Glioblastoma Cell Populations Research Articles

DNAR-14. MODULATING POPULATIONAL VARIANCE OF METHYL-GUANINE METHYL TRANSFERASE (MGMT) EXPRESSION THROUGH MICRORNA DEGRADATION: A NOVEL MECHANISM OF TEMOZOLOMIDE RESISTANCE

Abstract O6 methyl-guanine methyltransferase (MGMT) restores alkylated DNA to its undamaged form in a stochiometric manner to prevent the cytotoxic effects of temozolomide (TMZ), a DNA alkylating agent used as a standard-of-care treatment for glioblastomas. We previously demonstrated that he microRNA, miR-181d, post-transcriptionally repressed MGMT expression. Here, we show that treatment of glioblastoma cells with TMZ induced a feed-forward cascade resulting in an ataxia telangiectasia and Rad3 (ATR) and polyribonucleotide nucleotidyl transferase 1 (PNPT1)-dependent degradation of miR-181d. Single-cell analyses revealed such miR-181d degradation induced an: 1) increased mean level of MGMT expression and 2) widened the variance in MGMT expression in the cell population. While the former is known to confer increased population fitness to temozolomide, the importance of the latter is unknown. By mixing cultures derived from isogenic glioblastoma subclones with distinct MGMT expression levels, we generated glioblastoma cell populations with comparable mean MGMT expression levels while differing in the populational variance in MGMT expression (classified as “wide” or “narrow”). Despite comparable mean levels of MGMT expression, the glioblastoma population with wide variance exhibited increased temozolomide resistance relative to the population with the narrow variance, in vitro and in vivo. Transfection of miR-181d into the wide-variance population narrows the variance of MGMT expression and restores TMZ sensitivity to levels comparable to that observed in miR-181d transfected narrow-variance populations. These findings suggest that TMZ-induced miR-181d degradation enhanced TMZ resistance through modulation of both the mean and variance of MGMT expression, mechanisms which may be extrapolated to microRNA biology in general.

Protumoral lipid droplet-loaded macrophages are enriched in human glioblastoma and can be therapeutically targeted.

Glioblastoma presents a formidable clinical challenge because of its complex microenvironment. Here, we characterized tumor-associated foam cells (TAFs), a type of lipid droplet-loaded macrophage, in human glioblastoma. Through extensive analyses of patient tumors, together with in vitro and in vivo investigations, we found that TAFs exhibit distinct protumorigenic characteristics related to hypoxia, mesenchymal transition, angiogenesis, and impaired phagocytosis, and their presence correlates with worse outcomes for patients with glioma. We further demonstrated that TAF formation is facilitated by lipid scavenging from extracellular vesicles released by glioblastoma cells. We found that targeting key enzymes involved in lipid droplet formation, such as diacylglycerol O-acyltransferase or long-chain acyl-CoA synthetase, effectively disrupted TAF functionality. Together, these data highlight TAFs as a prominent immune cell population in glioblastoma and provide insights into their contribution to the tumor microenvironment. Disrupting lipid droplet formation to target TAFs may represent an avenue for future therapeutic development for glioblastoma.

Abstract IA004: Targeting clonal heterogeneity in treatment-refractory Glioblastoma with novel and empiric immunotherapies

Abstract Despite aggressive multimodal therapy, glioblastoma (GBM) remains the most common malignant primary brain tumor in adults. With the advent of therapies that revitalize the anti-tumor immune response, several immunotherapeutic modalities have been developed for the treatment of GBM. In this talk, we summarize recent clinical and pre-clinical efforts to develop immunotherapeutic and chimeric antigen receptor (CAR) T cell treatment strategies for GBM recurrence, including challenges in the form of fundamental mechanisms of therapy evasion by tumor cells, such as immense intratumoral heterogeneity, suppression of the tumor immune microenvironment, and low mutational burden. Insights from these advances and challenges have shaped our development of a translational research pipeline from initial target discovery, through target validation and exploration of mechanism, to building new biotherapeutics against novel cancer targets, and preclinical testing in our patient-derived animal models of treatment-resistant GBM. We have tracked GBM cell populations that undergo clonal evolution as a result of selective pressures exerted by standard treatments to identify the cellular composition of the recurrence, to allow us to determine the intracellular signaling pathways that drive GBM relapse. Ultimately, we will design rational combinations of therapeutics that can reignite the anti-tumor immune response, effectively and specifically target tumor cells, and reliably decrease tumor burden for GBM patients. Citation Format: Sheila K Singh. Targeting clonal heterogeneity in treatment-refractory Glioblastoma with novel and empiric immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr IA004.

