bims-malgli 2020-07-12 papers

bims-malgli

Biomed News

on Biology of malignant gliomas

Issue of 2020–07–12
thirty-two papers selected by
Oltea Sampetrean, Keio University

Cell Lineage-Based Stratification for Glioblastoma.
Low-dose oncolytic adenovirus therapy overcomes tumor-induced immune suppression and sensitizes intracranial gliomas to anti-PD-1 therapy.
Soluble protein tyrosine phosphatase receptor type Z (PTPRZ) in cerebrospinal fluid is a potential diagnostic marker for glioma.
Downregulation of PD-L1 via FKBP5 by celecoxib augments antitumor effects of PD-1 blockade in a malignant glioma model.
MerTK inhibition decreases immune suppressive glioblastoma-associated macrophages and neoangiogenesis in glioblastoma microenvironment.
Genetic driver mutations introduced in identical cell-of-origin in murine glioblastoma reveal distinct immune landscapes but similar response to checkpoint blockade.
Gliomas display distinct sex-based differential methylation patterns based on molecular subtype.
Advances in histone deacetylase inhibitors in targeting glioblastoma stem cells.
Conventional Treatment of Glioblastoma Reveals Persistent CD44+ Subpopulations.
TROY signals through JAK1-STAT3 to promote glioblastoma cell migration and resistance.
Synaptic Input to Brain Tumors: Clinical Implications.
Circulating angiogenic cells in glioblastoma: toward defining crucial functional differences in CAC-induced neoplastic versus reactive neovascularization.
Glioblastoma Myeloid-Derived Suppressor Cell Subsets Express Differential Macrophage Migration Inhibitory Factor Receptor Profiles That Can Be Targeted to Reduce Immune Suppression.
Overexpression of HGF/MET axis along with p53 inhibition induces de novo glioma formation in mice.
The soluble form of pan-RTK inhibitor and tumor suppressor LRIG1 mediates downregulation of AXL through direct protein-protein interaction in glioblastoma.
The Role of Liquid Biopsies in Detecting Molecular Tumor Biomarkers in Brain Cancer Patients.
Dissecting and rebuilding the glioblastoma microenvironment with engineered materials.
Biomarkers and smart intracranial devices for the diagnosis, treatment, and monitoring of high-grade gliomas: a review of the literature and future prospects.
A cytoskeleton regulator AVIL drives tumorigenesis in glioblastoma.
MicroRNA-93 acts as an "anti-inflammatory tumor suppressor" in glioblastoma.
Suicide gene therapy for the treatment of high-grade glioma: past lessons, present trends, and future prospects.
Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.
Sustained inhibition of PARP-1 activity delays glioblastoma recurrence by enhancing radiation-induced senescence.
Combined targeting of PI3K and MEK effector pathways via CED for DIPG therapy.
Sexually dimorphic impact of the iron-regulating gene, HFE, on survival in glioblastoma.
Proteins inform survival-based differences in patients with glioblastoma.
Expansion of effector and memory T cells is associated with increased survival in recurrent glioblastomas treated with dendritic cell immunotherapy.
The invasive proteome of glioblastoma revealed by laser-capture microdissection.
Multivariate analysis reveals differentially expressed genes among distinct subtypes of diffuse astrocytic gliomas: diagnostic implications.
Identification of functionally distinct and interacting cancer cell subpopulations from glioblastoma with intratumoral genetic heterogeneity.
Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes.
EGFR mutations are associated with response to depatux-m in combination with temozolomide and result in a receptor that is hypersensitive to ligand.

Cancer Cell. 2020 Jun 23. pii: S1535-6108(20)30307-X. [Epub ahead of print]

Cell Lineage-Based Stratification for Glioblastoma.

Zilai Wang, Daochun Sun, Yu-Jung Chen, Xuanhua Xie, Yufeng Shi, Viviane Tabar, Cameron W Brennan, Tejus A Bale, Chenura D Jayewickreme, Dan R Laks, Sheila Alcantara Llaguno, Luis F Parada.

  Glioblastoma, the predominant adult malignant brain tumor, has been computationally classified into molecular subtypes whose functional relevance remains to be comprehensively established. Tumors from genetically engineered glioblastoma mouse models initiated by identical driver mutations in distinct cells of origin portray unique transcriptional profiles reflective of their respective lineage. Here, we identify corresponding transcriptional profiles in human glioblastoma and describe patient-derived xenografts with species-conserved subtype-discriminating functional properties. The oligodendrocyte lineage-associated glioblastoma subtype requires functional ERBB3 and harbors unique therapeutic sensitivities. These results highlight the importance of cell lineage in glioblastoma independent of driver mutations and provide a methodology for functional glioblastoma classification for future clinical investigations.

Keywords:  Erbb3; GBM; PDX; cell of origin; glioblastoma; molecular classification; molecular subtype; mouse model; neural stem cell; oligodendrocyte lineage cell

DOI:  https://doi.org/10.1016/j.ccell.2020.06.003
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa011

Low-dose oncolytic adenovirus therapy overcomes tumor-induced immune suppression and sensitizes intracranial gliomas to anti-PD-1 therapy.

Zineb Belcaid, Cor Berrevoets, John Choi, Edward van Beelen, Eftychia Stavrakaki, Tessa Pierson, Jenneke Kloezeman, Denis Routkevitch, Mariëlle van der Kaaij, Alicia van der Ploeg, Dimitrios Mathios, Stefan Sleijfer, Clemens Dirven, Michael Lim, Reno Debets, Martine L M Lamfers.

   Background: The tumor-selective human adenovirus Delta24-RGD is currently under investigation in phase II clinical trials for patients with recurrent glioblastoma (GBM). To improve treatments for patients with GBM, we explored the potential of combining Delta24-RGD with antibodies targeting immune checkpoints.
Methods: C57BL/6 mice were intracranially injected with GL261 cells and treated with a low dose of Delta24-RGD virus. The expression dynamics of 10 co-signaling molecules known to affect immune activity was assessed in tumor-infiltrating immune cells by flow cytometry after viral injection. The antitumor activity was measured by tumor cell killing and IFNγ production in co-cultures. Efficacy of the combination viro-immunotherapy was tested in vitro and in the GL261 and CT2A orthotopic mouse GBM models. Patient-derived GBM cell cultures were treated with Delta24-RGD to assess changes in PD-L1 expression induced by virus infection.
Results: Delta24-RGD therapy increased intratumoral CD8+ T cells expressing Inducible T-cell co-stimulator (ICOS) and PD-1. Functionality assays confirmed a significant positive correlation between tumor cell lysis and IFNγ production in ex vivo cultures (Spearman r = 0.9524; P < .01). Co-cultures significantly increased IFNγ production upon treatment with PD-1 blocking antibodies. In vivo, combination therapy with low-dose Delta24-RGD and anti-PD-1 antibodies significantly improved outcome compared to single-agent therapy in both syngeneic mouse glioma models and increased PD-1+ tumor-infiltrating CD8+ T cells. Delta24-RGD infection induced tumor-specific changes in PD-L1 expression in primary GBM cell cultures.
Conclusions: This study demonstrates the potential of using low-dose Delta24-RGD therapy to sensitize glioma for combination with anti-PD-1 antibody therapy.

Keywords:  Delta24-RGD; PD-1; glioma; immunotherapy; oncolytic virotherapy

DOI:  https://doi.org/10.1093/noajnl/vdaa011
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa055

Soluble protein tyrosine phosphatase receptor type Z (PTPRZ) in cerebrospinal fluid is a potential diagnostic marker for glioma.

Yu Yamanoi, Masazumi Fujii, Yuta Murakami, Kenichiro Nagai, Kyoka Hoshi, Yasuhiro Hashimoto, Takashi Honda, Kiyoshi Saito, Shinobu Kitazume.

