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.

An expanded safety/feasibility study of the EMulate Therapeutics Voyager™ System in patients with recurrent glioblastoma.

Aim: The EMulate Therapeutics Voyager™ is a simple, wearable, home-use device that uses an alternating electromagnetic field to alter biologic signaling within cells. Objective: To assess the safety/feasibility of the Voyager in the treatment of recurrent glioblastoma (rGBM). Methods: In this study, patients with rGBM were treated with Voyager as monotherapy or in combination with standard chemotherapy at the Investigator's discretion. Safety was assessed by incidence of adverse events associated with the Voyager. Patients were followed until death. Results: A total of 75 patients were enrolled and treated for at least one day with the Voyager (safety population). Device-related adverse events were uncommon and generally did not result in interruption or withdrawal from treatment. There were no serious adverse events associated with Voyager. A total of 60 patients were treated for at least one month (clinical utility population). The median progression-free survival (PFS) was 17 weeks (4.3 months) in the Voyager only group (n = 24) and 21 weeks (5.3 months) in the Voyager + concurrent therapy group (n = 36). The median overall survival (OS) was 7 months in the Voyager only group and 9 months in the Voyager + concurrent therapy group. In patients treated with Voyager + concurrent therapy, the median OS for patients enrolled with their 1st or 2nd recurrence (n = 26) was 10 months, while in patients enrolled with their 3rd or 4th recurrence (n = 10) OS was 7 months. Conclusion: The data support the safety and feasibility of the Voyager for the treatment of rGBM. Further prospective study of the device is warranted. Trial Registration Number: NCT02296580 (ClinicalTrials.gov).

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.

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.

Aberrant mesenchymal differentiation of glioma stem-like cells: implications for therapeutic targeting.

Differentiation has been proposed as a therapeutic strategy for glioblastoma (GBM) in part due to observations of stem-like cells in GBM that have been shown to undergo terminal differentiation in response to growth factor withdrawal and BMP activation. However, the effects of long term exposure to serum culture conditions on glioma sphere cultures/glioma stem-like cells (GSCs) have not been examined. Here we show that GSCs retained both neurosphere formation and tumor initiation abilities after short or long term serum exposure. Under these conditions, GSCs expressed both neural lineage and stem cell markers, highlighting the aberrant pseudo-differentiation state. GSCs maintained under adherent serum cultured conditions continued to proliferate and initiate tumor formation with efficiencies similar to GSCs maintained under proliferating (neurosphere) conditions. Proneural (PN) GSCs under serum exposure showed an induction of mesenchymal (MES) gene expression signatures. Our data indicate that exposure to serum containing media result in aberrant differentiation (e.g. toward MES lineage) and activation of alternative oncogenic pathways in GSCs.

Experimental validation of 5 in-silico predicted glioma biomarkers.

BACKGROUND: Glioblastoma multiforme (GBM) is a high-grade glioma with poor prognosis. Identification of new biomarkers specific to GBM could help in disease diagnosis. We have developed and validated a bioinformatics method to predict proteins likely to be suitable as glioma biomarkers via a global microarray meta-analysis to identify uncharacterized genes consistently coexpressed with known glioma-associated genes. METHODS: A novel bioinformatics method was implemented called global microarray meta-analysis, using approximately 16,000 microarray experiments to identify uncharacterized genes consistently coexpressed with known glioma-associated genes. These novel biomarkers were validated as proteins highly expressed in human gliomas varying in tumor grades using immunohistochemistry. Glioma gene databases were used to assess delineation of expression of these markers in varying glioma grades and subtypes of GBM. RESULTS: We have identified 5 potential biomarkers-spondin1, Plexin-B2, SLIT3, fibulin-1, and LINGO1-that were validated as proteins highly expressed on the surface of human gliomas using immunohistochemistry. Expression of spondin1, Plexin-B2, and SLIT3 was significantly higher (P < .01) in high-grade gliomas than in low-grade gliomas. These biomarkers were significant discriminators in grade IV gliomas compared with either grade III or II tumors and also distinguished between GBM subclasses. CONCLUSIONS: This study strongly suggests that this type of bioinformatics approach has high translational potential to rapidly discern which poorly characterized proteins may be of clinical relevance.

Mesenchymal differentiation mediated by NF-κB promotes radiation resistance in glioblastoma.

Despite extensive study, few therapeutic targets have been identified for glioblastoma (GBM). Here we show that patient-derived glioma sphere cultures (GSCs) that resemble either the proneural (PN) or mesenchymal (MES) transcriptomal subtypes differ significantly in their biological characteristics. Moreover, we found that a subset of the PN GSCs undergoes differentiation to a MES state in a TNF-α/NF-κB-dependent manner with an associated enrichment of CD44 subpopulations and radioresistant phenotypes. We present data to suggest that the tumor microenvironment cell types such as macrophages/microglia may play an integral role in this process. We further show that the MES signature, CD44 expression, and NF-κB activation correlate with poor radiation response and shorter survival in patients with GBM.

Regulation of HGF expression by ΔEGFR-mediated c-Met activation in glioblastoma cells.

The hepatocyte growth factor receptor (c-Met) and a constitutively active mutant of the epidermal growth factor receptor (ΔEGFR/EGFRvIII) are frequently overexpressed in glioblastoma (GBM) and promote tumorigenesis. The mechanisms underlying elevated hepatocyte growth factor (HGF) production in GBM are not understood. We found higher, coordinated mRNA expression levels of HGF and c-Met in mesenchymal (Mes) GBMs, a subtype associated with poor treatment response and shorter overall survival. In an HGF/c-Met-dependent GBM cell line, HGF expression declined upon silencing of c-Met using RNAi or by inhibiting its activity with SU11274. Silencing c-Met decreased anchorage-independent colony formation and increased the survival of mice bearing intracranial GBM xenografts. Consistent with these findings, c-Met activation by ΔEGFR also elevated HGF expression, and the inhibition of ΔEGFR with AG1478 reduced HGF levels. Interestingly, c-Met expression was required for ΔEGFR-mediated HGF production, anchorage-independent growth, and in vivo tumorigenicity, suggesting that these pathways are coupled. Using an unbiased mass spectrometry-based screen, we show that signal transducer and activator of transcription 3 (STAT3) Y705 is a downstream target of c-Met signaling. Suppression of STAT3 phosphorylation with WP1193 reduced HGF expression in ΔEGFR-expressing GBM cells, whereas constitutively active STAT3 partially rescued HGF expression and colony formation in c-Met knockdown cells expressing ΔEGFR. These results suggest that the c-Met/HGF signaling axis is enhanced by ΔEGFR through increased STAT3-dependent HGF expression and that targeting c-Met in Mes GBMs may be an important strategy for therapy.

ELTD1, a potential new biomarker for gliomas.

BACKGROUND: Glioblastoma multiforme (GBM), a high-grade glioma, is characterized by being diffuse, invasive, and highly angiogenic and has a very poor prognosis. Identification of new biomarkers could help in the further diagnosis of GBM. OBJECTIVE: To identify ELTD1 (epidermal growth factor, latrophilin, and 7 transmembrane domain-containing protein 1 on chromosome 1) as a putative glioma-associated marker via a bioinformatic method. METHODS: We used advanced data mining and a novel bioinformatics method to predict ELTD1 as a potential novel biomarker that is associated with gliomas. Validation was done with immunohistochemistry, which was used to detect levels of ELTD1 in human high-grade gliomas and rat F98 glioma tumors. In vivo levels of ELTD1 in rat F98 gliomas were assessed using molecular magnetic resonance imaging. RESULTS: ELTD1 was found to be significantly higher (P = .03) in high-grade gliomas (50 patients) compared with low-grade gliomas (21 patients) and compared well with traditional immunohistochemistry markers including vascular endothelial growth factor, glucose transporter 1, carbonic anhydrase IX, and hypoxia-inducible factor 1α. ELTD1 gene expression indicates an association with grade, survival across grade, and an increase in the mesenchymal subtype. Significantly high (P < .001) in vivo levels of ELTD1 were additionally found in F98 tumors compared with normal brain tissue. CONCLUSION: Results of this study strongly suggests that associative analysis was able to accurately identify ELTD1 as a putative glioma-associated biomarker. The detection of ELTD1 was also validated in both rodent and human gliomas and may serve as an additional biomarker for gliomas in preclinical and clinical diagnosis of gliomas.

A prognostic gene expression signature in infratentorial ependymoma.

Patients with ependymoma exhibit a wide range of clinical outcomes that are currently unexplained by clinical or histological factors. Little is known regarding molecular biomarkers that could predict clinical behavior. Since recent data suggest that these tumors display biological characteristics according to their location (cerebral vs. infratentorial vs. spinal cord), rather than explore a broad spectrum of ependymoma, we focused on molecular alterations in ependymomas arising in the infratentorial compartment. Unsupervised clustering of available gene expression microarray data revealed two major subgroups of infratentorial ependymoma. Group 1 tumors over expressed genes that were associated with mesenchyme, Group 2 tumors showed no distinct gene ontologies. To assess the prognostic significance of these gene expression subgroups, real-time reverse transcriptase polymerase chain reaction assays were performed on genes defining the subgroups in a training set. This resulted in a 10-gene prognostic signature. Multivariate analysis showed that the 10-gene signature was an independent predictor of recurrence-free survival after adjusting for clinical factors. Evaluation of an external dataset describing subgroups of infratentorial ependymomas showed concordance of subgroup definition, including validation of the mesenchymal subclass. Importantly, the 10-gene signature was validated as a predictor of recurrence-free survival in this dataset. Taken together, the results indicate a link between clinical outcome and biologically identified subsets of infratentorial ependymoma and offer the potential for prognostic testing to estimate clinical aggressiveness in these tumors.

The transcriptional coactivator TAZ regulates mesenchymal differentiation in malignant glioma.

Recent molecular classification of glioblastoma (GBM) has shown that patients with a mesenchymal (MES) gene expression signature exhibit poor overall survival and treatment resistance. Using regulatory network analysis of available expression microarray data sets of GBM, including The Cancer Genome Atlas (TCGA), we identified the transcriptional coactivator with PDZ-binding motif (TAZ), to be highly associated with the MES network. TAZ expression was lower in proneural (PN) GBMs and lower-grade gliomas, which correlated with CpG island hypermethylation of the TAZ promoter compared with MES GBMs. Silencing of TAZ in MES glioma stem cells (GSCs) decreased expression of MES markers, invasion, self-renewal, and tumor formation. Conversely, overexpression of TAZ in PN GSCs as well as murine neural stem cells (NSCs) induced MES marker expression and aberrant osteoblastic and chondrocytic differentiation in a TEAD-dependent fashion. Using chromatin immunoprecipitation (ChIP), we show that TAZ is directly recruited to a majority of MES gene promoters in a complex with TEAD2. The coexpression of TAZ, but not a mutated form of TAZ that lacks TEAD binding, with platelet-derived growth factor-B (PDGF-B) resulted in high-grade tumors with MES features in a murine model of glioma. Our studies uncover a direct role for TAZ and TEAD in driving the MES differentiation of malignant glioma.

Treatment of spinal cord tumors.

Tumors associated with the spinal cord can have devastating effects on patient function and quality of life. Most of these tumors are from metastatic disease, usually to the epidural space. Less frequently, the tumors are intrinsic to the spinal cord itself (ie, primary tumor). Regardless of the etiology, spinal cord tumors often present first with progressive local or radicular pain, or both. Other symptoms include weakness, sensory changes, or sphincter dysfunction. The timeliness of diagnosis of spinal cord tumors and promptness of treatment are important, as they directly affect outcome. Dexamethasone, a corticosteroid, is used as a temporizing measure to improve or stabilize neurologic function until definitive treatment. For nonambulatory patients with epidural metastatic tumors, surgery followed by radiation therapy maximizes neurologic function and modestly lengthens survival. However, palliative radiotherapy alone is recommended for those with neurologic deficits lasting longer than 48 hours, survival prognosis less than 3 months, inability to tolerate surgery, multiple areas of compression, or radiosensitive tumors. An ambulatory patient with a stable spine should be considered for radiation treatment only. The role of chemotherapy for epidural metastatic tumors is not well established. For intramedullary metastases, the role of surgery and chemotherapy remains controversial and radiation is the mainstay. For low-grade or benign primary spinal cord tumors, resective surgery is of benefit and can be curative. For high-grade tumors, the benefit of resection is less clear, and radiotherapy and/or chemotherapy may be helpful. The use of chemotherapy for primary spinal cord tumors has rarely been assessed. Agents reported in the literature for treatment of spinal cord gliomas include temozolomide, irinotecan, cisplatin, and carboplatin. A multidisciplinary approach is often required to maximize the therapeutic and functional outcome of patients with metastatic and primary spinal cord tumors.

Antineutrophil cytoplasmic antibody-associated active scleritis.

OBJECTIVE: To determine whether antineutrophil cytoplasmic antibody (ANCA) testing provides prognostic information in evaluating scleritis. METHODS: Retrospective medical record review of patients evaluated at a tertiary care center from January 1, 1995, to June 30, 2006, was performed to compare clinical features, treatments, and associated systemic disease in patients who test positive for ANCA vs patients whose ANCA tests are negative. RESULTS: Among 78 patients identified, 14 tested positive for ANCA. Patients with positive ANCA test results were more likely to have an associated systemic disorder (10 of 14 or 71%) than were patients who tested negative for ANCA (26 of 64 or 41%; P = .04), and the disorder was more likely to have been diagnosed as a result of scleritis work-up (2 of 10 or 20% vs 19 of 26 or 73%; P = .007). Patients with positive ANCA test results had significantly more ocular complications (21 of 14 or 86% vs 20 of 64 or 31%; P < .001), including keratopathy (5 of 14 or 36% vs 6 of 64 or 9%; P = .02), visual acuity of 20/50 or worse (8 of 14 or 57% vs 11 of 64 or 17%; P = .001), and vascular pannus (3 of 14 or 21% vs 1 of 64 or 2%; P = .02). Aggressive therapy, such as chronic systemic corticosteroids (9 of 14 or 64% vs 9 of 64 or 14%; P < .001) and alkylator therapy (8 of 14 or 57% vs 7 of 64 or 11%; P < .001), was more likely to be recommended for patients who tested positive for ANCA. CONCLUSIONS: A substantial subset of patients with scleritis are also positive for ANCA. These patients are more likely to have severe ocular disease and undiagnosed primary vasculitic disease, thereby requiring more aggressive therapy. An ANCA test may be useful in the evaluation and treatment of patients with scleritis.