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Despite decades of research, the prognosis of high-grade pediatric brain tumors (PBTs) remains dismal; however, recent cases of favorable clinical responses were documented in clinical trials using oncolytic viruses (OVs). In the current study, we employed four different species of OVs: adenovirus Delta24-RGD, herpes simplex virus rQNestin34.5v1, reovirus R124, and the non-virulent Newcastle disease virus rNDV-F0-GFP against three entities of PBTs (high-grade gliomas, atypical teratoid/rhabdoid tumors, and ependymomas) to determine their in vitro efficacy. These four OVs were screened on 14 patient-derived PBT cell cultures and the degree of oncolysis was assessed using an ATP-based assay. Subsequently, the observed viral efficacies were correlated to whole transcriptome data and Gene Ontology analysis was performed. Although no significant tumor type-specific OV efficacy was observed, the analysis revealed the intrinsic biological processes that associated with OV efficacy. The predictive power of the identified expression profiles was further validated in vitro by screening additional PBTs. In summary, our results demonstrate OV susceptibility of multiple patient-derived PBT entities and the ability to predict in vitro responses to OVs using unique expression profiles. Such profiles may hold promise for future OV preselection with effective oncolytic potency in a specific tumor, therewith potentially improving OV responses.

BackgroundThe GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy.MethodsTo decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics.ResultsThe transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants.ConclusionAlthough the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1β [IL-1β], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409’s efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

RationaleCAN-2409 is a locally delivered oncolytic therapy, which results in vaccination against the injected tumor. CAN-2409 consists of a non-replicating adenovirus armed with the Herpes virus thymidine kinase, which metabolizes ganciclovir into a phosphorylated nucleotide that is incorporated into the tumor cell’s genome, thereby inflicting immunogenic cancer cell death. While CAN-2409’s immunological impact has been well characterized, its effects on the tumor cells transcriptome remains unknown. We compared the transcriptomic landscape after treatment of glioblastoma models with CAN-2409 in vitro and in vivo to assess how the interplay with the tumor microenvironment influences CAN-2409-mediated transcriptome alterations.MethodsWe performed RNA-Seq with CAN-2409 treated patient-derived glioma stem-like cells and tumors of C57/BL6 mice and compared KEGG pathway usage and differential gene expression focusing on immune cell and cytokine profiles. T-cell -killing assays were performed to assess candidate effectors.ResultsPCA analysis showed distinct clustering of control and CAN-2409 samples under both conditions. KEGG pathway analysis revealed significant enrichment for p53 signaling and cell cycle pathway, with similar dynamics for key regulators of both pathways in vitro and in vivo, including MYC, CCNB1, PLK1 and CDC20. Selected alterations (PLK1 and CCNB1) were validated at the protein level. Cytokine expression analysis revealed upregulation of pro-inflammatory IL12a under both conditions; immune cell gene profiling showed reduction of myeloid associated genes. T-cell-killing assays showed increased killing in the presence of IL-12.ConclusionCAN-2409 significantly alters the transcriptome both in vitro and in vivo. Comparison of pathway enrichment revealed mutual and differential utilization of pathways under both conditions, suggesting a modulating influence on the cell cycle in tumor cells, and of the tumor microenvironment on the transcriptome in vivo. IL-12 synthesis likely depends on interactions with the tumor microenvironment, and it facilitates CAN-2409 cell killing. This dataset provides potential to understand resistance mechanisms and identify potential biomarkers for future studies.

Abstract Cellular therapies offer a promising therapeutic strategy for the highly malignant brain tumor, glioblastoma (GBM). However, their clinical translation is limited by the lack of effective target identification and stringent testing in pre-clinical models that replicate standard treatment in GBM patients. In this study, we show the detection of cell surface death receptor (DR) target on CD146-enriched circulating tumor cells (CTC) captured from the blood of mice bearing GBM and patients diagnosed with GBM. Next, we developed allogeneic “off-the-shelf” clinical-grade bifunctional mesenchymal stem cells (MSCBif) expressing DR-targeted ligand and a safety kill switch. We show that biodegradable hydrogel encapsulated MSCBif (EnMSCBif) has a profound therapeutic efficacy in mice bearing patient-derived invasive, primary and recurrent GBM tumors following surgical resection. Activation of the kill switch enhances the efficacy of MSCBif and results in their elimination post-tumor treatment which can be tracked by positron emission tomography (PET) imaging. This study establishes a foundation towards a clinical trial of EnMSCBif in primary and recurrent GBM patients.

Oncolytic herpes simplex virus-1 lyses cancer cells while increases their immunogenicity. Blocking the CD47 “don’t eat me” signal on cancer cells promotes their phagocytosis by macrophages. Authors here show that oncolytic viruses expressing anti-CD47 antibodies improve glioblastoma survival in mouse models.

Oncolytic herpes simplex viruses (oHSVs) have demonstrated efficient lytic replication in human glioblastoma tumors using immunodeficient mouse models, but early-phase clinical trials have reported few complete responses. Potential reasons for the lack of efficacy are limited vector potency and the suppressive glioma tumor microenvironment (TME). Here we compare the oncolytic activity of two HSV-1 vectors, a KOS-strain derivative KG4:T124 and an F-strain derivative rQNestin34.5v.1, in the CT2A and GL261N4 murine syngeneic glioma models. rQNestin34.5v1 generally demonstrated a greater in vivo viral burden compared to KG4:T124. However, both vectors were rapidly cleared from CT2A tumors, while virus remained ensconced in GL261N4 tumors. Immunological evaluation revealed that the two vectors induced similar changes in immune cell recruitment to either tumor type at 2 days after infection. However, at 7 days after infection, the CT2A microenvironment displayed the phenotype of an untreated tumor, while GL261N4 tumors exhibited macrophage and CD4+/CD8+ T cell accumulation. Furthermore, the CT2A model was completely resistant to virus therapy, while in the GL261N4 model rQNestin34.5v1 treatment resulted in enhanced macrophage recruitment, impaired tumor progression, and long-term survival of a few animals. We conclude that prolonged intratumoral viral presence correlates with immune cell recruitment, and both are needed to enhance anti-tumor immunity.

Summary: Oncolytic viruses (OVs) are emerging cancer immunotherapy. Despite notable successes in the treatment of some tumors, OV therapy for central nervous system cancers has failed to show efficacy. We used an ex vivo tumor model developed from human glioblastoma tissue to evaluate the infiltration of herpes simplex OV rQNestin (oHSV-1) into glioblastoma tumors. We next leveraged our data to develop a computational, model of glioblastoma dynamics that accounts for cellular interactions within the tumor. Using our computational model, we found that low stromal density was highly predictive of oHSV-1 therapeutic success, suggesting that the efficacy of oHSV-1 in glioblastoma may be determined by stromal-to-tumor cell regional density. We validated these findings in heterogenous patient samples from brain metastatic adenocarcinoma. Our integrated modeling strategy can be applied to suggest mechanisms of therapeutic responses for central nervous system cancers and to facilitate the successful translation of OVs into the clinic.

rQNestin34.5v.2 is an oncolytic herpes simplex virus 1 (oHSV) that retains expression of the neurovirulent ICP34.5 gene under glioma-selective transcriptional regulation. To prepare an investigational new drug (IND) application, we performed toxicology and efficacy studies of rQNestin34.5v.2 in mice in the presence or absence of the immunomodulating drug cyclophosphamide (CPA). ICP34.5 allows HSV1 to survive interferon and improves viral replication by dephosphorylation of the eIF-2α translation factor. rQNestin34.5v.2 dephosphorylated eIF-2α in human glioma cells, but not in human normal cells, resulting in significantly higher cytotoxicity and viral replication in the former compared to the latter. In vivo toxicity of rQNestin34.5v.2 was compared with that of wild-type F strain in immunocompetent BALB/c mice and athymic mice by multiple routes of administration in the presence or absence of CPA. A likely no observed adverse effect level (NOAEL) dose for intracranial rQNestin34.5v.2 was estimated, justifying a phase 1 clinical trial in recurrent glioma patients (ClinicalTrials.gov: NCT03152318), after successful submission of an IND.

Abstract Meningioma represents the most common primary brain tumor in adults. Recently several non-NF2 mutations in meningioma have been identified and correlated with certain pathological subtypes, locations and clinical observations. Alterations of cellular pathways due to these mutations, however, have largely remained elusive. Here we report that the Krueppel like factor 4 (KLF4)-K409Q mutation in skull base meningiomas triggers a distinct tumor phenotype. Transcriptomic analysis of 17 meningioma samples revealed that KLF4 K409Q mutated tumors harbor an upregulation of hypoxia dependent pathways. Detailed in vitro investigation further showed that the KLF4 K409Q mutation induces HIF-1α through the reduction of prolyl hydroxylase activity and causes an upregulation of downstream HIF-1α targets. Finally, we demonstrate that KLF4 K409Q mutated tumors are susceptible to mTOR inhibition by Temsirolimus. Taken together, our data link the KLF4K409Q mediated upregulation of HIF pathways to the clinical and biological characteristics of these skull base meningiomas possibly opening new therapeutic avenues for this distinct meningioma subtype.

The use of mutant strains of oncolytic herpes simplex virus (oHSV) in early-phase human clinical trials for the treatment of glioblastoma multiforme (GBM) has proven safe, but limited efficacy suggests that more potent vector designs are required for effective GBM therapy. Inadequate vector performance may derive from poor intratumoral vector replication and limited spread to uninfected cells. Vector replication may be impaired by mutagenesis strategies to achieve vector safety, and intratumoral virus spread may be hampered by vector entrapment in the tumor-specific extracellular matrix (ECM) that in GBM is composed primarily of type IV collagen. In this report, we armed our previously described epidermal growth factor receptor (EGFR)vIII-targeted, neuronal microRNA-sensitive oHSV with a matrix metalloproteinase (MMP9) to improve intratumoral vector distribution. We show that vector-expressed MMP9 enhanced therapeutic efficacy and long-term animal survival in a GBM xenograft model. Keywords: matrix metalloproteinase 9 = MMP9, extracellular matrix = ECM, Oncolytic vectors = OVs, Glioblastoma multiforme = GBM, OVs derived from herpes simplex virus = oHSV

The delivery of single therapeutic microRNAs in brain cancers is challenging. Here, the authors engineer three neuronal microRNAs (miR-124, 128 and 137) into a cluster that, targeting oncogenic chromatin repressors, increases survival of GBM-bearing mice when combined with chemotherapy.

In vitroblood-brain barrier (BBB) models are crucial tools for screening brain-penetrating compounds. Here the authors develop a self-assembling BBB spheroid model with superior performance to the standard transwell BBB model, and use their platform to identify cell-penetrating peptides that can cross the BBB.

Despite the importance of molecular subtype classification of glioblastoma (GBM), the extent of extracellular vesicle (EV)-driven molecular and phenotypic reprogramming remains poorly understood. To reveal complex subpopulation dynamics within the heterogeneous intratumoral ecosystem, we characterized microRNA expression and secretion in phenotypically diverse subpopulations of patient-derived GBM stem-like cells (GSCs). As EVs and microRNAs convey information that rearranges the molecular landscape in a cell type-specific manner, we argue that intratumoral exchange of microRNA augments the heterogeneity of GSC that is reflected in highly heterogeneous profile of microRNA expression in GBM subtypes.

Large-scale transcriptomic profiling of glioblastoma (GBM) into subtypes has provided remarkable insight into the pathobiology and heterogeneous nature of this disease. The mechanisms of speciation and inter-subtype transitions of these molecular subtypes require better characterization to facilitate the development of subtype-specific targeting strategies. The deregulation of microRNA expression among GBM subtypes and their subtype-specific targeting mechanisms are poorly understood. To reveal the underlying basis of microRNA-driven complex subpopulation dynamics within the heterogeneous intra-tumoral ecosystem, we characterized the expression of the subtype-enriched microRNA-128 (miR-128) in transcriptionally and phenotypically diverse subpopulations of patient-derived glioblastoma stem-like cells. Because microRNAs are capable of re-arranging the molecular landscape in a cell-type-specific manner, we argue that alterations in miR-128 levels are a potent mechanism of bidirectional transitions between GBM subpopulations, resulting in intermediate hybrid stages and emphasizing highly intricate intra-tumoral networking.

Cells release heterogeneous nano-sized vesicles either as exosomes, being derived from endosomal compartments, or through budding from the plasma membrane as so-called microvesicles, commonly referred to as extracellular vesicles (EVs). EVs are known for their important roles in mammalian physiology and disease pathogenesis and provide a potential biomarker source in cancer patients. EVs are generally often analysed in bulk using Western blotting or by bead-based flow-cytometry or, with limited parameters, through nanoparticle tracking analysis. Due to their small size, single EV analysis is technically highly challenging. Here we demonstrate imaging flow cytometry (IFCM) to be a robust, multiparametric technique that allows analysis of single EVs and the discrimination of distinct EV subpopulations. We used IFCM to analyse the tetraspanin (CD9, CD63, CD81) surface profiles on EVs from human and murine cell cultures as well as plasma samples. The presence of EV subpopulations with specific tetraspanin profiles suggests that EV-mediated cellular responses are tightly regulated and dependent on cell environment. We further demonstrate that EVs with double positive tetraspanin expression (CD63+/CD81+) are enriched in cancer cell lines and patient plasma samples. In addition, we used IFCM to detect tumour-specific GFP-labelled EVs in the blood of mice bearing syngeneic intracerebral gliomas, indicating that this technique allows unprecedented disease modelling. In summary, our study highlights the heterogeneous and adaptable nature of EVs according to their marker profile and demonstrates that IFCM facilitates multiparametric phenotyping of EVs not only in vitro but also in patient plasma at a single EV level, with the potential for future functional studies and clinically relevant applications. Abbreviation: EDTA = ethylenediamine tetraacetic acid

Despite advances in surgical resection and chemoradiation, high-grade brain tumors continue to be associated with significant morbidity/mortality. Novel therapeutic strategies and approaches are, therefore, desperately needed for patients and their families. Given the success experienced in treating multiple other forms of cancer, immunotherapy and, in particular, immunovirotherapy are at the forefront amongst novel therapeutic strategies that are currently under investigation for incurable brain tumors. Accordingly, herein, we provide a focused mini review of pertinent oncolytic herpes viruses (oHSV) that are being investigated in clinical trials.

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

Abstract MicroRNA‐10b (miR‐10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes, while absent in normal neuroglial cells of the brain. miR‐10b inhibition strongly impairs proliferation and survival of cultured glioma cells, including glioma‐initiating stem‐like cells (GSC). Although several miR‐10b targets have been identified previously, the common mechanism conferring the miR‐10b‐sustained viability of GSC is unknown. Here, we demonstrate that in heterogeneous GSC, miR‐10b regulates cell cycle and alternative splicing, often through the non‐canonical targeting via 5′UTRs of its target genes, including MBNL1‐3, SART3, and RSRC1. We have further assessed the inhibition of miR‐10b in intracranial human GSC‐derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR‐10b antisense oligonucleotide inhibitors (ASO), direct intratumoral injections, continuous osmotic delivery, and systemic intravenous injections, have been explored. In all cases, the treatment with miR‐10b ASO led to targets’ derepression, and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR‐10b is a promising candidate for the development of targeted therapies against all GBM subtypes.