Triggering receptor expressed on myeloid cells 2 (TREM2) regulates phagocytosis in glioblastoma.

BACKGROUND: Glioblastomas (GBMs) are central nervous system tumors that resist standard-of-care interventions and even immune checkpoint blockade. Myeloid cells in the tumor microenvironment can contribute to GBM progression; therefore, emerging immunotherapeutic approaches include reprogramming these cells to achieve desirable antitumor activity. Triggering receptor expressed on myeloid cells 2 (TREM2) is a myeloid signaling regulator that has been implicated in a variety of cancers and neurological diseases with contrasting functions, but its role in GBM immunopathology and progression is still under investigation. METHODS: Our reverse translational investigations leveraged single-cell RNA sequencing and cytometry of human gliomas to characterize TREM2 expression across myeloid subpopulations. Using 2 distinct murine glioma models, we examined the role of Trem2 on tumor progression and immune modulation of myeloid cells. Furthermore, we designed a method of tracking phagocytosis of glioma cells in vivo and employed in vitro assays to mechanistically understand the influence of TREM2 signaling on tumor uptake. RESULTS: We discovered that TREM2 expression does not correlate with immunosuppressive pathways, but rather showed strong a positive association with the canonical phagocytosis markers lysozyme (LYZ) and macrophage scavenger receptor (CD163) in gliomas. While Trem2 deficiency was found to be dispensable for gliomagenesis, Trem2+ myeloid cells display enhanced tumor uptake compared to Trem2- cells. Mechanistically, we demonstrate that TREM2 mediates phagocytosis via Syk signaling. CONCLUSIONS: These results indicate that TREM2 is not associated with immunosuppression in gliomas. Instead, TREM2 is an important regulator of phagocytosis that may be exploited as a potential therapeutic strategy for brain tumors.

Gut epithelial Interleukin-17 receptor A signaling can modulate distant tumors growth through microbial regulation.

Microbes influence cancer initiation, progression and therapy responsiveness. IL-17 signaling contributes to gut barrier immunity by regulating microbes but also drives tumor growth. A knowledge gap remains regarding the influence of enteric IL-17-IL-17RA signaling and their microbial regulation on the behavior of distant tumors. We demonstrate that gut dysbiosis induced by systemic or gut epithelial deletion of IL-17RA induces growth of pancreatic and brain tumors due to excessive development of Th17, primary source of IL-17 in human and mouse pancreatic ductal adenocarcinoma, as well as B cells that circulate to distant tumors. Microbial dependent IL-17 signaling increases DUOX2 signaling in tumor cells. Inefficacy of pharmacological inhibition of IL-17RA is overcome with targeted microbial ablation that blocks the compensatory loop. These findings demonstrate the complexities of IL-17-IL-17RA signaling in different compartments and the relevance for accounting for its homeostatic host defense function during cancer therapy.

Liquid biopsy: creating opportunities in brain space.

In recent years, liquid biopsy has emerged as an alternative method to diagnose and monitor tumors. Compared to classical tissue biopsy procedures, liquid biopsy facilitates the repetitive collection of diverse cellular and acellular analytes from various biofluids in a non/minimally invasive manner. This strategy is of greater significance for high-grade brain malignancies such as glioblastoma as the quantity and accessibility of tumors are limited, and there are collateral risks of compromised life quality coupled with surgical interventions. Currently, blood and cerebrospinal fluid (CSF) are the most common biofluids used to collect circulating cells and biomolecules of tumor origin. These liquid biopsy analytes have created opportunities for real-time investigations of distinct genetic, epigenetic, transcriptomics, proteomics, and metabolomics alterations associated with brain tumors. This review describes different classes of liquid biopsy biomarkers present in the biofluids of brain tumor patients. Moreover, an overview of the liquid biopsy applications, challenges, recent technological advances, and clinical trials in the brain have also been provided.

Triggering receptor expressed on myeloid cells 2 (TREM2) regulates phagocytosis in glioblastoma.

Glioblastomas (GBMs) are tumors of the central nervous system that remain recalcitrant to both standard of care chemo-radiation and immunotherapies. Emerging approaches to treat GBMs include depletion or re-education of innate immune cells including microglia (MG) and macrophages (MACs). Here we show myeloid cell restricted expression of triggering receptor expressed on myeloid cells 2 (TREM2) across low- and high-grade human gliomas. TREM2 expression did not correlate with immunosuppressive pathways, but rather showed strong positive association with phagocytosis markers such as lysozyme (LYZ) and CD163 in gliomas. In line with these observations in patient tumors, Trem2-/- mice did not exhibit improved survival compared to wildtype (WT) mice when implanted with mouse glioma cell lines, unlike observations previously seen in peripheral tumor models. Gene expression profiling revealed pathways related to inflammation, adaptive immunity, and autophagy that were significantly downregulated in tumors from Trem2-/- mice compared to WT tumors. Using ZsGreen-expressing CT-2A orthotopic implants, we found higher tumor antigen engulfment in Trem2+ MACs, MG, and dendritic cells. Our data uncover TREM2 as an important immunomodulator in gliomas and inducing TREM2 mediated phagocytosis can be a potential immunotherapeutic strategy for brain tumors.

PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells.

Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs in vivo is sufficient to improve overall survival in an intracranial xenograft mouse model. PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.

Cell death in development, maintenance, and diseases of the nervous system.

Cell death, be it of neurons or glial cells, marks the development of the nervous system. Albeit relatively less so than in tissues such as the gut, cell death is also a feature of nervous system homeostasis-especially in context of adult neurogenesis. Finally, cell death is commonplace in acute brain injuries, chronic neurodegenerative diseases, and in some central nervous system tumors such as glioblastoma. Recent studies are enumerating the various molecular modalities involved in the execution of cells. Intimately linked with cell death are mechanisms of disposal that remove the dead cell and bring about a tissue-level response. Heretofore, the association between these methods of dying and physiological or pathological responses has remained nebulous. It is envisioned that careful cartography of death and disposal may reveal novel understandings of disease states and chart new therapeutic strategies in the near future.

Autism-Associated Vigilin Depletion Impairs DNA Damage Repair.

Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, and mRNA stability and is associated with autism spectrum disorders and cancer: vigilin, for example, can suppress proto-oncogene c-fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K-type homology (KH) domains. Here, we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ-H2AX and elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 focus formation, DNA end resection, and double-strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU)-induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in the transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer-related disorders.

NMR Spectroscopy-Based Metabolomics of Platelets to Analyze Brain Tumors.

"Tumor-educated platelets" have recently generated substantial interest for the diagnosis of cancer. We hypothesized that tumor educated platelets from patients with brain tumors will reflect altered metabolism compared to platelets from healthy volunteers. Here, in a pilot study, we have employed nuclear magnetic resonance (NMR) spectroscopy in platelets from brain tumor patients to demonstrate altered metabolism compared to the platelets obtained from healthy volunteers.

Tunneling Nanotubes Mediate Adaptation of Glioblastoma Cells to Temozolomide and Ionizing Radiation Treatment.

Glioblastoma (GBM) is the most prevalent and aggressive tumor in the central nervous system. Surgical resection followed by concurrent radiotherapy (ionizing radiation [IR]) and temozolomide (TMZ) is the standard of care for GBM. However, a large subset of patients offer resistance or become adapted to TMZ due mainly to the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Thus, alternative mechanisms of MGMT deregulation have been proposed but are heretofore unproven. We show that heterogeneous GBM cells express tunneling nanotubes (TNTs) upon oxidative stress and TMZ/IR treatment. We identified that MGMT protein diffused from resistant to sensitive cells upon exposure to TMZ/IR, resulting in protection against cytotoxic therapy in a TNT-dependent manner. In vivo analysis of resected GBM tumors support our hypothesis that the MGMT protein, but not its mRNA, was associated with TNT biomarkers. We propose that targeting TNT formation could be an innovative strategy to overcome treatment resistance in GBM.

Targeting glioma-initiating cells via the tyrosine metabolic pathway.

OBJECTIVE: Despite an aggressive multimodal therapeutic regimen, glioblastoma (GBM) continues to portend a grave prognosis, which is driven in part by tumor heterogeneity at both the molecular and cellular levels. Accordingly, herein the authors sought to identify metabolic differences between GBM tumor core cells and edge cells and, in so doing, elucidate novel actionable therapeutic targets centered on tumor metabolism. METHODS: Comprehensive metabolic analyses were performed on 20 high-grade glioma (HGG) tissues and 30 glioma-initiating cell (GIC) sphere culture models. The results of the metabolic analyses were combined with the Ivy GBM data set. Differences in tumor metabolism between GBM tumor tissue derived from within the contrast-enhancing region (i.e., tumor core) and that from the peritumoral brain lesions (i.e., tumor edge) were sought and explored. Such changes were ultimately confirmed at the protein level via immunohistochemistry. RESULTS: Metabolic heterogeneity in both HGG tumor tissues and GBM sphere culture models was identified, and analyses suggested that tyrosine metabolism may serve as a possible therapeutic target in GBM, particularly in the tumor core. Furthermore, activation of the enzyme tyrosine aminotransferase (TAT) within the tyrosine metabolic pathway influenced the noted therapeutic resistance of the GBM core. CONCLUSIONS: Selective inhibition of the tyrosine metabolism pathway may prove highly beneficial as an adjuvant to multimodal GBM therapies.

Immune biology of glioma-associated macrophages and microglia: functional and therapeutic implications.

CNS immune defenses are marshaled and dominated by brain resident macrophages and microglia, which are the innate immune sentinels and frontline host immune barriers against various pathogenic insults. These myeloid lineage cells are the predominant immune population in gliomas and can constitute up to 30-50% of the total cellular composition. Parenchymal microglial cells and recruited monocyte-derived macrophages from the periphery exhibit disease-specific phenotypic characteristics with spatial and temporal distinctions and are heterogeneous subpopulations based on their molecular signatures. A preponderance of myeloid over lymphoid lineage cells during CNS inflammation, including gliomas, is a contrasting feature of brain immunity relative to peripheral immunity. Herein we discuss glioma-associated macrophage and microglia immune biology in the context of their identity, molecular drivers of recruitment, nomenclature and functional paradoxes, therapeutic reprogramming and polarization strategies, relevant challenges, and our perspectives on therapeutic modulation.

The dystroglycan receptor maintains glioma stem cells in the vascular niche.

Glioblastomas (GBMs) are malignant central nervous system (CNS) neoplasms with a very poor prognosis. They display cellular hierarchies containing self-renewing tumourigenic glioma stem cells (GSCs) in a complex heterogeneous microenvironment. One proposed GSC niche is the extracellular matrix (ECM)-rich perivascular bed of the tumour. Here, we report that the ECM binding dystroglycan (DG) receptor is expressed and functionally glycosylated on GSCs residing in the perivascular niche. Glycosylated αDG is highly expressed and functional on the most aggressive mesenchymal-like (MES-like) GBM tumour compartment. Furthermore, we found that DG acts to maintain an MES-like state via tight control of MAPK activation. Antibody-based blockade of αDG induces robust ERK-mediated differentiation leading to reduced GSC potential. DG was shown to be required for tumour initiation in MES-like GBM, with constitutive loss significantly delaying or preventing tumourigenic potential in-vivo. These findings reveal a central role of the DG receptor, not only as a structural element, but also as a critical factor promoting MES-like GBM and the maintenance of GSCs residing in the perivascular niche.

Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation.

Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133+ PN signature, transition to a CD109+ MES subtype in a C/EBP-ß-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133-/CD109+ cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM.

Current clinical management of patients with glioblastoma.

BACKGROUND: Glioblastoma (GB) is the most aggressive primary brain tumor, historically resistant to treatment, and with overall fatal outcome. RECENT FINDINGS: Recently, several molecular subgroups and rare genetic alterations have been described in GB. In this review article, we will describe the current clinical management of patients with GB in the United States, discuss selected next-generation molecular-targeted therapies in GB, and present ongoing clinical trials for patients with GB. This review is intended for clinical and preclinical researchers who conduct work on GB and would like to understand more about the current standard of treatment of GB patients, historical perspectives, current challenges, and ongoing and upcoming clinical trials. CONCLUSIONS: GB is an extremely complex disease, and despite recent progress and advanced therapeutic strategies, the overall patient's prognosis remains dismal. Innovative strategies and integrative ways of approach to disease are urgently needed.

The molecular landscape of glioma in patients with Neurofibromatosis 1.

Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome in which glioma is one of the prevalent tumors. Gliomagenesis in NF1 results in a heterogeneous spectrum of low- to high-grade neoplasms occurring during the entire lifespan of patients. The pattern of genetic and epigenetic alterations of glioma that develops in NF1 patients and the similarities with sporadic glioma remain unknown. Here, we present the molecular landscape of low- and high-grade gliomas in patients affected by NF1 (NF1-glioma). We found that the predisposing germline mutation of the NF1 gene was frequently converted to homozygosity and the somatic mutational load of NF1-glioma was influenced by age and grade. High-grade tumors harbored genetic alterations of TP53 and CDKN2A, frequent mutations of ATRX associated with Alternative Lengthening of Telomere, and were enriched in genetic alterations of transcription/chromatin regulation and PI3 kinase pathways. Low-grade tumors exhibited fewer mutations that were over-represented in genes of the MAP kinase pathway. Approximately 50% of low-grade NF1-gliomas displayed an immune signature, T lymphocyte infiltrates, and increased neo-antigen load. DNA methylation assigned NF1-glioma to LGm6, a poorly defined Isocitrate Dehydrogenase 1 wild-type subgroup enriched with ATRX mutations. Thus, the profiling of NF1-glioma defined a distinct landscape that recapitulates a subset of sporadic tumors.

IL-8 associates with a pro-angiogenic and mesenchymal subtype in glioblastoma.

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by a high rate of vascularization. However, therapeutic targeting of the vasculature through anti-vascular endothelial growth factor (VEGF) treatment has been disappointing, for which Angiopoietin-2 (Ang-2) upregulation has partly been held accountable. In this study we therefore explored the interplay of Ang-2 and VEGFA and their effect on angiogenesis in GBM, especially in the context of molecular subclasses. In a large patient cohort we identified that especially combined high expression of Ang-2 and VEGFA predicted poor overall survival of GBM patients. The high expression of both factors was also associated with increased IL-8 expression in GBM tissues, but in vitro stimulation with Ang-2 and/or VEGFA did not indicate tumor or endothelial cell-specific IL-8 responses. Glioblastoma stem cells (GSCs) of the mesenchymal (MES) subtype showed dramatically higher expression of IL8 when compared to proneural (PN) GSCs. Secreted IL-8 derived from MES GSCs induced endothelial proliferation and tube formation, and the MES GBMs had increased counts of proliferating endothelial cells. Our results highlight a critical pro-angiogenic role of IL-8 in MES GBMs.

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo.

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.

GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma.

A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both in vitro and in vivo. Accordingly, a low GPR56-associated signature is prognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappa B (NF-κB) signaling pathway, thereby providing the rationale by which this receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM.

A relative increase in circulating platelets following chemoradiation predicts for poor survival of patients with glioblastoma.

BACKGROUND: Thrombocytosis is triggered by and promotes tumor growth. The relationship between the change in circulating platelets after chemoradiation therapy (CRT) or adjuvant temozolomide (TMZ) and survival in glioblastoma remains unclear. We hypothesized that an increase in platelets after these treatments would be predictive of a shorter survival. METHODS: We retrospectively reviewed data on 122 patients with newly diagnosed, pathologically proven glioblastoma who had been treated with surgery, followed by CRT and adjuvant TMZ, from 2007 to 2016. The association between the changes in blood count levels and survival was analyzed by the log-rank test. To adjust for confounding, we performed a multivariate analysis using known prognostic co-variates. RESULTS: Patients were dichotomized on the basis of the relative change in platelets after CRT from the baseline: ≤30% increase, low (n = 101) vs >30% increase, high (n = 12). The median survival for high vs. low platelets were 11 vs 28 months (p = 0.0062). No significant survival differences were observed on the basis of platelet changes during adjuvant TMZ. Similarly, changes in lymphocyte counts were not significantly prognostic. On multivariate analysis, MGMT, performance status, and an increase in platelets after CRT were significantly associated with survival (HR for platelets, 4.5; 95% confidence interval, 1.6-12.6). CONCLUSIONS: Increased platelet counts after CRT are predictive of poor survival in glioblastoma. The effect is platelet specific and does not reflect bone marrow changes, as lymphocyte changes were not significantly prognostic. These results suggest an interaction between platelets and tumor aggressiveness. Thus, platelets serve as a novel, minimally invasive liquid biopsy for predicting outcome.