   Background: High-grade glioma is the most pervasive and lethal of all brain malignancies. Despite advances in imaging technologies, discriminating between gliomas and other brain diseases such as multiple sclerosis (MS) often requires brain biopsy. Several reports show that protein tyrosine phosphatase receptor Z (PTPRZ) is highly expressed in glioblastoma, and we found that a soluble cleaved form of PTPRZ (sPTPRZ) was present in the cerebrospinal fluid (CSF). The aim of this study was to determine whether the sPTPRZ level in CSF has utility as a diagnostic marker for glioma.
Methods: Microarray datasets from normal brain tissue and brain tumors were obtained from the Gene Expression Omnibus. PTPRZ protein expression in clinical specimens was evaluated by immunohistochemistry. Semiquantitative western blotting was used to measure sPTPRZ levels in CSF samples from patients with glioma, schwannoma, MS, or nontumor disorders.
Results: Expression of PTPRZ mRNA and protein was markedly increased in glioblastoma, astrocytoma, oligodendroglioma, and schwannoma tissues compared with control brain tissue. sPTPRZ was present at significantly elevated levels in the CSF of patients with glioma (grades 1-4), but not in patients with schwannoma or MS, compared with the control samples. Receiver operating characteristic curve analysis showed that sPTPRZ in CSF could discriminate between glioma and MS patients (area under the curve 0.9676; P < .0001).
Conclusions: sPTPRZ in CSF is a promising diagnostic biomarker for glioma and could reduce the need for a surgical biopsy.

Keywords:  PTPRZ; cerebrospinal fluid; diagnostic marker; glioma; glycosylation

DOI:  https://doi.org/10.1093/noajnl/vdaa055
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdz058

Downregulation of PD-L1 via FKBP5 by celecoxib augments antitumor effects of PD-1 blockade in a malignant glioma model.

Izumi Yamaguchi, Kohei Nakajima, Kenji Shono, Yoshifumi Mizobuchi, Toshitaka Fujihara, Eiji Shikata, Tadashi Yamaguchi, Keiko Kitazato, Oltea Sampetrean, Hideyuki Saya, Yasushi Takagi.

   Background: Antitumor therapies targeting programmed cell death-1 (PD-1) or its ligand-1 (PD-L1) are used in various cancers. However, in glioblastoma (GBM), the expression of PD-L1 varies between patients, and the relationship between this variation and the efficacy of anti-PD-1 antibody therapy remains unclear. High expression levels of PD-L1 affect the proliferation and invasiveness of GBM cells. As COX-2 modulates PD-L1 expression in cancer cells, we tested the hypothesis that the COX-2 inhibitor celecoxib potentiates anti-PD-1 antibody treatment via the downregulation of PD-L1.
Methods: Six-week-old male C57BL/6 mice injected with murine glioma stem cells (GSCs) were randomly divided into four groups treated with vehicle, celecoxib, anti-PD-1 antibody, or celecoxib plus anti-PD-1 antibody and the antitumor effects of these treatments were assessed. To verify the mechanisms underlying these effects, murine GSCs and human GBM cells were studied in vitro.
Results: Compared with that with each single treatment, the combination of celecoxib and anti-PD-1 antibody treatment significantly decreased tumor volume and prolonged survival. The high expression of PD-L1 was decreased by celecoxib in the glioma model injected with murine GSCs, cultured murine GSCs, and cultured human GBM cells. This reduction was associated with post-transcriptional regulation of the co-chaperone FK506-binding protein 5 (FKBP5).
Conclusions: Combination therapy with anti-PD-1 antibody plus celecoxib might be a promising therapeutic strategy to target PD-L1 in glioblastoma. The downregulation of highly-expressed PD-L1 via FKBP5, induced by celecoxib, could play a role in its antitumor effects.

Keywords:  FKBP5; PD-1; PD-L1; celecoxib; glioblastoma

DOI:  https://doi.org/10.1093/noajnl/vdz058
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa065

MerTK inhibition decreases immune suppressive glioblastoma-associated macrophages and neoangiogenesis in glioblastoma microenvironment.

Yu-Ting Su, Madison Butler, Meili Zhang, Wei Zhang, Hua Song, Lee Hwang, Andy D Tran, Ryan E Bash, Allison N Schorzman, Ying Pang, Guangyang Yu, William C Zamboni, Xiaodong Wang, Stephen V Frye, Christopher Ryan Miller, Dragan Maric, Masaki Terabe, Mark R Gilbert, Henry Shelton Earp Iii, Jing Wu.

   Background: Glioblastoma-associated macrophages and microglia (GAMs) are the predominant immune cells in the tumor microenvironment. Activation of MerTK, a receptor tyrosine kinase, polarizes GAMs to an immunosuppressive phenotype, promoting tumor growth. Here, the role of MerTK inhibition in the glioblastoma microenvironment is investigated in vitro and in vivo.
Methods: Effects of MRX-2843 in glioblastoma microenvironment regulation were determined in vitro by cell viability, cytokine array, in vitro tube formation, Western blotting, and wound healing assays. A syngeneic GL261 orthotopic glioblastoma mouse model was used to evaluate the survival benefit of MRX-2843 treatment. Multiplex fluorescent immunohistochemistry was used to evaluate the expression of CD206, an anti-inflammatory marker on GAMs, and angiogenesis in murine brain tumor tissues.
Results: MRX-2843 inhibited cell growth and induced apoptosis in human glioblastoma cells and decreased protein expression of phosphorylated MerTK, AKT, and ERK, which are essential for cell survival signaling. Interleukin-8 and C-C motif chemokine ligand 2, the pro-glioma and pro-angiogenic cytokines, were decreased by MRX-2843. Decreased vascular formation and numbers of immunosuppressive (CD206+) GAMs were observed following MRX-2843 treatment in vivo, suggesting that in addition to alleviating immunosuppression, MRX-2843 also inhibits neoangiogenesis in the glioma microenvironment. These results were supported by a prolonged survival in the syngeneic mouse orthotopic GL261 glioblastoma model following MRX-2843 treatment.
Conclusion: Our findings suggest that MRX-2843 has a therapeutic benefit via promoting GAM polarization away from immunosuppressive condition, inhibiting neoangiogenesis in the glioblastoma microenvironment and inducing tumor cell death.

Keywords:  MRX-2843; MerTK; glioblastoma; immune modulation; neoangiogenesis

DOI:  https://doi.org/10.1093/noajnl/vdaa065
Glia. 2020 Jul 08.

Genetic driver mutations introduced in identical cell-of-origin in murine glioblastoma reveal distinct immune landscapes but similar response to checkpoint blockade.

Zhihong Chen, Cameron J Herting, James L Ross, Ben Gabanic, Montse Puigdelloses Vallcorba, Frank Szulzewsky, Megan L Wojciechowicz, Patrick J Cimino, Ravesanker Ezhilarasan, Erik P Sulman, Mingyao Ying, Avi Ma'ayan, Renee D Read, Dolores Hambardzumyan.

  Glioblastoma (GBM) is the most aggressive primary brain tumor. In addition to being genetically heterogeneous, GBMs are also immunologically heterogeneous. However, whether the differences in immune microenvironment are driven by genetic driver mutation is unexplored. By leveraging the versatile RCAS/tv-a somatic gene transfer system, we establish a mouse model for Classical GBM by introducing EGFRvIII expression in Nestin-positive neural stem/progenitor cells in adult mice. Along with our previously published Nf1-silenced and PDGFB-overexpressing models, we investigate the immune microenvironments of the three models of human GBM subtypes by unbiased multiplex profiling. We demonstrate that both the quantity and composition of the microenvironmental myeloid cells are dictated by the genetic driver mutations, closely mimicking what was observed in human GBM subtypes. These myeloid cells express high levels of the immune checkpoint protein PD-L1; however, PD-L1 targeted therapies alone or in combination with irradiation are unable to increase the survival time of tumor-bearing mice regardless of the driver mutations, reflecting the outcomes of recent human trials. Together, these results highlight the critical utility of immunocompetent mouse models for preclinical studies of GBM, making these models indispensable tools for understanding the resistance mechanisms of immune checkpoint blockade in GBM and immune cell-targeting drug discovery.

