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High vascularization is a biological characteristic of glioblastoma (GBM); however, an in-vitro experimental model to verify the mechanism and physiological role of vasculogenesis in GBM is not well-established. Recently, we established a self-organizing vasculogenic model using human umbilical vein endothelial cells (HUVECs) co-cultivated with human lung fibroblasts (hLFs). Here, we exploited this system to establish a realistic model of vasculogenesis in GBM. We developed two polydimethylsiloxane (PDMS) devices, a doughnut-hole dish and a 5-lane microfluidic device to observe the contact-independent effects of glioblastoma cells on HUVECs. We tested five patient-derived and five widely used GBM cell lines. Confocal fluorescence microscopy was used to observe the morphological changes in Red Fluorescent Protein (RFP)-HUVECs and fluorescein isothiocyanate (FITC)-dextran perfusion. The genetic and expression properties of GBM cell lines were analyzed. The doughnut-hole dish assay revealed KNS1451 as the only cells to induce HUVEC transformation to vessel-like structures, similar to hLFs. The 5-lane device assay demonstrated that KNS1451 promoted the formation of a vascular network that was fully perfused, revealing the functioning luminal construction. Microarray analysis revealed that KNS1451 is a mesenchymal subtype of GBM. Using a patient-derived mesenchymal GBM cell line, mature de-novo vessel formation could be induced in HUVECs by contact-independent co-culture with GBM in a microfluidic device. These results support the development of a novel in vitro research model and provide novel insights in the neovasculogenic mechanism of GBM and may potentially facilitate the future detection of unknown molecular targets. Supplementary Information The online version of this article (10.1007/s11033-020-06061-7) contains supplementary material, which is available to authorized users.

Background Glioblastoma (GBM) is a complex disease with extensive molecular and transcriptional heterogeneity. GBM can be subcategorized into four distinct subtypes; tumors that shift towards the mesenchymal phenotype upon recurrence are generally associated with treatment resistance, unfavorable prognosis, and the infiltration of pro-tumorigenic macrophages. Results We explore the transcriptional regulatory networks of mesenchymal-associated tumor-associated macrophages (MA-TAMs), which drive the malignant phenotypic state of GBM, and identify macrophage receptor with collagenous structure (MARCO) as the most highly differentially expressed gene. MARCOhigh TAMs induce a phenotypic shift towards mesenchymal cellular state of glioma stem cells, promoting both invasive and proliferative activities, as well as therapeutic resistance to irradiation. MARCOhigh TAMs also significantly accelerate tumor engraftment and growth in vivo. Moreover, both MA-TAM master regulators and their target genes are significantly correlated with poor clinical outcomes and are often associated with genomic aberrations in neurofibromin 1 (NF1) and phosphoinositide 3-kinases/mammalian target of rapamycin/Akt pathway (PI3K-mTOR-AKT)-related genes. We further demonstrate the origination of MA-TAMs from peripheral blood, as well as their potential association with tumor-induced polarization states and immunosuppressive environments. Conclusions Collectively, our study characterizes the global transcriptional profile of TAMs driving mesenchymal GBM pathogenesis, providing potential therapeutic targets for improving the effectiveness of GBM immunotherapy.

Background Programmed cell death protein-1 (PD-1) blockade therapy is one of the most remarkable immunotherapy strategies in many solid tumors, excluding glioma. The PD-1 expression, immune characteristics, and prognosis relevance in glioma remain poorly understood. Patients and methods RNA sequencing (RNA-seq) and mRNA microarray data were obtained for 325 and 301 glioma patients, respectively, from the Chinese Glioma Genome Atlas (CGGA) database. We analyzed the expression profile of PDCD1 (encoding PD-1) according to the different grade, isocitrate dehydrogenase (IDH) mutation status, and molecular subtype of glioblastoma. Gene ontology (GO) analyses were performed to explore biological processes of PD-1-related genes. Survival analysis was conducted using the Kaplan-Meier method. The findings were validated using The Cancer Genome Atlas (TCGA) RNA-seq data from 697 glioma samples. We also confirmed the PDCD1 gene expression feature and survival relevance in our own cohort of 73 glioma patients. R language was used for statistical analysis and generating figures. Results PDCD1 was enriched in glioblastoma (WHO, grade IV), IDH wild-type glioma and mesenchymal glioblastoma in CGGA and TCGA datasets; similar results were validated in our own patient cohort. GO analysis revealed that PDCD1-related genes were involved in inflammation immune responses and T cell-mediated immune responses in glioma. Circos plots indicated that PDCD1 was positively associated with CD28, ICOS, and the inhibitory checkpoint molecules CTLA4, HAVCR2, TIGIT, and LAG3. Patients with PDCD1 upregulation had much shorter overall survival. Conclusion PDCD1 upregulation was found in more malignant phenotypes of glioma and indicated a worse prognosis. Immunotherapy of targeting PD-1 or combined with other checkpoint molecules (eg, TIM-3, LAG-3, or TIGIT) blockade may represent a promising treatment strategy for glioma.

Mesenchymal glioblastoma stem cells (GSCs), a subpopulation in glioblastoma that are responsible for therapy resistance and tumor spreading in the brain, reportedly upregulate aldehyde dehydrogenase isoform-1A3 (ALDH1A3) which can be inhibited by disulfiram (DSF), an FDA-approved drug formerly prescribed in alcohol use disorder. Reportedly, DSF in combination with Cu2+ ions exerts multiple tumoricidal, chemo- and radio-therapy-sensitizing effects in several tumor entities. The present study aimed to quantify these DSF effects in glioblastoma stem cells in vitro, regarding dependence on ALDH1A3 expression. To this end, two patient-derived GSC cultures with differing ALDH1A3 expression were pretreated (in the presence of CuSO4, 100 nM) with DSF (0 or 100 nM) and the DNA-alkylating agent temozolomide (0 or 30 µM) and then cells were irradiated with a single dose of 0–8 Gy. As read-outs, cell cycle distribution and clonogenic survival were determined by flow cytometry and limited dilution assay, respectively. As a result, DSF modulated cell cycle distribution in both GSC cultures and dramatically decreased clonogenic survival independently of ALDH1A3 expression. This effect was additive to the impairment of clonogenic survival by radiation, but not associated with radiosensitization. Of note, cotreatment with temozolomide blunted the DSF inhibition of clonogenic survival. In conclusion, DSF targets GSCs independent of ALDH1A3 expression, suggesting a therapeutic efficacy also in glioblastomas with low mesenchymal GSC populations. As temozolomide somehow antagonized the DSF effects, strategies for future combination of DSF with the adjuvant standard therapy (fractionated radiotherapy and concomitant temozolomide chemotherapy followed by temozolomide maintenance therapy) are not supported by the present study.

CD204 is a specific marker of tumor‐associated macrophages (TAMs) in glioma. However, the expression levels of CD204 and its involvement in glioma are not fully understood. In this large‐scale study, we assessed the expression and function of CD204 in whole‐grade glioma molecularly and clinically. In total, 1323 glioma samples, including 301 microarray data and 325 RNA‐seq data from the Chinese Glioma Genome Atlas (CGGA) dataset and 697 RNA‐seq data from The Cancer Genome Atlas (TCGA) dataset, were utilized. The statistical analysis and graphical work were mainly performed using the R software. Univariate and multivariate Cox analysis demonstrated that CD204 was an independent prognosticator in glioma patients. CD204 expression was positively correlated with the grade of malignancy. CD204 was consistently upregulated in wild‐type isocitrate dehydrogenase glioma and highly expressed in mesenchymal glioblastoma. Gene ontology of CD204‐related genes showed that CD204 was most enriched in inflammatory response and immune response. It was associated with the stromal and immune populations, especially the monocytic lineage, fibroblasts, and T cells. Circos plots revealed that CD204 was closely associated with many immune checkpoint regulators, especially TIM‐3. CD204 expression is consistent with the malignant phenotype of glioma and independently predicts poor outcomes in glioma patients. Additionally, CD204+ TAMs, collaborating with other checkpoint members, may contribute to the dysfunction of T cells. These findings suggest that CD204 may be a promising target for glioma immunotherapy.

