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Simple Summary Extracellular vesicles (EVs) are a pivotal mechanism for long-distance intercellular communication and facilitate the stable transport of biological information. Conventional methods for profiling EVs are focused on the biological cargo obtained from large populations of cells and cannot map the secretion of specific subsets of EVs onto their cell of origin. We developed a high-throughput single-cell cloning method that can identify the kinetics of secretion of specific subsets of EVs. With the aid of this methodology, we illustrate that secretion of specific subsets of EVs can be an inheritable property of cancer cells. Our single-cell methodology enables the direct integration of EV secretion with multiple cellular functions and can enable new insights into cell and disease biology. Abstract Extracellular vesicles (EVs) mediate communication in health and disease. Conventional assays are limited in profiling EVs secreted from large populations of cells and cannot map EV secretion onto individual cells and their functional profiles. We developed a high-throughput single-cell technique that enabled the mapping of dynamics of EV secretion. By utilizing breast cancer cell lines, we established that EV secretion is heterogeneous at the single-cell level and that non-metastatic cancer cells can secrete specific subsets of EVs. Single-cell RNA sequencing confirmed that pathways related to EV secretion were enriched in the non-metastatic cells compared with metastatic cells. We established isogenic clonal cell lines from non-metastatic cells with differing propensities for CD81+CD63+EV secretion and showed for the first time that specificity in EV secretion is an inheritable property preserved during cell division. Combined in vitro and animal studies with these cell lines suggested that CD81+CD63+EV secretion can impede tumor formation. In human non-metastatic breast tumors, tumors enriched in signatures of CD81+CD63+EV have a better prognosis, higher immune cytolytic activity, and enrichment of pro-inflammatory macrophages compared with tumors with low CD81+CD63+EVs signatures. Our single-cell methodology enables the direct integration of EV secretion with multiple cellular functions and enables new insights into cell/disease biology.

Few studies implicate immunoregulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout in tumor cells did not affect tumor-cell proliferation in vitro or tumor progression in immunodeficient mice but completely impaired GBM progression in immune-competent mice. This impairment was reversed in mice with a defect in dendritic cells (DCs) or CD103+ DC differentiation in the brain and in tumor-draining lymph nodes. The presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors., Fibrinogen-like protein 2 (FGL2) mediates immune suppression in glioblastoma (GBM). Here, the authors show that FGL-2 expressed by GBM cancer cells acts by suppressing the differentiation of CD103+ DC cells required to activate the anti-tumor CD8+ T cell response via blocking GM-CSF signalling at NFKB, STAT1/5 and p38 level.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1–4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity. Primary macrophages engineered to express chimeric antigen receptors have anti-tumor activity in humanized mice.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Glioblastoma expression subtypes have been previously been associated with genomic abnormalities, treatment response, and differences in tumor microenvironment. We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical, a revision of the previously reported TCGA subtypes. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and the presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Comparison of matching primary and recurrent gliomas elucidated treatment-induced phenotypic tumor evolution, including expression subtype switching, in 45% of our cohort as well as associations between microenvironmental components and treatment response. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence was associated with CD8+ T cell enrichment. Frequency of M2 macrophage detection was associated with short-term relapse after radiation therapy. Our study provides a comprehensive transcriptional and cellular landscape of IDH wild-type glioblastoma during treatment modulated tumor evolution. Characterization of the evolving glioblastoma transcriptome and tumor microenvironment aids in designing more effective immunotherapy trials.

The original version of this Article contained errors in the author affiliations. Qingnan Zhao, Xueqing Xia, Longfei Huo and Shulin Li were incorrectly associated with Beijing Institute for Brain Disorders, 100069, Beijing, China.This has now been corrected in both the PDF and HTML versions of the Article.

Membrane-bound enzyme relocates to the cell nucleus to modify chromatin, inducing cancer resistance to radiotherapy., DNA repair pathways enable cancer cells to survive DNA damage induced after genotoxic therapies. Tyrosine kinase receptors (TKRs) have been reported as regulators of the DNA repair machinery. TIE2 is a TKR overexpressed in human gliomas at levels that correlate with the degree of increasing malignancy. Following ionizing radiation, TIE2 translocates to the nucleus, conferring cells with an enhanced nonhomologous end-joining mechanism of DNA repair that results in a radioresistant phenotype. Nuclear TIE2 binds to key components of DNA repair and phosphorylates H4 at tyrosine 51, which, in turn, is recognized by the proto-oncogene ABL1, indicating a role for nuclear TIE2 as a sensor for genotoxic stress by action as a histone modifier. H4Y51 constitutes the first tyrosine phosphorylation of core histones recognized by ABL1, defining this histone modification as a direct signal to couple genotoxic stress with the DNA repair machinery.

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

High-grade gliomas are notoriously heterogeneous regarding antigen expression, effector responses, and immunosuppressive mechanisms. Therefore, combinational immune therapeutic approaches are more likely to impact a greater number of patients and result in longer, durable responses. We have previously demonstrated the monotherapeutic effects of miR-124, which inhibits the signal transducer and activator of transcription 3 (STAT3) immune suppressive pathway, and immune stimulatory 4-1BB aptamers against a variety of malignancies, including genetically engineered immune competent high-grade gliomas. To evaluate potential synergy, we tested an immune stimulatory aptamer together with microRNA-124 (miRNA-124), which blocks tumor-mediated immune suppression, and found survival to be markedly enhanced, including beyond that produced by monotherapy. The synergistic activity appeared to be not only secondary to enhanced CD3(+) cell numbers but also to reduced macrophage immune tumor trafficking, indicating that a greater therapeutic benefit can be achieved with approaches that both induce immune activation and inhibit tumor-mediated immune suppression within the central nervous system (CNS) tumors.

BACKGROUND: Therapeutic targeting of the immune checkpoints CTLA-4 and PD-1/PD-L1 have demonstrated tumor regression in clinical trials, and phase II trials are ongoing in glioblastoma. Previous reports have suggested that responses are more frequent in patients with tumors expressing PD-L1; however, this has been disputed. At issue is the validation of PD-L1 biomarker assays and prognostic impact. METHODS: Using immunohistochemical analysis, we measured the incidence of PD-L1 expression in 94 patients with glioblastomas. We determined the total number of PD-L1-expressing cells within the glioblastomas and validated this finding using ex vivo glioblastoma flow cytometry, with further analysis of T cell populations. We evaluated the association between PD-L1 expression and median survival time using the protein expression data sets and mRNA from The Cancer Genome Atlas (TCGA). RESULTS: The median percentage of PD-L1-expressing cells in glioblastoma by cell surface staining is 2.77% (range 0-86.6%, n = 92), similar to the percentage found by ex vivo flow cytometry. The majority of glioblastoma patients (61%) had tumors with at least 1% PD-L1-positive cells, and 38% had at least 5% PD-L1 expression. PD-L1 and PD-1 are commonly expressed on glioblastoma-infiltrating T cells. Expression of PD-L1 is prognostic by multivariate analysis for a negative glioblastoma outcome by both immunohistochemistry (p = 0.018) and mRNA expression analysis of TCGA data (p = 0.009). Expression of PD-1 mRNA is correlated with perforin 1 (P 0.0001), both markers of cytolytic activity, as well as the T cell markers CD3 (P < 0.0001), CD4 (P < 0.0001) and CD8 (P < 0.0001). PD-L1 mRNA expression is correlated with granzyme A (P < 0.0001), CD3 (P = 0.0099) and CD4 (P < 0.001). CONCLUSIONS: The incidence of PD-L1 expression in glioblastoma patients is frequent but is confined to a minority subpopulation-similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome.

INTRODUCTION: Exosomes secreted by cancer cells have pleiotropic functions, and can promote autocrine signaling to distant cells. Elucidating the mechanistic modulation of the immune system by these exosomes provides insight into potential biomarkers for detection, recurrence, and response, and identifies potential new therapeutic targets. METHODS: Exosomes were isolated from human glioblastoma stem cells (GSC) and fibroblasts (control) using differential centrifugation. Fluorescent-labeled exosomes were co-cultured with human peripheral blood mononuclear cells (PBMCs). Flow cytometry and confocal microscopy methods were utilized to determine the ability of immune cells to uptake exosomes and to evaluate subsequent intracellular trafficking. The exosomal protein and RNA content was analyzed by mass spectrometry and Nanostring Counter System, respectively. The phenotypic and functional skewing of the monocyte lineage was analyzed after exposing these cells to exosomes. The cytokine array was used to analyze the cytokines generated following treatment of human normal monocytes with exosomes. RESULTS: The GSC-secreted exosomes were preferentially absorbed by CD14+ monocytes and Gr-1+ derived myeloid cells isolated from healthy volunteers and/or glioblastoma patients. When activated, CD4+ and CD8+ T cells could also uptake GSC-secreted exosomes. Confocal microscopy revealed that only monocytes could internalize GSC-secreted exosomes but not fibroblast-secreted exosomes. The exposure to GSC-secreted exosomes induces a phenotypic change in monocytes and prevents them from undergoing apoptosis. GSC-secreted exosomes, but not the fibroblast-secreted exosomes, increased expression of CD163, CD206, and decreased expression of MHC class II. Monocytes treated with GSC-secreted exosomes release IL-6, IL-1RA, CCL3, and CCL4 when compared to cells exposed to fibroblast exosomes. GSC-secreted exosomes contained distinct protein composition in contrast to fibroblast-secreted exosomes that may affect the anti-tumor function of monocyte-derived macrophages. CONCLUSIONS: Monocytes demonstrated a preferential uptake of GSC-secreted exosomes which then induced a glioma-supportive M2 phenotype. GSC-secreted exosomes can be a contributing factor in the M2 skewing within glioma microenvironment.

BACKGROUND: The addition of anti-angiogenic therapy to the few treatments that are available to patients with malignant gliomas was based on the fact that these tumors are highly vascularized and on findings from initial preclinical and clinical studies which showed encouraging results. However, tumors that initially respond to this therapy invariably recur with the acquisition of highly aggressive and invasive phenotype. Although several myeloid populations have been associated to the recurrence of these tumors, a specific targetable population has not been yet identified as responsible for the heightened invasion observed upon antiangiogenesis therapies. METHODS: Immunocompromised mice and immunocompetent GFP macrophage Fas-induced apoptosis (MAFIA) transgenic mice were used to model the response of malignant gliomas to several antiangiogenesis therapies, such as bevacizumab, VEGF Trap, DC101, and to temozolomide. Analysis of tumors and monocytic populations were performed by immunohistochemistry and double immunofluorescence. In vitro studies encompass the use of isolated monocytes from donors and a novel experimental model based on a THP1 human monocytic cell line. Data was corroborated in surgical specimens from human malignant gliomas RESULTS: Here we present evidence for the accumulation of Tie2-expressing cells at the tumor/normal brain interface of immunocompromised mice treated with anti-VEGF therapies in regions with heightened tumoral invasion. This cell population was further characterized as having markers of M2 polarized monocytes/macrophages compatible with Tie2-expressing monocytes (TEMs). We validated these results using the immunocompetent MAFIA transgenic mice treated with an anti-VEGFR compound. In contrast, TEMs were almost undetectable in the brains of glioma-bearing mice treated with temozolomide or anti-VEGF therapies that did not result in an invasive tumoral pattern. In vitro studies showed that TEMs enhanced the invasive properties of glioma cells compared with monocytes that did not express Tie2 (non-TEMs). Moreover, TEMs displayed a higher gelatinase enzymatic activity and, specifically, secreted higher levels of MMP2 and MMP9 than non-TEMs did. Consistently Tie2/MMP9 monocytic cells were detected in the invasive tumoral edge of mice treated with anti-VEGF therapies. Of clinical relevance, we detected the presence of TEMs in human surgical specimens after anti-VEGF therapy. CONCLUSIONS: Collectively, our results described the association of a specific myeloid/monocytic subpopulation characterized as Tie2-expressing monocytes with the heightened invasion observed after antiangiogenic therapy. Our results suggest that his distinctive population might explain the mechanism of escape of malignant gliomas to anti-angiogenesis therapies. Therefore, combination of antiangiogenesis therapies with approaches to decrease the tumor recruitment of TEMs might be required to obtain a therapeutic effect. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

The immune therapeutic potential of microRNAs (miRNAs) in the context of tumor-mediated immune suppression has not been previously described for monocyte-derived glioma-associated macrophages, which are the largest infiltrating immune cell population in glioblastomas and facilitate gliomagenesis.An miRNA microarray was used to compare expression profiles between human glioblastoma-infiltrating macrophages and matched peripheral monocytes. The effects of miR-142-3p on phenotype and function of proinflammatory M1 and immunosuppressive M2 macrophages were determined. The therapeutic effect of miR-142-3p was ascertained in immune-competent C57BL/6J mice harboring intracerebral GL261 gliomas and in genetically engineered Ntv-a mice bearing high-grade gliomas. Student t test was used to evaluate the differences between ex vivo datasets. Survival was analyzed with the log-rank test and tumor sizes with linear mixed models and F test. All statistical tests were two-sided.miR-142-3p was the most downregulated miRNA (approximately 4.95-fold) in glioblastoma-infiltrating macrophages. M2 macrophages had lower miR-142-3p expression relative to M1 macrophages (P = .03). Overexpression of miR-142-3p in M2 macrophages induced selective modulation of transforming growth factor beta receptor 1, which led to subsequent preferential apoptosis in the M2 subset (P = .01). In vivo miR-142-3p administration resulted in glioma growth inhibition (P = .03, n = 5) and extended median survival (miR-142-3p-treated C57BL/6J mice vs scramble control: 31 days vs 23.5 days, P = .03, n = 10; miR-142-3p treated Ntv-a mice vs scramble control: 32 days vs 24 days, P = .03, n = 9), with an associated decrease in infiltrating macrophages (R (2) = .303).These data indicate a unique role of miR-142-3p in glioma immunity by modulating M2 macrophages through the transforming growth factor beta signaling pathway.

Malignant gliomas contain stroma and a variety of immune cells including abundant activated microglia/macrophages. Mounting evidence indicates that the glioma microenvironment converts the glioma-associated microglia/macrophages (GAMs) into glioma-supportive, immunosuppressive cells; however, GAMs can retain intrinsic anti-tumor properties. Here, we review and discuss this duality and the potential therapeutic strategies that may inhibit their glioma-supportive and propagating functions.

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b(+) cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b(+) cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas.

Meeting abstracts The immune checkpoints, CTLA-4 and PD-1, are negative regulators of T cell activation and are inducers of FoxP3+ Tregs. Monoclonal antibody therapy to each has demonstrated tumor regression in clinical trials and potent therapeutic synergy has been observed when used in

Monocytes/macrophages are an influential component of the glioma microenvironment. However, understanding their diversity and plasticity constitute one of the most challenging areas of research due to the paucity of models to study these cells' inherent complexity. Herein, we analyzed the role of monocytes/macrophages in glioma growth by using a transgenic model that allows for conditional ablation of this cell population. We modeled glioma using intracranial GL261-bearing CSF-1R–GFP+ macrophage Fas-induced apoptosis (MAFIA) transgenic mice. Conditional macrophage ablation was achieved by exposure to the dimerizer AP20187. Double immunofluorescence was used to characterize M1- and M2-like monocytes/macrophages during tumor growth and after conditional ablation. During glioma growth, the monocyte/macrophage population consisted predominantly of M2 macrophages. Conditional temporal depletion of macrophages reduced the number of GFP+ cells, targeting mainly the repopulation of M2-polarized cells, and altered the appearance of M1-like monocytes/macrophages, which suggested a shift in the M1/M2 macrophage balance. Of interest, compared with control-treated mice, macrophage-depleted mice had a lower tumor mitotic index, microvascular density, and reduced tumor growth. These results demonstrated the possibility of studying in vivo the role and phenotype of macrophages in gliomas and suggested that transitory depletion of CSF-1R+ population influences the reconstitutive phenotypic pool of these cells, ultimately suppressing tumor growth. The MAFIA model provides a much needed advance in defining the role of macrophages in gliomas.