Keywords:  EGFRvIII; GEMM of GBM; PD-L1; glioblastoma; microenvironment

DOI:  https://doi.org/10.1002/glia.23883
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa002

Gliomas display distinct sex-based differential methylation patterns based on molecular subtype.

Mette L Johansen, L C Stetson, Vachan Vadmal, Kristin Waite, Michael E Berens, James R Connor, Justin Lathia, Joshua B Rubin, Jill S Barnholtz-Sloan.

   Background: Gliomas are the most common type of primary brain tumor and one of many cancers where males are diagnosed with greater frequency than females. However, little is known about the sex-based molecular differences in glioblastomas (GBMs) or lower grade glioma (non-GBM) subtypes. DNA methylation is an epigenetic mechanism involved in regulating gene transcription. In glioma and other cancers, hypermethylation of specific gene promoters downregulates transcription and may have a profound effect on patient outcome. The purpose of this study was to determine if sex-based methylation differences exist in different glioma subtypes.
Methods: Molecular and clinical data from glioma patients were obtained from The Cancer Genome Atlas and grouped according to tumor grade and molecular subtype (IDH1/2 mutation and 1p/19q chromosomal deletion). Sex-specific differentially methylated probes (DMPs) were identified in each subtype and further analyzed to determine if they were part of differentially methylated regions (DMRs) or associated with differentially methylated DNA transcription regulatory binding motifs.
Results: Analysis of methylation data in 4 glioma subtypes revealed unique sets of both sex-specific DMPs and DMRs in each subtype. Motif analysis based on DMP position also identified distinct sex-based sets of DNA-binding motifs that varied according to glioma subtype. Downstream targets of 2 of the GBM-specific transcription binding sites, NFAT5 and KLF6, showed differential gene expression consistent with increased methylation mediating downregulation.
Conclusion: DNA methylation differences between males and females in 4 glioma molecular subtypes suggest an important, sex-specific role for DNA methylation in epigenetic regulation of gliomagenesis.

Keywords:  DNA methylation; glioblastoma; glioma; sex-specific differences; survival

DOI:  https://doi.org/10.1093/noajnl/vdaa002
Cancer Chemother Pharmacol. 2020 Jul 07.

Advances in histone deacetylase inhibitors in targeting glioblastoma stem cells.

R Gajendra Reddy, Unis Ahmad Bhat, Sumana Chakravarty, Arvind Kumar.

  Glioblastoma multiforme (GBM) is a lethal grade IV glioma (WHO classification) and widely prevalent primary brain tumor in adults. GBM tumors harbor cellular heterogeneity with the presence of a small subpopulation of tumor cells, described as GBM cancer stem cells (CSCs) that pose resistance to standard anticancer regimens and eventually mediate aggressive relapse or intractable progressive GBM. Existing conventional anticancer therapies for GBM do not target GBM stem cells and are mostly palliative; therefore, exploration of new strategies to target stem cells of GBM has to be prioritized for the development of effective GBM therapy. Recent developments in the understanding of GBM pathophysiology demonstrated dysregulation of epigenetic mechanisms along with the genetic changes in GBM CSCs. Altered expression/activity of key epigenetic regulators, especially histone deacetylases (HDACs) in GBM stem cells has been associated with poor prognosis; inhibiting the activity of HDACs using histone deacetylase inhibitors (HDACi) has been promising as mono-therapeutic in targeting GBM and in sensitizing GBM stem cells to an existing anticancer regimen. Here, we review the development of pan/selective HDACi as potential anticancer agents in targeting the stem cells of glioblastoma as a mono or combination therapy.

Keywords:  Cancer stem cell; Epigenetic therapeutics; Glioblastoma multiforme; Histone deacetylase inhibitors

DOI:  https://doi.org/10.1007/s00280-020-04109-w
Mol Neurobiol. 2020 Jul 06.

Conventional Treatment of Glioblastoma Reveals Persistent CD44+ Subpopulations.

Johann Mar Gudbergsson, Esben Christensen, Serhii Kostrikov, Torben Moos, Meg Duroux, Andreas Kjær, Kasper Bendix Johnsen, Thomas Lars Andresen.

  Glioblastoma (GBM) is the most frequent and devastating primary tumor of the central nervous system with a median survival of 12 to 15 months after diagnosis. GBM is highly difficult to treat due to its delicate location, inter- and intra-tumoral heterogeneity, and high plasticity in response to treatment. In this study, we intracranially implanted primary GBM cells into mice which underwent conventional GBM treatments, including irradiation, temozolomide, and a combination. We obtained single cell suspensions through a combination of mechanical and enzymatic dissociation of brain tissue and investigated in detail the changes in GBM cells in response to conventional treatments in vivo using multi-color flow cytometry and cluster analysis. CD44 expression was elevated in all treatment groups, which was confirmed by subsequent immunohistochemistry. High CD44 expression was furthermore shown to correlate with poor prognosis of GBM and low-grade glioma (LGG) patients. Together, these results indicate a key role for CD44 in glioma pathogenesis.

Keywords:  CD133; CD44; Cancer; Glioblastoma; Plasticity; Population; Resistance; Stem cells; Subpopulation; Treatment; Tumor

DOI:  https://doi.org/10.1007/s12035-020-02004-2
Neoplasia. 2020 Jul 03. pii: S1476-5586(20)30127-5. [Epub ahead of print]22(9): 352-364

TROY signals through JAK1-STAT3 to promote glioblastoma cell migration and resistance.

Zonghui Ding, Jean M Kloss, Serdar Tuncali, Nhan L Tran, Joseph C Loftus.

  Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and carries a discouraging prognosis. Its aggressive and highly infiltrative nature renders the current standard treatment of maximal surgical resection, radiation, and chemotherapy relatively ineffective. Identifying the signaling pathways that regulate GBM migration/invasion and resistance is required to develop more effective therapeutic regimens to treat GBM. Expression of TROY, an orphan receptor of the TNF receptor superfamily, increases with glial tumor grade, inversely correlates with patient overall survival, stimulates GBM cell invasion in vitro and in vivo, and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in a preclinical intracranial xenograft model. Here, we have identified for the first time that TROY interacts with JAK1. Increased TROY expression increases JAK1 phosphorylation. In addition, increased TROY expression promotes STAT3 phosphorylation and STAT3 transcriptional activity that is dependent upon JAK1. TROY-mediated activation of STAT3 is independent of its ability to stimulate activity of NF-κB. Inhibition of JAK1 activity by ruxolitinib or knockdown of JAK1 expression by siRNA significantly inhibits TROY-induced STAT3 activation, GBM cell migration, and decreases resistance to temozolomide. Taken together, our data indicate that the TROY signaling complex may represent a potential therapeutic target with the distinctive capacity to exert effects on multiple pathways mediating GBM cell invasion and resistance.

Keywords:  Glioblastoma; JAK1; Migration; STAT3; TROY

DOI:  https://doi.org/10.1016/j.neo.2020.06.005
Neuro Oncol. 2020 Jul 05. pii: noaa158. [Epub ahead of print]

Synaptic Input to Brain Tumors: Clinical Implications.

Varun Venkataramani, Dimitar Ivanov Tanev, Thomas Kuner, Wolfgang Wick, Frank Winkler.

The recent discovery of synaptic connections between neurons and brain tumor cells fundamentally challenges our understanding of gliomas and brain metastases, and shows how these tumors can integrate into complex neuronal circuits. Here, we provide an overview on glutamatergic neuron-to-brain tumor synaptic communication (NBTSC) and explore novel therapeutic avenues. First, we summarize current concepts of direct synaptic interactions between presynaptic neurons and postsynaptic glioma cells, and indirect perisynaptic input to metastatic breast cancer cells. We explain how these novel structures drive brain tumor growth and invasion. Second, a vicious cycle of enhanced neuronal activity, including tumor-related epilepsy, and glioma progression is described. Finally, we discuss which future avenues to target NBTSC appear most promising. All in all, further characterization of NBTSC and the exploration of NBTSC-inhibiting therapies have the potential to reveal critical vulnerabilities of yet incurable brain tumors.