Mesenchymal glioblastoma (GBM) is the most aggressive subtype of GBM. Our previous study found that neurotrophic factor prosaposin (PSAP) is highly expressed and secreted in glioma and can promote the growth of glioma. The role of PSAP in mesenchymal GBM is still unclear. In this study, bioinformatic analysis, western blotting and RT-qPCR were used to detect the expression of PSAP in different GBM subtypes. Human glioma cell lines and patient-derived glioma stem cells were studied in vitro and in vivo, revealing that mesenchymal GBM expressed and secreted the highest level of PSAP among four subtypes of GBM, and PSAP could promote GBM invasion and epithelial-mesenchymal transition (EMT)-like processes in vivo and in vitro. Bioinformatic analysis and western blotting showed that PSAP mainly played a regulatory role in GBM invasion and EMT-like processes via the TGF-β1/Smad signaling pathway. In conclusion, the overexpression and secretion of PSAP may be an important factor causing the high invasiveness of mesenchymal GBM. PSAP is therefore a potential target for the treatment of mesenchymal GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Low-passage, serum-free cell lines cultured from patient tumour tissue are the gold-standard for preclinical studies and cellular investigations of glioblastoma (GBM) biology, yet entrenched, poorly-representative cell line models are still widely used, compromising the significance of much GBM research. We submit that greater adoption of these critical resources will be promoted by the provision of a suitably-sized, meaningfully-described reference collection along with appropriate tools for working with them. Consequently, we present a curated panel of 12 readily-usable, genetically-diverse, tumourigenic, patient-derived, low-passage, serum-free cell lines representing the spectrum of molecular subtypes of IDH-wildtype GBM along with their detailed phenotypic characterisation plus a bespoke set of lentiviral plasmids for bioluminescent/fluorescent labelling, gene expression and CRISPR/Cas9-mediated gene inactivation. The cell lines and all accompanying data are readily-accessible via a single website, Q-Cell (qimrberghofer.edu.au/q-cell/) and all plasmids are available from Addgene. These resources should prove valuable to investigators seeking readily-usable, well-characterised, clinically-relevant, gold-standard models of GBM.

Glioblastoma is the most malignant tumor occurring in the human central nervous system with overall median survival time

The molecular basis underlying glioblastoma (GBM) heterogeneity and plasticity is not fully understood. Using transcriptomic data of human patient-derived brain tumor stem cell lines (BTSCs), classified based on GBM-intrinsic signatures, we identify the AP-1 transcription factor FOSL1 as a key regulator of the mesenchymal (MES) subtype. We provide a mechanistic basis to the role of the neurofibromatosis type 1 gene (NF1), a negative regulator of the RAS/MAPK pathway, in GBM mesenchymal transformation through the modulation of FOSL1 expression. Depletion of FOSL1 in NF1-mutant human BTSCs and Kras-mutant mouse neural stem cells results in loss of the mesenchymal gene signature and reduction in stem cell properties and in vivo tumorigenic potential. Our data demonstrate that FOSL1 controls GBM plasticity and aggressiveness in response to NF1 alterations.

Background Gliomas represent the most common brain tumors within the central nervous system, with glioblastoma being the most aggressive type.1 Conventional treatment combines several approaches including surgery, chemotherapy, and radiation.2 However, the prognosis for glioblastoma remains unfavorable, with only 5% of patients surviving more than 5 years post-diagnosis.3 Thus, new treatment approaches are urgently needed. Natural killer (NK) cells directly lyse malignantly transformed or virally infected cells and secrete inflammatory cytokines that polarize cytotoxic immunity. Allogeneic NK cell adoptive transfer has shown clinical benefit in patients with advanced cancer.4–7 However, limitations of this approach include relatively low numbers of donor NK cells that can be isolated during an apheresis and variability in the quality of NK cells between donors. To overcome these limitations, we have developed a GMP manufacturing strategy to mass produce NK cells from induced pluripotent stem cells (iPSCs) as an approach to off-the-shelf cancer immunotherapy. We refer to these cells as ‘iNK’ (iPSC-derived NK) cells. Here, we provide preclinical data demonstrating the efficacy of iNK cells for immunotherapy against glioblastoma. Methods We generated iNK cells using previously published methods.8–10 iNK cells were used as effectors against an array of patient-derived glioblastoma lines in 2-dimensional live imaging IncuCyte assays where iNK cell-mediated killing was observed over the course of 48 hours. To investigate iNK cell infiltration and cytotoxicity in a more physiological context that accounts for the 3-dimensional architecture of the tumor, we also performed live imaging IncuCyte assays using iNK cells as effectors against glioblastoma spheroids. To test the anti-tumor function of iNK cells in vivo, we implanted patient-derived glioblastoma cells into mice via intracranial injection. Seven days later, 5 mice received intratumoral injections of iNK cells, and 5 mice received vehicle alone (as a control; figure 1A). All mice were monitored for weight and survival over 100 days. Results iNK cells exhibited strong and sustained cytotoxicity against 6 primary patient-derived mesenchymal glioblastoma lines in 2-dimensional IncuCyte assays and complete infiltration and destruction of glioblastoma spheroids in 3-dimensional IncuCyte assays. In xenogeneic adoptive transfer experiments, all mice receiving intratumoral injections of iNK cells survived out to day 100, while all mice in the vehicle group became moribund and had to be sacrificed by day 60 (figure 1B). Conclusions iNK cells are highly cytotoxic against glioblastoma cells, and our preclinical in vivo data provides proof-of-concept for future clinical trials. Ethics Approval This project has been approved by the University of Minnesota IACUC. Approval ID: 1812-36595A References Louis D N, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee W K, Ohgaki H, Wiestler O D, Kleihues P, Ellison D W. The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 2016;131:803–820. Stupp R, Mason W P, van den Bent M J, Weller M, Fisher B, Taphoorn M J B, Belanger K, Brandes A A, Marosi C, Bogdahn U, Curschmann J, Janzer R C, Ludwin S K, Gorlia T, Allgeier A, Lacombe D, Cairncross J G, Eisenhauer E, Mirimanoff R O, European Organization for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352:987–996. Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Barnholz-Sloan JS, Villano JL. Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol Biomarkers Prev 2017;23:1985–1996. Miller J S, Soignier Y, Panoskaltsis-Mortari A, McNearney S A, Yun G H, Fautsch S K, McKenna D, Le C, Defor T E, Burns L J, Orchard P J, Blazar B R, Wagner J E, Slungaard A, Weisdorf D J, Okazaki J, McGlave P B. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005;105:3051–3057. Bachanova V, Cooley S, Defor T E, Verneris M R, Zhang B, McKenna D H, Curtsinger J, Panoskaltsis-Mortari A, Lewis D, Hippen K, McGlave P, Weisdorf D J, Blazar B R, Miller J S. Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein. Blood 2014;123:3855. Ciurea S O, Schafer J R, Bassett R, Denman C J, Cao K, Willis D, Rondon G, Chen J, Soebbing D, Kaur I, Gulbis A, Ahmed S, Rezvani K, Scpall E J, Lee D A, Champlin R E. Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived NK cells after haploidentical transplant. Blood 2017;130:1857–1868. Romee R, Rosario M, Berrien-Elliott M M, Wagner J A, Jewell B A, Schappe T, Leong J W, Abdel-Latif S, Schneider S E, Willey S, Neal C C, Yu L, Oh T, Lee S, Mulder A, Cooper M A, Fehniger T A. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukiemia. Sci Transl Med2016:8;375ra123. Valamehr B, Abujarour R, Robinson M, Le T, Robbins D, Shoemaker D, Flynn P. A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs. Sci Rep2012:2;213. Valamehr B, Robinson M, Abujarour R, Rezner B, Vranceanu F, Le T, Medcalf A, Lee T T, Fitch M, Robbins D, Flynn P. Platform for induction and maintenance of transgene-free hiPSCs resembling ground state pluripotent stem cells. Stem Cell Reports 2014;2:366–381. Zhu H, Blum R H, Bjordahl R, Gaidarova S, Rogers P, Lee T T, Abujarour R, Bonello G B, Wu J, Tsai P-F, Miller J S, Walcheck B, Valamehr B, Kaufman D S. Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor immunity. Blood 2020;135:399–410.

Inhibitor of nuclear factor kappa‐B kinase subunit epsilon (IKBKE) plays critical roles in the proliferation, invasion, and epithelial‐mesenchymal transition (EMT) of glioblastoma (GBM). However, as an immune response factor, few studies have focused on the role of IKBKE in the glioblastoma‐mediated immunosuppressive microenvironment. Here, we found a higher IKBKE expression level in gliomas corresponding to higher malignancy of the tumor. The highest level of IKBKE expression was examined in the core region of GBM tissues as well as the mesenchymal subtype, which are featured with necrosis, immunocyte infiltration, and immunosuppression. Further in silico analysis demonstrated that the JAK/STAT as the signaling pathway most associated with IKBKE in mesenchymal GBM. The co‐expression patterns of IKBKE, pSTAT3, and PD‐L1 were detected within GBM tissues. Mechanistically, IKBKE could interact with STAT3 and thus enhancing the phosphorylation level of STAT3 and its nuclear translocation. In addition, pSTAT3 could transcriptionally regulate the expression of PD‐L1 through binding to its promoter. In vivo results further confirmed the inhibitory effect of the IKBKE downregulation on tumor growth. Collectively, our findings suggest IKBKE as the central node in the crosstalk between NF‐κB and STAT3 signaling within mesenchymal GBM. Targeting GBM through inhibiting IKBKE could restrain tumor growth and tumor‐mediated immunosuppressive environment., 1. Higher IKBKE expression was detected in higher grades of glioma. 2. IKBKE is enriched in mesenchymal GBM, which confers immunosuppressive signature. 3. IKBKE correlates with JAK/STAT signaling in MES GBM. 4. IKBKE activates the phosphorylation of STAT3, which transcriptionally regulates PD‐L1 expression.

Glioblastoma multiforme is one of the most malignant types of cancer. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine which has been associated with increased chemoresistance, migration, and invasion in glioblastoma. In this study, we attempted to elucidate the mechanisms that control extracellular adenosine levels in GSC subtypes. By using primary and U87MG-derived GSCs, we associated increased extracellular adenosine with the mesenchymal phenotype. [3H]-adenosine uptake occurred mainly through the equilibrative nucleoside transporters (ENTs) in GSCs, but mesenchymal GSCs have lower expression and ENT1-mediated uptake activity than proneural GSCs. By analyzing expression and enzymatic activity, we determined that ecto-5&prime, nucleotidase (CD73) is predominantly expressed in proneural GSCs, driving AMPase activity. While in mesenchymal GSCs, both CD73 and Prostatic Acid Phosphatase (PAP) contribute to the AMP (adenosine monophosphate) hydrolysis. We did not observe significant differences between the expression of proteins involved in the metabolization of adenosine among the GCSs subtypes. In conclusion, the lower expression and activity of the ENT1 transporter in mesenchymal GSCs contributes to the high level of extracellular adenosine that these GSCs present.

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene–deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin α(v)β(5) (ITGαvβ5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8(+) T cell cytotoxicity. Systemic administration in mice of 4-1BB–OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

Background Several tumor entities including brain tumors aberrantly overexpress intermediate conductance Ca2+ activated KCa3.1 K+ channels. These channels contribute significantly to the transformed phenotype of the tumor cells. Method PubMed was searched in order to summarize our current knowledge on the molecular signaling upstream and downstream and the effector functions of KCa3.1 channel activity in tumor cells in general and in glioblastoma cells in particular. In addition, KCa3.1 expression and function for repair of DNA double strand breaks was determined experimentally in primary glioblastoma cultures in dependence on the abundance of proneural and mesenchymal stem cell markers. Results By modulating membrane potential, cell volume, Ca2+ signals and the respiratory chain, KCa3.1 channels in both, plasma and inner mitochondrial membrane, have been demonstrated to regulate many cellular processes such as migration and tissue invasion, metastasis, cell cycle progression, oxygen consumption and metabolism, DNA damage response and cell death of cancer cells. Moreover, KCa3.1 channels have been shown to crucially contribute to resistance against radiotherapy. Futhermore, the original in vitro data on KCa3.1 channel expression in subtypes of glioblastoma stem(-like) cells propose KCa3.1 as marker for the mesenchymal subgroup of cancer stem cells and suggest that KCa3.1 contributes to the therapy resistance of mesenchymal glioblastoma stem cells. Conclusion The data suggest KCa3.1 channel targeting in combination with radiotherapy as promising new tool to eradicate therapy-resistant mesenchymal glioblastoma stem cells.

Rationale: Competitive endogenous RNA (ceRNA) networks play important roles in posttranscriptional regulation. Their dysregulation is common in cancer. However, ceRNA signatures have been poorly examined in the invasive and aggressive phenotypes of mesenchymal glioblastoma (GBM). This study aims to characterize mesenchymal glioblastoma at the mRNA-miRNA level and identify the mRNAs in ceRNA networks (micNET) markers and their mechanisms in tumorigenesis. Methods: The mRNAs in ceRNA networks (micNETs) of glioblastoma were investigated by constructing a GBM ceRNA network followed by integration with a STRING protein interaction network. The prognostic micNET markers of mesenchymal GBM were identified and validated across multiple datasets. ceRNA interactions were identified between micNETs and miR181 family members. LY2109761, an inhibitor of TGFBR2, demonstrated tumor-suppressive effects on both primary cultured cells and a patient-derived xenograft intracranial model. Results: We characterized mesenchymal glioblastoma at the mRNA-miRNA level and reported a ceRNA network that could separate the mesenchymal subtype from other subtypes. Six genes (TGFBR2, RUNX1, PPARG, ACSL1, GIT2 and RAP1B) that interacted with each other in both a ceRNA-related manner and in terms of their protein functions were identified as markers of the mesenchymal subtype. The coding sequence (CDS) and 3'-untranslated region (UTR) of TGFBR2 upregulated the expression of these genes, whereas TGFBR2 inhibition by siRNA or miR-181a/d suppressed their expression levels. Furthermore, mesenchymal subtype-related genes and the invasion phenotype could be reversed by suppressing the six mesenchymal marker genes. Conclusions: This study suggests that the micNETs may have translational significance in the diagnosis of mesenchymal GBM and may be novel therapeutic targets.

The tumor microenvironment is being increasingly recognized as a key factor in cancer aggressiveness. In this study, we characterized the inflammatory gene signatures altered in glioma cell lines and tumor specimens of differing histological and molecular subtypes. The results showed that glioblastoma multiforme (GBM) shows upregulation of a subset of inflammatory genes when compared to astrocytomas and oligodendrogliomas. With molecular subtypes of GBM, the expression of inflammatory genes is heterogeneous, being enriched in mesenchymal and downregulated in Proneural/GCIMP. Other inflammation-associated processes such as tumor-associated macrophage (TAM) signatures are upregulated in mesenchymal, and a subset of 33 mesenchymal-enriched inflammatory and TAM markers showed correlation with poor survival. We found that various GBM tumor-upregulated genes such as IL6, IL8 and CCL2 are also actively expressed in glioma cell lines, playing differential and cooperative roles in promoting proliferation, invasion, angiogenesis and macrophage polarization in vitro. These genes can be stimulated by pathways typically altered in GBM, including the EGFR, PDGFR, MEK1/2-ERK1/2, PI3K/Akt and NFκB cascades. Taken together, the results presented herein depict some inflammatory pathways altered in gliomas and highlight potentially relevant targets to therapy improvement.

// Yuan Pan 1 , Laura J. Smithson 1 , Yu Ma 1 , Dolores Hambardzumyan 2 , David H. Gutmann 1 1 Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA 2 Department of Oncology, Aflac Cancer and Blood Disorders Center, Emory University, GA, USA Correspondence to: David H. Gutmann, email: gutmannd@wustl.edu Keywords: chemokine, glioma, neurofibromin, mTOR, CD44 Received: October 24, 2016 Accepted: March 13, 2017 Published: March 23, 2017 ABSTRACT Glioblastoma (GBM) is the most common malignant brain tumor in adults, with a median survival of 15 months. These poor clinical outcomes have prompted the development of drugs that block neoplastic cancer cell growth; however, non-neoplastic cell-derived signals (chemokines and cytokines) in the tumor microenvironment may also represent viable treatment targets. One such chemokine, Ccl5, produced by low-grade tumor-associated microglia, is responsible for maintaining neurofibromatosis type 1 (NF1) mouse optic glioma growth in vivo . Since malignant gliomas may achieve partial independence from growth regulatory factors produced by non-neoplastic cells in the tumor microenvironment by producing the same cytokines secreted by the stromal cells in their low-grade counterparts, we tested the hypothesis that CCL5/CCL5-receptor signaling in glioblastoma creates an autocrine circuit important for high-grade glioma growth. Herein, we demonstrate that increased CCL5 expression was restricted to both human and mouse mesenchymal GBM (M-GBM), a molecular subtype characterized by NF1 loss. We further show that the NF1 protein, neurofibromin, negatively regulates Ccl5 expression through suppression of AKT/mTOR signaling. Consistent with its role as a glioblastoma growth regulator, Ccl5 knockdown in M-GBM cells reduces M-GBM cell survival in vitro , and increases mouse glioblastoma survival in vivo . Finally, we demonstrate that Ccl5 operates through an unconventional CCL5 receptor, CD44, to inhibit M-GBM apoptosis. Collectively, these findings reveal an NF1 -dependent CCL5-mediated pathway that regulates M-GBM cell survival, and support the concept that paracrine factors important for low-grade glioma growth can be usurped by high-grade tumors to create autocrine regulatory circuits that maintain malignant glioma survival.

Interconversion of transformed non-stem cells to cancer stem cells, termed cancer cell plasticity, contributes to intra-tumor heterogeneity and its molecular mechanisms are currently unknown. Here, we have identified Tenascin C (TNC) to be upregulated and secreted in mesenchymal glioblastoma (MES GBM) subtype with high NF-κB signaling activity. Silencing TNC decreases proliferation, migration and suppresses self-renewal of glioma stem cells. Loss of TNC in MES GBM compromises de-differentiation of transformed astrocytes and blocks the ability of glioma stem cells to differentiate into tumor derived endothelial cells (TDEC). Inhibition of NF-κB activity or TNC knockdown in tumor cells decreased their tumorigenic potential in vivo. Our results uncover a link between NF-κB activation in MES GBM and high levels of TNC in GBM extracellular matrix. We suggest that TNC plays an important role in the autocrine regulation of glioma cell plasticity and hence can be a potential molecular target for MES GBM.

Purpose: Cellular immunotherapies are currently being explored to eliminate highly invasive and chemoradioresistant glioblastoma (GBM) cells involved in rapid relapse. We recently showed that concomitant stereotactic injections of nonalloreactive allogeneic Vγ9Vδ2 T lymphocytes eradicate zoledronate-primed human GBM cells. In the present study, we investigated the spontaneous reactivity of allogeneic human Vγ9Vδ2 T lymphocytes toward primary human GBM cells, in vitro and in vivo, in the absence of any prior sensitization. Experimental Design: Through functional and transcriptomic analyses, we extensively characterized the immunoreactivity of human Vγ9Vδ2 T lymphocytes against various primary GBM cultures directly derived from patient tumors. Results: We evidenced that GBM cells displaying a mesenchymal signature are spontaneously eliminated by allogeneic human Vγ9Vδ2 T lymphocytes, a reactivity process being mediated by γδ T-cell receptor (TCR) and tightly regulated by cellular stress–associated NKG2D pathway. This led to the identification of highly reactive Vγ9Vδ2 T lymphocyte populations, independently of a specific TCR repertoire signature. Moreover, we finally provide evidence of immunotherapeutic efficacy in vivo, in the absence of any prior tumor cell sensitization. Conclusions: By identifying pathways implicated in the selective natural recognition of mesenchymal GBM cell subtypes, accounting for 30% of primary diagnosed and 60% of recurrent GBM, our results pave the way for novel targeted cellular immunotherapies.

Summary Long non-coding RNAs (lncRNAs) have an undefined role in the pathobiology of glioblastoma multiforme (GBM). These tumors are genetically and phenotypically heterogeneous with transcriptome subtype-specific GBM stem-like cells (GSCs) that adapt to the brain tumor microenvironment, including hypoxic niches. We identified hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) as a subtype-specific hypoxia-inducible lncRNA, upregulated in mesenchymal GSCs. Its deregulation affects GSC growth, self-renewal, and hypoxia-dependent molecular reprogramming. Among the HIF1A-AS2 interactome, IGF2BP2 and DHX9 were identified as direct partners. This association was needed for maintenance of expression of their target gene, HMGA1. Downregulation of HIF1A-AS2 led to delayed growth of mesenchymal GSC tumors, survival benefits, and impaired expression of HMGA1 in vivo. Our data demonstrate that HIF1A-AS2 contributes to GSCs' speciation and adaptation to hypoxia within the tumor microenvironment, acting directly through its interactome and targets and indirectly by modulating responses to hypoxic stress depending on the subtype-specific genetic context.

The overall survival for adults with malignant glioma (glioblastoma) remains poor despite advances in radiation and chemotherapy. One of the mechanisms by which cancer cells develop relative resistance to treatment is through de-regulation of endoplasmic reticulum (ER) homeostasis. We have recently shown that ABCG1, an ATP-binding cassette transporter, maintains ER homeostasis and suppresses ER stress-induced apoptosis in low-grade glioma. Herein, we demonstrate that ABCG1 expression is increased in human adult glioblastoma, where it correlates with poor survival in individuals with the mesenchymal subtype. Leveraging a mouse model of mesenchymal glioblastoma (NPcis), shRNA-mediated Abcg1 knockdown (KD) increased CHOP ER stress protein expression and resulted in greater NPcis glioma cell death in vitro. Moreover, Abcg1 KD reduced NPcis glioma growth and increased mouse survival in vivo. Collectively, these results demonstrate that ABCG1 is critical for malignant glioma cell survival, and might serve as a future therapeutic target for these deadly brain cancers.

GBM is a devastating disease with peak incidence in the seventh decade. Pre-clinical models are essential for studying resistance mechanisms and screening novel therapies. However, historically these models have failed to predict response in humans. Current models seldom incorporate surgical resection, and commonly use young animals whose immune contexture differs from older patients. Here, we have established an orthotopic model employing the syngeneic mesenchymal-NFpp10a-cell line which incorporates surgical resection in aged mice. We further characterise response to ICI and temozolomide monotherapy. NFpp10a and GL261-cell lines were exposed in vitro to irradiation (0Gy/2Gy/5Gy) and response assessed using colony formation assays. NFpp10a formed significantly more colonies at 5Gy compared to GL261 at both day 10 (NFpp10a 5.167 vs GL261 1.4; p=0.0017) and day 14 (NFpp10a 3.5 vs GL261 0; p< 0.0001). Hence, NFpp10a displays increased radioresistance. Next, NFpp10a-luciferin expressing cells were orthotopically implanted into young (6-8weeks;n=16) and aged (18months;n=16) C57BL/6-mice. Weekly bioluminescence imaging (BLI) was performed to monitor growth. Mice undergoing resection showed a median 18.47-fold drop in BLI signal. We demonstrated resection survival advantage in aged mice (Resection:33.5 days vs Non-Resection:18 days, p= 0.0166) and showed young age to be a positive prognostic factor (Young:62 days vs Aged:22 days, p=0.0002). Subsequently, we orthotopically implanted NFpp10a-Luc2 cells into C57BL/6 mice and treated with temozolomide (n=24) or PBS control (n=23), and anti-PD1 (n=24) or IgG (n=23). We observed that temozolomide and anti-PD1 monotherapy had no impact on NFpp10-Luc2 growth (temozolomide-overall:p=0.9001, anti-PD1-overall:p=0.7933) or survival (temozolomide-overall:p=0.3035, anti-PD1-overall:p=0.6328). Overall, we have established an NFpp10-Luc2 mesenchymal-GBM model in aged mice which incorporates surgical resection and accurately displays significant resistance to temozolomide and anti-PD1 monotherapy. We are currently employing this model to study the efficacy of neoadjuvant anti-PD1 therapy. Mechanistic analyses with multiplex-immunohistochemistry, scRNA and whole exome sequencing are planned to interrogate treatment effects on the tumor microenvironment.

Summary The molecular basis underlying Glioblastoma (GBM) heterogeneity and plasticity are not fully understood. Using transcriptomic data of patient-derived brain tumor stem cell lines (BTSCs), classified based on GBM-intrinsic signatures, we identify the AP-1 transcription factor FOSL1 as a master regulator of the mesenchymal (MES) subtype. We provide a mechanistic basis to the role of the Neurofibromatosis type 1 gene ( NF1 ), a negative regulator of the RAS/MAPK pathway, in GBM mesenchymal transformation through the modulation of FOSL1 expression. Depletion of FOSL1 in NF1-mutant human BTSCs and Kras-mutant mouse neural stem cells results in loss of the mesenchymal gene signature, reduction in stem cell properties and in vivo tumorigenic potential. Our data demonstrate that FOSL1 controls GBM plasticity and aggressiveness in response to NF1 alterations. Significance Glioblastoma (GBM) is a very heterogenous disease for which multiple transcriptional subtypes have been described. Among these subtypes, the Mesenchymal (MES) GBMs have the worst prognosis. Here we provide the first causal evidence linking Neurofibromatosis type 1 gene (NF1) signalling and the acquisition of a MES gene expression program through the regulation of the AP-1 transcription factor FOSL1 . Using patient expression datasets, combined with in vitro and in vivo gain- and loss-of function mouse models, we show that FOSL1 is an important modulator of GBM that is required and sufficient for the activation of a MES program. Our work sheds light on the mechanisms that control the tumorigenicity of the most aggressive adult brain tumor type.

Glioblastoma (GBM), the deadliest and most common primary CNS malignancy in adults, is highly heterogeneous with variable prognoses and treatment responses. The molecular underpinnings for such variability remain largely unclear. Mesenchymal (MES)-GBMs, associated with poorest prognosis and highest treatment resistance, exhibit hyperactivity of transcriptional coactivator with PDZ-binding motif (TAZ), the major effector of the Hippo tumor suppressive pathway. Additionally, the tumor microenvironment (TME) of MES-GBMs appears to be altered, containing more infiltrative tumor-associated macrophages and microglia than GBMs of other subtypes. Moreover, the Hippo pathway has recently been shown to be involved in suppressing tumor immunogenicity. Yet, it remains elusive whether TAZ dysregulation distorts the TME to contribute to MES transition and tumor progression of MES-GBMs. We suspected that such TME distortion facilitates tumor immune evasion, MES transition, and tumor progression, therefore worsening prognoses and treatment responses. To test this, we generated three GBM cell lines expressing various forms of TAZ: 1) GBM(4SA) expressing constitutively-active TAZ 2) GBM(4SA-S51A) expressing TAZ incapable of binding to its downstream effector, TEAD, and 3) GBM(vector), vector control. We found mice orthotopically implanted with GBM(4SA) phenocopied MES-GBM patients and exhibited significantly shorter survival. Histopathology revealed that GBM(4SA) highly resembles human GBMs, with high cellular and nuclear anaplasia, microvascular proliferation, and pseudopalisading necrosis. Moreover, TME analyses from these GBMs suggested that GBM(4SA) contains significantly more infiltrative myeloid cells than GBM(4SA-S51A) or GBM(vector), suggesting that hyperactivity of TAZ leads to an altered TME. While most infiltrative myeloid cells in the GBM(vector) or GBM(4SA-S51A) TME were of monocyte lineage, those in the GBM(4SA) TME were predominantly granulocytic, likely representing neutrophils. We will further delineate the roles of each myeloid cell type in the MES-GBM TME in driving tumor progression and treatment resistance, and we hope to draw implications for future GBM clinical management and novel TME-targeted anti-GBM therapies.

The homeodomain transcription factor PROX1 has been linked to several cancer types, including gliomas, but its functions remain to be further elucidated. Here we describe a functional role and the prognostic value of PROX1 in glioblastoma. Low expression of PROX1 correlated with poor overall survival and the mesenchymal glioblastoma subtype signature. The latter finding was recapitulated in vitro, where suppression or overexpression of PROX1 in glioma cell cultures transitioned cells to a mesenchymal or to a nonmesenchymal glioblastoma gene expression signature, respectively. PROX1 modulation affected proliferation rates that coincided with changes in protein levels of CCNA1 and CCNE1 as well as the cyclin inhibitors CDKN1A, CDKN1B, and CDKN1C. Overexpression of SOX2 increased PROX1 expression, but treatment with a CDK2 inhibitor subsequently decreased PROX1 expression, which was paralleled by decreased SOX2 levels. The THRAP3 protein was a novel binding partner for PROX1, and suppression of THRAP3 increased both transcript and protein levels of PROX1. Together, these findings highlight the prognostic value of PROX1 and its role as a regulator of glioblastoma gene expression subtypes, intratumoral heterogeneity, proliferation, and cell-cycle control. Significance: These findings demonstrate the role and prognostic value of PROX1 in glioblastomas; low PROX1 levels correlate with a mesenchymal gene expression subtype and shorter survival in glioblastoma tumors. Cancer Res; 78(20); 5901–16. ©2018 AACR.

The most common and aggressive form of primary brain tumor in adults is glioblastoma (GBM). From the global DNA methylation profiling study, previously published from our laboratory, we identified Guanine Nucleotide binding-protein Gamma subunit 4 (GNG4) to be one of the most hyper methylated and down regulated genes in GBM. GBM derived cell lines showed reduced GNG4 transcript levels, which could be reversed by methylation inhibitor treatment. Bisulphite sequencing confirmed the methylation status in glioblastoma tumor tissue and GBM derived cell lines. Overexpression of GNG4 was found to inhibit proliferation and colony formation of GBM cell lines and in vitro transformation of immortalized human astrocytes, thus suggesting a potential tumor suppressor role of GNG4 in GBM. Correlation of GNG4 transcript levels with that of all GPCRs from TCGA data revealed chemokine receptors as the potential target of GNG4. Furthermore, exogenous over expression of GNG4 inhibited SDF1α/CXCR4-dependent chemokine signaling as seen by reduced pERK and pJNK and GBM cell migration. The inhibitory association between GNG4 and SDF1α/CXCR4 was more evident in mesenchymal subtype of GBM. Thus, this study identifies GNG4 as an inhibitor of SDF1α/CXCR4-dependent signaling and emphasizes the significance of epigenetic inactivation of GNG4 in glioblastoma, especially in mesenchymal subtype.

Gliomatosis cerebri (GC) is presently considered a distinct astrocytic glioma entity according to the WHO classification for CNS tumors. It is characterized by widespread, typically bilateral infiltration of the brain involving three or more lobes. Genetic studies of GC have to date been restricted to the analysis of individual glioma-associated genes, which revealed mutations in the isocitrate dehydrogenase 1 (IDH1) and tumor protein p53 (TP53) genes in subsets of patients. Here, we report on a genome-wide analysis of DNA methylation and copy number aberrations in 25 GC patients. Results were compared with those obtained for 105 patients with various types of conventional, i.e., non-GC gliomas including diffuse astrocytic gliomas, oligodendrogliomas and glioblastomas. In addition, we assessed the prognostic role of methylation profiles and recurrent DNA copy number aberrations in GC patients. Our data reveal that the methylation profiles in 23 of the 25 GC tumors corresponded to either IDH mutant astrocytoma (n = 6), IDH mutant and 1p/19q codeleted oligodendroglioma (n = 5), or IDH wild-type glioblastoma including various molecular subgroups, i.e., H3F3A-G34 mutant (n = 1), receptor tyrosine kinase 1 (RTK1, n = 4), receptor tyrosine kinase 2 (classic) (RTK2, n = 2) or mesenchymal (n = 5) glioblastoma groups. Two tumors showed methylation profiles of normal brain tissue due to low tumor cell content. While histological grading (WHO grade IV vs. WHO grade II and III) was not prognostic, the molecular classification as classic/RTK2 or mesenchymal glioblastoma was associated with worse overall survival. Multivariate Cox regression analysis revealed MGMT promoter methylation as a positive prognostic factor. Taken together, DNA-based large-scale molecular profiling indicates that GC comprises a genetically and epigenetically heterogeneous group of diffuse gliomas that carry DNA methylation and copy number profiles closely matching the common molecularly defined glioma entities. These data support the removal of GC as a distinct glioma entity in the upcoming revision of the WHO classification.

The molecular pathways driving mesenchymal glioblastoma (GBM) are still not well understood. We report here that truncated glioma-associated oncogene homolog 1 (tGLI1) is a tumor-specific transcription factor that facilitates GBM growth, is enriched in the mesenchymal subtype of GBM and glioma stem cells (GSC), and promotes mesenchymal GSC by upregulating transcription of CD44. In an orthotopic GBM xenograft mouse model, tGLI1-overexpressing tumors grew more aggressively with increased proliferation and angiogenesis compared with control and GLI1-overexpressing xenografts. tGLI1 was highly expressed in GBM clinical specimens but undetectable in normal brains, whereas GLI1 was expressed in both tissues. A tGLI1 activation signature (tGAS) correlated with glioma grade, tumor angiogenesis, and poor overall survival, and GBMs with high tGAS were enriched with mesenchymal GBM/GSC gene signatures. Neurospheres contained increased levels of tGLI1, but not GLI1, compared with the monolayer culture; mesenchymal GSC expressed more tGLI1 than proneural GSC. Ectopic tGLI1 expression enhanced the ability of mesenchymal GSC to yield neurospheres in vitro and to form tumors in mouse brains. Selective tGLI1 knockdown reduced neurosphere formation of GBM cells. tGLI1 bound to and transactivated the promoter of the CD44 gene, a marker and mediator for mesenchymal GSC, leading to its expression. Collectively, these findings advance our understanding of GBM biology by establishing tGLI1 as a novel transcriptional activator of CD44 and a novel mediator of mesenchymal GBM and GSC. Significance: These findings highlight the role of a tumor-specific gain-of-function transcription factor tGLI1 in mesenchymal glioma stem cell maintenance and mesenchymal GBM growth. Cancer Res; 78(10); 2589–600. ©2018 AACR.

Intertumoral molecular heterogeneity in glioblastoma identifies four major subtypes based on expression of molecular markers. Among them, the two clinically interrelated subtypes, proneural and mesenchymal, are the most aggressive with proneural liable for conversion to mesenchymal upon therapy. Using two patient-derived novel primary cell culture models (MTA10 and KW10), we developed a minimal but unique four-gene signature comprising genes vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B) and angiopoietin 1 (ANG1), angiopoietin 2 (ANG2) that effectively segregated the proneural (MTA10) and mesenchymal (KW10) glioblastoma subtypes. The cell culture preclassified as mesenchymal showed elevated expression of genes VEGF-A, VEGF-B and ANG1, ANG2 as compared to the other cell culture model that mimicked the proneural subtype. The differentially expressed genes in these two cell culture models were confirmed by us using TCGA and Verhaak databases and we refer to it as a minimal multigene signature (MMS). We validated this MMS on human glioblastoma tissue sections with the use of immunohistochemistry on preclassified (YKL-40 high or mesenchymal glioblastoma and OLIG2 high or proneural glioblastoma) tumor samples (n = 30). MMS segregated mesenchymal and proneural subtypes with 83% efficiency using a simple histopathology scoring approach (p = 0.008 for ANG2 and p = 0.01 for ANG1). Furthermore, MMS expression negatively correlated with patient survival. Importantly, MMS staining demonstrated spatiotemporal heterogeneity within each subclass, adding further complexity to subtype identification in glioblastoma. In conclusion, we report a novel and simple sequencing-independent histopathology-based biomarker signature comprising genes VEGF-A, VEGF-B and ANG1, ANG2 for subtyping of proneural and mesenchymal glioblastoma.

Metabolic dysregulation promotes cancer growth through not only energy production, but also epigenetic reprogramming. Here, we report that a critical node in methyl donor metabolism, nicotinamide N-methyltransferase (NNMT), ranked among the most consistently overexpressed metabolism genes in glioblastoma relative to normal brain. NNMT was preferentially expressed by mesenchymal glioblastoma stem cells (GSCs). NNMT depletes S-adenosyl methionine (SAM), a methyl donor generated from methionine. GSCs contained lower levels of methionine, SAM, and nicotinamide, but they contained higher levels of oxidized nicotinamide adenine dinucleotide (NAD+) than differentiated tumor cells. In concordance with the poor prognosis associated with DNA hypomethylation in glioblastoma, depletion of methionine, a key upstream methyl group donor, shifted tumors toward a mesenchymal phenotype and accelerated tumor growth. Targeting NNMT expression reduced cellular proliferation, self-renewal, and in vivo tumor growth of mesenchymal GSCs. Supporting a mechanistic link between NNMT and DNA methylation, targeting NNMT reduced methyl donor availability, methionine levels, and unmethylated cytosine, with increased levels of DNA methyltransferases, DNMT1 and DNMT3A. Supporting the clinical significance of these findings, NNMT portended poor prognosis for glioblastoma patients. Collectively, our findings support NNMT as a GSC-specific therapeutic target in glioblastoma by disrupting oncogenic DNA hypomethylation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SummaryTo understand the relationships between the non-GCIMP glioblastoma (GBM) subgroups, we performed mathematical modeling to predict the temporal sequence of driver events during tumorigenesis. The most common order of evolutionary events is 1) chromosome (chr) 7 gain and chr10 loss, followed by 2) CDKN2A loss and/or TP53 mutation, and 3) alterations canonical for specific subtypes. We then developed a computational methodology to identify drivers of broad copy number changes, identifying PDGFA (chr7) and PTEN (chr10) as driving initial nondisjunction events. These predictions were validated using mouse modeling, showing that PDGFA is sufficient to induce proneural-like gliomas and that additional NF1 loss converts proneural to the mesenchymal subtype. Our findings suggest that most non-GCIMP mesenchymal GBMs arise as, and evolve from, a proneural-like precursor.

The prognosis of glioblastoma, the most malignant type of glioma, is still poor, with only a minority of patients showing long-term survival of more than three years after diagnosis. To elucidate the molecular aberrations in glioblastomas of long-term survivors, we performed genome- and/or transcriptome-wide molecular profiling of glioblastoma samples from 94 patients, including 28 long-term survivors with >36 months overall survival (OS), 20 short-term survivors with

Glioblastoma multiforme (GBM), the most common and deadliest brain tumor with a median survival of 12–15 months, has been characterized by robust angiogenesis, high invasiveness, and universal recurrence. Genomics and transcriptomics studies defined three GBM subtypes (proneuronal, classical, mesenchymal), which are associated with genomic abnormalities, treatment response, and diversity in tumor microenvironment. However, molecular mechanisms of subtype-specific genotype in contribution to tumor phenotype are less understood. We have previously generated a de novo human GBM model by inactivation of p53 and activation of AKT signaling pathways in human neural stem/progenitor cells (hNSCs). Further characterization of this model indicates that the tumors recapitulate GBM’s classical histopathogical features and exhibit a molecular profile resembling the mesenchymal subtype. Comparative transcriptomic analysis revealed that YKL-40/CHI3L1, a secreted glycoprotein, is significantly upregulated during AKT activation induced malignant transformation of hNSCs in vitro and in vivo. Pharmacological inhibition of PI3K/AKT/mTOR signaling pathway results in decreasing YKL-40 mRNA expression and protein secretion level. Mechanistically, motif enrichment analysis and functional validation revealed that YKL-40 is bidirectionally regulated by a gene regulatory network with two transcription factors, BACH2 and CEBPB. Furthermore, YKL-40 significantly activates AKT-S6 and MAPK-ERK kinase signaling pathways, resulting in enhancing tumor cells proliferation, neurosphere and soft-agar colony formation, and tumor progression. In vivo silencing YKL-40 attenuates tumor angiogenesis and prolongs animal survival in the orthotopic xenograft mouse models. Interestingly, knocking down YKL-40 expression decreases microglia/macrophages infiltration in tumor mass. Both in vitro and in vivo validation indicate that PI3K/AKT/YKL-40 mediates tumor-associated microglia/macrophages recruitment and activation during tumor progression. Taken together, these results suggest that auto- and paracrine signaling of PI3K/AKT/YKL-40 affect tumor cells and tumor microenvironment contributing to glioblastoma progression, indicating YKL-40 as a predictive biomarker for evaluation of anti- PI3K/AKT/mTOR therapy, and a potential drug target for the treatment of this devastating disease.

Glioblastoma (GBM), the deadliest and most common adult brain malignancy, is molecularly and clinically heterogeneous. The most common subtype (both primary and recurrent), mesenchymal (MES)-GBM, has the worst prognosis and highest treatment resistance. MES-GBM exhibits hyperactive transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo tumor suppressive pathway effector whose expression in GBMs predicts short survival. Yet, how Hippo-TAZ dysregulation might drive GBM MES transition remains elusive, precluding subtype-specific treatments. Tumor evolution requires signaling dysregulation and co-opting the tumor microenvironment (TME). Understanding GBM heterogeneity was recently complicated by the notion that subtypes vary in TME immune composition. The MES-GBM TME is differentially-distorted in silico, with more tumor-associated macrophages/microglia (TAMs) and neutrophils (TANs). Yet, how TAZ hyperactivity, MES transition, and GBM TME distortion interrelate and impact tumor progression remains unknown. We suspected that TME distortion facilitates immune evasion, MES transition, and tumor progression, worsening treatment responses. To test this, we devised an orthotopic xenograft mouse model phenotypically and histopathologically recapitulating human MES-GBM by expressing constitutively-active TAZ (TAZ4SA) in human GBM cells lacking MES signatures (GBM4SA). GBM4SA mice lived significantly shorter compared to mice with GBM expressing vector (GBMvector) or mutant TAZ unable to bind its effector, TEAD (GBM4SA-S51A). Moreover, more myeloid cells infiltrate the GBM4SA TME than the GBMvector or GBM4SA-S51A TMEs. While most myeloid cells infiltrating the GBMvector and GBM4SA-S51A TMEs were TAMs, most infiltrating the GBM4SA TME were TANs, suggesting TAZ hyperactivation differentially distorts the TME. Next, to delineate the roles of TANs in GBM4SA tumor progression, mice were depleted of neutrophils by administering Ly6G antibody. Serial blood smears and flow cytometry revealed effective depletion was achieved. We are currently investigating the impact of systemic neutrophil depletion on GBM mesenchymal transition and tumor progression in hopes of informing future GBM clinical management and novel TME-targeted immunotherapies.

Despite multimodal therapy and ever-improving surgical technique, glioblastoma (GBM) maintains its poor prognosis due to the chemotherapeutic resistance it builds over time, eventually rendering existing agents ineffective. A well-characterized phenomenon that takes place particularly in mesenchymal GBM tissue is the Warburg Effect, characterized by a shift from oxidative phosphorylation to glycolysis as a primary means to derive energy. Pyruvate Dehydrogenase Kinase 1 (PDK1) is one upregulated molecule in this shift toward anaerobic metabolism that is known for phosphorylating pyruvate dehydrogenase, the “gate keeper” of the TCA cycle. We sought to characterize further the role of PDK1 in terms of its metabolic role and any extra-metabolic functions it may serve. By silencing PDK1 and considering changes in downstream expression, we hope to understand what other factors serve as contributors to metabolic alterations seen in GBM. Elucidating the mechanism of this crucial shift more thoroughly, especially without the prominently cited role of PDK1, can begin to expose new targets for interrupting tumor maintenance and proliferation by affecting its metabolism. In this study we used differential expression of two vectors in which PDK1 was silenced, shPDK1 and SCBT-PDK1. GSEA analysis was done and analyzed to identify classes of genes or proteins that are over-represented and to identify significantly enriched or depleted groups of targets whose expression was vastly changed by silencing of PDK1. Using GSEA heat map data, gene targets whose expression was noted to be consistently changed with silencing of PKD1 targets were identified. Datamining and immunoblot analyses were then conducted to evaluate for expression of these genes in correlating GBM specimens. Ultimately, we further characterize PDK1’s global role in GBM and evaluate non-metabolic effects of PDK1 that may still affect survival and proliferation of GBM tumors. Citation Format: Neha Jain, Maheedhara Guda, Collin Labak, Chase Smith, Simon Park, Andrew Tsung, Kiran Velpula. The role of PDK1 in metabolic reprogramming of mesenchymal glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1848.

Background: Radiation is a mainstay of treatment in patients diagnosed with glioblastoma. Despite maximal surgical and chemo-radiotherapeutic treatment, the disease remains uniformly fatal, with local disease recurrence within the previously radiated field comprising the major pattern of progression. According to the molecular subtyping of GBM, there is a predominance of mesenchymal GBM cells within the recurrent tumor mass, with characteristic increased expression of CD44, N-cadherin, L1CAM, TWIST1 and phosphorylated STAT3. Moreover, these populations of cells are known to be resistant to additional salvage radiation. The current study aims to evaluate the role of N-cadherin in the relative radiation resistance of mesenchymal subtype GBM. Methods: Overall survival data from the TCGA was analyzed using Cox regression hazard ratios based on N-cadherin expression across multiple molecular subtypes of GBM along with age and MGMT covariates. Using the U3035 and U87MG cells, the analyses of N-cadherin expression or exogenous exposure in the setting of external beam radiation were performed via stable shRNA knockdowns or the use of recombinant human N-cadherin ectodomain-Fc fusion proteins in clonogenic survival assays. Orthotopic implantations were performed in NSG mice with the endpoints of overall survival and tumor size, as determined by luciferase imaging. Subcellular localization of RAD51 was accomplished by confocal microscopy. Statistical analyses were performed using SPSS and GraphPad Prism with significance at p Results: N-cadherin expression above the median among the mesenchymal subtype of GBM in the TCGA dataset correlated with a significant decrease in overall survival (HR 1.924, p=0.029). The elimination of N-cadherin expression in normally high expressing U3035 GBM cells led to a significant increase in the ability of radiation to induce cell death in clonogenic assay (17.2% U3035 vs 2% U3035 Δ N-cadherin at 8Gy; 12.7% U3035 vs 1.75% U3035 Δ N-cadherin at 20Gy) and an increase in the presence of double stranded DNA breaks (DSBs) on TUNEL assay. N-cadherin ectodomain-Fc fusion protein exposure in U87 cells resulted in a decrease in NSG mouse mean OS (17.6±0.25 days) compared to control radiated mice (22.3±1.1 days). Conversely, N-cadherin knockdown in U3035 cells resulted in further decrease in tumor bioluminescence beyond that seen in 4Gy x 4 radiation treatment of native U3035 xenografts (21% vs 4% of baseline). Conclusions: The reduction of N-cadherin expression in mesenchymal U3035 GBM cells increases the efficacy of radiation-induced cell death. This finding is accompanied by a decrease in the nuclear localization of RAD51 following DSBs induced by ionizing radiation, indicating a potential crosstalk between DNA damage repair mechanisms and calcium dependent cell-cell adhesion. Citation Format: Christopher P. Cifarelli, J. Austin Vargo, Ian MacFawn, Steven M. Frisch. N-cadherin(CDH2) expression in mesenchymal glioblastoma modulates radiation sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3928.

SummaryWe describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

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 nnesenchymal (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-alpha/NF-kappa B-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-kappa B activation correlate with poor radiation response and shorter survival in patients with GBM.

Brain invasion by glioblastoma determines prognosis, recurrence, and lethality in patients, but no master factor coordinating the invasive properties of glioblastoma has been identified. Here we report evidence favoring such a role for the noncanonical WNT family member Wnt5a. We found the most invasive gliomas to be characterized by Wnt5a overexpression, which correlated with poor prognosis and also discriminated infiltrating mesenchymal glioblastoma from poorly motile proneural and classical glioblastoma. Indeed, Wnt5a overexpression associated with tumor-promoting stem-like characteristics (TPC) in defining the character of highly infiltrating mesenchymal glioblastoma cells (Wnt5aHigh). Inhibiting Wnt5a in mesenchymal glioblastoma TPC suppressed their infiltrating capability. Conversely, enforcing high levels of Wnt5a activated an infiltrative, mesenchymal-like program in classical glioblastoma TPC and Wnt5aLow mesenchymal TPC. In intracranial mouse xenograft models of glioblastoma, inhibiting Wnt5a activity blocked brain invasion and increased host survival. Overall, our results highlight Wnt5a as a master regulator of brain invasion, specifically TPC, and they provide a therapeutic rationale to target it in patients with glioblastoma. Cancer Res; 77(4); 996–1007. ©2016 AACR.

Somatic mutations activating MAPK and PI3K signalling play a pivotal role in both tumours and brain developmental disorders. We developed a zebrafish model of brain tumours based on somatic expression of oncogenes that activate MAPK and PI3K signalling in neural progenitor cells and found that HRASV12 was the most effective in inducing both heterotopia and invasive tumours. Tumours, but not heterotopias, require persistent activation of phospho (p)-ERK and express a gene signature similar to the mesenchymal glioblastoma subtype, with a strong YAP component. Application of an eight-gene signature to human brain tumours establishes that YAP activation distinguishes between mesenchymal glioblastoma and low grade glioma in a wide The Cancer Genome Atlas (TCGA) sample set including gliomas and glioblastomas (GBMs). This suggests that the activation of YAP might be an important event in brain tumour development, promoting malignant versus benign brain lesions. Indeed, co-expression of dominant-active YAP (YAPS5A) and HRASV12 abolishes the development of heterotopias and leads to the sole development of aggressive tumours. Thus, we have developed a model proving that neurodevelopmental disorders and brain tumours might originate from the same activation of oncogenes through somatic mutations, and established that YAP activation is a hallmark of malignant brain tumours., Editors' choice: A new brain tumour model in zebrafish allows simultaneous analysis of malignant tumours and heterotopias, suggesting that both lesions originate from similar events, with Yap1 as a driving force in tumour development.

International audience; BACKGROUND: Cellular self-renewal capacity in glioblastomas is heterogeneous, with only stem-like cells having this property. These cells generate a specific tumor phenotype, but no link with tumor location or molecular characteristics has ever been made. METHODS: Two cells lines, established from cell-dissociated glioblastomas and A2B5+ magnetic cell sorting, were used to decipher the mechanisms of cell migration in glioblastomas. GBM6 was derived from a glioblastoma close to the subventricular zone, whereas GBM9 was derived from a cortical glioblastoma and contained a high number of CD133(+) cells. RESULTS: Orthotopic injections in both the subventricular zone and the cortex of nude mice showed that GBM6 and GBM9 cells had a differential pattern of migration that mirrored that of adult and fetal normal neural stem cells, respectively. GBM6 demonstrated higher tumorigenicity than GBM9, and whichever cell line was injected, subventricular zone-implanted tumors were larger than cortical ones. In vitro, GBM6 and GBM9 displayed high autorenewal and proliferation rates, and their expression profiles and genomic status showed that they had distinctive molecular signatures: GBM6 was classified as a mesenchymal glioblastoma and GBM9 as a proneural glioblastoma. CONCLUSIONS: Altogether, our findings suggest that tumor location in addition to molecular signature influence tumor growth and migration pattern.

ceRNA networks play important roles in post-transcriptional regulation. Their dysregulation is common in cancer. However, ceRNA signatures are barely examined for mesenchymal glioblastoma characteristic of invasive and aggressive phenotypes. Here, we investigated the mRNAs in ceRNA networks (micNET) of glioblastoma by constructing a GBM ceRNA network, followed by being integrated with STRING protein interaction network. We report that six micNETs (TGFBR2, RUNX1, PPARG, ACSL1, GIT2 and RAP1B) signify mesenchymal GBM across multiple datasets. Patients with highly expressed micNET markers have poor response to TMZ chemotherapy. Mechanistically, the ceRNA interaction were identified between micNETs and miR181s family members. The inhibitor of TGFBR2, LY2109761, demonstrated tumor-suppressive effect on both primary cultured cell and PDX intracranial model. Thereby, the micNETs provide a promising translational significance in diagnosis of mesenchymal GBM as well as novel therapeutic targets. In conclusion, our study revealed that the ceRNA signature of mesenchymal glioblastoma was enriched in the TGF-β pathway and characterized this subtype in the mRNA-miRNA dimension. We identified that key micNETs signatures could be used to diagnose mesenchymal GBM and discovered that the TGF-β pathway is a potential therapeutic target for mesenchymal glioblastoma.

Introduction. Glioblastoma (GBM) is one of the most aggressive cancer types, accounting for more than 60% of malignant brain tumors in adults. Genomic profiling defined four subtypes of GBM termed proneural, neural, classical, and mesenchymal. The mesenchymal subtype is characterized by epithelial-mesenchymal-transition (EMT) and is associated with poor prognosis. Our aim in this study is to identify the mesenchymal subtype-specific biomarkers that may drive EMT, resulting in worse clinical outcome. Materials and methods. We investigated 433 primary GBM patients from TCGA that have both gene and microRNA (miRNA) expression profiles. A prediction analysis for microarrays (PAM) classifier was built based on the predefined 141 patient labels and 738 gene signatures. The classifier was then used to classify the remaining unlabeled patients into one of the four subtypes. Comparisons between the mesenchymal and non-mesenchymal subtypes were performed using two-sided t-test statistics. Anti-correlated miRNA-mRNA interactions were used for network inferred by ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks), and the network were visualized by RedeR package. We tested the statistically significant over-representation of EMT signature genes in the regulon of each miRNA. MiRNAs of top significance (false discovery rate, FDR < 0.05) were selected as master regulators. Results and Discussion. We classified the remaining unlabeled 292 GBM patients by using the PAM classifier. Gene set enrichment analysis (GSEA) and survival analysis were performed to do molecular and clinical characterization of the four subtypes, respectively, which all align with previous characteristics. Such as, mesenchymal subtype is enriched for EMT signatures, and proneural subtype has the best clinical outcomes. We also classified the 43 GBM cell lines from the Cancer Cell Line Encyclopedia (CCLE) into one of the four subtypes. In total, we identified 54 miRNAs downregulated, and 2,520 genes upregulated in mesenchymal compared with the non-mesenchymal subtypes. ARACNe predicted 28,586 miRNA-mRNA interactions, 2,784 of which were stable and therefore used for network construction. A miRNA regulatory network was subsequently built to describe the regulatory relations among miRNAs and target genes. Master regulator analysis identified 6 miRNAs, namely miR-9, miR-488, miR-95, miR-551b, miR-30c and miR-101, which may functioning as master regulators of the EMT program. Conclusion. We used a relatively large GBM cohort to infer a miRNA-mRNA regulatory network associated with EMT. Further in vitro and in vivo experiments are ongoing to access the 6 miRNAs role in the EMT program. Citation Format: Lan Zhao, William Cho, Hu Zhu, Bing Shi, Hong Yan. Identification of master microRNA regulatory networks for the mesenchymal glioblastoma subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 497.

International audience; We have profiled promoter DNA methylation alterations in 272 glioblastoma tumors in the context of The Cancer Genome Atlas (TCGA). We found that a distinct subset of samples displays concerted hypermethylation at a large number of loci, indicating the existence of a glioma-CpG island methylator phenotype (G-CIMP). We validated G-CIMP in a set of non-TCGA glioblastomas and low-grade gliomas. G-CIMP tumors belong to the proneural subgroup, are more prevalent among lower-grade gliomas, display distinct copy-number alterations, and are tightly associated with IDH1 somatic mutations. Patients with G-CIMP tumors are younger at the time of diagnosis and experience significantly improved outcome. These findings identify G-CIMP as a distinct subset of human gliomas on molecular and clinical grounds.

The inference of transcriptional networks that regulate transitions into physiological or pathological cellular states remains a central challenge in systems biology. A mesenchymal phenotype is the hallmark of tumour aggressiveness in human malignant glioma, but the regulatory programs responsible for implementing the associated molecular signature are largely unknown. Here we show that reverse-engineering and an unbiased interrogation of a glioma-specific regulatory network reveal the transcriptional module that activates expression of mesenchymal genes in malignant glioma. Two transcription factors (C/EBPbeta and STAT3) emerge as synergistic initiators and master regulators of mesenchymal transformation. Ectopic co-expression of C/EBPbeta and STAT3 reprograms neural stem cells along the aberrant mesenchymal lineage, whereas elimination of the two factors in glioma cells leads to collapse of the mesenchymal signature and reduces tumour aggressiveness. In human glioma, expression of C/EBPbeta and STAT3 correlates with mesenchymal differentiation and predicts poor clinical outcome. These results show that the activation of a small regulatory module is necessary and sufficient to initiate and maintain an aberrant phenotypic state in cancer cells.

Proneural and mesenchymal are two subtypes of glioblastoma identified by gene expression profiling. In this study, the primary aim was to detect markers to develop a clinically applicable method for distinguishing proneural and mesenchymal glioblastoma. The secondary aims were to investigate the temporospatial dynamics of these markers and to explore the association between these markers and the expression of chromatin-modifying genes. One hundred thirty-three glioma samples (grade II: 14 samples, grade III: 18, grade IV: 101) were analyzed. We quantified the expression of 6 signature genes associated with proneural and mesenchymal glioblastoma by quantitative reverse transcription-polymerase chain reaction. We assigned proneural (PN) and mesenchymal (MES) scores based on the average of the 6 markers and calculated a predominant metagene (P-M) score by subtracting the MES from the PN score. We used these scores to analyze correlations with malignant transformation, tumor recurrence, tumor heterogeneity, chromatin-modifying gene expression, and HDAC7 expression. The MES score positively correlated with tumor grade, whereas the PN score did not. The P-M score was able to distinguish the proneural and mesenchymal subtypes. It was decreased in cases of tumor recurrence and malignant transformation and showed variability within a tumor, suggesting intratumoral heterogeneity. The PN score correlated with the expression of multiple histone-modifying genes, whereas the MES score was associated only with HDAC7 expression. Thus, we demonstrated a simple and straightforward method of quantifying proneural/mesenchymal markers in glioblastoma. Of note, HDAC7 expression might be a novel therapeutic target in glioblastoma treatment.

Summary Glioblastoma is a highly lethal cancer for which novel therapeutics are urgently needed. Two distinct subtypes of glioblastoma stem-like cells (GSCs) were recently identified: mesenchymal (MES) and proneural (PN). To identify mechanisms to target the more aggressive MES GSCs, we combined transcriptomic expression analysis and kinome-wide short hairpin RNA screening of MES and PN GSCs. In comparison to PN GSCs, we found significant upregulation and phosphorylation of the receptor tyrosine kinase AXL in MES GSCs. Knockdown of AXL significantly decreased MES GSC self-renewal capacity in vitro and inhibited the growth of glioblastoma patient-derived xenografts. Moreover, inhibition of AXL with shRNA or pharmacologic inhibitors also increased cell death significantly more in MES GSCs. Clinically, AXL expression was elevated in the MES GBM subtype and significantly correlated with poor prognosis in multiple cancers. In conclusion, we identified AXL as a potential molecular target for novel approaches to treat glioblastoma and other solid cancers., Highlights • shRNA screen identified kinases that alter GSC viability in a subtype-dependent manner • AXL is highly expressed in mesenchymal GSCs • Targeting AXL decreases mesenchymal GSC self-renewal, viability, and tumorigenicity • AXL expression predicts poor prognosis in several tumor types, To identify mechanistic differences between the mesenchymal and proneural glioblastoma subtypes, Goidts, Nakano, and colleagues performed a phenotypic screen, silencing the whole kinome in patient-derived mesenchymal and proneural glioblastoma stem-like cells (GSCs). AXL was identified as an important regulator of mesenchymal GSC viability and tumorigenicity, making it an attractive therapeutic target.