Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding.

The development and maintenance of an epithelium requires finely balanced rates of growth and cell death. However, the mechanical and biochemical mechanisms that ensure proper feedback control of tissue growth, which when deregulated contribute to tumorigenesis, are poorly understood. Here we use the fly notum as a model system to identify a novel process of crowding-induced cell delamination that balances growth to ensure the development of well-ordered cell packing. In crowded regions of the tissue, a proportion of cells undergo a serial loss of cell-cell junctions and a progressive loss of apical area, before being squeezed out by their neighbours. This path of delamination is recapitulated by a simple computational model of epithelial mechanics, in which stochastic cell loss relieves overcrowding as the system tends towards equilibrium. We show that this process of delamination is mechanistically distinct from apoptosis-mediated cell extrusion and precedes the first signs of cell death. Overall, this analysis reveals a simple mechanism that buffers epithelia against variations in growth. Because live-cell delamination constitutes a mechanistic link between epithelial hyperplasia and cell invasion, this is likely to have important implications for our understanding of the early stages of cancer development.

Repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab show no efficacy in glioblastoma: a phase 1 trial.

We previously showed that chimeric antigen receptor (CAR) T-cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces upregulation of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Here we conducted a phase 1 trial (NCT03726515) of CAR T-EGFRvIII cells administered concomitantly with the anti-PD1 (aPD1) monoclonal antibody pembrolizumab in patients with newly diagnosed, EGFRvIII+ glioblastoma (GBM) (n = 7). The primary outcome was safety, and no dose-limiting toxicity was observed. Secondary outcomes included median progression-free survival (5.2 months; 90% confidence interval (CI), 2.9-6.0 months) and median overall survival (11.8 months; 90% CI, 9.2-14.2 months). In exploratory analyses, comparison of the TME in tumors harvested before versus after CAR + aPD1 administration demonstrated substantial evolution of the infiltrating myeloid and T cells, with more exhausted, regulatory, and interferon (IFN)-stimulated T cells at relapse. Our study suggests that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicates a need to consider alternative strategies.

Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma.

Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.

Cdc42, Par6, and aPKC regulate Arp2/3-mediated endocytosis to control local adherens junction stability.

BACKGROUND: By acting as a dynamic link between adjacent cells in a monolayer, adherens junctions (AJs) maintain the integrity of epithelial tissues while allowing for neighbor exchange. Although it is not currently understood how this combination of AJ stability and plasticity is achieved, junctionally associated actin filaments are likely to play a role, because actin-based structures have been implicated in AJ organization and in the regulation of junctional turnover. RESULTS: Here, through exploring the role of actin cytoskeletal regulators in the developing Drosophila notum, we have identified a critical role for Cdc42-aPKC-Par6 in the maintenance of AJ organization. In this system, the loss or inhibition of Cdc42-aPKC-Par6 leads to junctional discontinuities, the formation of ectopic junctional structures, and defects in apical actin cytoskeletal organization. Affected cells also undergo progressive apical constriction and, frequently, delamination. Surprisingly, this Cdc42-aPKC-Par6-dependent regulation of junctional stability was found to be independent of several well-known targets of Cdc42-aPKC-Par6: Baz, Lgl, Rac, and SCAR. However, similar AJ defects are observed in wasp, arp2/3, and dynamin mutant cells, suggesting a requirement for actin-mediated endocytosis in the maintenance of junctional stability downstream of Cdc42. This was confirmed in endocytosis assays, which revealed a requirement for Cdc42, Arp2/3, and Dynamin for normal rates of E-cadherin internalization. CONCLUSIONS: By focusing on the molecular mechanisms required to maintain an epithelium, this analysis reveals a novel role for the epithelial polarity machinery, Cdc42-Par6-aPKC, in local AJ remodeling through the control of Arp2/3-dependent endocytosis.

Treating ICB-resistant glioma with anti-CD40 and mitotic spindle checkpoint controller BAL101553 (lisavanbulin).

Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might affect the tumor microenvironment and sensitize cancer cells to immunotherapy. We used 2 glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti-programmed cell death 1 and anti-cytotoxic T lymphocyte-associated protein 4; anti-PD-1 and anti-CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade-resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T cell-independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti-PD-1 and anti-CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.

Clonal Evolution of a High-Grade Pediatric Glioma With Distant Metastatic Spread.

OBJECTIVE: High-grade glioma (HGG) rarely spreads outside the CNS. To test the hypothesis that the lesions were metastases originating from an HGG, we sequenced the relapsing HGG and distant extraneural lesions. METHODS: We performed whole-exome sequencing of an HGG lesion, its local relapse, and distant lesions in bone and lymph nodes. RESULTS: Phylogenetic reconstruction and histopathologic analysis confirmed the common glioma origin of the secondary lesions. The mutational profile revealed an IDH1/2 wild-type HGG with an activating mutation in EGFR and biallelic focal loss of CDKN2A (9p21). In the metastatic samples and the local relapse, we found an activating PIK3CA mutation, further copy number gains in chromosome 7 (EGFR), and a putative pathogenic driver mutation in a canonical splice site of FLNA. CONCLUSIONS: Our findings demonstrate tumor spread outside the CNS and identify potential genetic drivers of metastatic dissemination outside the CNS, which could be leveraged as therapeutic targets or potential biomarkers.

Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells.

Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and ß1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles' heel for a significant and unique subset of GBM patients.

Seladin-1 and testicular germ cell tumours: new insights into cisplatin responsiveness.

The molecular basis for the exquisite sensitivity of testicular germ cell tumours of adolescents and adults (TGCTs), ie seminomas and non-seminomatous germ cell tumours, to chemo/radiotherapy has not been fully clarified so far. It has been suggested that it may be dependent on factors involved in the regulation of apoptosis. Seladin-1 is a multi-functional protein involved in various biological processes, including apoptosis. The aim of our study was to assess the expression of seladin-1 in different histological types of TGCTs, known to have varying treatment sensitivity, in order to establish whether this protein may influence cisplatin responsiveness in vitro. Seladin-1 expression levels, both at the mRNA and at the protein level, were higher in the adjacent normal parenchyma than in the pathological counterparts. In tumoural tissues, the level of expression differed among TGCT histological types. The highest tumour-expression level was found in teratoma, whereas the lowest was detected in seminoma, corresponding to the different chemo/and radiosensitivities of these tumour types. In common with other cancers, in TGCT-derived cell lines seladin-1 showed anti-apoptotic properties through inhibition of caspase-3 activation. We confirmed our results using a non-seminomatous cell line model (NT2) before and after differentiation with retinoic acid. Significantly higher seladin-1 expression was observed in the differentiated derivatives (teratoma) and an inverse relationship was found between seladin-1 expression and the amount of cleaved caspase-3. Seladin-1 silencing or overexpression in this cell line supports involvement of seladin-1 in cisplatin responsiveness. Seladin-1 silencing was associated with greater cisplatin responsiveness demonstrated by decreased cell viability and increased expression of apoptotic markers. In contrast, overexpression of seladin-1 was associated with a higher survival rate and a clear anti-apoptotic effect. In conclusion, we have demonstrated for the first time an important role for seladin-1 in the biology of TGCTs and provided new insights into cisplatin responsiveness of these tumours.

Inflammation and lymphocyte infiltration are associated with shorter survival in patients with high-grade glioma.

