Glioma-associated Macrophages Research Articles

Blood-Brain Barrier Opening with Ultrasound Augments Doxorubicin and Anti-PD-1 Therapy Efficacy, Intracranial Immune Activation, and Prolongs Survival in Preclinical Glioma Models

INTRODUCTION: The limited drug penetration across the blood-brain barrier (BBB) represents a challenge for the treatment of glioblastoma patients. A novel drug delivery technology based on low-intensity pulsed ultrasound combined with intravenous microbubbles (LIPU/MB) overcomes this challenge by opening the BBB. In this study, we hypothesize that LIPU/MB enhances the therapeutic effect of both liposomal doxorubicin and anti-PD-1 therapy in gliomas by eliciting local immune responses. METHODS: We utilized LIPU/MB to deliver doxorubicin and anti-PD-1 blocking antibodies to murine models and evaluated drug concentrations in the brain. Flow cytometry evaluated IFN-g production by glioma-associated microglia and macrophages in murine gliomas treated with doxorubicin and anti-PD-1 therapy delivered by LIPU/MB. Furthermore, we assessed the efficacy of this drug combination delivered by LIPU/MB in glioma-bearing mice. RESULTS: LIPU/MB induced a 4-fold increase in doxorubicin concentrations in sonicated brains compared to those not sonicated (p = 0.003). In addition, doxorubicin delivered with LIPU/MB elicited an IFN-g phenotype (p < 0.0001) as well as MHC I and PD-L1 expression in glioma-associated microglia and macrophages (p < 0.05). Increased brain concentrations of doxorubicin led to long-term survivors in the majority of glioma-bearing mice. On the other hand, LIPU/MB-mediated BBB opening increased brain concentrations of anti-PD-1 therapy in mice (6.3-fold increase, p < 0.01). The combination of doxorubicin and anti-PD-1 therapy delivered with LIPU/MB resulted in long-term survival (p < 0.0001). This effect was abrogated in CD8-/- mice suggesting that T cells are important for the efficacy of doxorubicin and anti-PD-1 therapy delivered with LIPU/MB. CONCLUSIONS: This preclinical study demonstrates the immense potential of LIPU/MB technology in enhancing intracranial immune responses and drug delivery for glioblastoma treatment using doxorubicin and anti-PD-1 therapy, paving the way for more effective treatment strategies for this brain cancer.

390 Modelling Glioma Associated Macrophages Using Human Embryonic Stem Cell Derived Microglia

INTRODUCTION: Glioma associated macrophages/microglia (GAMs) are the most abundant immune cell type in glioblastoma and play a key role in immunotherapeutic resistance. The tumor microenvironment (TME) is thought to modulate GAM phenotype and function leading to a more pro-tumorigenic immune milieu however the exact nature of this crosstalk is unclear. Existing systems to study human GAMs are suboptimal as primary human GAMs are difficult to culture. There is therefore a need to develop human model systems of sufficient fidelity to probe GAM function. Here we report on the development of a human embryonic stem cell (hES)-based platform to derive microglia without recourse to immortalizing oncogenic transduction. This technology can assess the impact of tumor derived factors on the microglial phenotype and represents a novel, scalable model system to investigate GAM function. METHODS: hES-microglia were generated using a stepwise cytokine-based differentiation protocol. Upon exposure to conditioned media obtained from cultures of a panel of patient-derived glioblastoma cell lines (n=6 biological replicates), hES-GAMs were analyzed using flow cytometry, single cell RNA sequencing (scRNA). Functional assays included phagocytosis of pH rhodo particles, wound scratch migration assays and intracellular proteome analysis. RESULTS: Exposure to tumor media leads to transcriptional changes similar to those seen in primary GAMs with increased chemotactic genes such as CCL2(MCP1), CCL3 and CCL4, with corresponding changes in cytokine profile at the protein level. Other upregulated cytokines of note include VEGF, OSM, CCL4 and IL10RB. Functional impact of co-culture with tumor media included a dramatic increase in GAM motility (>30 fold) and a statistically significant decrease in phagocytic ability (29.1% p < 0.001). CONCLUSIONS: The hES-derived GAM model is a promising novel platform to dissect the impact of the TME on tumor-derived GAMs.