Correlation of MR-Based Metabolomics and Molecular Profiling in the Tumor Microenvironment of Temozolomide-Treated Orthotopic GL261 Glioblastoma in Mice.

The tumor microenvironment in glioblastoma (GB) is considered to be "cold", i.e., the fraction of cytotoxic T cells, for instance, is low. Instead, macrophages are the major immune cell population in GB, which stem either from tissue response (resident microglia) or recruitment of macrophages from the periphery, thereby undergoing tumor-dependent "imprinting" mechanisms by which macrophages can adapt a tumor-supportive phenotype. In this regard, it is important to describe the nature of macrophages associated with GB, in particular under therapy conditions using the gold standard chemotherapy drug temozolomide (TMZ). Here, we explored the suitability of combining information from in vivo magnetic resonance spectroscopic (MRS) approaches (metabolomics) with in vitro molecular analyses to assess therapy response and characterize macrophage populations in mouse GB using an isogenic GL261 model. For macrophage profiling, expression levels of matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) were determined, since their gene products affect macrophage-tumor cell communication by extensive cleavage of immunomodulatory membrane proteins, such as PD-L1. In tumor mice with an overall therapy response, expression of genes encoding the proteases ADAM8, ADAM10, and ADAM17 was increased and might contribute to the immunosuppressive phenotype of GB and immune cells. In tumors responding to therapy, expression levels of ADAM8 were upregulated by TMZ, and higher levels of PD-L1 were correlated significantly. Using a CRISPR/Cas9 knockout of ADAM8 in GL261 cells, we demonstrated that soluble PD-L1 (sPD-L1) is only generated in the presence of ADAM8. Moreover, primary macrophages from WT and ADAM8-deficient mice showed ADAM8-dependent release of sPD-L1, independent of the macrophage polarization state. Since ADAM8 expression is induced in responding tumors and PD-L1 shedding is likely to decrease the anti-tumor activities of T-cells, we conclude that immunotherapy resistance is caused, at least in part, by the increased presence of proteases, such as ADAM8.

Translocator protein (18kDA) (TSPO) marks mesenchymal glioblastoma cell populations characterized by elevated numbers of tumor-associated macrophages

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing.We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages.In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions.

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.

Radiation-Induced Cellular Senescence Reduces Susceptibility of Glioblastoma Cells to Oncolytic Vaccinia Virus.

Glioblastoma (GBM) is a malignant brain cancer refractory to the current standard of care, prompting an extensive search for novel strategies to improve outcomes. One approach under investigation is oncolytic virus (OV) therapy in combination with radiotherapy. In addition to the direct cytocidal effects of radiotherapy, radiation induces cellular senescence in GBM cells. Senescent cells cease proliferation but remain viable and are implicated in promoting tumor progression. The interaction of viruses with senescent cells is nuanced; some viruses exploit the senescent state to their benefit, while others are hampered, indicating senescence-associated antiviral activity. It is unknown how radiation-induced cellular senescence may impact the oncolytic properties of OVs based on the vaccinia virus (VACV) that are used in combination with radiotherapy. To better understand this, we induced cellular senescence by treating GBM cells with radiation, and then evaluated the growth kinetics, infectivity, and cytotoxicity of an oncolytic VACV, ∆F4LΔJ2R, as well as wild-type VACV in irradiated senescence-enriched and non-irradiated human GBM cell lines. Our results show that both viruses display attenuated oncolytic activities in irradiated senescence-enriched GBM cell populations compared to non-irradiated controls. These findings indicate that radiation-induced cellular senescence is associated with antiviral activity and highlight important considerations for the combination of VACV-based oncolytic therapies with senescence-inducing agents such as radiotherapy.

Gut microbiota modulation by high glucose diet invigorates anti-tumor immune response to glioblastoma multiforme

Abstract Most studies about the relationship between high sugar diet and tumor progression state that the pro-tumoral effects of high sugar diet are induced by metabolic diseases such as obesity and diabetes caused by long-term, excessive sugar diet. However, there are still many unknowns about the effect of high sugar diet on tumors when it lasts for a relatively short period that does not cause metabolic diseases. In this background, we find that survival rate of glioblastoma (GBM) is increased through supplement of high glucose diet (HGD) for several weeks. We find that gut microbiota modulation by HGD plays a crucial role in enhancement of anti-tumor immune response. Through single cell RNA sequencing analysis, modulation of gut microbiota by HGD results in an increase in T cell-mediated anti-tumor immune response in GBM. In addition, we find the novel cytotoxic CD4+ T cell population in GBM is increased due to synergistic action with anti PD-1 and the change occurs only through the synergistic action of anti PD-1 treatment and HGD. As a result, we identify that HGD can enhance anti-tumor immune response in GBM through changes in gut microbiota and suggest that the role of glucose diet on GBM needs to be elucidated from a different perspective.