Glioma represents a serious health burden in terms of morbidity and mortality. The prognostic significance of the lymphoid and myeloid infiltrates in glioma is not clearly determined. Moreover, the characterization of different leukocyte subsets in the tumor microenvironment relies mainly on immunohistochemistry observations, and data about their association with prognosis are contradictory. Here, we performed acomprehensive study of both the tumor-infiltrating and circulating immune compartments of patients with high-grade glioma. Nineteen tumor biopsies and 30 PBMC samples were analyzed by RNA sequencing. Validation was performed on The Cancer Genome Atlas (TCGA) RNA sequencing data from glioma and on additional 39 tumor biopsies analyzed by flow cytometry. We identified prognostic tumor and peripheral immune signatures, which associate increased inflammation, immune infiltration and activation with shorter overall survival in high-grade glioma patients. Importantly, we confirmed our observations by flow cytometry analysis and validated the tumor-signature using the TCGA dataset. In addition, both tumor genotype and grade associated with the degree of glioma immune infiltration. Unlike in the majority of cancers, lymphocyte infiltration at the tumor site is anegative prognostic factor in glioma, suggesting the ambivalent pro-tumorigenic role of immune responses in glioma.

Impact of Radiochemotherapy on Immune Cell Subtypes in High-Grade Glioma Patients.

Glioblastoma is a dreadful disease with very poor prognosis, median overall survival being <2 years despite standard-of-care treatment. This has led to the development of alternative strategies, among which immunotherapy is being actively tested. In particular, many clinical trials of therapeutic vaccination using peptides or tumor cells are ongoing. A major issue in implementing therapeutic vaccines in patients with high-grade glioma is that immune responses have to be elicited in the context of immunosuppressive treatments. Indeed, radiotherapy, chemotherapy, and steroids, which are part of the standard of care for patients with glioblastoma, are known to deplete leukocytes. Whether lymphopenia is beneficial or detrimental to elicitation of efficient immune responses is still debated. Here, in order to determine the impact of standard radiochemotherapy on immune cell subsets, we analyzed the phenotype and function of immune populations in 25 patients with high-grade glioma along concomitant radiochemotherapy and adjuvant chemotherapy with temozolomide. Thirteen healthy individuals were studied along the same period. We show that absolute T and B cell counts are reduced upon concomitant radiochemotherapy. Importantly, T cell counts were not restored long-term after discontinuation of treatment. In addition, the percentage of T regulatory cells among CD4 T cells was increased during the same period and was not decreased upon treatment discontinuation. Finally, we show that the ability of T cells to proliferate is transiently reduced after concomitant radiochemotherapy but is restored at the time of adjuvant TMZ cycles. Although not experimentally validated, transient reduction in proliferation associated with strong lymphopenia during radiochemotherapy may suggest that vaccine-induced T cell stimulation would be suboptimal in that period and that therapeutic vaccination should be performed outside radiochemotherapy administration. In addition, strategies aiming at depleting Treg cells should be implemented in future trials.

An Experimentally Defined Hypoxia Gene Signature in Glioblastoma and Its Modulation by Metformin.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, characterized by a high degree of intertumoral heterogeneity. However, a common feature of the GBM microenvironment is hypoxia, which can promote radio- and chemotherapy resistance, immunosuppression, angiogenesis, and stemness. We experimentally defined common GBM adaptations to physiologically relevant oxygen gradients, and we assessed their modulation by the metabolic drug metformin. We directly exposed human GBM cell lines to hypoxia (1% O2) and to physioxia (5% O2). We then performed transcriptional profiling and compared our in vitro findings to predicted hypoxic areas in vivo using in silico analyses. We observed a heterogenous hypoxia response, but also a common gene signature that was induced by a physiologically relevant change in oxygenation from 5% O2 to 1% O2. In silico analyses showed that this hypoxia signature was highly correlated with a perinecrotic localization in GBM tumors, expression of certain glycolytic and immune-related genes, and poor prognosis of GBM patients. Metformin treatment of GBM cell lines under hypoxia and physioxia reduced viable cell number, oxygen consumption rate, and partially reversed the hypoxia gene signature, supporting further exploration of targeting tumor metabolism as a treatment component for hypoxic GBM.

Exploratory Study of the Effect of IMA950/Poly-ICLC Vaccination on Response to Bevacizumab in Relapsing High-Grade Glioma Patients.

