personalized immunotherapeutic strategies should be designed to effectively target glioblastomas.

Increased apoptotic sensitivity of glioblastoma enables therapeutic targeting by BH3-mimetics

Glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. We investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti-apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non-malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti-apoptotic BCL-2 family members. High anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein-targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3-mimetics.

CBIO-12. KAT5 ACTIVITY CONTROLS GLIOBLASTOMA CELL CYCLE DYNAMICS AND TUMOR GROWTH

Abstract Current standard of care therapy for glioblastoma (GB) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how GB cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GB stem-like cells (GSCs) harboring a G0 reporter construct, which is stabilized when cells enter a G0-like state. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that knockout of KAT5 in vitro and in vivo dramatically increases G0 subpopulations in GSC cultures and GSC-induced tumors. Using genetically engineered GSC harboring KAT5 under the control of a Doxycyclin-titratable promoter, we establish that incrementally down regulating KAT5 activity is sufficient to slow cell cycle dynamics causing a build-up G0-like cells; and that partial inhibition of KAT5 leads to extended (mouse) patient survival. Further, in primary tumors, cell-based KAT5 activity assays revealed that high grade tumors harbor larger cell subpopulations with higher KAT5 activity than lower grade tumors. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors, may contribute to tumor progression, and may provide novel therapeutic opportunities.

Targeted Liposomes Encapsulating miR-603 Complexes Enhance Radiation Sensitivity of Patient-Derived Glioblastoma Stem-Like Cells.

Despite potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a strategy for miRNA delivery in the treatment of glioblastoma, the most common form of adult brain cancer, that involves complexation of miRNA with polyethylenimine (PEI) and encapsulation in targeted liposomes. miRNA 603 (miR-603) is a master regulatory miRNA that suppresses glioblastoma radiation resistance through down-regulation of insulin-like growth factor 1 (IGF1) signaling. miR-603 was complexed with PEI, a cationic polymer, and encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α5β1 integrin that is overexpressed in glioblastomas. Cultured patient-derived glioblastoma cells internalized PR_b-functionalized liposomes but not the non-targeted liposomes. The integrin targeting and complexation of the miRNA with PEI were associated with a 22-fold increase in intracellular miR-603 levels, and corresponding decreases in IGF1 and IGF1 receptor (IGF1R) mRNA expression. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI sensitized the cells to ionizing radiation (IR), a standard of care treatment for glioblastomas. These results suggest that PR_b-functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a therapeutic platform for glioblastomas.

Abstract 3114: Olfactory ensheathing glia as a cell-based therapy for glioblastomas

Abstract Introduction: Olfactory ensheathing glia (OEG) is a fully differentiated cell that promotes the continuous regeneration of odor receptors from the nasal mucosa to the olfactory bulb during adult lifespan. OEG secretes several factors making the environment more permissive to regeneration, and promotes differentitation of progenitor/neural stem cells. Recently, our group showed for the first time that OEG can target glioblastoma (GBM) cells and GBM stem-like cells (GSC) in the mice brain via intranasal delivery, the natural route of OEG to the brain. Here, we test the potential of OEG in inhibiting the proliferation and stemness of GSC, leading to their differentiation (similar to their neural stem cells counterpart), making them susceptible to conventional therapy. Methods: OEG derived from mice OB (1.5e+5 cells) were co-cultured with GSC at 1:1 ratio. We used a dual secreted luciferase reporter that can be multiplexed together to monitor both OEG and GSCs viability and proliferation in the same well. For in vivo analysis, mice-bearing patient-derived xenograft GSCs were treated with a single injection of 1e+5 OEG intranasally or control. One week later, each group was subdivided into two groups which either received ionizing radiation (IR, one dose of 4Gy) or control. Results: GSC or their corresponding differentiated glioma cells (cultured in serum for 10 days) displayed a decrease in cell viability when co-cultured with OEG compared to control. Analysis of mRNA levels and immunofluorescence images from these co-cultures (using a Transwell) showed a significant decrease in markers associated with stemness and aggressiveness, and an increase in both neuronal and astrocytic markers compared to control. When assessed tumor formation, GSC cultured with OEG conditioned media (OEGCM) failed to form tumors and became more susceptible to IR and Temozolomide. In mice bearing PDX, intranasal injection of non-modified OEG lead to inhibition of tumor growth prolonged mouse survival (48 days vs 34 days for control; P<0.0001). IR had a modest effect on this model (38 days; P<0.005 vs control). The combination of a single OEG injection and IR had the most therapeutic effect on tumor growth and animal survival (54 days; P<0.0001 vs control). Conclusion: Our findings show that OEG inhibits GBM stemness and malignancy, induces differentiation of GSCs, making them prone to conventional therapy. OEG have the advantage over other typical cell therapies since they are terminally differentiated and can be easily obtained from the olfactory mucosa, a simple procedure typically done for patients with spinal cord injury allowing autologous transplantation. Citation Format: Litia Carvalho, Renata Fleming, Gulsah Erel-Akbaba, Ghazal Lashgari, Daniel Ryan, Elie Tabet, Max Zinter, Jian Teng, Bakhos Tannous. Olfactory ensheathing glia as a cell-based therapy for glioblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3114.

