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Background Extracellular vesicles (EVs) play an important role in cell–cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. Here, we investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic, and proteomic alterations of original tumors. Methods EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis. Results Levels of circulating EVs were elevated in meningioma patients compared to healthy individuals, and the plasma EV concentration correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection. Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in almost all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins that could potentially be utilized to enrich meningioma EVs from biofluids. Conclusions Elevated EV levels in meningioma patient plasma could aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.

Flow-cytometry gating strategy for T-cell memory/activation and ectoenzymatic exhaustion markers.

Supplemental data of next-generation T-cell receptor sequencing.

Supplementary Figure and Table Legends

Additional cytokines determined by multiplex flow-cytometry after PHA stimulation of PBL.

Purpose: Immunotherapeutic treatment strategies for glioblastoma (GBM) are under investigation in clinical trials. However, our understanding of the immune phenotype of GBM-infiltrating T cells (tumor-infiltrating lymphocytes; TILs) and changes during disease progression is limited. Deeper insight is urgently needed to therapeutically overcome tumor-induced immune exhaustion.Experimental Design: We used flow cytometry and cytokine assays to profile TILs and peripheral blood lymphocytes (PBLs) from patients with GBM, comparing newly diagnosed or recurrent GBM to long-term survivors (LTS) and healthy donors. TCR sequencing was performed on paired samples of newly diagnosed and recurrent GBM.Results: We identified a clear immune signature of exhaustion and clonal restriction in the TILs of patients with GBM. Exhaustion of CD8+ TILs was defined by an increased prevalence of PD-1+, CD39+, Tim-3+, CD45RO+, HLA-DR+ marker expression, and exhibition of an effector-/transitional memory differentiation phenotype, whereas KLRG1 and CD57 were underrepresented. Immune signatures were similar in primary and recurrent tumors; however, restricted TCR repertoire clonality and a more activated memory phenotype were observed in TILs from recurrent tumors. Moreover, a reduced cytokine response to PHA stimulation in the blood compartment indicates a dysfunctional peripheral T-cell response in patients with GBM. LTS displayed a distinct profile, with abundant naïve and less exhausted CD8+ T cells.Conclusions: TILs and PBLs exhibit contrasting immune profiles, with a distinct exhaustion signature present in TILs. While the exhaustion profiles of primary and recurrent GBM are comparable, TCR sequencing demonstrated a contracted repertoire in recurrent GBM, concomitant with an increased frequency of activated memory T cells in recurrent tumors. Clin Cancer Res; 24(17); 4187–200. ©2018 AACR.See related commentary by Jackson and Lim, p. 4059

Supplemental Table 1. Patient characteristics for flow-cytometry analysis. IDH=isocitrate dehydrogenase, MGMT=O6-Methylguanin-DNA-Methyltransferase. Supplemental Table 2. Matched-paired GBM patient samples for TCR repertoire sequencing analysis. T=radiotherapy, CT=chemotherapy with temozolomide Supplemental Table 3. Antibodies used to polychromatic flow-cytometry analysis of T-cells

Flow-cytometry gating strategy for T-cell differentiation and exhaustion markers.

BACKGROUND Primary CNS lymphoma represents a malignant disease with dismal prognosis. Standard of care is high dose chemotherapy and radiation. However, this combination cannot be applied to the elderly and fragile population. Immunotherapy holds great promise to be effective in these patients. This study therefore aims to explore the phenotype of tumor-infiltrating lymphocytes (TIL) in order to analyze the potential for immune checkpoint inhibition. MATERIAL AND METHODS We performed ex vivo multicolor flow-cytometry on surgical specimens of nine patients with intracerebral lymphoma, including seven with primary CNS lymphoma after isolation of TILs following standard protocols. Data was analyzed using a Fortessa LSR flow cytometer and Diva software. The study was approved by the local ethics committee (PV4904). RESULTS Our ex vivo phenotyping demonstrated a predominant infiltration of CD8+ T cells, which outnumber CD4+ T cells by a ratio of 2:1 (p CONCLUSION Taken together, our study demonstrates a strong infiltration of cytotoxic CD8+ T cells into cerebral lymphoma, which potentially can be disinhibited using checkpoint immunotherapy. Our profiling suggests that PD-1 and TIGIT present appealing targets for such kind of immune disinhibition.

Immunotherapeutic strategies are increasingly important in neuro-oncology, and the elucidation of escape mechanisms that lead to treatment resistance is crucial. We investigated the impact of immune pressure on the clonal dynamics and immune escape signature by comparing glioma growth in immunocompetent versus immunodeficient mice. Glioma-bearing WT and Pd-1(–/–) mice survived significantly longer than immunodeficient Pfp(–/–) Rag2(–/–) mice. While tumors in Pfp(–/–) Rag2(–/–) mice were highly polyclonal, immunoedited tumors in WT and Pd-1(–/–) mice displayed reduced clonality with emergence of immune escape clones. Tumor cells in WT mice were distinguished by an IFN-γ–mediated response signature with upregulation of genes involved in immunosuppression. Tumor-infiltrating stromal cells, which include macrophages/microglia, contributed even more strongly to the immunosuppressive signature than the actual tumor cells. The identified murine immune escape signature was reflected in human patients and correlated with poor survival. In conclusion, immune pressure profoundly shapes the clonal composition and gene regulation in malignant gliomas.

BACKGROUND We previously demonstrated that extracellular vesicles (EV) from CNS tumors reflect the molecular subtype of the original tumor and mediate an exchange of pro-oncogenic signals. EVs are commonly characterized by nanoparticle analysis (NTA), electron microscopy and tetraspanin markers, including CD9, CD81 and CD63. It is unclear, however, to what extent circulating tumor EVs are utilizable for diagnostic purposes and how their marker profile overlaps with EVs derived from other cell types. Aiming to define markers that allow distinction and enrichment of glioma EVs from patient blood, we utilized Imaging Flow Cytometry (IFC) to discriminate single EVs via multiple surface markers. METHODS EVs were isolated from blood of patients suffering from glioblastoma (n=24), anaplastic astrocytoma (n=8), brain metastasis (n=7), meningioma (n=12), pituitary gland tumor (n=11), epilepsy (n=11) and from healthy controls (n=18) and were analyzed by IFC, immunoblotting, electron microscopy and NTA. In addition, circulating EVs from PALM-GFP-GL261 and PALM-GFP-CT2A tumor-bearing mice (n=5) as well as from glioblastoma stem-like (GSC) cultures (n=4), neural stem cells (NSC), cerebral endothelial cells (cEC) and T-cells (n=4) were characterized. RESULTS CNS tumor patients have significantly elevated levels of circulating EVs (P < 0.001), as measured by NTA and IFC. In particular, the proportion of double positive CD9+/CD81+, CD9+/CD63+and CD63+/CD81+EVs is increased in glioblastoma patients (p=0.018) compared with healthy controls[L1]. In accordance, cultured GSCs secrete increased levels of CD9+/CD81+EVs in vitro. In the two syngeneic murine PALM-GFP glioma models, only 0.1-0.01% of circulating plasma EVs were found to be derived from intracranial tumors, underlining the need to identify markers that can enrich tumor-specific EVs for molecular profiling. CONCLUSION Glioma patients display increased levels of circulating plasma EVs that can be profiled by IFC, which is a unique and novel technique that facilitates discrimination of different EV subpopulations.

BACKGROUND Extracellular vesicles (EVs) play an important role in cell-cell communication in different types of tumors, carrying multiple layers of biological functional molecules, including proteins, RNA, DNA and lipids. Their implication as biomarkers in tumor disease is under current investigation. We previously showed that EVs in glioblastoma reflect the tumor subtype and that glioblastoma patients have elevated circulating particle counts. Regarding to meningioma, it is not known to what extent these usually benign tumors secrete EVs and how these EVs reflect the tumor. Here we report the first study that analyzed meningioma cell-derived EVs. METHODS Meningioma tissue, short-term cell cultures and cell culture-derived EVs (menEVs), (n=4) were analyzed by global mass-spectromety, immunoblotting and imaging flow cytometry and compared to EVs from glioblastoma short-term cell cultures (gEVs), (n=4). Plasma EVs from meningioma patients (n = 12) were analyzed for their tetraspanin marker expression (CD9, CD81 and CD63). EVs were further analyzed by nanoparticle analysis (NTA) and electron microscopy. RESULTS menEVs were 110-140nm in size and exhibited vesicular structures by electron microscopy. We identified 269 proteins in menEVs through mass spectometry. 45 proteins were upregulated in menEVs compared to short-term cell culture and original tumor tissue. 99 proteins were exclusively found in menEVs compared to gEVs, with osteopontin being the top highly expressed protein within the mEV fraction. Both meningioma and glioblastoma patients have elevated circulating plasma EV counts (p< 0.01), as measured by NTA. CONCLUSION The increase in circulating plasma EVs in meningioma patients suggests that tumor cell-derived EVs augment the pool of circulating EVs and could be utilized to obtain information on the tumor by liquid biopsy. Osteopontin is known to be expressed at high levels in meningiomas and its association with menEVs may facilitate isolation of circulating meningioma-specific EVs for analysis.

Background. The dichotomy between glioblastoma cell migration and proliferation is regulated by various parameters including oxygen tension. In glioblastoma stem-like cells, hypoxia induces downregulation of pentose phosphate pathway (PPP) enzymes and a flux shift towards glycolysis. We investigated whether the 2 parallel glucose metabolic pathways are intrinsically linked with cell function and whether these pathways are mechanistically involved in regulating functional programs. Methods. Enzyme expression, migration, and proliferation under hypoxia were studied in multiple cell types. Rapidly and slowly dividing or migrating glioblastoma cells were separated, and enzyme profiles were compared. Glucose-6-phosphate dehydrogenase (G6PD) and Aldolase C (ALDOC), the most strongly inversely regulated PPP and glycolysis enzymes, were knocked down by short hairpin RNA. Results. Hypoxia caused downregulation of PPP enzymes and upregulation of glycolysis enzymes in a broad spectrum of cancer and nonneoplastic cells and consistently stimulated migration while reducing proliferation. PPP enzyme expression was increased in rapidly dividing glioblastoma cells, whereas glycolysis enzymes were decreased. Conversely, glycolysis enzymes were elevated in migrating cells, whereas PPP enzymes were diminished. Knockdown of G6PD reduced glioblastoma cell proliferation, whereas ALDOC knockdown decreased migration. Enzyme inhibitors had similar effects. G6PD knockdown in a highly proliferative but non-invasive glioblastoma cell line resulted in prolonged survival of mice with intracerebral xenografts, whereas ALDOC knockdown shortened survival. In a highly invasive glioblastoma xenograft model, tumor burden was unchanged by either knockdown. Conclusions. Cell function and metabolic state are coupled independently of hypoxia, and glucose metabolic pathways are causatively involved in regulating "go or grow" cellular programs.

BACKGROUND Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumor classification in central nervous system (CNS) tumors. Extracellular vesicles (EVs) are released by CNS tumor cells and contain high molecular weight tumor DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioma-derived EVs reflects genome-wide tumor methylation profiles and allows tumor subtype classification. METHODS DNA was isolated from EVs secreted by cultured glioma stem-like cells (GSC) as well as from the cells of origin and from the original tumor samples (n=3 patients). EVs were classified by nanoparticle analysis (NTA), immunoblotting, imaging flow cytometry (IFCM), multiplex EV assays and electron microscopy. Genome-wide DNA methylation profiling was performed using an 850k Illumina EPIC array and results were classified according to the DKFZ brain tumor classifier. RESULTS The size range of GSC-derived EVs was 120–150 nm, as measured by NTA. The majority of secreted EVs exhibited high expression of common EV markers (i.e. CD9, CD63 and CD81), as characterized by IFCM and multiplex EV assays. Genome-wide methylation profiling of GSC-derived EVs correctly identified the methylation class of the original tumor, including information on the IDH mutation status and subclass classification (RTK1, RTK2). In addition, copy number alterations and the MGMT metyhlation status matched the pattern of the parental GSCs and original tumor samples. CONCLUSION EV DNA faithfully reflects the tumor methylation class as well as the MGMT methylation status and copy number variations present in the parental cells and the original tumor. Methylation profiling of circulating tumor EV DNA could become a useful tool to detect and classify CNS tumors.

Background Aerobic glycolysis confers several advantages to tumor cells, including shunting of metabolites into anabolic pathways. In glioblastoma cells, hypoxia induces a flux shift from the pentose phosphate pathway toward glycolysis and a switch from proliferation to migration. The mechanistic link between glycolysis and migration is poorly understood. Since glucose-6-phosphate isomerase (GPI) is identical to the secreted cytokine autocrine motility factor (AMF), we investigated whether GPI/AMF regulates glioblastoma cell invasion. Methods The expression and hypoxic regulation of GPI/AMF and its receptor AMFR were analyzed in glioblastoma tissue and cell lines. Functional effects were studied in vitro and in xenograft models. Results High GPI/AMF expression in glioblastomas was found to be associated with a worse patient prognosis, and levels were highest in hypoxic pseudopalisades. Hypoxia upregulated both GPI/AMF and AMFR expression as well as GPI/AMF secretion in vitro. GPI/AMF stimulated cell migration in an autocrine fashion, and GPI/AMF expression was upregulated in migratory cells but reduced in rapidly proliferating cells. Knockdown or inhibition of GPI/AMF reduced glioblastoma cell migration but in part stimulated proliferation. In a highly invasive orthotopic glioblastoma model, GPI/AMF knockdown reduced tumor cell invasion but did not prolong survival. In a highly proliferative model, knockdown tumors were even larger and more proliferative than controls; however, perivascular invasion, provoked by simultaneous bevacizumab treatment, was reduced. Conclusions GPI/AMF is a potent motogen for glioblastoma cells, explaining in part the association between glycolysis and migration. Targeting GPI/AMF is, however, problematic, since beneficial anti-invasive effects may be outweighed by unintended mitogenic effects. Key points 1.Increased glycolysis is linked with increased cell migration and invasion in glioblastoma cells. 2.The glycolysis enzyme GPI/AMF may serve as a target for antimetabolic and anti-invasive therapy. 3.Despite reducing tumor invasion, GPI/AMF targeting may have unwanted growth stimulatory effects.

BACKGROUND: Extracellular vesicles (EVs) are known for their important role in cancer progression and hold considerable potential as tumor biomarkers. However, purification of tumor-specific EVs from plasma is still a pressing need since contamination by normal host cell-EVs, results in compromised analytical sensitivity. Fatty acid synthase (FASN) was recently shown by us to be present on EVs from cultured glioma cells. Here we analyzed circulating patient EVs for the expression of FASN, CD9, CD81 and CD63. METHODS: Plasma EVs from patients with glioblastoma (n=24), anaplastic astrocytoma (n=6) and healthy controls (n=16) as well as EVs from early passage glioma stem cells (GSCs) were analyzed for FASN, CD9, CD81, CD63 by imaging flow cytometry (IFC). EVs were further investigated by nanoparticle tracking analysis (NTA), electron microscopy and immunoblotting. Glioblastomas, anaplastic astrocytomas and normal brain tissue specimens were analyzed for FASN by Western blotting and immunohistochemistry. RESULTS: FASN is elevated in glioblastoma tissue compared to noncancerous brain and FASN expression levels correlate with WHO grade (p+/CD81(+)and FASN(+)/CD63(+)EVs are present in plasma from patients with glioblastoma and anaplastic astrocytoma as determined by IFC (p+EVs and double positive (CD9(+)/CD63(+), CD9(+)/CD81(+)or CD63(+)/CD81(+)) EVs (p CONCLUSION: FASN expression is elevated on circulating EVs from patients with malignant gliomas and FASN is a promising marker for the identification and enrichment of glioma-derived plasma EVs, an important prerequisite for in-depth genetic, epigenetic and transcriptional analyses that could inform clinicians on molecular alterations and help monitoring treatment efficacy.

Fluctuations in oxygen tension during tissue remodeling impose a major metabolic challenge in human tumors. Stem-like tumor cells in glioblastoma, the most common malignant brain tumor, possess extraordinary metabolic flexibility, enabling them to initiate growth even under non-permissive conditions. We identified a reciprocal metabolic switch between the pentose phosphate pathway (PPP) and glycolysis in glioblastoma stem-like (GS) cells. Expression of PPP enzymes is upregulated by acute oxygenation but downregulated by hypoxia, whereas glycolysis enzymes, particularly those of the preparatory phase, are regulated inversely. Glucose flux through the PPP is reduced under hypoxia in favor of flux through glycolysis. PPP enzyme expression is elevated in human glioblastomas compared to normal brain, especially in highly proliferative tumor regions, whereas expression of parallel preparatory phase glycolysis enzymes is reduced in glioblastomas, except for strong upregulation in severely hypoxic regions. Hypoxia stimulates GS cell migration but reduces proliferation, whereas oxygenation has opposite effects, linking the metabolic switch to the "go or grow" potential of the cells. Our findings extend Warburg's observation that tumor cells predominantly utilize glycolysis for energy production, by suggesting that PPP activity is elevated in rapidly proliferating tumor cells but suppressed by acute severe hypoxic stress, favoring glycolysis and migration to protect cells against hypoxic cell damage.

BACKGROUND: The "go or grow" hypothesis states that glioma cells either favor proliferation at the expense of migration or migration at the expense of proliferation. This dichotomy is regulated by multiple factors, including ECM components, microRNAs and transcription factors. We recently found that glucose metabolism also contributes to this dichotomy and discovered an oxygen-dependent metabolic switch between the pentose phosphate pathway (PPP) and the preparatory phase of glycolysis in glioma stem-like cells and tissues, which is associated with distinct phenotypes. Expression of glycolysis enzymes and glycolytic flux are increased by hypoxia and associated with cell migration, whereas the PPP is favored by oxygenation and associated with proliferation (Kathagen et al., Acta Neuropathol 2013). Building on these findings, we now studied the causal regulatory involvement of glycolysis versus the PPP in migration versus proliferation. METHODS: Enzyme inhibitors and lentiviral shRNA transduction were used to modulate the expression and activity of enzymes of glycolysis and the PPP. Proliferation, migration and clonogenicity were analyzed in vitro. Tumor initiation and growth were studied in an orthotopic nude mouse model. RESULTS: We identified G6PD and ALDOC as the most strongly inversely regulated enzymes of the PPP and glycolysis, respectively. Stable knockdown of G6PD, the first and key regulatory PPP enzyme, resulted in strikingly reduced proliferation but increased migration of GS cells. Conversely, knockdown of ALDOC inhibited migration while favoring proliferation. Similar results were obtained using enzyme inhibitors. The G6PD inhibitor 6-aminonicotinamide inhibited proliferation while stimulating migration, whereas erythrose-4-phosphate, an inhibitor of the glycolysis enzyme glucose-6-phosphate-isomerase (GPI) decreased migration. 2-deoxyglucose, a hexokinase inhibitor inhibited both proliferation and migration. Knockdown of G6PD in a highly proliferative but non-invasive conventional serum-grown glioblastoma cell line resulted in prolonged survival in vivo, whereas knockdown of ALDOC shortened survival. Interestingly, GPI is also known as a secreted factor termed autocrine motility factor (AMF), which binds to the surface receptor AMFR. Hypoxia strongly increased the secretion of GPI/AMF from GS cells as well as the expression of AMFR. Recombinant human GPI/AMF stimulated the migration of GS cells even more strongly than hepatocyte growth factor, a known potent glioma cell motogen. CONCLUSIONS: By interference with key enzymes of glycolysis and the PPP we demonstrated a direct causal relevance of the two pathways for the "go or grow" behaviour of GS cells, respectively. The identification of GPI/AMF as an extraordinarily potent motogen for GS cells, explains in part the pro-migratory effect of glycolysis. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.

The Warburg effect describes the metabolic redirection in tumor cells away from oxidative phosphorylation to aerobic glycolysis, a status even enhanced by hypoxia. We recently discovered an oxygen concentration-dependent reciprocal metabolic switch between glycolysis and the pentose phosphate pathway, which is associated with a pro-migratory as opposed to a pro-proliferative phenotype, respectively (Kathagen et al., Acta Neuropathol 2013). However, the mechanistic link between glycolysis and cell migration is poorly understood. Since the hypoxia-induced glycolytic enzyme glucose 6-phosphate isomerase (GPI) is identical to autocrine motility factor (AMF), a secreted cytokine that binds to the cell surface receptor AMFR, we studied the functional effects of GPI/AMF on glioblastoma cells. Recombinant GPI/AMF strongly stimulated the migration of glioblastoma stem-like (GS) cells in modified Boyden chamber assays and was even more powerful than Hepatocyte Growth Factor/Scatter Factor, a known highly potent glioma cell motogen. In contrast, GPI/AMF moderately reduced the proliferation of GS cells. Erythrose 4-phosphate, an inhibitor of GPI/AMF activity, abrogated the pro-migratory effect of GPI/AMF while having no effect on GS cell proliferation. Gene expression profiling, qPCR and Western Blot analyses revealed a strong upregulation of GPI/AMF as well as of AMFR expression in response to hypoxia. Furthermore, the amount of secreted GPI/AMF in supernatants of GS cells was strikingly increased under hypoxia. GS cell-conditioned medium had a pro-migratory effect on cells, which was abrogated by immunodepletion of GPI/AMF from the culture medium. Knockdown of GPI/AMF expression by shRNA reduced GS cell migration but had no effect on proliferation. Immunostaining of human glioblastoma sections revealed increased expression of both GPI/AMF and AMFR in hypoxic pseudopalisading regions. To conclude, GPI/AMF is an extraordinarily potent motogen for GS cells, which explains in part the association between increased glycolytic activity. GPI/AMF may be thus be a valuable target for inhibiting glioblastoma cell invasion.