Abstract 2650: IGFBP2 assists EGFRvIII-positive glioblastoma to evade immune elimination

Abstract In recent years, immunotherapy has achieved significant success in the treatment of a variety of cancer types, including melanoma, non-small cell lung cancer, and colorectal cancer. However, in stark contrast, patients with glioblastoma (GBM) show remarkably poor responses to immunotherapy, failing to derive any therapeutic benefit. Thus, an understanding of the immunosuppressive microenvironment of GBM, which is considered an immunotherapy-cold tumor, is of paramount importance. One common and distinctive genetic mutation found in gliomas is that of EGFRvIII, which is linked with enhanced cell proliferation, invasion, migration, and angiogenesis in GBM. Intriguingly, we found that this mutation induced a significantly more severe immunosuppressive microenvironment compared with wild-type EGFR. To unravel the mechanisms underlying EGFRvIII mutation-induced immunosuppression in GBM, we posited that the mutant variant secretes an array of immunosuppression-related proteins into the glioma microenvironment. To investigate this, we conducted proteomic analysis of conditioned media from EGFRvIII mutant cell lines and discovered significant changes in the expression of insulin-like growth factor-binding protein-2 (IGFBP2), a well-documented oncogenic protein. Although IGFBP2 is known to be upregulated in various tumor types and is associated with malignant behaviors such as proliferation and invasion in gliomas, its role in glioma immunology remains largely unexplored. In line with our earlier bioinformatic analyses, our study revealed significant enrichment of IGFBP2 in pathways related to immunosuppression in GBM. Thus, in the present investigation, we analyzed the mechanisms by which IGFBP2 induces an immunosuppressive microenvironment. Using co-culture models, we found that IGFBP2-overexpressing glioma cells as well as glioma cells treated with exogenous IGFBP2 recruited immune cells through binding to integrin αvβ5. Furthermore, we demonstrated that IGFBP2 promoted the secretion of the classical macrophage polarization factor IL-10 by phosphorylation of STAT3. These findings indicate the significant influence of IGFBP2 in steering glioma-associated macrophages toward a pro-tumor M2-like phenotype. We also observed that IGFBP2 upregulated the expression of PD-L1 on tumor cell surfaces. These results were verified in animal experiments. Intriguingly, our preliminary data indicated that exogenous IGFBP2 translocated to the cell nucleus within hours, and immunoprecipitation assays confirmed its interaction with STAT3 in vitro. These findings collectively underscore the pivotal role of IGFBP2 in the creation of the immunosuppressive microenvironment induced by EGFRvIII mutation. Consequently, targeting IGFBP2 holds great promise for advancing immunotherapeutic approaches in EGFRvIII-mutant glioblastomas, offering an exciting avenue for potential treatments of GBM. Citation Format: Jikang Fan, Xisen Wang, Tao Li, Xuejun Yang, Wei Zhang. IGFBP2 assists EGFRvIII-positive glioblastoma to evade immune elimination [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 2650.

BIOM-09. OPPOSING EFFECTS OF LINKED CX3CR1 GERMLINE VARIANTS ON SURVIVAL AND IMMUNE ACTIVATION IN GLIOMA

Abstract Linkage-disequilibrium connects CX3CR1 V249I and T280M single-nucleotide polymorphisms; V249I occurs as a single-variant (SV) and T280M exclusively as a double-variant (DV) concurrent with V249I. In low-grade gliomas (LGG), we found improved median overall-survival (mOS) in SV (n = 14, mOS 21.03yrs), compared to WT (n = 45, mOS 9.41yrs) and DV (n = 31, mOS 10.49yrs; p = 0.037 Log-rank). In TCGA glioblastoma (GBM), SV (n = 69, mOS 15.65mo) improved survival over DV (n = 106, mOS 11.74mo; p = 0.044 Log-rank). In a separate GBM cohort, bulk RNA-sequencing revealed MMP9 over-expression in DV (n = 15) compared to SV (n = 10; log2FC 2.87, p-adj 0.079). On a single-gene basis, MMP9 expression was decreased in SV compared to DV and WT (n = 33; p = 0.044, Kruskal-Wallis). MMP9 promotes tumor angiogenesis and invasion. SV showed SPP1 over-expression compared to DV (log2FC -1.75, p-adj 0.026), with decreased SPP1 expression in both WT and DV compared to SV (p = 0.004, Kruskal-Wallis). SPP1 is a pro-inflammatory cytokine enriched in glioma-associated macrophages. Gene Ontology revealed enrichment of biological processes: neutrophil migration, granulocyte chemotaxis, and neutrophil chemotaxis in SV (p< 0.05). We single-cell RNA-sequenced IDH mutant gliomas (51777 cells). We subset and re-clustered myeloid-derived cells (microglia/macrophages, 8944 cells), identifying thirteen clusters. Myeloid-derived cells clustered by CX3CR1 status, with 50.8% of SV cells in cluster five and 85.3% of DV cells in clusters six or seven. Cluster five over-expressed IL1B (average-log2FC 3.23), a tumor-suppressive M1 macrophage marker. Clusters six and seven were characterized by IFIT genes – immunomodulatory in myeloid-derived cells (IFIT3 average-log2FC 2.65 and 2.06). Assessing CX3CR1 status in normal population – 74.2% are WT, 10.0% SV, and 15.7% DV. In glioma patients, we identify 59.9% WT, 14.9% SV, and 25.2% DV – an increase of 49% in SV and 61% in DV (p = 1.95e-6, Chi-square). Our findings demonstrate opposing effects of linked CX3CR1 germline variants on glioma survival and indicate enhanced immune activation in SV.

TMIC-65. PP2A/STRN4 NEGATIVELY REGULATES STING-TYPE I IFN SIGNALING IN GLIOMA ASSOCIATED MACROPHAGES THROUGH YAP/TAZ

Abstract Glioma associated Macrophages/microglia (GAMs) are the predominant immune cells in the tumor microenvironment and drive immunosuppression. Strategies depleting GAMs have not demonstrated efficacy in clinical trials for GBM. It is critical to identify the underlying mechanisms how GAMs mediate immunosuppression. STING agonist, which activates the Type I IFN signaling in myeloid cells, is a promising strategy but also have shown limited efficacy in clinical trials. Here, we report that protein phosphatase 2A (PP2A), with its specific B regulatory subunit Striatin 4 (STRN4), negatively regulated STING-Type I IFN in GAMs. Mice with macrophage PP2A deficiency exhibited improved glioma survival with radiation, which is known to activate STING. The glioma microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that Hippo kinase MST1/2 was required for STING activation in macrophages. STING agonists induced dissociation of PP2A from MST1/2 in normal macrophages, but not in glioma conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. We are also the first to demonstrate that in human GBM patients, YAP/TAZ signature was highly expressed in GAMs but not in non tumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized glioma -conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4- YAP/TAZ has, in our opinion, been an unappreciated mechanism that mediates immunosuppression in glioma-associated macrophages, and targeting the PP2A/STRN4-YAP/TAZ axis can sensitize glioma to immunotherapy.

Clinical characterization of EFHD2 (swiprosin-1) in Glioma-associated macrophages and its role in regulation of immunosuppression

Glioblastoma has been extensively studied due to its high mortality and short survival. The evolution mechanism of tumor-associated macrophages (TAMs) to Glioma-associated microglia and macrophages (GAMs) in the tumor microenvironment (TME) remains to be elucidated. The tumor cell-to-cell interaction patterns have not been well defined yet. The EF-Hand Domain Family Member D2 (EFHD2) has been reported to be differentially expressed as an immunomodulatory molecule in a variety of cancers. But large-scale clinical data from multiple ethnic communities have not been used to investigate the role of EFHD2 in glioma. RNA-seq data from 313 or 657 glioma patients from the Chinese Glioma Genome Atlas (CGGA) database and 603 glioma patients from the Cancer Genome Atlas (TCGA) database were analyzed retrospectively. Cell localization was performed using single-cell sequencing data from the CGGA database and the GSE131928 dataset. Mouse glioma cell lines and primary macrophages isolated from Efhd2 knockout mice were co-cultured to validate the immunomodulatory effects of EFHD2 on macrophages and the remodeling of TME of glioblastoma. EFHD2 is enriched in high-grade gliomas, isocitrate dehydrogenase wild-type, and 1p/19q non-co-deficient gliomas. It is a potential biomarker of glioma-proneuronal subtypes and an independent prognostic factor for overall survival in patients with malignant glioblastoma. EFHD2 regulates the monocyte-macrophage system function and positively correlates with immunosuppressive checkpoints. Further experimental data demonstrates that Efhd2 influences the polarization state of GAMs and inhibits the secretion of TGF-β1. In vitro experiments have revealed that macrophages lacking Efhd2 suppress the vitality of two glioma cell lines and decelerate the growth of glioma xenografts. In conclusion, EFHD2 promises to be a key target for TME-related immunotherapy.

LMAP-07 POTENTIAL MECHANISM OF IMMUNE ESCAPE RELATED TO TUMOR-ASSOCIATED MACROPHAGE IN GLIOBLASTOMA AFTER COMBINED THERAPY WITH RADIOTHERAPY AND IMMUNE CHECKPOINT BLOCKER

Abstract Immune checkpoint blockers (ICBs) have revolutionized the treatment of cancer. Radiotherapy (RT) has been demonstrated to increase the survival benefit of ICBs in some cancers through its local and systemic immunomodulation, although its efficacy in glioblastoma (GBM) is very limited. With a thorough investigation of the immune responses to single and dual treatment utilizing single-cell RNA-seq analyses on the synergy of RT and anti-PD-L1 antibody (aPD-L1), we studied the potential mechanism of immune escape in GBM even when RT and ICB are combined. Our findings demonstrated a statistically significant longer survival of mice treated with RT and aPD-L1 combined than either single modality of treatment in an orthotopic syngeneic GBM model achieving a 70% cure rate. In the tumor tissues collected from the mouse brain that failed dual treatment, i.e., immune escaped, immune cell profiling still demonstrated much lower T regulatory cells and higher CD8+ cytotoxic T cells infiltrated to tumor, and elevated CD8+/CD4+ and CD8+/Treg ratios in the dual therapy group compared to single therapies, as expected with synergistic effects of dual treatment. RT with aPD-L1 markedly reduced CD8+ and CD4+ T exhaustion cells and elevated T-memory cells in comparison to single modality groups. Nevertheless, compared to the RT alone or no treatment control group, dual therapy failed to decrease the proportion of Arginase1-positive glioma-associated macrophages (GAM) in macrophage/monocytes in tumor microenvironment, which were considerably increased if treated with aPD-L1 only. Moreover, the combination of RT with aPD-L1 did not alter immunosuppressive gene expression in the macrophage/monocyte groups. In conclusion, our research demonstrated that RT and aPD-L1 together may elicit a potent and durable antitumor immune response in the GBM mouse model through regulating lymphocytes but not macrophages. Our findings point to the urgent need for research into GAM-modulating drug and their potential to prevent immunological escape.

MiR-146a-5p deficiency in extracellular vesicles of glioma-associated macrophages promotes epithelial-mesenchymal transition through the NF-κB signaling pathway

Glioma-associated macrophages (GAMs) are pivotal chains in the tumor immune microenvironment (TIME). GAMs mostly display M2-like phenotypes with anti-inflammatory features related to the malignancy and progression of cancers. Extracellular vesicles derived from immunosuppressive GAMs (M2-EVs), the essential components of the TIME, greatly impact the malignant behavior of GBM cells. M1- or M2-EVs were isolated in vitro, and human GBM cell invasion and migration were reinforced under M2-EV treatment. Signatures of the epithelial-mesenchymal transition (EMT) were also enhanced by M2-EVs. Compared with M1-EVs, miR-146a-5p, considered the key factor in TIME regulation, was deficient in M2-EVs according to miRNA-sequencing. When the miR-146a-5p mimic was added, EMT signatures and the invasive and migratory abilities of GBM cells were correspondingly weakened. Public databases predicted the miRNA binding targets and interleukin 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were screened as miR-146a-5p binding genes. Bimolecular fluorescent complementation and coimmunoprecipitation confirmed interactions between TRAF6 and IRAK1. The correlation between TRAF6 and IRAK1 was evaluated with immunofluorescence (IF)-stained clinical glioma samples. The TRAF6-IRAK1 complex is the switch and the brake that modulates IKK complex phosphorylation and NF-κB pathway activation, as well as the EMT behaviors of GBM cells. Furthermore, a homograft nude mouse model was explored and mice transplanted with TRAF6/IRAK1-overexpressing glioma cells had shorter survival times while mice transplanted with glioma cells with miR-146a-5p overexpression or TRAF6/IRAK1 knockdown lived longer. This work indicated that in the TIME of GBM, the deficiency of miR-146a-5p in M2-EVs enhances tumor EMT through disinhibition of the TRAF6-IRAK1 complex and IKK-dependent NF-κB signaling pathway providing a novel therapeutic strategy targeting the TIME of GBM.

Isolation and Purification of Fungal β-Glucan as an Immunotherapy Strategy for Glioblastoma.

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor microenvironment. Immunotherapy has emerged as an effective strategy to turn the immune system response against tumor cells. Glioma-associated macrophages and microglia (GAMs) are major drivers of such anti-inflammatory scenarios. Therefore, enhancing the anti-cancerous response in GAMs may represent a potential co-adjuvant therapy to treat glioblastoma patients. In that vein, fungal β-glucan molecules have long been known as potent immune modulators. Their ability to stimulate the innate immune activity and improve treatment response has been described. Those modulating features are partly attributed to their ability to bind to pattern recognition receptors, which, interestingly, are greatly expressed in GAMs. Thus, this work is focused on the isolation, purification, and subsequent use of fungal β-glucans to enhance the tumoricidal response of microglia against glioblastoma cells. The mouse glioblastoma (GL261) and microglia (BV-2) cell lines are used to test the immunomodulatory properties of four different fungal β-glucans extracted from mushrooms heavily used in the current biopharmaceutical industry: Pleurotus ostreatus, Pleurotus djamor, Hericium erinaceus,and Ganoderma lucidum. To test these compounds, co-stimulation assays were performed to measure the effect of a pre-activated microglia-conditioned medium on the proliferation and apoptosis activation in glioblastoma cells.