Immunotherapy, including therapeutic vaccines, is increasingly being developed for patients with high-grade glioma, and combinations of immunotherapies and synergy with standard of care are being investigated. In this regard, bevacizumab (BEV) has been shown to synergize with immunotherapy in preclinical studies of glioma and in other tumour entities. Here, we conducted a post-hoc exploratory study to evaluate the effect of the IMA950/poly-ICLC peptide vaccine on subsequent BEV administration in high-grade glioma patients. 16 IMA950-vaccinated and 40 non-vaccinated patients were included. At initial diagnosis, patients benefited from surgery and chemoradiation. At first or subsequent recurrence, patients received 10mg/kg of BEV every 2-3 weeks. Primary endpoints were overall survival (OS) and progression-free survival (PFS) from BEV initiation. IMA950-vaccinated patients did not show improved response to BEV as compared to non-vaccinated patients: there was no difference in median PFS (2.6 vs. 4.2 months for vaccinated and control patients, respectively, p = 0.50) nor in median OS (7.8 vs. 10.0 months for vaccinated and control patients, respectively, p = 0.69). In conclusion, potential synergy of BEV and therapeutic vaccines, when administered sequentially, has yet to be established in the clinical setting of GBM recurrence. Potential synergy of concomitant administration should be tested in future trials.

Phase I/II trial testing safety and immunogenicity of the multipeptide IMA950/poly-ICLC vaccine in newly diagnosed adult malignant astrocytoma patients.

BACKGROUND: Peptide vaccines offer the opportunity to elicit glioma-specific T cells with tumor killing ability. Using antigens eluted from the surface of glioblastoma samples, we designed a phase I/II study to test safety and immunogenicity of the IMA950 multipeptide vaccine adjuvanted with poly-ICLC (polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose) in human leukocyte antigen A2+ glioma patients. METHODS: Adult patients with newly diagnosed glioblastoma (n = 16) and grade III astrocytoma (n = 3) were treated with radiochemotherapy followed by IMA950/poly-ICLC vaccination. The first 6 patients received IMA950 (9 major histocompatibility complex [MHC] class I and 2 MHC class II peptides) intradermally and poly-ICLC intramuscularly (i.m.). After protocol amendment, IMA950 and poly-ICLC were mixed and injected subcutaneously (n = 7) or i.m. (n = 6). Primary endpoints were safety and immunogenicity. Secondary endpoints were overall survival, progression-free survival at 6 and 9 months, and vaccine-specific peripheral cluster of differentiation (CD)4 and CD8 T-cell responses. RESULTS: The IMA950/poly-ICLC vaccine was safe and well tolerated. Four patients presented cerebral edema with rapid recovery. For the first 6 patients, vaccine-induced CD8 T-cell responses were restricted to a single peptide and CD4 responses were absent. After optimization of vaccine formulation, we observed multipeptide CD8 and sustained T helper 1 CD4 T-cell responses. For the entire cohort, CD8 T-cell responses to a single or multiple peptides were observed in 63.2% and 36.8% of patients, respectively. Median overall survival was 19 months for glioblastoma patients. CONCLUSION: We provide, in a clinical trial, using cell surface-presented antigens, insights into optimization of vaccines generating effector T cells for glioma patients. TRIAL REGISTRATION: Clinicaltrials.gov NCT01920191.

The leucine-rich repeat-containing G protein-coupled receptor 8 gene T222P mutation does not cause cryptorchidism.