DOI: https://doi.org/10.1093/neuonc/noaa158
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa040

Circulating angiogenic cells in glioblastoma: toward defining crucial functional differences in CAC-induced neoplastic versus reactive neovascularization.

Karin Huizer, Andrea Sacchetti, Sigrid Swagemakers, Peter J van der Spek, Wim Dik, Dana A Mustafa, Johan M Kros.

   Background: In order to identify suitable therapeutic targets for glioma anti-angiogenic therapy, the process of neovascularization mediated by circulating angiogenic cells (CACs) needs to be scrutinized.
Methods: In the present study, we compared the expression of neovascularization-related genes by 3 circulating CAC subsets (hematopoietic progenitor cells [HPCs], CD34+, and KDR+ cells; internal controls: peripheral blood mononuclear cells and circulating endothelial cells) of treatment-naïve patients with glioblastoma (GBM) to those of patients undergoing reactive neovascularization (myocardial infarction (MI). CACs from umbilical cord (representing developmental neovascularization) and healthy subjects served as controls. Fluorescent-activated cell sorting was used to isolate CACs, RT-PCR to determine the expression levels of a panel of 48 neovascularization-related genes, and Luminex assays to measure plasma levels of 21 CAC-related circulating molecules.
Results: We found essential differences in gene expression between GBM and MI CACs. GBM CACs had a higher expression of proangiogenic factors (especially, KITL, CXCL12, and JAG1), growth factor and chemotactic receptors (IGF1R, TGFBR2, CXCR4, and CCR2), adhesion receptor monomers (ITGA5 and ITGA6), and matricellular factor POSTN. In addition, we found major differences in the levels of neovascularization-related plasma factors. A strong positive correlation between plasma MMP9 levels and expression of CXCR4 in the CAC subset of HPCs was found in GBM patients.
Conclusions: Our findings indicate that CAC-mediated neovascularization in GBM is characterized by more efficient CAC homing to target tissue and a more potent proangiogenic response than in physiologic tissue repair in MI. Our findings can aid in selecting targets for therapeutic strategies acting against GBM-specific CACs.

Keywords:  angiogenesis; circulating angiogenic cell; endothelial progenitor cell; glioma; hematopoietic progenitor cell; myocardial infarction; neovascularization

DOI:  https://doi.org/10.1093/noajnl/vdaa040
Front Immunol. 2020 ;11 1191

Glioblastoma Myeloid-Derived Suppressor Cell Subsets Express Differential Macrophage Migration Inhibitory Factor Receptor Profiles That Can Be Targeted to Reduce Immune Suppression.

Tyler J Alban, Defne Bayik, Balint Otvos, Anja Rabljenovic, Lin Leng, Leu Jia-Shiun, Gustavo Roversi, Adam Lauko, Arbaz A Momin, Alireza M Mohammadi, David M Peereboom, Manmeet S Ahluwalia, Kazuko Matsuda, Kyuson Yun, Richard Bucala, Michael A Vogelbaum, Justin D Lathia.

  The application of tumor immunotherapy to glioblastoma (GBM) is limited by an unprecedented degree of immune suppression due to factors that include high numbers of immune suppressive myeloid cells, the blood brain barrier, and T cell sequestration to the bone marrow. We previously identified an increase in immune suppressive myeloid-derived suppressor cells (MDSCs) in GBM patients, which correlated with poor prognosis and was dependent on macrophage migration inhibitory factor (MIF). Here we examine the MIF signaling axis in detail in murine MDSC models, GBM-educated MDSCs and human GBM. We found that the monocytic subset of MDSCs (M-MDSCs) expressed high levels of the MIF cognate receptor CD74 and was localized in the tumor microenvironment. In contrast, granulocytic MDSCs (G-MDSCs) expressed high levels of the MIF non-cognate receptor CXCR2 and showed minimal accumulation in the tumor microenvironment. Furthermore, targeting M-MDSCs with Ibudilast, a brain penetrant MIF-CD74 interaction inhibitor, reduced MDSC function and enhanced CD8 T cell activity in the tumor microenvironment. These findings demonstrate the MDSC subsets differentially express MIF receptors and may be leveraged for specific MDSC targeting.

Keywords:  MDSC; MIF–macrophage migration inhibitory factor; glioma; immunesuppresion; immunetherapy

DOI:  https://doi.org/10.3389/fimmu.2020.01191
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa067

Overexpression of HGF/MET axis along with p53 inhibition induces de novo glioma formation in mice.

Yuan Qin, Anna Musket, Jianqun Kou, Johanna Preiszner, Barbara R Tschida, Anna Qin, Craig A Land, Ben Staal, Liang Kang, Kirk Tanner, Yong Jiang, John B Schweitzer, David A Largaespada, Qian Xie.

   Background: Aberrant MET receptor tyrosine kinase (RTK) activation leads to invasive tumor growth in different types of cancer. Overexpression of MET and its ligand hepatocyte growth factor (HGF) occurs more frequently in glioblastoma (GBM) than in low-grade gliomas. Although we have shown previously that HGF-autocrine activation predicts sensitivity to MET tyrosine kinase inhibitors (TKIs) in GBM, whether it initiates tumorigenesis remains elusive.
Methods: Using a well-established Sleeping Beauty (SB) transposon strategy, we injected human HGF and MET cDNA together with a short hairpin siRNA against Trp53 (SB-hHgf.Met.ShP53) into the lateral ventricle of neonatal mice to induce spontaneous glioma initiation and characterized the tumors with H&E and immunohistochemistry analysis. Glioma sphere cells also were isolated for measuring the sensitivity to specific MET TKIs.
Results: Mixed injection of SB-hHgf.Met.ShP53 plasmids induced de novo glioma formation with invasive tumor growth accompanied by HGF and MET overexpression. While glioma stem cells (GSCs) are considered as the tumor-initiating cells in GBM, both SB-hHgf.Met.ShP53 tumor sections and glioma spheres harvested from these tumors expressed GSC markers nestin, GFAP, and Sox 2. Moreover, specific MET TKIs significantly inhibited tumor spheres' proliferation and MET/MAPK/AKT signaling.
Conclusions: Overexpression of the HGF/MET axis along with p53 attenuation may transform neural stem cells into GSCs, resulting in GBM formation in mice. These tumors are primarily driven by the MET RTK pathway activation and are sensitive to MET TKIs. The SB-hHgf.Met.ShP53 spontaneous mouse glioma model provides a useful tool for studying GBM tumor biology and MET-targeting therapeutics.

Keywords:  HGF/MET signaling; genetically engineered mouse model; glioblastoma; glioma initiating cells; targeted therapy

DOI:  https://doi.org/10.1093/noajnl/vdaa067
Neurooncol Adv. 2019 May-Dec;1(1):1(1): vdz024

The soluble form of pan-RTK inhibitor and tumor suppressor LRIG1 mediates downregulation of AXL through direct protein-protein interaction in glioblastoma.

Virginie Neirinckx, Ann-Christin Hau, Anne Schuster, Sabrina Fritah, Katja Tiemann, Eliane Klein, Petr V Nazarov, André Matagne, Martyna Szpakowska, Max Meyrath, Andy Chevigné, Mirko H H Schmidt, Simone P Niclou.

   Background: Targeted approaches for inhibiting epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases (RTKs) in glioblastoma (GBM) have led to therapeutic resistance and little clinical benefit, raising the need for the development of alternative strategies. Endogenous LRIG1 (Leucine-rich Repeats and ImmunoGlobulin-like domains protein 1) is an RTK inhibitory protein required for stem cell maintenance, and we previously demonstrated the soluble ectodomain of LRIG1 (sLRIG1) to potently inhibit GBM growth in vitro and in vivo.
Methods: Here, we generated a recombinant protein of the ectodomain of LRIG1 (sLRIG1) and determined its activity in various cellular GBM models including patient-derived stem-like cells and patient organoids. We used proliferation, adhesion, and invasion assays, and performed gene and protein expression studies. Proximity ligation assay and NanoBiT complementation technology were applied to assess protein-protein interactions.
Results: We show that recombinant sLRIG1 downregulates EGFRvIII but not EGFR, and reduces proliferation in GBM cells, irrespective of their EGFR expression status. We find that sLRIG1 targets and downregulates a wide range of RTKs, including AXL, and alters GBM cell adhesion. Mechanistically, we demonstrate that LRIG1 interferes with AXL but not with EGFR dimerization.
Conclusions: These results identify AXL as a novel sLRIG1 target and show that LRIG1-mediated RTK downregulation depends on direct protein interaction. The pan-RTK inhibitory activity of sLRIG1 warrants further investigation for new GBM treatment approaches.

Keywords:  AXL; EGFR; LRIG1; glioblastoma; receptor tyrosine kinase

DOI:  https://doi.org/10.1093/noajnl/vdz024
Cancers (Basel). 2020 Jul 08. pii: E1831. [Epub ahead of print]12(7):

The Role of Liquid Biopsies in Detecting Molecular Tumor Biomarkers in Brain Cancer Patients.

Heena Sareen, Celine Garrett, David Lynch, Branka Powter, Daniel Brungs, Adam Cooper, Joseph Po, Eng-Siew Koh, Joey Yusof Vessey, Simon McKechnie, Renata Bazina, Mark Sheridan, James van Gelder, Balsam Darwish, Mathias Jaeger, Tara L Roberts, Paul De Souza, Therese M Becker.

  Glioblastoma multiforme (GBM) is one of the most lethal primary central nervous system cancers with a median overall survival of only 12-15 months. The best documented treatment is surgical tumor debulking followed by chemoradiation and adjuvant chemotherapy with temozolomide, but treatment resistance and therefore tumor recurrence, is the usual outcome. Although advances in molecular subtyping suggests GBM can be classified into four subtypes, one concern about using the original histology for subsequent treatment decisions is that it only provides a static snapshot of heterogeneous tumors that may undergo longitudinal changes over time, especially under selective pressure of ongoing therapy. Liquid biopsies obtained from bodily fluids like blood and cerebro-spinal fluid (CSF) are less invasive, and more easily repeated than surgery. However, their deployment for patients with brain cancer is only emerging, and possibly suppressed clinically due to the ongoing belief that the blood brain barrier prevents the egress of circulating tumor cells, exosomes, and circulating tumor nucleic acids into the bloodstream. Although brain cancer liquid biopsy analyses appear indeed challenging, advances have been made and here we evaluate the current literature on the use of liquid biopsies for detection of clinically relevant biomarkers in GBM to aid diagnosis and prognostication.

Keywords:  EGFR; IDHI; MGMT; biomarker; glioma

DOI:  https://doi.org/10.3390/cancers12071831
Nat Rev Mater. 2019 Oct;4(10): 651-668

Dissecting and rebuilding the glioblastoma microenvironment with engineered materials.

Kayla J Wolf, Joseph Chen, Jason Coombes, Manish K Aghi, Sanjay Kumar.

Glioblastoma (GBM) is the most aggressive and common form of primary brain cancer. Several decades of research have provided great insight into GBM progression; however, the prognosis remains poor with a median patient survival time of ~ 15 months. The tumour microenvironment (TME) of GBM plays a crucial role in mediating tumour progression and thus is being explored as a therapeutic target. Progress in the development of treatments targeting the TME is currently limited by a lack of model systems that can accurately recreate the distinct extracellular matrix composition and anatomic features of the brain, such as the blood-brain barrier and axonal tracts. Biomaterials can be applied to develop synthetic models of the GBM TME to mimic physiological and pathophysiological features of the brain, including cellular and ECM composition, mechanical properties, and topography. In this Review, we summarize key features of the GBM microenvironment and discuss different strategies for the engineering of GBM TME models, including 2D and 3D models featuring chemical and mechanical gradients, interfaces and fluid flow. Finally, we highlight the potential of engineered TME models as platforms for mechanistic discovery and drug screening as well as preclinical testing and precision medicine.

DOI: https://doi.org/10.1038/s41578-019-0135-y
Neurooncol Adv. 2019 May-Dec;1(1):1(1): vdz013

Biomarkers and smart intracranial devices for the diagnosis, treatment, and monitoring of high-grade gliomas: a review of the literature and future prospects.

Umbreen Hafeez, Lawrence M Cher.

  Glioblastoma (GBM) is the most common primary brain neoplasm with median overall survival (OS) around 15 months. There is a dearth of effective monitoring strategies for patients with high-grade gliomas. Relying on magnetic resonance images of brain has its challenges, and repeated brain biopsies add significant morbidity. Hence, it is imperative to establish a less invasive way to diagnose, monitor, and guide management of patients with high-grade gliomas. Currently, multiple biomarkers are in various phases of development and include tissue, serum, cerebrospinal fluid (CSF), and imaging biomarkers. Here we review and summarize the potential biomarkers found in blood and CSF, including extracellular macromolecules, extracellular vesicles, circulating tumor cells, immune cells, endothelial cells, and endothelial progenitor cells. The ability to detect tumor-specific biomarkers in blood and CSF will potentially not only reduce the need for repeated brain biopsies but also provide valuable information about the heterogeneity of tumor, response to current treatment, and identify disease resistance. This review also details the status and potential scope of brain tumor-related cranial devices and implants including Ommaya reservoir, microelectromechanical systems-based depot device, Alzet mini-osmotic pump, Metronomic Biofeedback Pump (MBP), ipsum G1 implant, ultra-thin needle implant, and putative devices. An ideal smart cranial implant will overcome the blood-brain barrier, deliver various drugs, provide access to brain tissue, and potentially measure and monitor levels of various biomarkers.

Keywords:  Biomarkers; high-grade gliomas; intracranial devices; intracranial implants

DOI:  https://doi.org/10.1093/noajnl/vdz013
Nat Commun. 2020 Jul 10. 11(1): 3457

A cytoskeleton regulator AVIL drives tumorigenesis in glioblastoma.

Zhongqiu Xie, Pawel Ł Janczyk, Ying Zhang, Aiqun Liu, Xinrui Shi, Sandeep Singh, Loryn Facemire, Kristopher Kubow, Zi Li, Yuemeng Jia, Dorothy Schafer, James W Mandell, Roger Abounader, Hui Li.

Glioblastoma is a deadly cancer, with no effective therapies. Better understanding and identification of selective targets are urgently needed. We found that advillin (AVIL) is overexpressed in all the glioblastomas we tested including glioblastoma stem/initiating cells, but hardly detectable in non-neoplastic astrocytes, neural stem cells or normal brain. Glioma patients with increased AVIL expression have a worse prognosis. Silencing AVIL nearly eradicated glioblastoma cells in culture, and dramatically inhibited in vivo xenografts in mice, but had no effect on normal control cells. Conversely, overexpressing AVIL promoted cell proliferation and migration, enabled fibroblasts to escape contact inhibition, and transformed immortalized astrocytes, supporting AVIL being a bona fide oncogene. We provide evidence that the tumorigenic effect of AVIL is partly mediated by FOXM1, which regulates LIN28B, whose expression also correlates with clinical prognosis. AVIL regulates the cytoskeleton through modulating F-actin, while mutants disrupting F-actin binding are defective in its tumorigenic capabilities.

DOI: https://doi.org/10.1038/s41467-020-17279-1
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa047

MicroRNA-93 acts as an "anti-inflammatory tumor suppressor" in glioblastoma.

Max Hübner, Nicholas Moellhoff, David Effinger, Christian Ludwig Hinske, Simon Hirschberger, Tingting Wu, Martin Bernhard Müller, Gabriele Strauß, Friedrich-Wilhelm Kreth, Simone Kreth.

Background: Inflammation is an important driver of malignant glioma disease. Inflammatory mediators are not only produced by immune cells in the tumor microenvironment, but also by glioblastoma (GBM) cells themselves creating a mutually reinforcing loop. We here aimed at identifying an "anti-inflammatory switch" that allows to dampen inflammation in GBM.
Methods: We used human GBM specimens, primary cultures, and cell lines. The response of GBM cells toward inflammatory stimuli was tested by incubation with supernatant of stimulated human immune cells. Expression levels were measured by whole transcriptome microarrays and qRT-PCR, and protein was quantified by LUMINEX and SDS-PAGE. MicroRNA binding to 3'UTRs was analyzed by luciferase assays. Proliferation rates were determined by flow cytometry, and invasion and angiogenesis were studied using migration and endothelial tube formation assays.
Results: We demonstrated GBM cells to secrete high amounts of proinflammatory mediators in an inflammatory microenvironment. We found miR-93 as a potential "anti-inflammatory tumor suppressor" dramatically downregulated in GBM. Concordantly, cytokine secretion dropped after miR-93 re-expression. Transfection of miR-93 in GBM cells led to down-regulation of hubs of the inflammatory networks, namely, HIF-1α and MAP3K2 as well as IL-6, G-CSF, IL-8, LIF, IL-1β, COX2, and CXCL5. We showed only COX2 and CXCL5 to be indirectly regulated by miR-93 while all other genes are true targets. Phenotypically, re-expression of miR-93 in GBM cells substantially suppressed proliferation, migration, and angiogenesis.
Conclusions: Alleviating GBM-derived inflammation by re-expression of miR-93 may be a powerful tool to mitigate these tumors' aggressiveness and holds promise for new clinical approaches.

DOI: https://doi.org/10.1093/noajnl/vdaa047
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa013

Suicide gene therapy for the treatment of high-grade glioma: past lessons, present trends, and future prospects.

Jubayer A Hossain, Antonio Marchini, Boris Fehse, Rolf Bjerkvig, Hrvoje Miletic.

  Suicide gene therapy has represented an experimental cancer treatment modality for nearly 40 years. Among the various cancers experimentally treated by suicide gene therapy, high-grade gliomas have been the most prominent both in preclinical and clinical settings. Failure of a number of promising suicide gene therapy strategies in the clinic pointed toward a bleak future of this approach for the treatment of high-grade gliomas. Nevertheless, the development of new vectors and suicide genes, better prodrugs, more efficient delivery systems, and new combinatorial strategies represent active research areas that may eventually lead to better efficacy of suicide gene therapy. These trends are evident by the current increasing focus on suicide gene therapy for high-grade glioma treatment both in the laboratory and in the clinic. In this review, we give an overview of different suicide gene therapy approaches for glioma treatment and discuss clinical trials, delivery issues, and immune responses.

Keywords:  glioma; immunotherapy; stem cells; suicide gene therapy; viral vectors

DOI:  https://doi.org/10.1093/noajnl/vdaa013
Nat Commun. 2020 Jul 08. 11(1): 3406

Single-cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy.

Charles P Couturier, Shamini Ayyadhury, Phuong U Le, Javad Nadaf, Jean Monlong, Gabriele Riva, Redouane Allache, Salma Baig, Xiaohua Yan, Mathieu Bourgey, Changseok Lee, Yu Chang David Wang, V Wee Yong, Marie-Christine Guiot, Hamed Najafabadi, Bratislav Misic, Jack Antel, Guillaume Bourque, Jiannis Ragoussis, Kevin Petrecca.

Cancer stem cells are critical for cancer initiation, development, and treatment resistance. Our understanding of these processes, and how they relate to glioblastoma heterogeneity, is limited. To overcome these limitations, we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells, and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells, and, using RNA velocity, is often the originator of the other cell types. Finally, we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development, suggests a possible origin for glioblastoma hierarchy, and helps to identify cancer stem cell-specific targets.

DOI: https://doi.org/10.1038/s41467-020-17186-5
Cancer Lett. 2020 Jul 06. pii: S0304-3835(20)30353-0. [Epub ahead of print]

Sustained inhibition of PARP-1 activity delays glioblastoma recurrence by enhancing radiation-induced senescence.

Atanu Ghorai, Tejashree Mahaddalkar, Rahul Thorat, Shilpee Dutt.

  Glioblastoma (GBM) is the most common primary brain tumor and is highly aggressive with a median survival of 15 months. We have previously shown that residual cells of GBM form multinucleated giant cells (MNGCs) showing a senescent phenotype, but eventually escape from therapy induced senescence (TIS), resulting in GBM recurrence. Here we demonstrate the role of PARP-1 in TIS and its recovery. We show that genetic and pharmacological inhibition of PARP-1 has an anti-proliferative effect on GBM cell lines and primary cultures derived from patient samples. Furthermore, the PARP-1 inhibitor olaparib, in combination with radiation increased MNGCs formation and senescence as assessed by β-galactosidase activity, and macroH2A1 levels in residual cells. Additionally, we found that reduced PARP-1 activity and not protein levels in residual cells was crucial for MNGCs formation and their maintenance in the senescent state. PARP-1 activity was restored to higher levels in recurrent cells that escaped from TIS. Importantly, olaparib + radiation treatment significantly delayed recurrence in vitro as well in vivo in orthotopic GBM mouse models with a significant increase in overall survival of mice. Overall, this study demonstrates that sustained inhibition of PARP-1 activity during radiation treatment significantly delays GBM recurrence.

Keywords:  Glioblastoma; MNGCs; PARylation; Radiation resistance & recurrence; Therapy induced senescence

DOI:  https://doi.org/10.1016/j.canlet.2020.06.023
Neurooncol Adv. 2019 May-Dec;1(1):1(1): vdz004

Combined targeting of PI3K and MEK effector pathways via CED for DIPG therapy.

Raymond Chang, Umberto Tosi, Julia Voronina, Oluwaseyi Adeuyan, Linda Y Wu, Melanie E Schweitzer, David J Pisapia, Oren J Becher, Mark M Souweidane, Uday B Maachani.

   Background: Midline gliomas like diffuse intrinsic pontine glioma (DIPG) carry poor prognosis and lack effective treatment options. Studies have implicated amplifications in the phosphatidylinositol 3-kinase (PI3K) signaling pathway in tumorigenesis; compensatory activation of parallel pathways (eg, mitogen-activated protein kinase [MEK]) may underlie the resistance to PI3K inhibition observed in the clinic.
Methods: Three patient-derived cell lines (SU-DIPG-IV, SU-DIPG-XIII, and SF8628) and a mouse-derived brainstem glioma cell line were treated with PI3K (ZSTK474) and MEK (trametinib) inhibitors, alone or in combination. Synergy was analyzed using Chou-Talalay combination index (CI). These agents were also used alone or in combination in a subcutaneous SU-DIPG-XIII tumor model and in an intracranial genetic mouse model of DIPG, given via convection-enhanced delivery (CED).
Results: We found that these agents abrogate cell proliferation in a dose-dependent manner. Combination treatments were found to be synergistic (CI < 1) across cell lines tested. They also showed significant tumor suppression when given systemically against a subcutaneous DIPG model (alone or in combination) or when given via direct intracranial injection (CED) in a intracranial DIPG mouse model (combination only, median survival 47 vs 35 days post-induction, P = .038). No significant short- or long-term neurotoxicity of ZSTK474 and trametinib delivered via CED was observed.
Conclusions: Our data indicate that ZSTK474 and trametinib combinatorial treatment inhibits malignant growth of DIPG cells in vitro and in vivo, prolonging survival. These results suggest a promising new combinatorial approach using CED for DIPG therapy, which warrants further investigation.

Keywords:  MEK; PI3K; combinatorial therapy; convection-enhanced delivery; midline glioma

DOI:  https://doi.org/10.1093/noajnl/vdz004
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa001

Sexually dimorphic impact of the iron-regulating gene, HFE, on survival in glioblastoma.

Darya S Nesterova, Vishal Midya, Brad E Zacharia, Elizabeth A Proctor, Sang Y Lee, Lindsay C Stetson, Justin D Lathia, Joshua B Rubin, Kristin A Waite, Michael E Berens, Jill S Barnholtz-Sloan, James R Connor.

   Background: The median survival for patients with glioblastoma (GBM), the most common primary malignant brain tumor in adults, has remained approximately 1 year for more than 2 decades. Recent advances in the field have identified GBM as a sexually dimorphic disease. It is less prevalent in females and they have better survival compared to males. The molecular mechanism of this difference has not yet been established. Iron is essential for many biological processes supporting tumor growth and its regulation is impacted by sex. Therefore, we interrogated the expression of a key component of cellular iron regulation, the HFE (homeostatic iron regulatory) gene, on sexually dimorphic survival in GBM.
Methods: We analyzed TCGA microarray gene expression and clinical data of all primary GBM patients (IDH-wild type) to compare tumor mRNA expression of HFE with overall survival, stratified by sex.
Results: In low HFE expressing tumors (below median expression, n = 220), survival is modulated by both sex and MGMT status, with the combination of female sex and MGMT methylation resulting in over a 10-month survival advantage (P < .0001) over the other groups. Alternatively, expression of HFE above the median (high HFE, n = 240) is associated with significantly worse overall survival in GBM, regardless of MGMT methylation status or patient sex. Gene expression analysis uncovered a correlation between high HFE expression and expression of genes associated with immune function.
Conclusions: The level of HFE expression in GBM has a sexually dimorphic impact on survival. Whereas HFE expression below the median imparts a survival benefit to females, high HFE expression is associated with significantly worse overall survival regardless of established prognostic factors such as sex or MGMT methylation.

Keywords:  HFE; glioblastoma; iron; sex; tumor

DOI:  https://doi.org/10.1093/noajnl/vdaa001
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa039

Proteins inform survival-based differences in patients with glioblastoma.

L C Stetson, Quinn T Ostrom, Daniela Schlatzer, Peter Liao, Karen Devine, Kristin Waite, Marta E Couce, Peggy L R Harris, Amber Kerstetter-Fogle, Michael E Berens, Andrew E Sloan, Mohammad M Islam, Vilashini Rajaratnam, Shama P Mirza, Mark R Chance, Jill S Barnholtz-Sloan.

   Background: Improving the care of patients with glioblastoma (GB) requires accurate and reliable predictors of patient prognosis. Unfortunately, while protein markers are an effective readout of cellular function, proteomics has been underutilized in GB prognostic marker discovery.
Methods: For this study, GB patients were prospectively recruited and proteomics discovery using liquid chromatography-mass spectrometry analysis (LC-MS/MS) was performed for 27 patients including 13 short-term survivors (STS) (≤10 months) and 14 long-term survivors (LTS) (≥18 months).
Results: Proteomics discovery identified 11 941 peptides in 2495 unique proteins, with 469 proteins exhibiting significant dysregulation when comparing STS to LTS. We verified the differential abundance of 67 out of these 469 proteins in a small previously published independent dataset. Proteins involved in axon guidance were upregulated in STS compared to LTS, while those involved in p53 signaling were upregulated in LTS. We also assessed the correlation between LS MS/MS data with RNAseq data from the same discovery patients and found a low correlation between protein abundance and mRNA expression. Finally, using LC-MS/MS on a set of 18 samples from 6 patients, we quantified the intratumoral heterogeneity of more than 2256 proteins in the multisample dataset.
Conclusions: These proteomic datasets and noted protein variations present a beneficial resource for better predicting patient outcome and investigating potential therapeutic targets.

Keywords:  glioblastoma; mass spectrometry; proteomics; survival

DOI:  https://doi.org/10.1093/noajnl/vdaa039
Neurooncol Adv. 2019 May-Dec;1(1):1(1): vdz022

Expansion of effector and memory T cells is associated with increased survival in recurrent glioblastomas treated with dendritic cell immunotherapy.

Marica Eoli, Cristina Corbetta, Elena Anghileri, Natalia Di Ianni, Micaela Milani, Valeria Cuccarini, Silvia Musio, Rosina Paterra, Simona Frigerio, Sara Nava, Daniela Lisini, Sara Pessina, Luisa Maddaloni, Raffaella Lombardi, Maria Tardini, Paolo Ferroli, Francesco DiMeco, Maria Grazia Bruzzone, Carlo Antozzi, Bianca Pollo, Gaetano Finocchiaro, Serena Pellegatta.

   Background: The efficacy of dendritic cell (DC) immunotherapy as a single therapeutic modality for the treatment of glioblastoma (GBM) patients remains limited. In this study, we evaluated in patients with GBM recurrence the immune-mediated effects of DC loaded with autologous tumor lysate combined with temozolomide (TMZ) or tetanus toxoid (TT).
Methods: In the phase I-II clinical study DENDR2, 12 patients were treated with 5 DC vaccinations combined with dose-dense TMZ. Subsequently, in eight patients, here defined as Variant (V)-DENDR2, the vaccine site was preconditioned with TT 24 hours before DC vaccination and TMZ was avoided. As a survival endpoint for these studies, we considered overall survival 9 months (OS9) after second surgery. Patients were analyzed for the generation of effector, memory, and T helper immune response.
Results: Four of 12 DENDR2 patients reached OS9, but all failed to show an immunological response. Five of eight V-DENDR2 patients (62%) reached OS9, and one patient is still alive (OS >30 months). A robust CD8+ T-cell activation and memory T-cell formation were observed in V-DENDR2 OS>9. Only in these patients, the vaccine-specific CD4+ T-cell activation (CD38+/HLA-DR+) was paralleled by an increase in TT-induced CD4+/CD38low/CD127high memory T cells. Only V-DENDR2 patients showed the formation of a nodule at the DC injection site infiltrated by CCL3-expressing CD4+ T cells.
Conclusions: TT preconditioning of the vaccine site and lack of TMZ could contribute to the efficacy of DC immunotherapy by inducing an effector response, memory, and helper T-cell generation.

Keywords:  T-cell memory; dendritic cells; glioblastoma; immunotherapy; tetanus toxoid

DOI:  https://doi.org/10.1093/noajnl/vdz022
Neurooncol Adv. 2019 May-Dec;1(1):1(1): vdz029

The invasive proteome of glioblastoma revealed by laser-capture microdissection.

Thomas Daubon, Joris Guyon, Anne-Aurélie Raymond, Benjamin Dartigues, Justine Rudewicz, Zakaria Ezzoukhry, Jean-William Dupuy, John M J Herbert, Frédéric Saltel, Rolf Bjerkvig, Macha Nikolski, Andreas Bikfalvi.

   Background: Glioblastomas are heterogeneous tumors composed of a necrotic and tumor core and an invasive periphery.
Methods: Here, we performed a proteomics analysis of laser-capture micro-dissected glioblastoma core and invasive areas of patient-derived xenografts.
Results: Bioinformatics analysis identified enriched proteins in central and invasive tumor areas. Novel markers of invasion were identified, the genes proteolipid protein 1 (PLP1) and Dynamin-1 (DNM1), which were subsequently validated in tumors and by functional assays.
Conclusions: In summary, our results identify new networks and molecules that may play an important role in glioblastoma development and may constitute potential novel therapeutic targets.

Keywords:  glioblastoma; intratumor heterogeneity; invasion; patient-derived xenograft; proteomics analysis

DOI:  https://doi.org/10.1093/noajnl/vdz029
Sci Rep. 2020 Jul 09. 10(1): 11270

Multivariate analysis reveals differentially expressed genes among distinct subtypes of diffuse astrocytic gliomas: diagnostic implications.

Nerea González-García, Ana Belén Nieto-Librero, Ana Luisa Vital, Herminio José Tao, María González-Tablas, Álvaro Otero, Purificación Galindo-Villardón, Alberto Orfao, María Dolores Tabernero.

Diagnosis and classification of gliomas mostly relies on histopathology and a few genetic markers. Here we interrogated microarray gene expression profiles (GEP) of 268 diffuse astrocytic gliomas-33 diffuse astrocytomas (DA), 52 anaplastic astrocytomas (AA) and 183 primary glioblastoma (GBM)-based on multivariate analysis, to identify discriminatory GEP that might support precise histopathological tumor stratification, particularly among inconclusive cases with II-III grade diagnosed, which have different prognosis and treatment strategies. Microarrays based GEP was analyzed on 155 diffuse astrocytic gliomas (discovery cohort) and validated in another 113 tumors (validation set) via sequential univariate analysis (pairwise comparison) for discriminatory gene selection, followed by nonnegative matrix factorization and canonical biplot for identification of discriminatory GEP among the distinct histological tumor subtypes. GEP data analysis identified a set of 27 genes capable of differentiating among distinct subtypes of gliomas that might support current histological classification. DA + AA showed similar molecular profiles with only a few discriminatory genes overexpressed (FSTL5 and SFRP2) and underexpressed (XIST, TOP2A and SHOX2) in DA vs AA and GBM. Compared to DA + AA, GBM displayed underexpression of ETNPPL, SH3GL2, GABRG2, SPX, DPP10, GABRB2 and CNTN3 and overexpression of CHI3L1, IGFBP3, COL1A1 and VEGFA, among other differentially expressed genes.

DOI: https://doi.org/10.1038/s41598-020-67743-7
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa061

Identification of functionally distinct and interacting cancer cell subpopulations from glioblastoma with intratumoral genetic heterogeneity.

Min Guo, Marjolein van Vliet, Jian Zhao, Teresita Díaz de Ståhl, Mikael S Lindström, Huaitao Cheng, Susanne Heller, Monica Nistér, Daniel Hägerstrand.

   Background: Glioblastomas display a high level of intratumoral heterogeneity with regard to both genetic and histological features. Within single tumors, subclones have been shown to communicate with each other to affect overall tumor growth. The aim of this study was to broaden the understanding of interclonal communication in glioblastoma.
Methods: We have used the U-343 model, consisting of U-343 MG, U-343 MGa, U-343 MGa 31L, and U-343 MGa Cl2:6, a set of distinct glioblastoma cell lines that have been derived from the same tumor. We characterized these with regard to temozolomide sensitivity, protein secretome, gene expression, DNA copy number, and cancer cell phenotypic traits. Furthermore, we performed coculture and conditioned media-based experiments to model cell-to-cell signaling in a setting of intratumoral heterogeneity.
Results: Temozolomide treatment of a coculture composed of all 4 U-343 cell lines presents a tumor relapse model where the least sensitive population, U-343 MGa 31L, outlives the others. Interestingly, the U-343 cell lines were shown to have distinct gene expression signatures and phenotypes although they were derived from a single tumor. The DNA copy number analysis revealed both common and unique alterations, indicating the evolutionary relationship between the cells. Moreover, these cells were found to communicate and affect each other's proliferation, both via contact-dependent and -independent interactions, where NOTCH1, TGFBI, and ADAMTS1 signaling effects were involved, respectively.
Conclusions: These results provide insight into how complex the signaling events may prove to be in a setting of intratumoral heterogeneity in glioblastoma and provide a map for future studies.

Keywords:  NOTCH; TGFBI; cell-to-cell interaction; glioblastoma heterogeneity; secretome; temozolomide

DOI:  https://doi.org/10.1093/noajnl/vdaa061
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdaa056

Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes.

Juliana Hofstatter Azambuja, Nils Ludwig, Saigopalakrishna Yerneni, Aparna Rao, Elizandra Braganhol, Theresa L Whiteside.

   Background: Glioblastoma is one of the most immunosuppressive human tumors. Emerging data suggest that glioblastoma-derived exosomes (GBex) reprogram the tumor microenvironment into a tumor-promoting milieu by mechanisms that not yet understood.
Methods: Exosomes were isolated from supernatants of glioblastoma cell lines by size exclusion chromatography. The GBex endosomal origin, size, protein cargos, and ex vivo effects on immune cell functions were determined. GBex were injected intravenously into mice to evaluate their ability to in vivo modulate normal immune cell subsets.
Results: GBex carried immunosuppressive proteins, including FasL, TRAIL, CTLA-4, CD39, and CD73, but contained few immunostimulatory proteins. GBex co-incubated with primary human immune cells induced simultaneous activation of multiple molecular pathways. In CD8+ T cells, GBex suppressed TNF-α and INF-γ release and mediated apoptosis. GBex suppressed natural killer (NK) and CD4+ T-cell activation. GBex activated the NF-κB pathway in macrophages and promoted their differentiation into M2 cells. Inhibition of the NF-κB pathway in macrophages reversed the GBex-mediated effects. GBex-driven reprogramming of macrophages involved the release of soluble factors that promoted tumor proliferation in vitro. In mice injected with GBex, the frequency of splenic CD8+ T cells, NK cells, and M1-like macrophages was reduced, while that of naïve and M2-like macrophages increased (P < .05).
Conclusions: GBex reprogrammed functions of all types of immune cells in vitro and altered their frequency in vivo. By creating and sustaining a highly immunosuppressive environment, GBex play a key role in promoting tumor progression.

Keywords:   tumor microenvironment; exosomes; glioblastoma; immune system; macrophages

DOI:  https://doi.org/10.1093/noajnl/vdaa056
Neurooncol Adv. 2020 Jan-Dec;2(1):2(1): vdz051

EGFR mutations are associated with response to depatux-m in combination with temozolomide and result in a receptor that is hypersensitive to ligand.

Youri Hoogstrate, Wies Vallentgoed, Johan M Kros, Iris de Heer, Maurice de Wit, Marica Eoli, Juan Manuel Sepulveda, Annemiek M E Walenkamp, Jean-Sebastien Frenel, Enrico Franceschi, Paul M Clement, Micheal Weller, Martin E van Royen, Peter Ansell, Jim Looman, Earle Bain, Marie Morfouace, Thierry Gorlia, Vassilis Golfinopoulos, Martin van den Bent, Pim J French.

   Background: The randomized phase II INTELLANCE-2/EORTC_1410 trial on EGFR-amplified recurrent glioblastomas showed a trend towards improved overall survival when patients were treated with depatux-m plus temozolomide compared with the control arm of alkylating chemotherapy only. We here performed translational research on material derived from this clinical trial to identify patients that benefit from this treatment.
Methods: Targeted DNA-sequencing and whole transcriptome analysis was performed on clinical trial samples. High-throughput, high-content imaging analysis was done to understand the molecular mechanism underlying the survival benefit.
Results: We first define the tumor genomic landscape in this well-annotated patient population. We find that tumors harboring EGFR single-nucleotide variations (SNVs) have improved outcome in the depatux-m + TMZ combination arm. Such SNVs are common to the extracellular domain of the receptor and functionally result in a receptor that is hypersensitive to low-affinity EGFR ligands. These hypersensitizing SNVs and the ligand-independent EGFRvIII variant are inversely correlated, indicating two distinct modes of evolution to increase EGFR signaling in glioblastomas. Ligand hypersensitivity can explain the therapeutic efficacy of depatux-m as increased ligand-induced activation will result in increased exposure of the epitope to the antibody-drug conjugate. We also identified tumors harboring mutations sensitive to "classical" EGFR tyrosine-kinase inhibitors, providing a potential alternative treatment strategy.
Conclusions: These data can help guide treatment for recurrent glioblastoma patients and increase our understanding into the molecular mechanisms underlying EGFR signaling in these tumors.

Keywords:  EGFR; amphiregulin; depatux-m; extracellular domain mutations; ligand hypersensitivity

DOI:  https://doi.org/10.1093/noajnl/vdz051