Ultrahigh-plex Spatial Phenotyping of the Glioblastoma Multiforme Immune Microenvironment

Abstract The glioblastoma multiforme (GBM) microenvironment is highly immunosuppressive, leading to poor clinical outcomes and median survival of less than 15 months. Information about the nature of immunosuppressive and immunostimulatory cell types in GBM is fragmented, and a better understanding is urgently needed. We have developed a spatial phenotyping application that permits comprehensive characterization of the human GBM immune microenvironment. The Phenocycler-Fusion is a fast spatial biology solution that affords ultrahigh-plex single-cell spatial readouts. We used this solution for deep immune phenotyping of >50 proteins, comprising immune cell lineages, activation states and checkpoints, as well as markers for tumor, vascular and neuronal landscapes of GBM. Via single cell spatial phenotyping, we then isolated spatial signatures within the GBM tissue immune microenvironment. We focused on large numbers of glioma-associated macrophages that were identified via the combinatorial expression of key markers CD68, CD163, STAT1, STAT6, Iba-1 and SIRPα. The macrophages and their biomarker expression profiles displayed significant inter- and intra-tumoral heterogeneity, indicative of the complex biology of GBM. In addition, we profiled other immunosuppressive elements, including Tregs and exhausted T cells. Our data provide a comprehensive account of diverse immune cell populations in GBM and they may uncover systematic differences among GBM patient samples. Our application has enormous potential to further our understanding of the GBM immune microenvironment and is ready to be deployed by other laboratories.

Macrophages in Recurrent Glioblastoma as a Prognostic Factor in the Synergistic System of the Tumor Microenvironment.

Glioblastoma (GBM) is a common and highly malignant primary tumor of the central nervous system in adults. Ever more recent papers are focusing on understanding the role of the tumor microenvironment (TME) in affecting tumorigenesis and the subsequent prognosis. We assessed the impact of macrophages in the TME on the prognosis in patients with recurrent GBM. A PubMed, MEDLINE and Scopus review was conducted to identify all studies dealing with macrophages in the GBM microenvironment from January 2016 to December 2022. Glioma-associated macrophages (GAMs) act critically in enhancing tumor progression and can alter drug resistance, promoting resistance to radiotherapy and establishing an immunosuppressive environment. M1 macrophages are characterized by increased secretion of proinflammatory cytokines, such as IL-1ß, tumor necrosis factor (TNF), IL-27, matrix metalloproteinase (MMPs), CCL2, and VEGF (vascular endothelial growth factor), IGF1, that can lead to the destruction of the tissue. In contrast, M2 is supposed to participate in immunosuppression and tumor progression, which is formed after being exposed to the macrophage M-CSF, IL-10, IL-35 and the transforming growth factor-ß (TGF-β). Because there is currently no standard of care in recurrent GBM, novel identified targeted therapies based on the complex signaling and interactions between the glioma stem cells (GSCs) and the TME, especially resident microglia and bone-marrow-derived macrophages, may be helpful in improving the overall survival of these patients in the near future.

Recruitment mechanisms and therapeutic implications of tumor-associated macrophages in the glioma microenvironment.

As one of the main components of the glioma immune microenvironment, glioma-associated macrophages (GAMs) have increasingly drawn research interest. Primarily comprised of resident microglias and peripherally derived mononuclear macrophages, GAMs are influential in a variety of activities such as tumor cell resistance to chemotherapy and radiotherapy as well as facilitation of glioma pathogenesis. In addition to in-depth research of GAM polarization, study of mechanisms relevant in tumor microenvironment recruitment has gradually increased. Suppression of GAMs at their source is likely to produce superior therapeutic outcomes. Here, we summarize the origin and recruitment mechanism of GAMs, as well as the therapeutic implications of GAM inhibition, to facilitate future glioma-related research and formulation of more effective treatment strategies.

525 Diffuse Microglia Inflammation is Associated with Anti-Tumor Efficacy in Polio Virotherapy for Glioblastoma

INTRODUCTION: Glioblastomas (GBM) harbor abundant inflammatory infiltrates with glioma-associated macrophages and microglia (GAMM) actively promoting tumor progression. Intratumor virotherapy with the highly attenuated rhino:poliovirus chimera, PVSRIPO, causes sublethal infection and activation of GAMM via the constitutively expressed human poliovirus receptor, CD155. METHODS: We optimized immune-competent CT2A murine glioblastoma models for blinded neuropathological analysis of therapy responses. Samples (n = 55) were collected in a time series after PVSRIPO or mock intratumor infusion for systemic review of histological features related to therapy. Immune cells were defined both by morphology and immunohistochemical (IHC) staining patterns. Multiplex IHC and RNA sequencing were performed to study the immune infiltrate and PD1/PD-L1 immune checkpoint expression. The role of the PD1:PD-L1 axis in restricting the antitumor efficacy of PVSRIPO was tested using survival studies. RESULTS: PVSRIPO treatment caused substantial, but transient tumor regression with intense engagement of the GAMM infiltrate. This was accompanied by diffuse microglia activation and inflammtion in the CNS surrounding the tumor, extending to the ipsilateral and even the contralateral hemispheres. PVSRIPO-instigated pervasive microglia activation occurred against a backdrop of sustained innate antiviral inflammation, associated with induction of the PD-L1 immune checkpoint on GAMM. Combining PVSRIPO with PD1/PD-L1 blockade led to more durable remissions in CT2A model. CONCLUSIONS: Our work implicates GAMM as active drivers of PVSRIPO induced antitumor inflammation and reveals profound and widespread neuroinflammatory activation of the CNS-resident myeloid compartment by PVSRIPO, which is closely associated with tumor regression.

The short isoform of MS4A7 is a novel player in glioblastoma microenvironment, M2 macrophage polarization, and tumor progression

BackgroundThe unique intracranial tumor microenvironment (TME) contributes to the immunotherapy failure for glioblastoma (GBM), thus new functional protein targets are urgently needed. Alternative splicing is a widespread regulatory mechanism by which individual gene can express variant proteins with distinct functions. Moreover, proteins located in the cell plasma membrane facilitate targeted therapies. This study sought to obtain functional membrane protein isoforms from GBM TME.MethodsWith combined single-cell RNA-seq and bulk RNA-seq analyses, novel candidate membrane proteins generated by prognostic splicing events were screened within GBM TME. The short isoform of MS4A7 (MS4A7-s) was selected for evaluation by RT-PCR and western blotting in clinical specimens. Its clinical relevance was evaluated in a GBM patient cohort. The function of MS4A7-s was identified by in vitro and in vivo experiments. MS4A7-s overexpression introduced transcriptome changes were analyzed to explore the potential molecular mechanism.ResultsThe main expression product, isoform MS4A7-s, generated by exon skipping, is an M2-specific plasma membrane protein playing a pro-oncogenic role in GBM TME. Higher expression of MS4A7-s correlates with poor prognosis in a GBM cohort. In vitro cell co-culture experiments, intracranial co-injection tumorigenesis assay, and RNA-seq suggest MS4A7-s promotes activation of glioma-associated macrophages’ (GAMs) PI3K/AKT/GSK3β pathway, leading to M2 polarization, and drives malignant progression of GBM.ConclusionsMS4A7-s, a novel splicing isoform of MS4A7 located on the surface of GAMs in GBM TME, is a predictor of patient outcome, which contributes to M2 polarization and the malignant phenotype of GBM. Targeting MS4A7-s may constitute a promising treatment for GBM.

Glioma-associated microglia/macrophages (GAMs) in glioblastoma: Immune function in the tumor microenvironment and implications for immunotherapy.

Glioma is a mixed solid tumor composed of neoplastic and non-neoplastic components. Glioma-associated macrophages and microglia (GAMs) are crucial elements of the glioma tumor microenvironment (TME), regulating tumor growth, invasion, and recurrence. GAMs are also profoundly influenced by glioma cells. Recent studies have revealed the intricate relationship between TME and GAMs. In this updated review, we provide an overview of the interaction between glioma TME and GAMs based on previous studies. We also summarize a series of immunotherapies targeting GAMs, including clinical trials and preclinical studies. Specifically, we discuss the origin of microglia in the central nervous system and the recruitment of GAMs in the glioma background. We also cover the mechanisms through which GAMs regulate various processes associated with glioma development, such as invasiveness, angiogenesis, immunosuppression, recurrence, etc. Overall, GAMs play a significant role in the tumor biology of glioma, and a better understanding of the interaction between GAMs and glioma could catalyze the development of new and effective immunotherapies for this deadly malignancy.

Polio virotherapy targets the malignant glioma myeloid infiltrate with diffuse microglia activation engulfing the CNS.

Malignant gliomas commandeer dense inflammatory infiltrates with glioma-associated macrophages and microglia (GAMM) promoting immune suppression, evasion, and tumor progression. Like all cells in the mononuclear phagocytic system, GAMM constitutively express the poliovirus receptor, CD155. Besides myeloid cells, CD155 is widely upregulated in the neoplastic compartment of malignant gliomas. Intratumor treatment with the highly attenuated rhino:poliovirus chimera, PVSRIPO, yielded long-term survival with durable radiographic responses in patients with recurrent glioblastoma (Desjardins et al. New England Journal of Medicine, 2018). This scenario raises questions about the contributions of myeloid versus neoplastic cells to polio virotherapy of malignant gliomas. We investigated PVSRIPO immunotherapy in immunocompetent mouse brain tumor models with blinded, board-certified neuropathologist review, a range of neuropathological, immunohistochemical, and immunofluorescence analyses, and RNAseq of the tumor region. PVSRIPO treatment caused intense engagement of the GAMM infiltrate associated with substantial, but transient tumor regression. This was accompanied by marked microglia activation and proliferation in normal brain surrounding the tumor, in the ipsilateral hemisphere and extending into the contralateral hemisphere. There was no evidence for lytic infection of malignant cells. PVSRIPO-instigated microglia activation occurred against a backdrop of sustained innate antiviral inflammation, associated with induction of the Programmed Cell Death Ligand 1 (PD-L1) immune checkpoint on GAMM. Combining PVSRIPO with PD1/PD-L1 blockade led to durable remissions. Our work implicates GAMM as active drivers of PVSRIPO-induced antitumor inflammation and reveals profound and widespread neuroinflammatory activation of the brain-resident myeloid compartment by PVSRIPO.

Cytohesin-4 Upregulation in Glioma-Associated M2 Macrophages Is Correlated with Pyroptosis and Poor Prognosis.

Cytohesin-4 (CYTH4) is a member of the PSCD family. Members of this family appear to mediate the regulation of protein sorting and membrane trafficking. In previous studies, CYTH4 has been linked with multiple brain diseases, but not glioma, the most common type of brain tumor. We utilized multiple glioma single-cell RNA sequencing datasets and bulk data from the TCGA and CGGA and conducted GSEA and KEGG and GO analyses. Biomarker potential was tested via ROC curve analysis. Radar plots were used to study TMB and MSI correlations. Immune cell studies were conducted using CIBERSORT. All statistical analyses were performed in R software and GraphPad Prism 9. CYTH4 was overexpressed in the glioma macrophage population in several single-cell RNA sequencing datasets and was most correlated with M2 macrophages. CYTH4 expression was higher in tumor tissues and was correlated with survival and WHO grade. ROC curves suggested CYTH4 overexpression to be a potential glioma biomarker. GSEA results indicated a relationship between CYTH4 and apoptosis, and PPI analysis supported a pyroptosis correlation. KEGG and GO analysis results linked CYTH4 with antigen processing and presentation and neutrophil activities. In summary, the study identified a CYTH4/pyroptosis/M2 macrophage axis. CYTH4 was upregulated in M2 macrophages in glioma and affected pyroptosis. CYTH4 overexpression is a potential biomarker predicting a poor prognosis.

Microglia and Brain Macrophages as Drivers of Glioma Progression

Evidence is accumulating that the tumour microenvironment (TME) has a key role in the progression of gliomas. Non-neoplastic cells in addition to the tumour cells are therefore finding increasing attention. Microglia and other glioma-associated macrophages are at the centre of this interest especially in the context of therapeutic considerations. New ideas have emerged regarding the role of microglia and, more recently, blood-derived brain macrophages in glioblastoma (GBM) progression. We are now beginning to understand the mechanisms that allow malignant glioma cells to weaken microglia and brain macrophage defence mechanisms. Surface molecules and cytokines have a prominent role in microglia/macrophage-glioma cell interactions, and we discuss them in detail. The involvement of exosomes and microRNAs forms another focus of this review. In addition, certain microglia and glioma cell pathways deserve special attention. These "synergistic" (we suggest calling them "Janus") pathways are active in both glioma cells and microglia/macrophages where they act in concert supporting malignant glioma progression. Examples include CCN4 (WISP1)/Integrin α6β1/Akt and CHI3L1/PI3K/Akt/mTOR. They represent attractive therapeutic targets.

DDEL-13. ULTRASOUND-ENHANCED DELIVERY OF LIPOSOMAL DOXORUBICIN ACROSS THE BLOOD BRAIN BARRIER INDUCES AN IFN-GPHENOTYPE IN MICROGLIA, MACROPHAGES, AND T CELLS AND IMPROVES RESPONSE TO PD-1 BLOCKADE IN GLIOMAS

Abstract INTRODUCTION Given the limited drug penetration across the blood-brain barrier (BBB), the therapeutic potential of new and existing therapies has not been fully exploited for the benefit of glioblastoma (GBM) patients. METHODS Here we employed a novel drug delivery technology based on low-intensity pulsed ultrasound combined with intravenous microbubbles (LIPU/MB) that temporarily opens the BBB to deliver liposomal doxorubicin (DOX) and anti-PD-1 therapy (aPD-1) in mouse glioma models and 3 recurrent GBM patients. Immunological variables were evaluated in tumor and immune cells as well as efficacy in glioma-bearing mice treated with DOX delivered by LIPU. These included measurement of HLA ABC and HLA DR protein expression by tumor cells, microglia, and macrophages and IFN-g production by glioma-associated microglia and macrophages in mouse and human tumors. We also assessed efficacy of LIPU/MB enhanced combination therapy in glioma-bearing mice. RESULTS Upregulation of HLA ABC and HLA DR was observed in GBM cell lines at low concentrations of DOX. Tumor cells from GBM patients treated with DOX, aPD-1 and LIPU/MB showed increased expression of HLA ABC and HLA DR compared to paired pretreatment samples. In both mice and humans, LIPU/MB liposomal DOX increased absolute brain drug concentrations and elicited a specific IFN-g phenotype and MHC I expression in glioma-associated microglia and macrophages in mice and humans. Furthermore, LIPU/MB-mediated BBB opening increased brain concentrations of aPD-1 in mice and in peritumoral regions of GBM patients. Combined treatment with liposomal DOX and aPD-1 delivered with LIPU/MB resulted in long-term survival of glioma-bearing mice that relied on the activity of CD8+ T cells for its efficacy. CONCLUSIONS Overall, this translational study demonstrates the utility of LIPU/MB to stimulate intracranial immune responses in the context of treatment with DOX and aPD-1 for gliomas.

Characteristics and regulatory mechanisms of lipid metabolism remodeling after malignant transformation of glioma-associated macrophages

Objective: To observe the lipid metabolism characteristics of tumor-associated macrophages (TAM) after malignant transformation in the glioma micro-environment, and analyze the biological phenotype changes and regulatory mechanisms after inhibiting the lipid metabolism remodeling. Methods: Twelve male Balb/c mice of 6-8 weeks were used in the study. Macrophages (Mφ) were derived from mouse bone marrow, and malignantly transformed macrophages (tMφ1 and tMφ2) were cloned from the model of glioma stem cell (GSC) through interaction with Mφ in vivo and in vitro. Intracellular lipid droplet formation and cellular cholesterol content were measured respectively in Mφ, tMφ1 and tMφ2. qRT-PCR was performed to detect the genes expression level related with lipid metabolism, including sterol regulatory element binding protein (SREBP), fatty acid synthase (FASN), and 3-hydroxy-3-methylglutarate monoacyl coenzyme A reductase (HMG-CoA). Simvastatin (SIM) was used to analyze the proliferation, immigration and invasiveness ability in tMφ1 and tMφ2 after inhibition of the lipid metabolism. Differential expression profiles of miRNAs after SIM treatment were constructed in t-Mφ1 and bio-informatics analysis was screened and verified for miR449a and its target gene sorting micro-tubule connectin 17 (SNX17) associated with lipid metabolism remodeling. The effect on SNX17 by up-regulated miR-449a were analyzed by qRT-PCR and Western blot, meanwhile, the biological phenotype and cholesterol content were observed after up-regulation of miR449a. Low-density lipoprotein receptor (LDLR) protein levels after SNX17 knockdown and intracellular cholesterol content after LDLR knockdown were detected respectively. Results: The numbers of intracellular lipid droplet formation in tMφ1 and tMφ2 were more than that in Mφ (P<0.001). Likewise, the relative contents of cholesterol (3.89±0.68 and 3.56±0.53), SREBP (4.78±0.60 and 2.84±0.41), FASN (4.65±0.70 and 3.01±0.45), and HMG-CoA (5.74±0.55 and 2.97±0.34) were significantly higher in tMφ1 and tMφ2 than those of Mφ (1.01 wel, 1.02 wel and 0.99 wel, respectively) (all P<0.001). The proliferation rates of tMφ1 and tMφ2 decreased from (47.06±5.88) % and (45.29±5.64)% to (23.53±4.70)% and (18.74±5.76)%, respectively after treatment with SIM (both P<0.05). The numbers of migrated cells decreased from 1 025±138 and 350±47 to 205±63 and 99±25, respectively (both P<0.001). And the numbers of invasiveness cells decreased from 919±45 and 527±34 to 220±23 and 114±21, respectively (both P<0.001). While the relative intracellular cholesterol content decreased to 0.52±0.08 and 0.58±0.07 (both P<0.05), respectively. MiR-449a was screened from tMφ1 by SIM, and the target gene was analyzed and verified to be SNX17. SNX17 expression was down-regulated, and the proliferation rate, the number of migration and invasiveness was significantly decreased after miR-449a over-expression (all P<0.05). Low-density lipoprotein receptor (LDLR) expression was down-regulated after knock-down of SNX17, while the cholesterol content was decreased after knock-down of LDLR in tMφ1 and tMφ2 (all P<0.05). Conclusions: Malignantly transformed TAMs undergo lipid metabolism remodeling characterized with enhanced lipid metabolism. MiR-449a regulates the LDLR by targeting SNX17, thereby affecting the lipid metabolism of malignantly transformed macrophages, and subsequently inhibiting its proliferation, migration, and invasion ability. Precise intervention with miR-449a/SNX17/LDLR axis could provide an experimental basis for reversing its tumor-promoting micro-environment remodeled by GSC through metabolic intervention.