CONTEXT: Insulin-like 3 and its receptor, leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8), are essential for the first phase of testicular descent. Homozygous loss of either of the two genes in mice leads to cryptorchidism. Although mutations in both homologous human genes are not a common cause of cryptorchidism. To date, only one missense mutation at codon 222 (T222P) of the LGR8 gene has been proposed as a causative mutation for cryptorchidism. This conclusion was based on both functional in vitro studies and the lack of mutation in a large group of controls. The geographical origin of the mutation carriers suggested a founder effect in the Mediterranean area. OBJECTIVES: We sought to define the frequency of the T222P mutation in four different countries to assess whether the screening for this mutation could be of use as a diagnostic genetic test. MATERIALS AND METHODS: A total of 822 subjects (359 with a history of cryptorchidism and 463 controls) from Italy, Spain, Hungary, and Egypt were genotyped for the T222P mutation by direct sequencing. RESULTS: The phenotypical expression of the mutation also included normal testicular descent. The mutation frequency was not significantly different in cryptorchid patients vs. noncryptorchid controls (3.6 vs. 1.7%, respectively). No significant geographical differences were observed in mutation frequencies. The haplotype analysis allowed us to predict three distinct haplotypes, i.e. three possible mutation events. CONCLUSIONS: Our results suggest that the T222P mutation cannot be considered either causative or a susceptibility factor for cryptorchidism. A true causative mutation in the LGR8 gene still remains to be identified.

Correction: Correction: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

[This corrects the article DOI: 10.1371/journal.pone.0176578.].

Responsiveness to anti-PD-1 and anti-CTLA-4 immune checkpoint blockade in SB28 and GL261 mouse glioma models.

Immune checkpoint blockade (ICB) is currently evaluated in patients with glioblastoma (GBM), based on encouraging clinical data in other cancers, and results from studies with the methylcholanthrene-induced GL261 mouse glioma. In this paper, we describe a novel model faithfully recapitulating some key human GBM characteristics, including low mutational load, a factor reported as a prognostic indicator of ICB response. Consistent with this observation, SB28 is completely resistant to ICB, contrasting with treatment sensitivity of the more highly mutated GL261. Moreover, SB28 shows features of a poorly immunogenic tumor, with low MHC-I expression and modest CD8+ T-cell infiltration, suggesting that it may present similar challenges for immunotherapy as human GBM. Based on these key features for immune reactivity, SB28 may represent a treatment-resistant malignancy likely to mirror responses of many human tumors. We therefore propose that SB28 is a particularly suitable model for optimization of GBM immunotherapy.

Antigenic expression and spontaneous immune responses support the use of a selected peptide set from the IMA950 glioblastoma vaccine for immunotherapy of grade II and III glioma.

Gliomas are lethal brain tumors that resist standard therapeutic approaches. Immunotherapy is a promising alternative strategy mostly developed in the context of glioblastoma. However, there is a need for implementing immunotherapy for grade II/III gliomas, as these are the most common CNS tumors in young adults with a high propensity for recurrence, making them lethal despite current treatments. We recently identified HLA-A2-restricted tumor-associated antigens by peptide elution from glioblastoma and formulated a multipeptide vaccine (IMA950) evaluated in phase I/II clinical trials with promising results. Here, we investigated expression of the IMA950 antigens in patients with grade II/III astrocytoma, oligodendroglioma or ependymoma, at the mRNA, protein and peptide levels. We report that the BCAN, CSPG4, IGF2BP3, PTPRZ1 and TNC proteins are significantly over-expressed at the mRNA (n = 159) and protein (n = 36) levels in grade II/III glioma patients as compared to non-tumor samples (IGF2BP3 being absent from oligodendroglioma). Most importantly, we detected spontaneous antigen-specific T cell responses to one or more of the IMA950 antigens in 100% and 71% of grade II and grade III patients, respectively (27 patients tested). These patients displayed T cell responses of better quality (higher frequency, broader epitope targeting) than patients with glioblastoma. Detection of spontaneous T cell responses to the IMA950 antigens shows that these antigens are relevant for tumor targeting, which will be best achieved by combination with CD4 epitopes such as the IDH1R132H peptide. Altogether, we provide the rationale for using a selective set of IMA950 peptides for vaccination of patients with grade II/III glioma.

Correction: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

[This corrects the article DOI: 10.1371/journal.pone.0149099.].

CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation.

Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing.

Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma.