Mesenchymal Glioblastoma Research Articles

Peroxisome proliferator-activated receptor gamma as a theragnostic target for mesenchymal-type glioblastoma patients

Glioblastomas (GBMs) are characterized by four subtypes, proneural (PN), neural, classical, and mesenchymal (MES) GBMs, and they all have distinct activated signaling pathways. Among the subtypes, PN and MES GBMs show mutually exclusive genetic signatures, and the MES phenotype is, in general, believed to be associated with more aggressive features of GBM: tumor recurrence and drug resistance. Therefore, targeting MES GBMs would improve the overall prognosis of patients with fatal tumors. In this study, we propose peroxisome proliferator-activated receptor gamma (PPARγ) as a potential diagnostic and prognostic biomarker as well as therapeutic target for MES GBM; we used multiple approaches to assess PPARγ, including biostatistics analysis and assessment of preclinical studies. First, we found that PPARγ was exclusively expressed in MES glioblastoma stem cells (GSCs), and ligand activation of endogenous PPARγ suppressed cell growth and stemness in MES GSCs. Further in vivo studies involving orthotopic and heterotopic xenograft mouse models confirmed the therapeutic efficacy of targeting PPARγ; compared to control mice, those that received ligand treatment exhibited longer survival as well as decreased tumor burden. Mechanistically, PPARγ activation suppressed proneural–mesenchymal transition (PMT) by inhibiting the STAT3 signaling pathway. Biostatistical analysis using The Cancer Genomics Atlas (TCGA, n = 206) and REMBRANDT (n = 329) revealed that PPARγ upregulation is linked to poor overall survival and disease-free survival of GBM patients. Analysis was performed on prospective (n = 2) and retrospective (n = 6) GBM patient tissues, and we finally confirmed that PPARγ expression was distinctly upregulated in MES GBM. Collectively, this study provides insight into PPARγ as a potential therapeutic target for patients with MES GBM.

TGFβ1-induced beta-site APP-cleaving enzyme 2 upregulation promotes tumorigenesis through the NF-κB signalling pathway in human gliomas.

Gliomas are the most common primary malignant tumours of the central nervous system, and new molecular biomarkers are urgently needed for diagnosis and targeted therapy. Here, we report that increased beta‐site APP‐cleaving enzyme 2 (BACE2) expression is associated with increases in the grade of human glioma, the incidence of the mesenchymal molecular glioblastoma multiforme subtype and the likelihood of poor prognoses for patients. BACE2 knockdown suppressed cell invasion, cell migration and tumour growth both in vitro and in vivo, while BACE2 overexpression promoted the mesenchymal transition and cell proliferation. Furthermore, TGFβ1 stimulated BACE2 expression through Smad‐dependent signalling, which modulated TNF‐α‐induced NF‐κB activity through the PP1A/IKK pathway to promote tumorigenesis in both U87MG and U251 cells. Our study indicated that BACE2 plays a significant role in glioma development. Therefore, BACE2 is a potential therapeutic target for human gliomas due to its function and ability to be regulated.

A molecularly distinct subset of glioblastoma requires serum-containing media to establish sustainable bona fide glioblastoma stem cell cultures.

Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra-tumor and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking and the majority of patients survive less than 1 year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models in target identification and drug screening studies. The current state-of-the-art method, to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 218 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The frequency of maintainable SF cultures (SFCs) was most successful, but for a subgroup of GBM specimens, a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of a GSC, that is, extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that would have evaded modeling in SF conditions only provide unique cell models of GBM inter-tumor heterogeneity.

The Phenotypes of Proliferating Glioblastoma Cells Reside on a Single Axis of Variation.

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identified. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are sufficient to generate the heterogeneity observed in GBM. We performed single-cell/single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity. SIGNIFICANCE: Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classic-subtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types.See related commentary by Fine, p. 1650.This article is highlighted in the In This Issue feature, p. 1631.

NKG2D Controls Natural Reactivity of Vγ9Vδ2 T Lymphocytes against Mesenchymal Glioblastoma Cells

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. 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. 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. 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.

Identification of Periostin as a Potential Biomarker in Gliomas by Database Mining

Bioinformatics analysis integrating microenvironmental factors and single cell analysis segregated the glioblastoma (GBM) subtype into 3 subtypes: proneural, classic, and mesenchymal. Mesenchymal GBM tends to have the worst survival but benefits from aggressive treatment protocols. Therefore, it is clinically meaningful to identify relevant biomarkers to distinguish the mesenchymal subtype. Moreover, in developing nations with limited resources, rigorous examinations are costly and inefficient for patient care. In this study, we analyzed The Cancer Genome Atlas (TCGA)-Glioblastoma and TCGA-Low-Grade Glioma RNA sequencing (RNAseq) cohorts and confirmed that the mesenchymal subtype was associated with the worst prognosis. We identified periostin (POSTN) as a mesenchymal subtype biomarker with prognostic value across histologic grades and confirmed the reliability of POSTN by gene expression meta-analysis combining TCGA, Chinese Glioma Genome Atlas (CGGA) and REMBRANDT (Repository for Molecular Brain Neoplasia Data) GBM cohorts (hazard ratio, 1.71 [range, 1.47-2.07], n= 693) and LGG cohorts (hazard ratio, 2.55 [range, 1.61-4.05], n= 1226). By using available online glioma databases, our study provided an insight into the expression of POSTN as an independent predictor for patients with glioma (GBM and LGG) and could be useful for diagnostic simplification to identify high-risk groups.

PATH-21. CLINICAL UTILITY OF DNA METHYLATION PROFILING FOR DIAGNOSIS OF CHALLENGING CENTRAL NERVOUS SYSTEM TUMORS: THE TORONTO EXPERIENCE

Abstract The update on the WHO classification of central nervous system (CNS) tumors incorporated molecular signatures for a more accurate diagnosis. Recently, DKFZ has demonstrated the utility of DNA methylation profiling(MP) for molecular classification of CNS tumors. We performed a prospective clinical study over the last three years to evaluate the clinical utility ofDNA MP on FFPE samples of 66 challenging CNS tumor cases using online DKFZ classifier. Eleven samples were excluded due to low tumor DNA content or low calibration(predictive) scores(CS)< 0.3.DNA MP confirmed the original pathology diagnoses in 15(27%)cases. The integrated molecular diagnoses were changed in 38/55(70%) including establishment of a new diagnostic entity, change in molecular signature and subtyping. TheWHO grades were changed in 16(27%) of the tumors; about two-thirds resulted in upgrading. We detected non-canonical IDH mutations in 9 diffuse gliomas and the CNV plots revealed false positive FISH results for 1p/19q co-deletion in two diffuse gliomas. The CNV plots contributed to the final diagnosis in 40(72%) patients. The molecular subtypes of medulloblastoma, ependymoma and glioblastoma subclasses were determined in 36(65%) cases. Seventy-five percent of cases with confirmation of initial diagnosis or change in molecular diagnosis had CS > 0.5, among which 51% had a CS >0.9. The median and range CS of cases with new diagnostic entity and confirmed cases were 0.86(0.37–0.99) and 0.98(0.42–0.99), respectably. Furthermore, we detected higher CS in IDH-mutant gliomas in comparison to glioblastoma IDH-wild type(P=0.04). We also observed lower CS in mesenchymal glioblastoma in comparison to other subclasses. The MGMT promoter methylation was determined in 17/20(85%) glioblastoma cases. While the DKFZ group established CS of 0.9 as a cut-off for matching to methylation classes, our findings suggest lower threshold values in challenging CNS tumor cases. Our experience indicates clinical utility of MP of challenging CNS tumors as a reliable ancillary diagnostic tool in routine neuropathology practice.

COMP-04. MODELING PRECISION ONCOLOGY FOR GLIOBLASTOMA THROUGH INTEGRATION OF DESCRIPTIVE, FUNCTIONAL, AND NETWORK-BASED GENOMICS

Abstract Precision oncology is largely based on the notion that identification and targeting of oncogenic drivers will lead to improved clinical outcomes. However, the promise of precision oncology awaits to be fulfilled for many cancers, including Glioblastoma (GBM), where identification of oncogenic drivers has yet to improve survival rates. Here, we have attempted to systematically identify GBM vulnerabilities by performing genome-wide CRISRP-Cas9 lethality screens in patient-derived GBM stem-like cells (GSCs). In validation studies, we comprehensively retested GSC-specific hits in multiple GSC isolates, which were also genomically profiled (e.g. RNA-seq, exome-seq, CNV), and further integrated these data with CRISPR-Cas9 lethality screens from over 500 human cell lines from the Broad Institute’s CRISPR Avana dataset. As a result, we have begun making GBM dependency predictions and functional associations for top scoring hits, including: tumor developmental subtype; loss of functional redundancy with other genes/proteins; cancer-specific subnetworks of genes involved in mitochondrial protein turnover and membrane trafficking; and genes of unknown function essential for subset of GBMs. A few examples of these categories include the following scenarios. We find ADAR (Adenosine Deaminase RNA Specific) gene dependency is associated with the mesenchymal GBM subtype. The EFR3Agene, which has roles in maintaining active pools of phosphatidylinositol 4-kinase, appears required when the expression of its paralog EFR3Bis low or absent in tumor cells. The F-box protein-encoding gene FBXO42appears non-essential to most human cells lines and neural stem cells, but when knocked out in sensitive GSCs causes mitotic arrest, mitotic catastrophe, and cell death. While still a work in progress, we hope to use these results as a foundation for exploring and illuminating patient-specific molecular vulnerabilities for brain tumors. The results also underscore the need for integration of functional genetic approaches, where gene activities are inhibited, into precision oncology paradigms.

SCIDOT-26. THE ROLE OF TUMOR-DERIVED GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF/CSF2) IN REGULATION OF MICROGLIA-DEPENDENT INVASION IN GLIOMAS

Abstract Brain resident immune cells (microglia) and peripheral macrophages accumulate in malignant gliomas and constitute for 30–50% of the tumor mass. These immune cells are polarized by factors released by glioma and become the pro-invasive, immunosuppressive cells that support tumor progression. We have previously found that tumor-derived granulocyte macrophage colony stimulating factor (GM-CSF/Csf-2) is a crucial factor controlling accumulation of microglia and macrophages in murine gliomas. The analysis of TCGA dataset revealed overexpression of the CSF2 gene (encoding GM-CSF) in a set of mesenchymal glioblastomas (most aggressive WHO grade IV gliomas) and its association with high immune gene expression. To study the role of GM-CSF in microglia-stimulated glioma invasion, we used a co-culture system, which mimics microglia interactions with tumor cells. We silenced the expression of CSF2 in glioma cells and found reduced microglia-dependent invasion of glioma cells. To translate those results into clinically relevant setting, we designed and tested humanized short peptides interfering with binding of GM-CSF to its receptor. Selected peptide effectively inhibited binding of GM-CSF to its receptor as demonstrated with different methods. We selected the non-cytotoxic peptides that potently blocked microglia-dependent glioma invasion in cell co-cultures. Blocking GM-CSF-receptor signaling pathway with a neutralizing antibody against a GM-CSF receptor also inhibited microglia-dependent invasion of glioma cells. Altogether, our results demonstrate that glioma-derived GM-CSF supports pro-tumorigenic polarization of microglia turning them into cells that facilitate glioma growth and shape the immune microenvironment. The study was supported by grant 2014/15/B/NZ3/04704 from The National Science Centre, Poland.

TMIC-64. THE ROLE OF TUMOR-DERIVED GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF/CSF2) IN REGULATION OF MICROGLIA-DEPENDENT INVASION IN GLIOMAS

Abstract Brain resident immune cells (microglia) and peripheral macrophages accumulate in malignant gliomas and constitute for 30–50% of the tumor mass. These immune cells are polarized by factors released by glioma and become the pro-invasive, immunosuppressive cells that support tumor progression. We have previously found that tumor-derived granulocyte macrophage colony stimulating factor (GM-CSF/CSF2) is a crucial factor controlling accumulation of microglia and macrophages in murine gliomas. The analysis of TCGA dataset revealed overexpression of the CSF2 gene (encoding GM-CSF) in a set of mesenchymal glioblastomas and its association with high immune gene expression. To study the role of GM-CSF in microglia-stimulated glioma invasion, we used a co-culture system, which mimics microglia interactions with tumor cells. We silenced the expression of CSF2 in glioma cells and found reduced microglia-dependent invasion of glioma cells. To translate those results into clinically relevant setting, we designed and tested humanized short peptides interfering with binding of GM-CSF to its receptor. Selected peptide effectively inhibited binding of GM-CSF to its receptor as demonstrated with different methods. We selected the non-cytotoxic peptides that potently blocked microglia-dependent glioma invasion in cell co-cultures. Blocking GM-CSF-receptor signaling pathway with a neutralizing antibody against a GM-CSF receptor also inhibited microglia-dependent invasion of glioma cells. Altogether, our results demonstrate that glioma-derived GM-CSF supports pro-tumorigenic polarization of microglia turning them into cells that facilitate glioma growth and shape the immune microenvironment. This work was supported by statutory budget of Nencki Institute of Experimental Biology and grant PBS3/B7/19/2015 from The National Centre for Research and Development, Poland.

Identification of SERPINE1 as a Regulator of Glioblastoma Cell Dispersal with Transcriptome Profiling.

High mortality rates of glioblastoma (GBM) patients are partly attributed to the invasive behavior of tumor cells that exhibit extensive infiltration into adjacent brain tissue, leading to rapid, inevitable, and therapy-resistant recurrence. In this study, we analyzed transcriptome of motile (dispersive) and non-motile (core) GBM cells using an in vitro spheroid dispersal model and identified SERPINE1 as a modulator of GBM cell dispersal. Genetic or pharmacological inhibition of SERPINE1 reduced spheroid dispersal and cell adhesion by regulating cell-substrate adhesion. We examined TGFβ as a potential upstream regulator of SERPINE1 expression. We also assessed the significance of SERPINE1 in GBM growth and invasion using TCGA glioma datasets and a patient-derived orthotopic GBM model. SERPINE1 expression was associated with poor prognosis and mesenchymal GBM in patients. SERPINE1 knock-down in primary GBM cells suppressed tumor growth and invasiveness in the brain. Together, our results indicate that SERPINE1 is a key player in GBM dispersal and provide insights for future anti-invasive therapy design.

SLUG Directs the Precursor State of Human Brain Tumor Stem Cells.

In glioblastoma (GBM), brain tumor stem cells (BTSCs) encompass heterogenous populations of multipotent, self-renewing, and tumorigenic cells, which have been proposed to be at the root of therapeutic resistance and recurrence. While the functional significance of BTSC heterogeneity remains to be fully determined, we previously distinguished relatively quiescent stem-like precursor state from the more aggressive progenitor-like precursor state. In the present study, we hypothesized that progenitor-like BTSCs arise from stem-like precursors through a mesenchymal transition and drive post-treatment recurrence. We first demonstrate that progenitor-like BTSCs display a more mesenchymal transcriptomic profile. Moreover, we show that both mesenchymal GBMs and progenitor-like BTSCs are characterized by over-activated STAT3/EMT pathways and that SLUG is the primary epithelial to mesenchymal transition (EMT) transcription factor directly regulated by STAT3 in BTSCs. SLUG overexpression in BTSCs enhances invasiveness, promotes inflammation, and shortens survival. Importantly, SLUG overexpression in a quiescent stem-like BTSC line enhances tumorigenesis. Finally, we report that recurrence is associated with SLUG-induced transcriptional changes in both BTSCs and GBM patient samples. Collectively, our findings show that a STAT3-driven precursor state transition, mediated by SLUG, may prime BTSCs to initiate more aggressive mesenchymal recurrence. Targeting the STAT3/SLUG pathway may maintain BTSCs in a quiescent stem-like precursor state, delaying recurrence and improving survival in GBM.

IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma.

Immunotherapy shows a promise for treating glioblastoma (GBM), the most malignant and immunosuppressive glioma. The mesenchymal phenotype of cancer cells was frequently reported to be associated with their induction of immunosuppression within the cancer microenvironment. Overexpressed insulin-like growth factor binding protein 2 (IGFBP2) promotes GBM cell migration and invasion, and contributes to glioma progression and cancer recurrence and poor survival in GBM. However, whether IGFBP2 can induce immunosuppression in GBM was not reported yet. Thus, the study applied a syngeneic mouse GBM model, human GBM samples, and cancer-immune cell co-culture experiments to investigate the effect of IGFBP2 on GBM exposed immune cells and its association with the mesenchymal induction. We found that IGFBP2 promoted the mesenchymal feature of GBM cells. The inhibition of IGFBP2 relieved immunosuppression by increasing CD8+ T and CD19+ B cells and decreasing CD163+ M2 macrophages. Further, the IGFBP2-promoted immunosuppression was associated with its induction of the mesenchymal feature of GBM cells and the inhibitory phosphorylated FcγRIIB of GBM exposed immune cells. Blocking IGFBP2 suppressed tumor growth and improved survival of tumor bearing mice in the mouse GBM model. These findings support the notion that targeting the IGFBP2 may present an effective immunotherapeutic strategy for mesenchymal GBMs.

Integrative Tumor Profiling Following a Ketogenic Diet Offers a Window into the Interplay between Metabolic Vulnerabilities and Radiation Sensitization in Glioblastoma.

Glioblastoma (GBM) continues to be an invariably fatal malignancy with limited treatment options. Although considerable progress has been made in understanding molecular alterations in these tumors, this level of knowledge has not translated to clinical improvements. Metabolic reprogramming represents a hallmark of cancer and may serve as a therapeutic target. A ketogenic diet (KD) is a clinical strategy designed to target tumor metabolism and represents an active area of research. Although both preclinical and clinical data demonstrating efficacy has been mixed, a recent report suggests an unprecedented potential of a KD to enhance radiation response (RT) in an orthotopic model using an established mouse GBM cell line. In this study, we sought to validate these findings by determining the capacity of a KD to enhance RT in an aggressive, patient-derived GBM stem cell model and perform an integrative analysis on tissue to both define metabolic changes related to a KD and provide a mechanistic framework of activity. Immune deficient mice (nu/nu) were injected orthotopically with patient derived mesenchymal GBM tumor stem cells and randomized to four treatment arms (n=8/arm). Mice were fed ad libitum standard diet (SD) or KD (Bio-Serve; 1:4) alone or in combination with hypofractionated RT (hfRT; 6 Gy x 3). LC/GC-MS will be used to perform global metabolomic and proteomic profiling of tumor, serum, and normal brain of treated mice. Blood glucose and ketones were measured using the Abbott Precision Xtra meter kit. A KD demonstrated improved survival in GBM tumor bearing mice when compared to SD (mOS 22d vs. 17d; p=0.001) and equivalent survival when compared to SD mice treated with hfRT (mOS 24d; p= 0.57). The combination of KD and hfRT led to marked improvement in overall survival (mOS not reached; p<0.001). Although studies remain ongoing, at Day 29, 7/8 mice treated with KD+hfRT remained alive, while all 8 mice treated with SD+hfRT required euthanization due to neurologic progression. Surprisingly, alterations in blood ketone levels or glucose were not observed in mice treated with a KD. Global metabolomic and proteomic profiling of tumor, serum, and normal brain are ongoing. The anti-tumor activity and potent radiation sensitization of a KD was validated in an aggressive patient-derived GBM model. Ongoing integrative profiling of tumor and serum in mice treated with a KD will be presented to provide a previously undescribed insight into mechanisms of anti-tumor activity. As a KD is challenging to implement clinically, these mechanistic-based studies will provide a unique opportunity to learn how metabolism may be therapeutically exploited to provide a framework for the identification of novel mechanisms of radiation sensitization.