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

Rhythmic Ca2+ Communication Promotes Glioma Cell Proliferation.

A plastic glioblastoma cell population exhibits rhythmic Ca2+ oscillations that drive brain tumor growth.

STEM-18. EPIGENETIC AND MOLECULAR COORDINATION BETWEEN HDAC2 AND SMAD3-SKI IS REQUIRED FOR GROWTH AND STEM CELL CHARACTERISTICS OF BRAIN TUMOUR STEM CELLS

Abstract Brain tumour stem cell population in glioblastoma (GBM) display key cancer stem cell characteristics of high self-renewal and drug resistance that are maintained by the coordinated functions of epigenetic and molecular regulators. Yet, specific epigenetic mechanisms that, in collaboration with relevant molecular pathways, help maintain a stem-like state in BTSCs remain poorly understood. Here, we identify HDAC2 as a foremost epigenetic regulator in BTSCs that specifically utilizes the transforming growth factor-β (TGF-β) pathway related proteins, SMAD3-SKI, for remodelling BTSC chromatin accessibility and transcriptional programs to facilitate their stemness and tumorigenic potentials. Our initial drug screening revealed that selective inhibition of HDAC1 and 2 with romidepsin was effective in targeting BTSC viability, cell proliferation and self-renewal in vitro. Using CRISPR-cas9 knockout and shRNA knockdown strategies, we further demonstrated that loss of HDAC2 disrupts an epigenetic and molecular coordination between HDAC2 and SMAD-SKI proteins, which negatively impacts BTSC survival, cell proliferation and self-renewal in vitro and improves median survival in orthotopic xenograft mouse models. Loss of HDAC2 showed reduction in the protein abundance of transcriptional regulator, SMAD3 and negative regulator protein, SKI. However, overexpression of SMAD3 in HDAC2 deficient BTSCs could partially rescues their cell functional deficits. These findings suggest that context-specific epigenetic regulations by HDAC2 and its interaction with the critical transcriptional regulators, SMAD3-SKI, maintains the stemness and growth characteristics of BTSCs. Further HDAC2 overexpression increases cell proliferation and self-renewal abilities in normal neural stem cells (NSCs). These findings thus support the role of HDAC2 as a key epigenetic determinant of stemness in normal NSCs and of cancer stem cell characteristics and tumorigenic potential in BTSCs. Collectively, our data raises the potential that disruption of the coordinated mechanisms regulated by HDAC2-SMAD3-SKI axis may be an effective therapeutic approach for targeting GBM BTSCs.

Alternative RNA splicing modulates ribosomal composition and determines the spatial phenotype of glioblastoma cells.

Glioblastoma (GBM) is characterized by exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here, we found that the compositions of ribosomes of GBM cells in the tumour core and edge differ due to alternative RNA splicing. The acidic pH in the core switches before messenger RNA splicing of the ribosomal gene RPL22L1 towards the RPL22L1b isoform. This allows cells to survive acidosis, increases stemness and correlates with worse patient outcome. Mechanistically, RPL22L1b promotes RNA splicing by interacting with lncMALAT1 in the nucleus and inducing its degradation. Contrarily, in the tumour edge region, RPL22L1a interacts with ribosomes in the cytoplasm and upregulates the translation of multiple messenger RNAs including TP53. We found that the RPL22L1 isoform switch is regulated by SRSF4 and identified a compound that inhibits this process and decreases tumour growth. These findings demonstrate how distinct GBM cell populations arise during tumour growth. Targeting this mechanism may decrease GBM heterogeneity and facilitate therapy.

P17.16.B Additional antitumoral activity of TTFields stimulation and Chloride Intracellular Channel 1 inhibition in human glioblastoma primary culture

Abstract Background Glioblastoma relapse inevitably occurs in the majority of patients after surgery. Chemo- and radiotherapy are in most cases ineffective, while Tumor Treating Fields (TTFields) therapy delays average tumor recurrence by a few months. It is widely accepted that tumor reappearance is a result of the proliferation of residual glioblastoma stem cell (GSC) subpopulation, which escaped surgery. Previous experiments demonstrated that inhibition of the membrane form of Chloride Intracellular Channel 1 (tmCLIC1) is particularly efficient to hit GSCs in glioblastoma human primary cultures. tmCLIC1 is an ideal target as it is express only in the tumor cell membranes. tmCLIC1 blockage slowdown GSC cell cycle at G1/S transition. On the contrary, TTFields act mainly during G2/M progression. The objective of our study is to test the therapeutic potential of combining TTFields exposure with the inhibition of the protein transmembrane Chloride Intracellular Channel 1 (tmCLIC1), which is crucial for GSCs proliferation. Material and Methods Cell preparation: human primary culture from patients’ biopsies. Stimulation device: NOVOCURE Inovitro system®. Stimulation procedure: long-term TTFields stimulation in in-vitro experiments mimics glioblastoma cells’ recrudescence. Analysis: cytofluorimetry for stem cell recognition, cell-cycle progression, and cell count. Results Cell growth is inhibited after the first three days of TTFields exposure. However, despite the chronic presence of TTFields stimulation in prolonged experiments, glioblastoma cells reestablish the ability to proliferate. At the end of twelve days, exponentially growing cells show an increase in stemness markers, a drop in the membrane resistance with a related increase of membrane current, and an average depolarized membrane potential. Twelve days of TTFields treatment resulted in a progressive increase in GSCs in the tumor cell population from 10% to more than 30%. Thus, experiments have been designed to combine TTFields stimulation with tmCLIC1 inhibition. This would include in the treatment of the vast majority of the glioblastoma cell population. After twelve days, the parallel antiproliferative activities show a complete impairment in cell proliferation. Conclusion The additive effect of TTFields stimulation and tmCLIC1 ion channel inhibition could represent an efficacious step forward to optimize an antitumoral therapy against glioblastoma expansion.

CD95 gene deletion may reduce clonogenic growth and invasiveness of human glioblastoma cells in a CD95 ligand-independent manner

CD95 (Fas/APO-1) is a multifunctional cell surface receptor with antithetic roles. First described to mediate cell death, interactions of CD95 with its natural ligand, CD95L, have also been described to induce tumor-promoting signaling leading to proliferation, invasion and stem cell maintenance, mainly in cancer cells that are resistant to CD95-mediated apoptosis. While activation of CD95-mediated apoptosis in cancer cells may not be clinically practicable due to toxicity, inhibition of tumor-promoting CD95 signaling holds therapeutic potential. In the present study, we characterized CD95 and CD95L expression in human glioma-initiating cells (GIC), a glioblastoma cell population with stem cell features, and investigated the consequences of CRISPR-Cas9-mediated CD95 or CD95L gene deletion. In vitro, GIC expressed CD95 but not CD95L and were sensitive to CD95-mediated apoptosis. Upon genetic deletion of CD95, GIC acquired resistance to CD95L-induced apoptosis but exhibited inferior clonogenic growth, sphere-forming capacity, and invasiveness compared with control cells, suggesting the existence of CD95L-independent constitutive CD95 signaling with tumor-promoting properties in GIC. In vivo, GIC expressed CD95 and a non-canonical form of CD95L lacking the CD95-binding region. CD95 genetic deletion did not prolong survival in immunocompromised GIC-bearing mice. Altogether, these data indicate that canonical CD95L may not be expressed in human GIC and suggest the existence of a CD95L-independent CD95-signaling pathway that maintains some malignancy traits of GIC. The lack of altered survival of tumor-bearing mice after genetic deletion of CD95 suggests that CD95 signaling is not essential to maintain the growth of human GIC xenografted into the brains of nude mice. The ligand-independent tumor-promoting role of constitutive CD95 in our GIC models in vitro highlights the complexity and challenges associated with targeting CD95 with therapeutic intent.

Abstract 2315: Synergic antitumoral activity of TTFields stimulation and Chloride Intracellular Channel 1 (CLIC1) inhibition in human glioblastoma primary culture

Abstract Glioblastoma relapse inevitably occurs in the majority of patients after surgery. Chemo- and radio- therapy are in most cases ineffective, while Tumor Treating Fields (TTFields) therapy delays average tumor recurrence by a few months. It is widely accepted that tumor reappearance is a result of the proliferation of residual glioblastoma stem cell (GSC) subpopulation, which escaped surgery. Long-term TTFields stimulation in in vitro experiments mimic glioblastoma cells recrudescence. After the first three days of TTFields exposure, cell growth is totally inhibited. However, in prolonged experiments, despite the chronic presence of TTFields stimulation, glioblastoma cells reestablish the ability to proliferate, even if at low rate. At the end of twelve days, exponentially growing cells show an increase of stemness markers, a drop of the membrane resistance with a related increase of membrane current, and an average depolarized membrane potential. Twelve days of TTFields treatment resulted in progressive increase in GSCs in the tumor cell population from 10% to more than 30%. Our long-term goal is to improve TTFields antitumoral activity by linking brain stimulation with a pharmacological strategy targeting GSC proliferation. The objective of our study is to test the therapeutic potential of combining TTFields exposure with the inhibition of the protein transmembrane Chloride Intracellular Channel 1 (tmCLIC1), which is crucial for GSCs proliferation. tmCLIC1 activity increases under oxidative stress conditions. Preliminary experiments using the INOVITRO-LIVE system showed a cytoplasmic oxidation increase under acute TTFields stimulation. tmCLIC1 impairment by ion channel specific antagonists affected the G1/S checkpoint and hence, tumor cell proliferation. Targeting synergically different glioblastoma cell cycle checkpoints, G2/M with TTFields stimulation and G1/S with tmCLIC1 inhibition, would include the vast majority of glioblastoma cell population. Combination TTFields exposure and tmCLIC1 ion channel inhibition could represent an efficacious step forward in the treatment of glioblastoma. Citation Format: Michele Mazzanti, Stefania Castiglione, Matteo Ranucci, Gaetano Cannavale, Francesca Cianci, Ivan Verduci. Synergic antitumoral activity of TTFields stimulation and Chloride Intracellular Channel 1 (CLIC1) inhibition in human glioblastoma primary culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2315.

A Conceptual Framework for Inducing T Cell-Mediated Immunity Against Glioblastoma.

Glioblastoma is a highly aggressive brain tumor with limited treatment options. Several major challenges have limited the development of novel therapeutics, including the extensive heterogeneity of tumor cell states within each glioblastoma and the ability of glioma cells to diffusely infiltrate into neighboring healthy brain tissue, including the contralateral hemisphere. A T cell-mediated immune response could deal with these challenges based on the ability of polyclonal T cell populations to recognize diverse tumor antigens and perform surveillance throughout tissues. Here we will discuss the major pathways that inhibit T cell-mediated immunity against glioblastoma, with an emphasis on receptor-ligand systems by which glioma cells and recruited myeloid cells inhibit T cell function. A related challenge is that glioblastomas tend to be poorly infiltrated by T cells, which is not only caused by inhibitory molecular pathways but also currently utilized drugs, in particular high-dose corticosteroids that kill activated, proliferating T cells. We will discuss innovative approaches to induce glioblastoma-directed T cell responses, including neoantigen-based vaccines and sophisticated CAR T cell approaches that can target heterogeneous glioblastoma cell populations. Finally, we will propose a conceptual framework for the future development of T cell-based immunotherapies for glioblastoma.

Spatial Heterogeneity of Invading Glioblastoma Cells Regulated by Paracrine Factors.

Glioblastoma (GBM) is the most common and lethal type of malignant primary brain tumor in adults. GBM displays heterogeneous tumor cell population comprising glioma-initiating cells (GICs) with stem cell-like characteristics and differentiated glioma cells. During GBM cell invasion into normal brain tissues, which is the hallmark characteristic of GBM, GICs at the invasion front retain stemness, while cells at the tumor core display cellular differentiation. However, the mechanism of cellular differentiation underlying the formation of spatial cellular heterogeneity in GBM remains unknown. In the present study, we first observed spatially heterogeneous GBM cell populations emerged from an isogenic clonal population of GICs during invasion into a 3D collagen hydrogel in a microfluidic device. Specifically, GICs at the invasion front maintained stemness, while trailing cells displayed astrocytic differentiation. The spatial cellular heterogeneity resulted from the difference in cell density between GICs at the invasion front and trailing cells. Trailing GICs at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they secreted, while cells at the invasion front of low cell density retained stemness due to the lack of paracrine factors. In addition, we demonstrated that interstitial flow suppressed astrocytic differentiation of trailing GICs by the clearance of paracrine factors. Our findings suggest that intercellular crosstalk between tumor cells is an essential factor in developing the spatial cellular heterogeneity of GBM cells with various differentiation statuses. It also provides insights into the development of novel therapeutic strategies targeting GBM cells with stem cell characteristics at the invasion front. Impact Statement We elucidated the mechanism of cellular differentiation underlying the spatial cellular heterogeneity of glioblastoma composed of glioma-initiating cells (GICs) and differentiated glioma cells during invasion in a microfluidic device. Trailing cells at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they produced, while cells at the invasion front of low cell density were shown to retain stemness due to the lack of paracrine factors. Our findings provide valuable knowledge for the development of effective therapeutic strategies targeting GICs at the invasion front.

Matrix Hyaluronic Acid and Hypoxia Influence a CD133+ Subset of Patient-Derived Glioblastoma Cells.

Glioblastoma (GBM) displays diffusive invasion throughout the brain microenvironment, which is partially responsible for its short median survival rate (<15 months). Stem-like subpopulations (GBM stem-like cells, GSCs) are believed to play a central role in therapeutic resistance and poor patient prognosis. Given the extensive tissue remodeling and processes such as vessel co-option and regression that occur in the tumor microenvironment, it is essential to understand the role of metabolic constraint such as hypoxia on GBM cell populations. This work describes the use of a multidimensional gelatin hydrogel to culture patient-derived GBM cells, to evaluate the influence of hypoxia and the inclusion brain-mimetic hyaluronic acid on the relative activity of GSCs versus overall GBM cells. Notably, CD133+ GBM cell fraction is crucial for robust formation of tumor spheroids in multidimensional cultures. In addition, while the relative size of the CD133+ GBM subpopulation increased in response to both hypoxia and matrix-bound hyaluronan, we did not observe cell subtype-specific changes in invasion signaling pathway activation. Taken together, this study highlights the potential of biomimetic culture systems for resolving changes in the population dynamics and behavior of subsets of GBM specimens for the future development of precision medicine applications. Impact Statement This study describes a gelatin hydrogel platform to investigate the role of extracellular hyaluronic acid and hypoxia on the behavior of a CD133+ subset of cells within patient-derived glioblastoma (GBM) specimens. We report that the relative expansion of the CD133+ GBM stem cell-like population is strongly responsive to extracellular cues, highlighting the significance of biomimetic hydrogel models of the tumor microenvironment to investigate invasion and therapeutic response.

Abstract 3064: Investigation of ionizing radiation response in new resistance models of glioblastoma

Abstract Glioblastoma is one of the most aggressive central nervous system tumors, which constitutes 50% of all primary brain tumors. Glioblastomas have a very poor prognosis, approximately 12-15 months even with standard therapy. Current treatment protocols include surgery, chemotherapy, and radiotherapy. High rates of recurrence after treatment, genetic heterogeneity, and developing resistance to chemotherapy and radiotherapy are challenges to overcome in glioblastoma treatment. Therefore, developing models of chemo- or radio-resistance is of priority to understand the molecular mechanisms that operate during the transition of cells to therapy-refractory state, and to develop new therapeutic approaches. In this study, we generated ionizing radiation (IR)-exposed glioblastoma cell populations to investigate molecular alterations associated with acquired radiotherapy resistance. To generate a clinically relevant radioresistance model, we have exposed glioblastoma cells to a total dose of 60 Gy IR over a 6-week period and cultured age-matched parental cells. We successfully generated sustainable radioresistant cells, as demonstrated by cell viability and colony formation assays. RNA sequencing, performed on these matched cell lines revealed major alterations in the expression of genes of the cell cycle, DNA damage recognition, and repair machinery in the radioresistant cells. Specifically, elements of the DNA double strand break (DSB) recognition and repair, such as ATM, ATR, and CHK1 were markedly upregulated in the radioresistant cell populations. In line with these findings, DNA repair capacity of the radioresistant cell populations was increased, as revealed by the faster clearance of H2AX and 53BP1 in the radioresistant populations. Complementing this approach, pharmacological inhibition of major DNA damage response elements, such as ATM, ATR, as well as CHK1 markedly sensitized the radioresistant cells. Our results prepare a groundwork for studying the molecular mechanisms of radiotherapy resistance and developing novel combination therapy strategies for glioblastomas. Citation Format: Nareg Pinarbasi, Tugba Bagci-Onder, Ilknur Sur Erdem, Ugur Selek. Investigation of ionizing radiation response in new resistance models of glioblastoma [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 3064.

Meclofenamate causes loss of cellular tethering and decoupling of functional networks in glioblastoma.

Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy. In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis. TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity. The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach.