Astrocytes in glioblastoma tumor microenvironment.

Glioblastoma (GBM) is often fatal within a year despite treatment, with immune checkpoint blockade therapies benefiting only about 10% of patients. Non-responsive GBMs exhibit an immunosuppressive profile and higher levels of tumor-associated myeloid cells (TAMs). Addressing this immunosuppressive microenvironment is a major challenge in GBM immunotherapy. Our recent studies have identified a positive correlation between Proline-Rich Tyrosine Kinase 2 (Pyk2) activation in GBM tumor cells and the cytokine expression profile of TAMs. This identified Pyk2 as a potential prognostic marker for the immune state in GBM tumor microenvironment. We hypothesize that Pyk2 and MEK/Erk signaling in GBM cells regulate the release of chemokines and cytokines through extracellular vesicles (EVs) by modulating the actin cytoskeleton, thereby impacting TAM activation. The study aimed to investigate the role of Pyk2/MEK/Erk signaling in EV release in GBM cells, potentially revealing new therapeutic targets. Two humans primary GBM cell lines, with and without Pyk2 CRISPR/Cas9 knockout (Pyk2KO), were used. Flow cytometric analysis of EVs, enriched from cell-conditioned medium, identified a shift towards larger in diameter EVs populations in Pyk2 KO cells, compared to wild-type (WT) cells. Analysis using Integrin as a plasma membrane marker showed that 84.70% of Integrin+ and 15.30% of Integrin- EVs were derived from WT cells, whereas Pyk2KO cells produced 89.07% Integrin+ and 10.93% Integrin- EVs. Treatment of WT cells with the Erk/MEK inhibitor Avutometinib (1µM) altered the EV population’s ratio to 79.11% Integrin+ and 20.89% Integrin-. Similarly, Avutometinib treatment of Pyk2KO cells resulted in 78.83% Integrin+ and 21.17% Integrin- EVs, similar to the ratio observed in Avutometinib-treated WT cells. Western blot and PCR analysis of EVs content revealed a significant reduction in CCL2, CCL5, tumor necrosis factor (TNF), and vascular endothelial growth factors (VEGF) in EVs from Pyk2KO GBM cells, compared to WT. In vivo studies using GL261/C57Bl/6 mouse glioma implantation model and flow cytometric analysis of TAMs from tumors generated with WT and Pyk2KO GL261 cells demonstrated increased infiltration of TNF+/IFNg+ CD8+ lymphocytes and Ly6C+/CD206- myeloid cells, alongside a reduction in CD45+/CD11b+/CD33+/MHCII- myeloid-derived suppressor cells. PCR analysis of TAM isolated from Pyk2KO tumors revealed lower gene expression of TNF, CCL5, CCL2, VEGFa, and epidermal growth factor (EGF) compared to WT tumors. The study highlights that Pyk2/MEK/Erk signaling regulates the immune microenvironment in GBM by influencing EV release, which impacts TAM cell activation. Pyk2 primarily regulates the release of Integrin- EVs, while MEK/Erk predominantly regulates EVs shed from the plasma membrane. The findings underscore the interplay between Pyk2 and MEK/Erk signaling in regulating EV biogenesis and composition. Citation Format: Neisha M. Ramirez. Pyk2 and MEK/ERK signaling regulate the extracellular vesicle release and tumor-associated myeloid Cells in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B046.

Mitochondria transfer from tumor-activated stromal cells (TASC) to primary Glioblastoma cells

Extracellular vesicles in the glioblastoma microenvironment: A diagnostic and therapeutic perspective.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, and causes profound immune suppression both locally and systemically, hindering immunotherapy efficacy. Myeloid-derived suppressor cells (MDSCs) are among the most abundant immune cells in the GBM tumor microenvironment (TME), which exert immune suppression primarily by dampening functional cytotoxic T cells. Extracellular vesicles (EVs) are key mediators of GBM immunosuppression and have been shown to polarize myeloid cells toward immunosuppressive phenotypes, including MDSCs. Inhibition of EV-myeloid cell interactions thus represents a novel strategy to mitigate GBM-mediated immunosuppression. However, the mechanisms of GBM-EVs uptake by myeloid cells are poorly understood. Here, we explored two pharmacological EV uptake inhibitors: Methyl-β-cyclodextrin (MβCD), which disrupts caveolin-mediated endocytosis by depleting membrane cholesterol, and cytochalasin D (CytoD), which impedes phagocytosis via actin depolymerization. Our results showed immunosuppressive myeloid cells (CD11b+/HLA-DRlow/-) and their subset, monocytic MDSCs (mMDSCs) (CD11b+/HLA-DRlow/-/CD14high/CD15-), were induced upon GBM-EV treatment of healthy donor CD11b+ cells. Both inhibitors reduced these populations, with CytoD showing a more pronounced effect (CytoD: 15.01%, MBCD: 16.24% vs. untreated 23.77%) along with partial restoration of T cell proliferation. To validate these findings in vivo, we retro-orbitally injected EVs isolated from the syngeneic murine glioma cell lines GL261 and CT2A into immunocompetent C57BL/6 mice, followed by treatment with either CytoD, MBCD, or vehicle. We observed thymic involution following EV treatment, accompanied by an increase in systemic immunosuppressive myeloid cells in PBMCs and spleen. Both pharmacological inhibitors attenuated these effects, reducing systemic MDSC populations and their suppressive profiles. Our findings highlight that GBM-EVs as key drivers of tumor-mediated immune suppression and demonstrate that targeting EV uptake in myeloid cells is a viable strategy to counteract GBM-mediated immunosuppression.

BackgroundAnoikis is a specialized form of programmed cell death induced by the loss of cell adhesion to the extracellular matrix (ECM). Acquisition of anoikis resistance is a significant marker for cancer cell invasion, metastasis, therapy resistance, and recurrence. Although current research has identified multiple factors that regulate anoikis resistance, the pathological mechanisms of anoikis-mediated tumor microenvironment (TME) in glioblastoma (GBM) remain largely unexplored.MethodsUtilizing single-cell RNA sequencing (scRNA-seq) data and employing non-negative matrix factorization (NMF), we identified and characterized TME cell clusters with distinct anoikis-associated gene signatures. Prognostic and therapeutic response analyses were conducted using TCGA and CGGA datasets to assess the clinical significance of different TME cell clusters. The spatial relationship between BRMS1 + microglia and tumor cells was inferred from spatial transcriptome RNA sequencing (stRNA-seq) data. To simulate the tumor immune microenvironment, co-culture experiments were performed with microglia (HMC3) and GBM cells (U118/U251), and microglia were transfected with a BRMS1 overexpression lentivirus. Western blot or ELISA were used to detect BRMS1, M2 macrophage-specific markers, PI3K/AKT signaling proteins, and apoptosis-related proteins. The proliferation and apoptosis capabilities of tumor cells were evaluated using CCK-8, colony formation, and apoptosis assays, while the invasive and migratory abilities of tumor cells were assessed using Transwell assays.ResultsNMF-based analysis successfully identified CD8 + T cell and microglia cell clusters with distinct gene signature characteristics. Trajectory analysis, cell communication, and gene regulatory network analyses collectively indicated that anoikis-mediated TME cell clusters can influence tumor cell development through various mechanisms. Notably, BRMS1 + AP-Mic exhibited an M2 macrophage phenotype and had significant cell communication with malignant cells. Moreover, high expression of BRMS1 + AP-Mic in TCGA and CGGA datasets was associated with poorer survival outcomes, indicating its detrimental impact on immunotherapy. Upregulation of BRMS1 in microglia may lead to M2 macrophage polarization, activate the PI3K/AKT signaling pathway through SPP1/CD44-mediated cell interactions, inhibit tumor cell apoptosis, and promote tumor proliferation and invasion.ConclusionThis pioneering study used NMF-based analysis to reveal the important predictive value of anoikis-regulated TME in GBM for prognosis and immunotherapeutic response. BRMS1 + microglial cells provide a new perspective for a deeper understanding of the immunosuppressive microenvironment of GBM and could serve as a potential therapeutic target in the future.

Nanoparticle-based drug delivery systems to modulate tumor immune response for glioblastoma treatment.

BACKGROUND Glioblastoma (GBM), the deadliest brain tumor in adults, is traditionally diagnosed and characterized using neuroimaging and brain biopsies. These methods, however, suffer from limited specificity and sensitivity. The surgical risks associated with tumor sampling further complicate the monitoring of GBM’s molecular progression. Consequently, the identification of circulating GBM biomarkers is crucial for non-invasive tracking of the disease from diagnosis to relapse. Extracellular vesicles (EVs) secreted by GBM cells, which cross the blood-brain barrier and carry tumor-derived molecules, present a promising avenue for the continuous evaluation of GBM presence and status. MATERIAL AND METHODS We isolated extracellular vesicles (EVs) using Size Exclusion Chromatography (SEC) from 2 mL of plasma and characterized them via immunoblotting, flow cytometry, and transmission electron microscopy. The concentration and size of EVs were measured using Tunable Resistive Pulse Sensing (TRPS). We compared EV levels in 50 glioblastoma (GBM) patients pre-surgery with those in non-GBM controls, which included 100 healthy individuals and 50 patients with brain malignancies that present similarly to GBM in neuroimaging studies. Longitudinal assessments of plasma EVs were performed in 44 GBM patients by comparing pre-operative and post-operative samples taken 72 hours after surgery. Additionally, we analyzed surface markers on GBM and non-GBM plasma EVs using MACSPlex and conducted proteomic analyses via mass spectrometry in 27 GBM patients and 38 healthy individuals. RESULTS We found higher concentrations and mean sizes of plasma EVs in GBM patients compared to controls, with a noticeable decrease post-operatively. Multiplex flow cytometry indicated universal expression of CD9, CD63, and CD81 across all samples, with significant enrichment of CD63 and CD81 in GBM plasma EVs. The expression profiles of the other 34 markers were similar across all samples, suggesting dilution of tumor-derived EVs among non-tumor EVs. However, T-cell markers CD8 and HLA-DRDPDQ were more prevalent on GBM-derived EVs. Proteomic analysis identified over 2,000 proteins in all samples, with 117 being upregulated in GBM samples, predominantly within the complement cascade pathways. CONCLUSION Our findings suggest that monitoring circulating EV levels provides a reliable, non-invasive method for the differential diagnosis and follow-up of GBM patients. The combined analysis of EV concentration, size, and proteome offers potential as promising biomarkers for longitudinal monitoring of GBM, supporting the implementation of liquid biopsy in GBM care.

BackgroundHighly multiplexed spatial biology technologies have become key to unveiling unique cellular phenotypes and cellular neighborhoods in healthy and diseased tissues. Glioblastoma (GBM), the most prevalent and aggressive brain tumor...

Glioblastoma (GBM) is the most common malignant brain tumor in adults, and immunotherapies and genetic therapies for GBM have evolved dramatically over the past decade, but GBM therapy is still facing a dilemma due to the high recurrence rate. The inflammatory microenvironment is a general signature of tumors that accelerates epigenetic changes in GBM and helps tumors avoid immunological surveillance. GBM tumor cells and glioma-associated microglia/macrophages are the primary contributors to the inflammatory condition, meanwhile the modification of epigenetic events including DNA methylation, non-coding RNAs, and histone methylation and deacetylases involved in this pathological process of GBM, finally result in exacerbating the proliferation, invasion, and migration of GBM. On the other hand, histone deacetylase inhibitors, DNA methyltransferases inhibitors, and RNA interference could reverse the inflammatory landscapes and inhibit GBM growth and invasion. Here, we systematically review the inflammatory-associated epigenetic changes and regulations in the microenvironment of GBM, aiming to provide a comprehensive epigenetic profile underlying the recognition of inflammation in GBM.

INTRODUCTION: Glioblastoma (GBM), the deadliest brain tumor in adults, is traditionally diagnosed and characterized using neuroimaging and brain biopsies. These methods, however, suffer from limited specificity and sensitivity. The surgical risks associated with tumor sampling further complicate the monitoring of GBM's molecular progression. Extracellular vesicles (EVs) secreted by GBM cells, which cross the blood-brain barrier and carry tumor-derived molecules, present a promising avenue for the continuous evaluation of GBM presence and status. METHODS: We isolated EVs using SEC from plasma and characterized them via immunoblotting, flowcytometry, and TEM. The concentration and size of EVs were measured using TRPS. We compared EV levels in 50 GBM patients pre-surgery with those in non-GBM controls, which included 100 healthy individuals and 50 patients with pther brain malignancies. Longitudinal assessments of plasma EVs were performed in 44 GBM patients by comparing pre-operative and post-operative samples taken 72 hours after surgery. We analyzed surface markers using MACSPlex and proteomic analyses RESULTS: We found higher concentrations and sizes of plasma EVs in GBM patients compared to controls, with a noticeable decrease post-operatively. Multiplex flow cytometry indicated universal expression of CD9, CD63, and CD81 across all samples, with significant enrichment of CD63 and CD81 in GBM. The expression profiles of the other 34 markers were similar across all samples, suggesting dilution of tumor-derived EVs among non-tumor EVs. However, T-cell markers CD8 and HLA-DRDPDQ were more prevalent on GBM-derived EVs. Proteomic analysis identified over 2,000 proteins in all samples, with 117 being upregulated in GBM samples. CONCLUSIONS: Our findings suggest that monitoring circulating EV levels provides a reliable, non-invasive method for the differential diagnosis and follow-up of GBM patients. The combined analysis of EV concentration, size, and proteome offers potential as promising biomarkers for longitudinal monitoring of GBM, supporting the implementation of liquid biopsy in GBM care.

Glioblastoma (GB) is the most common and aggressive primary brain tumor in adults and currently incurable. Despite multimodal treatment regimens, median survival in unselected patient cohorts is <1 year, and recurrence remains almost inevitable. Escape from immune surveillance is thought to contribute to the development and progression of GB. While GB tumors are frequently infiltrated by natural killer (NK) cells, these are actively suppressed by the GB cells and the GB tumor microenvironment. Nevertheless, ex vivo activation with cytokines can restore cytolytic activity of NK cells against GB, indicating that NK cells have potential for adoptive immunotherapy of GB if potent cytotoxicity can be maintained in vivo. NK cells contribute to cancer immune surveillance not only by their direct natural cytotoxicity which is triggered rapidly upon stimulation through germline-encoded cell surface receptors, but also by modulating T-cell mediated antitumor immune responses through maintaining the quality of dendritic cells and enhancing the presentation of tumor antigens. Furthermore, similar to T cells, specific recognition and elimination of cancer cells by NK cells can be markedly enhanced through expression of chimeric antigen receptors (CARs), which provides an opportunity to generate NK-cell therapeutics of defined specificity for cancer immunotherapy. Here, we discuss effects of the GB tumor microenvironment on NK-cell functionality, summarize early treatment attempts with ex vivo activated NK cells, and describe relevant CAR target antigens validated with CAR-T cells. We then outline preclinical approaches that employ CAR-NK cells for GB immunotherapy, and give an overview on the ongoing clinical development of ErbB2 (HER2)-specific CAR-NK cells currently applied in a phase I clinical trial in glioblastoma patients.

•Gliomas are primary brain tumors that develop from glial cells within the central nervous system (CNS) and are among the deadliest human cancers. Glioblastoma (GBM) is the most aggressive glioma. Innate immune cells such as microglia and macrophages account for &amp;gt;50% of the cellular population within the GBM microenvironment. Innate immune cells express pattern recognition receptors (PRRs). NLRX1 is a PRR that regulates diverse signaling pathways and cellular processes including antiviral signaling, ROS production, apoptosis, autophagy, metabolic homeostasis, etc. Additionally, the tumor-suppressive and tumor-promoting functions of NLRX1 have been observed in many cancer types. For example, the tumor-suppressive role of NLRX1 has been observed in colitis-associated carcinogenesis, pancreatic cancer, and primary breast cancer. In contrast, the tumor-promoting role of NLRX1 was found in basal-like and metastatic breast carcinoma. Hence, the effect of NLRX1 on cancer may be context-dependent on cancer type or cell type aided by differences in the microenvironment. Further, how NLRX1 regulates GBM pathophysiology remains largely unexplored. This study aims to determine the expression pattern of NLRX1 in GBM cells and GBM-associated innate immune cells and investigate how NLRX1 expression regulates various cellular processes in GBM cell lines to regulate GBM pathophysiology. We report that NLRX1 is differentially expressed in GBM cell lines, GBM-associated innate immune cells, and glioma patient tissues. si-RNA-mediated silencing of Nlrx1 decreases the ability of the GBM cell line, LN-229 to proliferate and migrate. Further, Nlrx1-/- GBM cells show increased tunneling nanotube (TNT) formation capabilities. TNTs help in metabolic homeostasis maintenance by enabling the exchange of subcellular organelles such as mitochondria and endoplasmic reticulum. Nlrx1-/- GBM cells exhibit attenuated ability to form 3D spheroids. This research will aid the understanding of GBM pathophysiology, leading to novel diagnostic/prognostic markers and the discovery of signaling pathways that, in turn, may help create better GBM therapy approaches and improve overall survival.

Tuning a bioengineered hydrogel for studying astrocyte reactivity in glioblastoma

Introduction: We have completed two CAR T cell clinical trials for glioblastoma (GBM) and have identified several key challenges to therapeutic efficacy, including the inherently heterogenous genomic landscape and the immunosuppressive tumor microenvironment (TME) found in GBM. Our previous study showed that EGFR variant III (EGFRvIII)-targeting monovalent CAR T cells reduced target-positive tumor cell populations, but tumor recurrence resulted from target-negative tumor cells, highlighting the limitation of single-target approaches in heterogenous tumors. With regards to the highly immunosuppressive TME in GBM, we found that transforming growth factor-β (TGFβ) was present in the GBM TME as a major driver of suppression of the anti-GBM response in clinical samples. TGFb is consistently highly expressed in both GBM tumor cell lines and patient tumor tissues. Methods: We used two parallel scFv constructs, independently targeting both IL13Rα2 and EGFRvIII, in combination with a truncated dominant negative (dn) TGFβ receptor II. This trivalent construct was designed to explore possible additive effects in both in vitro and in vivo GBM model systems to limit tumor escape and overcome the immunosuppressive GBM TME. The CART-EGFR-IL13Rα2-dnTGFb construct broadened the targeted tumor cell repertoire, blocked TGFβ signaling, and served as a sink for free TGFβ in the GBM TME to overcome the suppressive function of TGFβ. Results: The tri-modular CAR T construct had an enhanced proliferative response when compared with the CART-EGFR-IL13Rα2 construct, in vitro. In co-culture assays, this construct led to reduced PD-1 expression and increased central memory phenotype, when compared to the bicistronic CAR T construct, which suggested a lower fraction of exhausted T cells. Tri-modular CAR T cells blocked the suppressive pSmad2/3 signaling pathway, leading to the increased tumor killing activity in co-culture experiments with both adherent and suspension GBM cell lines. In an immunodeficient mouse model, tri-modular CAR T cells eradicated tumor cells efficiently and mice had a longer median survival when compared those treated with the bicistronic CART-EGFR-IL13Rα2 cells, lacking the dnTGFb receptor II. Conclusion: Overcoming the adaptive changes in the local TME and addressing antigen heterogeneity will be required to improve the clinical efficacy of CAR T-directed strategies. Our combination work showed that bicistronic CART constructs cooperate with truncated TGFβ receptor II efficiently. In summary, the dominant-negative TGFβ RII CART-EGFR-IL13Rα2 structure is a promising strategy to address the clinical challenges of antigenic heterogeneity and the immunosuppressive TME in GBM we have observed in our two GBM CART cell trials at UPenn. Citation Format: Nannan Li, Jesse Rodriguez, Zev Binder, Donald O’Rourke. Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB097.

Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most common primary brain tumor in adults. GBM has the poorest prognosis and no efficient cure to date due to diffusive growth into the surrounding brain, preventing complete surgical resection and leading to inevitable tumor relapse. Tumor microenvironment (TME) of GBM contains brain-residing microglia and bone-marrow derived macrophages (collectively known as glioma-associated microglia/macrophages, GAMs) that constitute up to 30% of the tumor mass and promote tumor invasion. Hypoxia (a shortage of oxygen) is a key factor in tumor progression of GBM as it can globally and rapidly alter the gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can enhance the pro-tumorigenic function of GAMs, e.g. by inducing expression of cytokines and cell surface receptors both in GAMs and glioma cells, but little is known about chromatin alterations of GBM under hypoxia. Since regulation of expression of such molecules could depend on the epigenetic alterations, we hypothesize that hypoxia may potently alter the chromatin accessibility and functions of GAMs and glioma cells.We determine the genome-wide changes in chromatin accessibility in GAMs and glioma cells in response to hypoxic stress using single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing), which allows simultaneous genome-wide assessment of chromatin accessibility and expression of intracellular protein markers in single cells, allowing faithful selection of hypoxic and non-hypoxic cells. Secondly, we are employing an oxygen-dependent co-culture model in vitro to study the mechanisms of chromatin alterations in GAMs and glioma cells under controlled hypoxic conditions and test how these changes depend on the glioma - GAMs cross-communication. In summary, we characterize the interactions between innate immune cells and glioma cells by looking at their chromatin alterations under hypoxia.Supported by the National Science Center grant (Poland) 2019/33/B/NZ1/01556 (KBL).