Nrf2 and SQSTM1/p62 jointly contribute to mesenchymal transition and invasion in glioblastoma.

Accumulating evidence suggests that constitutively active Nrf2 has a pivotal role in cancer as it induces pro-survival genes that promote cancer cell proliferation and chemoresistance. The mechanisms of Nrf2 dysregulation and functions in cancer have not been fully characterized. Here, we jointly analyzed the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) and the Cancer Genome Atlas (TCGA) multi-omics data in order to identify cancer types where Nrf2 activation is present. We found that Nrf2 is hyperactivated in a subset of glioblastoma (GBM) patients, whose tumors display a mesenchymal subtype, and uncover several different mechanisms contributing to increased Nrf2 activity. Importantly, we identified a positive feedback loop between SQSTM1/p62 and Nrf2 as a mechanism for activation of the Nrf2 pathway. We also show that autophagy and serine/threonine signaling regulates p62 mediated Keap1 degradation. Our results in glioma cell lines indicate that both Nrf2 and p62 promote proliferation, invasion and mesenchymal transition. Finally, Nrf2 activity was associated with decreased progression free survival in TCGA GBM patient samples, suggesting that treatments have limited efficacy if this transcription factor is overactivated. Overall, our findings place Nrf2 and p62 as the key components of the mesenchymal subtype network, with implications to tumorigenesis and treatment resistance. Thus, Nrf2 activation could be used as a surrogate prognostic marker in mesenchymal subtype GBMs. Furthermore, strategies aiming at either inhibiting Nrf2 or exploiting Nrf2 hyperactivity for targeted gene therapy may provide novel treatment options for this subset of GBM.

Human Mesenchymal glioblastomas are characterized by an increased immune cell presence compared to Proneural and Classical tumors

ABSTRACTGlioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults, with a median survival of 14.6 months. Recent efforts have focused on identifying clinically relevant subgroups to improve our understanding of pathogenetic mechanisms and patient stratification. Concurrently, the role of immune cells in the tumor microenvironment has received increasing attention, especially T cells and tumor-associated macrophages (TAM). The latter are a mixed population of activated brain-resident microglia and infiltrating monocytes/monocyte-derived macrophages, both of which express ionized calcium-binding adapter molecule 1 (IBA1). This study investigated differences in immune cell subpopulations among distinct transcriptional subtypes of GBM. Human GBM samples were molecularly characterized and assigned to Proneural, Mesenchymal or Classical subtypes as defined by NanoString nCounter Technology. Subsequently, we performed and analyzed automated immunohistochemical stainings for TAM as well as specific T cell populations. The Mesenchymal subtype of GBM showed the highest presence of TAM, CD8+, CD3+ and FOXP3+ T cells, as compared to Proneural and Classical subtypes. High expression levels of the TAM-related gene AIF1, which encodes the TAM-specific protein IBA1, correlated with a worse prognosis in Proneural GBM, but conferred a survival benefit in Mesenchymal tumors. We used our data to construct a mathematical model that could reliably identify Mesenchymal GBM with high sensitivity using a combination of the aforementioned cell-specific IHC markers. In conclusion, we demonstrated that molecularly distinct GBM subtypes are characterized by profound differences in the composition of their immune microenvironment, which could potentially help to identify tumors amenable to immunotherapy.

Kinins in Glioblastoma Microenvironment.

Tumour progression involves interactions among various cancer cell clones, including the cancer stem cell subpopulation and exogenous cellular components, termed cancer stromal cells. The latter include a plethora of tumour infiltrating immunocompetent cells, among which are also immuno-modulatory mesenchymal stem cells, which by vigorous migration to growing tumours and susequent transdifferentiation into various types of tumour-residing stromal cells, may either inhibit or support tumour progression. In the light of the scarce therapeutic options existing for the most malignant brain tumour glioblastoma, mesenchymal stem cells may represent a promising novel tool for cell therapy, e.g. drug delivery vectors. Here, we review the increasing number of reports on mutual interactions between mesenchymal stem cells and glioblastoma cells in their microenvironment. We particularly point out two novel aspects: the different responses of cancer cells to their microenvironmental cues, and to the signalling by kinin receptors that complement the immuno-modulating cytokine-signalling networks. Inflammatory glioblastoma microenvironment is characterised by increasing expression of kinin receptors during progressive glioma malignancy, thus making kinin signalling and kinins themselves rather important in this context. In general, their role in tumour microenvironment has not been explored so far. In addition, kinins also regulate blood brain barrier-related drug transfer as well as brain tumour angiogenesis. These studies support the on-going research on kinin antagonists as candidates in the development of anti-invasive agents for adjuvant glioblastoma therapy.

Abstract 5259: Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions

Abstract Glioblastoma represents an aggressive, primary brain tumor with limited treatment options. We performed an integrative, cross-platform analysis coupling global metabolomics and gene expression profiling in &amp;gt;100 patient-derived gliomas to begin to understand the diverse metabolic programs driving the aggressive phenotype of this malignancy. Alterations in fatty acid β-oxidation (FAO) emerged as a key metabolic node differentiating glioblastoma from low-grade astrocytoma, as demonstrated by an accumulation of acylcarnitines. Metabolic heterogeneity was observed within glioblastoma that could further define tumors as FAO ‘high’ and ‘low’. Integrative analyses identified these metabolic subtypes to be enriched with mesenchymal (MES) and proneural (PN) glioblastoma subtypes, respectively. These findings were metabolomically and functionally recapitulated in molecular subtype-specific preclinical models. Analysis of gene expression profiles from these lines uncovered an orchestrated transcriptional program designed to promote fatty acid uptake, activation, and mitochondrial oxidation. Studies designed to determine the biologic consequence of enhanced FAO in glioblastoma only identified a role in glucose-deprived conditions, where it served as a vital, alternate source for ATP synthesis. Based on these findings, we tested the hypothesis that dual targeting of glycolysis and FAO would elicit energetic stress-mediated cell death in glioblastoma. Accordingly, the glycolysis inhibitor 2DG and the FAO inhibitor etomoxir only demonstrated anti-proliferative activity in MES glioblastoma cell lines when used as single agents in vitro, while the combination resulted in robust, necroptosis-mediated cell death. Synergistic anti-tumor activity was observed following combined glycolysis and FAO inhibition when extended in vivo in an orthotopic model. Collectively, our findings suggest FAO provides metabolic plasticity in MES glioblastoma, allowing these cells to adapt to nutrient-deprived microenvironments. Combinatorial strategies designed to inhibit glycolysis and FAO represents an attractive therapeutic approach in glioblastoma. Citation Format: Shiva Kant, Antony Dayalan, Pravin Kesarwani, Prakash Chinnaiyan. Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions [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 5259.

Abstract 5175: Expression profile and role of LOXL3 in astrocytomas

Abstract Astrocytomas are the most common primary central nervous system tumors in adults. The WHO classification based on histologial characteristics according consider four malignant grades, glioblastoma (GBM) being the most malignant (grade IV). Molecular classification of GBM is based on the integration of somatic mutations, DNA methylation and specific transcript/protein expression and consider four subtypes: proneural, classic, mesenchymal and neural. Lysyl oxidase family is composed of five members (LOX, LOXL1, LOXL2, LOXL3 and LOXL4) which are enzymes responsible for catalyzing lysine-derived cross-links of collagen and elastin. The aim of the present work was to analyze the gene expression of LOXL3 in astrocytomas of different grades and different GBM molecular subtypes, the impact of LOXL3 expression level on overall survival in GBM cases of our cohort. These data were also analyzed in the TCGA database to validate our findings. LOXL3expression increased with astrocytoma malignant grades, and proved to be a prognostic factor, as GBM patients with lower LOXL3 expression presented longer survival time than those with higher expression (p&amp;lt;0.041). Analogous data was obtained in the TCGA database, when considering temozolamide-treated GBM patients (p=0.049). Additionally, LOXL3 expression was higher in mesenchymal GBM subtype than in proneural and classic GBM subtypes in TCGA cohort (p&amp;lt;0.001). To further understand LOXL3 functional role in gliomagenesis, LOXL3 was silenced with two distinct siRNA sequences in U87MG, a mesenchymal subtype of GBM cell line. LOXL3 down regulation was confirmed at gene and protein expression levels. Functional assays with silenced LOXL3 demonstrated a decrease in the cell proliferation (p&amp;lt;0.05), and an increase in the apoptosis associated to temozolamide treatment (p&amp;lt;0.05). Immunofluorescence analysis in U87MG cells showed LOXL3 colocalized with mitochondria. Interestingly, such colocalization decreased when LOXL3 was downregulated, associated to a clear morphological alteration in mitochondria shape. A rough increase in mitochondria volume was observed, suggesting fused mitochondria. In addition, such condition was associated to a 23% decrease in mitochondrial DNA copy number. Altogether, our findings suggest that LOXL3 is upregulated in GBM, with impact in prognosis and might play a role in mitochondria dynamics and/or mitophagy, modulating apoptosis. Transcriptome analysis, currently in progress, will further clarify the associated players in this signaling pathway. Citation Format: Talita de Souza Laurentinho, Roseli da Silva Soares, Suely Kazue Marie, Sueli Mieko Oba-Shinjo. Expression profile and role of LOXL3 in astrocytomas [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 5175.

Abstract 1900: A tension-mediated glycocalyx feedback loop promotes glioblastoma

Abstract A considerable challenge in glioblastoma (GBM) therapy is treatment resistance and tumor recurrence, resulting in dismal patient prognosis. GBM aggression and recurrence is often associated with a mesenchymal phenotype, increased expression of glycoproteins and the presence of glioma-initiating “stem-like” cells. Using patient-derived xenograft models, and immune competent syngeneic and transgenic glioma mouse models, we show that aggressive mesenchymal-like GBMs, reminiscent of recurrent GBM, are mechanically stiffer, present with a bulky glycocalyx and demonstrate a stem-like phenotype. We report that the more aggressive mesenchymal GBMs show increased mechanical signaling and contractility, and their tumors are surrounded by a stiffer ECM that maintains and even further enhances integrin mechanosignaling. Since a large proportion of these bulky glycoproteins are also stem markers, upregulation of the glycoproteins and their modulators leads to enhanced GBM stem-ness. This was evident both in our mesenchymal models of GBM as well as multiple patient datasets which compared paired primary and recurrent GBM RNA sequencing and protein data. In addition to stem-ness, we show that the mechanically “enhanced” GBMs also foster a differential immune landscape, infiltrated by type 2 macrophages, believed to be pro-tumorigenic and immune evasive. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignaling, the GBM immune landscape and a bulky glycocalyx, which promotes a mesenchymal, stem-like phenotype and likely recurrence in GBM patients. Thus, therapeutic strategies to target GBM tissue tension could prevent recurrence, reduce mortality and improve patient outcome. Citation Format: Shelly Kaushik, James Matthew Barnes, Russell O. Bainer, Jason K. Sa, Elliot C. Woods, Fuiboon Kai, Jonathon N. Lakins, Joanna J. Phillips, Valerie M. Weaver. A tension-mediated glycocalyx feedback loop promotes 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 1900.