Integrated Metabolomics and Transcriptomics Analysis of Monolayer and Neurospheres from Established Glioblastoma Cell Lines.

Simple SummaryGlioblastomas are very aggressive tumours without efficient treatment, where cancer stem-like cells are thought to be responsible for relapse. This pilot study investigated the metabolic discrepancies between monolayer and neurosphere cultures of two glioblastoma cell lines using transcriptomics and metabolomics. We show that the two culture systems display substantial differences regarding their metabolome and transcriptome. Specifically, we found that metabolic reactions connected to arginine biosynthesis are crucial to support the different metabolic needs of neurospheres from the two cell lines. By identifying metabolic vulnerabilities in different glioblastoma subpopulations, new therapeutic strategies may be emerging that can be explored to treat this disease. Moreover, this data set may be of great value as a resource for the scientific community.Altered metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Tumours are composed of different cell populations, with cancer stem-like cells being one of the most prominent examples. This specific pool of cells is thought to be responsible for cancer growth and recurrence and plays a particularly relevant role in glioblastoma (GBM), the most lethal form of primary brain tumours. Here, we have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells, and demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres, although individual nodes of this pathway were distinctly regulated in the two cellular systems. Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features, where arginine biosynthesis may be a key metabolic pathway. Additionally, TCGA data from GBM patients showed significant regulation of specific components of the arginine biosynthesis pathway, providing further evidence for the importance of this metabolic pathway in GBM.

On-chip perivascular niche supporting stemness of patient-derived glioma cells in a serum-free, flowable culture.

Glioblastoma multiforme (GBM) is the most common and the most aggressive type of primary brain malignancy. Glioblastoma stem-like cells (GSCs) can migrate in vascular niches within or away from the tumour mass, increasing tumour resistance to treatments and contributing to relapses. To study individual GSC migration and their interactions with the perivasculature of the tumour microenvironment, there is a need to develop a human organotypic in vitro model. Herein, we demonstrated a perivascular niche-on-a-chip, in a serum-free condition with gravity-driven flow, that supported the stemness of patient-derived GSCs and foetal neural stem cells grown in a three-dimensional environment (3D). Endothelial cells from three organ origins, (i) human brain microvascular endothelial cells (hCMEC/D3), (ii) human umbilical vein endothelial cells (HUVECs) and, (iii) human lung microvascular endothelial cells (HMVEC-L) formed rounded microvessels within the extracellular-matrix integrated microfluidic chip. By optimising cell extraction protocols, systematic studies were performed to evaluate the effects of serum-free media, 3D cell cultures, and the application of gravity-driven flow on the characteristics of endothelial cells and their co-culture with GSCs. Our results showed the maintenance of adherent and tight junction markers of hCMEC/D3 in the serum-free culture and that gravity-driven flow was essential to support adequate viability of both the microvessel and the GSCs in co-culture (>80% viability at day 3). Endpoint biological assays showed upregulation of neovascularization-related genes (e.g., angiopoietins, vascular endothelial growth factor receptors) in endothelial cells co-cultured with GSCs in contrast to the neural stem cell reference that showed insignificant changes. The on-chip platform further permitted live-cell imaging of GSC – microvessel interaction, enabling quantitative analysis of GSC polarization and migration. Overall, our comparative genotypic (i.e. qPCR) and phenotypic (i.e. vessel permeability and GSC migration) studies showed that organotypic (brain cancer cells–brain endothelial microvessel) interactions differed from those within non-tissue specific vascular niches of human origin. The development and optimization of this on-chip perivascular niche, in a serum-free flowable culture, could provide the next level of complexity of an in vitro system to study the influence of glioma stem cells on brain endothelium.

CBIO-19. LOW GLOBAL HISTONE H4 ACETYLATION LEVELS REVEAL CANDIDATE QUIESCENT CELL POPULATIONS IN GLIOMA AND DISTINGUISH TUMORS BY GRADE

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, the presence of quiescent-like/G0 states, therefore, represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a G0-reporter to identify genes that when inhibited trap GSCs in G0-like states. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, which targets both histones (e.g., H4) and non-histone proteins for acetylation. NuA4 functions as a transcriptional activator, whose activities are coordinated with MYC in certain contexts, and also participates in DNA double-strand break repair by facilitating chromatin opening. However, currently little is known about the roles for NuA4 complex in GBM biology. Through modeling KAT5 function in GSC in vitro cultures and in vivo tumors, we find that KAT5 inhibition causes cells to arrest in a G0-like state with high p27 levels, G1-phase DNA content, low protein synthesis rates, low rRNA rates, lower metabolic rate, suppression of cell cycle gene expression, and low histone H4 acetylation. Interestingly, partial inhibition of KAT5 activity slows highly aggressive tumor growth, while increasing p27hi H4-aclow populations. Remarkably, we that low grade gliomas have significantly higher H4-aclow subpopulations and generally lower H4-ac levels than aggressive grade IV tumors. Taken together, our results suggest that NuA4/KAT5 activity may play a key role in quiescence ingress/egress in glioma and that targeting its activity in high grade tumors may effectively “down grade” them, thus, increase patient survival.