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Glioblastoma (GBM) is the most prevalent malignant brain tumor. Current standard-of-care treatments offer limited benefits for patient survival. Virotherapy is emerging as a novel strategy to use oncolytic viruses (OVs) for the treatment of GBM. These engineered and non-engineered viruses infect and lyse cancer cells, causing tumor destruction without harming healthy cells. Recent advances in genetic modifications to OVs have helped improve their targeting capabilities and introduce therapeutic genes, broadening the therapeutic window and minimizing potential side effects. The efficacy of oncolytic virotherapy can be enhanced by combining it with other treatments such as immunotherapy, chemotherapy, or radiation. Recent studies suggest that manipulating the gut microbiome to enhance immune responses helps improve the therapeutic efficacy of the OVs. This narrative review intends to explore OVs and their role against solid tumors, especially GBM while emphasizing the latest technologies used to enhance and improve its therapeutic and clinical responses.

Significant gaps remain in our understanding of immunotherapy-related neurotoxicity in pediatric patients, largely because much of our knowledge comes from studies in adults. Accurately identifying the adverse effects of immunotherapy in children is also challenging, owing to variations in terminology and grading systems. Moreover, the manifestation of immunotherapy-related neurotoxicity differs greatly across different diseases, various modalities, dosages, and delivery methods. Combining immunotherapy with other treatments might improve outcomes but introduces new complexities and potential for increased toxicities. Additionally, pediatric patients with intracranial malignancy have unique responses to immunotherapies and distinct neurotoxicity compared to those with extracranial malignancy. Consequently, we must enhance our understanding of the pathophysiology, prevalence, severity, and management of immunotherapy's neurotoxic effects in this vulnerable group. This review consolidates the current knowledge of immunotherapy-related neurotoxicity in pediatric oncology, highlighting various types of neurotoxicity including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and tumor inflammation-associated neurotoxicity (TIAN), among others. Furthermore, we examine the unique features of neurotoxicity associated with adoptive cellular therapy (ACT), antibody-based therapies, immune checkpoint inhibitors (ICIs), oncolytic viruses (OV), and cancer vaccines., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Pediatric brain tumors are the most common solid tumors in children. Even to date, with the advances in multimodality therapeutic management, survival outcomes remain dismal in some types of tumors, such as pediatric-type diffuse high-grade gliomas or central nervous system embryonal tumors. Failure to understand the complex molecular heterogeneity and the elusive tumor and microenvironment interplay continues to undermine therapeutic efficacy. Developing a strategy that would improve survival for these fatal tumors remains unmet in pediatric neuro-oncology. Oncolytic viruses (OVs) are emerging as a feasible, safe, and promising therapy for brain tumors. The new paradigm in virotherapy implies that the direct cytopathic effect is followed, under certain circumstances, by an antitumor immune response responsible for the partial or complete debulking of the tumor mass. OVs alone or combined with other therapeutic modalities have been primarily used in adult neuro-oncology. A surge in encouraging preclinical studies in pediatric brain tumor models recently led to the clinical translation of OVs with encouraging results in these tumors. In this review, we summarize the different virotherapy tested in preclinical and clinical studies in pediatric brain tumors, and we discuss the limitations and future avenues necessary to improve the response of these tumors to this type of therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated., Methods: The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq, and multiplexed immunofluorescence staining., Results: The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype., Conclusions: The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Oncolytic viruses (OVs) are biological therapeutic agents that selectively destroy cancer cells while sparing normal healthy cells. Besides direct oncolysis, OV infection induces a proinflammatory shift in the tumor microenvironment and the release of tumor-associated antigens (TAAs) that might induce an anti-tumor immunity. Due to their immunostimulatory effect, OVs have been explored for cancer vaccination against specific TAAs. However, this approach usually requires genetic modification of the virus and the production of a new viral vector for each target, which is difficult to implement for low prevalent antigens. In a recent study, Chiaro et al. presented an elegant proof of concept on how to implement the PeptiCRAd vaccination platform to overcome this limitation for the treatment of mesothelioma. Authors showed the feasibility of identifying immunogenic TAAs in human mesothelioma and using them to coat oncolytic adenovirus particles. The result was a customized virus-based cancer vaccine that circumvents time and resource-consuming steps incurred from genetically engineering viruses. Although some questions remain to be addressed, this interesting approach suggests novel strategies for personalized cancer medicine using oncolytic virotherapy., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Oncolytic viruses are a promising treatment for patients with high-grade gliomas, but neutralizing antibodies can limit their efficacy in patients with prior virus exposure or upon repeated virus injections. Data from a previous clinical trial using the oncolytic adenovirus Delta-24-RGD showed that generation of anti-viral neutralizing antibodies may affect the long-term survival of glioma patients. Past studies have examined the effects of neutralizing antibodies during systemic virus injections, but largely overlooked their impact during local virus injections into the brain. We found that immunoglobulins colocalized with viral proteins upon local oncolytic virotherapy of brain tumors, warranting a strategy to prevent virus neutralization and maximize oncolysis. Thus, we generated a chimeric virus, Delta-24-RGD-H43m, by replacing the capsid protein HVRs from the serotype 5-based Delta-24-RGD with those from the rare serotype 43. Delta-24-RGD-H43m evaded neutralizing anti-Ad5 antibodies and conferred a higher rate of long-term survival than Delta-24-RGD in glioma-bearing mice. Importantly, Delta-24-RGD-H43m activity was significantly more resistant to neutralizing antibodies present in sera of glioma patients treated with Delta-24-RGD during a phase 1 clinical trial. These findings provide a framework for a novel treatment of glioma patients that have developed immunity against Delta-24-RGD., Competing Interests: Declaration of interests H.J., F.F.L., M.M.A., C.G.-M., and J.F. report intellectual property related to the Delta-24 platform. M.A. reports DNAtrix-sponsored research not related to this work., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Glioblastoma, the most common primary brain tumor, has a 6.8% survival rate 5 years post diagnosis. Our team developed an oncolytic adenovirus with an OX-40L expression cassette named Delta-24-RGDOX. While studies have revealed the interaction between the gut microbiota and immunotherapy agents, there are no studies linking the gut microbiota with viroimmunotherapy efficacy. We hypothesize that gut bacterial signatures will be associated with oncolytic viral therapy efficacy. To test this hypothesis, we evaluated the changes in gut microbiota in two mouse cohorts: (1) GSC-005 glioblastoma-bearing mice treated orally with indoximod, an immunotherapeutic agent, or with Delta-24-RGDOX by intratumoral injection and (2) a mouse cohort harboring GL261-5 tumors used to mechanistically evaluate the importance of CD4 + T cells in relation to viroimmunotherapy efficacy. Microbiota assessment indicated significant differences in the structure of the gut bacterial communities in viroimmunotherapy-treated animals with higher survival compared with control or indoximod-treated animals. Moreover, viroimmunotherapy-treated mice with prolonged survival had a higher abundance of Bifidobacterium . The CD4 + T cell depletion was associated with gut dysbiosis, lower mouse survival, and lower antitumor efficacy of the therapy. These findings suggest that microbiota modulation along the gut-glioma axis contributes to the clinical efficacy and patient survival of viroimmunotherapy treated animals., Competing Interests: C.G.-M. and J.F. have intellectual property related to this study., (© 2024 The Authors.)

Currently, there is a lack of effective therapies for the majority of glioblastomas (GBMs), the most common and malignant primary brain tumor. While immunotherapies have shown promise in treating various types of cancers, they have had limited success in improving the overall survival of GBM patients. Therefore, advancing GBM treatment requires a deeper understanding of the molecular and cellular mechanisms that cause resistance to immunotherapy. Further insights into the innate immune response are crucial for developing more potent treatments for brain tumors. Our review provides a brief overview of innate immunity. In addition, we provide a discussion of current therapies aimed at boosting the innate immunity in gliomas. These approaches encompass strategies to activate Toll-like receptors, induce stress responses, enhance the innate immune response, leverage interferon type-I therapy, therapeutic antibodies, immune checkpoint antibodies, natural killer (NK) cells, and oncolytic virotherapy, and manipulate the microbiome. Both preclinical and clinical studies indicate that a better understanding of the mechanisms governing the innate immune response in GBM could enhance immunotherapy and reinforce the effects of chemotherapy and radiotherapy. Consequently, a more comprehensive understanding of the innate immune response against cancer should lead to better prognoses and increased overall survival for GBM patients.

Background: Endovascular selective intra-arterial (ESIA) infusion of cellular oncotherapeutics is a rapidly evolving strategy for treating glioblastoma. Evaluation of ESIA infusion requires a unique animal model. Our goal was to create a rabbit human GBM model to test IA infusions of cellular therapies and to test its usefulness by employing clinical-grade microcatheters and infusion methods to deliver mesenchymal stem cells loaded with an oncolytic adenovirus, Delta-24-RGD (MSC-D24)., Methods: Rabbits were immunosuppressed with mycophenolate mofetil, dexamethasone, and tacrolimus. They underwent stereotactic xenoimplantation of human GBM cell lines (U87, MDA-GSC-17, and MDA-GSC-8-11) into the right frontal lobe. Tumor formation was confirmed on magnetic resonance imaging, histologic, and immunohistochemistry analysis. Selective microcatheter infusion of MSC-D24 was performed via the ipsilateral internal carotid artery to assess model utility and the efficacy and safety of this approach., Results: Twenty-five rabbits were implanted (18 with U87, 2 MDA-GSC-17, and 5 MDA-GSC-8-11). Tumors formed in 68% of rabbits (77.8% for U87, 50.0% for MDA-GSC-17, and 40.0% for MDA-GSC-8-11). On MRI, the tumors were hyperintense on T2-weighted image with variable enhancement (evidence of blood brain barrier breakdown). Histologically, tumors showed phenotypic traits of human GBM including varying levels of vascularity. ESIA infusion into the distal internal carotid artery of 2 ml of MSCs-D24 (107 cells) was safe in the model. Examination of post infusion specimens documented that MSCs-D24 homed to the implanted tumor at 24 hours., Conclusions: The intracranial immunosuppressed rabbit human GBM model allows testing of ESIA infusion of novel therapeutics (eg, MSC-D24) in a clinically relevant fashion., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cancer cell heterogeneity and immunosuppressive tumor microenvironment (TME) pose a challenge in treating solid tumors with adoptive cell therapies targeting limited tumor-associated antigens (TAA), such as chimeric antigen receptor T-cell therapy. We hypothesize that oncolytic adenovirus Delta-24-RGDOX activates the TME and promote antigen spread to potentiate the abscopal effect of adoptive TAA-targeting T cells in localized intratumoral treatment. Herein, we used C57BL/6 mouse models with disseminated tumors derived from B16 melanoma cell lines to assess therapeutic effects and antitumor immunity. gp100-specific pmel-1 or ovalbumin (OVA)-specific OT-I T cells were injected into the first subcutaneous tumor, followed by three injections of Delta-24-RGDOX. We found TAA-targeting T cells injected into one subcutaneous tumor showed tumor tropism. Delta-24-RGDOX sustained the systemic tumor regression mediated by the T cells, leading to improved survival rate. Further analysis revealed that, in mice with disseminated B16-OVA tumors, Delta-24-RGDOX increased CD8 + leukocyte density within treated and untreated tumors. Importantly, Delta-24-RGDOX significantly reduced the immunosuppression of endogenous OVA-specific CTLs while increasing that of CD8 + leukocytes and, to a lesser extent, adoptive pmel-1 T cells. Consequently, Delta-24-RGDOX drastically increased the density of the OVA-specific CTLs in both tumors, and the combination synergistically enhanced the effect. Consistently, the splenocytes from the combination group showed a significantly stronger response against other TAAs (OVA and TRP2) than gp100, resulted in higher activity against tumor cells. Therefore, our data demonstrate that, as an adjuvant therapy followed TAA-targeting T cells in localized treatment, Delta-24-RGDOX activates TME and promotes antigen spread, leading to efficacious systemic antitumor immunity to overcome tumor relapse., Significance: Adjuvant therapy with oncolytic viruses promotes antigen spread to potentiate localized intratumoral adoptive T-cell therapy with limited TAA targets, leading to sustainable systemic antitumor immunity to overcome tumor relapse., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2-20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1-69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7-13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05-0.87). A total of 56.2% (95% CI 41.1-70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406)., (© 2023. The Author(s).)

The coronavirus disease 2019 (COVID-19) pandemic has produced a new global challenge for patients with cancer. The disease and the immunosuppression induced by cancer therapies have generated a perfect storm of conditions to increase the severity of the symptoms and worsen the prognosis. However, a few clinical reports showcased the power of viruses to induce remission in some patients suffering from liquid tumors. Here, we reviewed six cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that resulted in cancer remission, simultaneously highlighting the strengths and the unique challenges of oncolytic virotherapy. Virotherapy has become a special case of cancer immunotherapy. This paradigm-shifting concept suggests that oncolytic viruses are not only promising agents to combat particularly immunologically suppressed, immunotherapy-resistant tumors but also that the trigger of local inflammation, such as SARS-CoV-2 infection of the respiratory pathways, may trigger an abscopal effect sufficient to induce the remission of systemic cancer., Competing Interests: C.G.-M. and J.F. report license agreements with DNAtrix, Inc. C.G.-M. and J.F. are shareholders of DNAtrix, Inc., (© 2022 The Authors.)

Background: Oncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials. Results from these studies have suggested that tumor microenvironment remodeling is required to achieve an effective response in solid tumors. Here, we assess the extent to which targeting specific mechanisms underlying the immunosuppressive tumor microenvironment optimizes viroimmunotherapy., Methods: We used RNA-seq analyses to analyze the transcriptome, and validated the results using Q-PCR, flow cytometry, and immunofluorescence. Viral activity was analyzed by replication assays and viral titration. Kyn and Trp metabolite levels were quantified using liquid chromatography-mass spectrometry. Aryl hydrocarbon receptor (AhR) activation was analyzed by examination of promoter activity. Therapeutic efficacy was assessed by tumor histopathology and survival in syngeneic murine models of gliomas, including Indoleamine 2,3-dioxygenase (IDO)-/- mice. Flow cytometry was used for immunophenotyping and quantification of cell populations. Immune activation was examined in co-cultures of immune and cancer cells. T-cell depletion was used to identify the role played by specific cell populations. Rechallenge experiments were performed to identify the development of anti-tumor memory., Results: Bulk RNA-seq analyses showed the activation of the immunosuppressive IDO-kynurenine-AhR circuitry in response to Delta-24-RGDOX infection of tumors. To overcome the effect of this pivotal pathway, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors. The combination therapy increased the frequency of CD8 + T cells and decreased the rate of myeloid-derived suppressor cell and immunosupressive Treg tumor populations in animal models of solid tumors. Functional studies demonstrated that IDO-blockade-dependent activation of immune cells against tumor antigens could be reversed by the oncometabolite kynurenine. The concurrent targeting of the effectors and suppressors of the tumor immune landscape significantly prolonged the survival in animal models of orthotopic gliomas., Conclusions: Our data identified for the first time the in vivo role of IDO-dependent immunosuppressive pathways in the resistance of solid tumors to oncolytic adenoviruses. Specifically, the IDO-Kyn-AhR activity was responsible for the resurface of local immunosuppression and resistance to therapy, which was ablated through IDO inhibition. Our data indicate that combined molecular and immune therapy may improve outcomes in human gliomas and other cancers treated with virotherapy., Competing Interests: Competing interests: MA, HJ, FL, CG-M, and JF report license agreements with DNAtrix. CG-M and JF are shareholders of DNAtrix. MA reports DNAtrix-sponsored research not related to this work., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Pediatric patients with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median survival of less than 1 year. Oncolytic viral therapy has been evaluated in patients with pediatric gliomas elsewhere in the brain, but data regarding oncolytic viral therapy in patients with DIPG are lacking., Methods: We conducted a single-center, dose-escalation study of DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells, in patients with newly diagnosed DIPG. The patients received a single virus infusion through a catheter placed in the cerebellar peduncle, followed by radiotherapy. The primary objective was to assess the safety and adverse-event profile of DNX-2401. The secondary objectives were to evaluate the effect of DNX-2401 on overall survival and quality of life, to determine the percentage of patients who have an objective response, and to collect tumor-biopsy and peripheral-blood samples for correlative studies of the molecular features of DIPG and antitumor immune responses., Results: A total of 12 patients, 3 to 18 years of age, with newly diagnosed DIPG received 1×10 10 (the first 4 patients) or 5×10 10 (the subsequent 8 patients) viral particles of DNX-2401, and 11 received subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. Hemiparesis and tetraparesis developed in 1 patient each. Over a median follow-up of 17.8 months (range, 5.9 to 33.5), a reduction in tumor size, as assessed on magnetic resonance imaging, was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months. Two patients were alive at the time of preparation of the current report, 1 of whom was free of tumor progression at 38 months. Examination of a tumor sample obtained during autopsy from 1 patient and peripheral-blood studies revealed alteration of the tumor microenvironment and T-cell repertoire., Conclusions: Intratumoral infusion of oncolytic virus DNX-2401 followed by radiotherapy in pediatric patients with DIPG resulted in changes in T-cell activity and a reduction in or stabilization of tumor size in some patients but was associated with adverse events. (Funded by the European Research Council under the European Union's Horizon 2020 Research and Innovation Program and others; EudraCT number, 2016-001577-33; ClinicalTrials.gov number, NCT03178032.)., (Copyright © 2022 Massachusetts Medical Society.)

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors, and patient survival has not changed despite many therapeutic efforts, emphasizing the urgent need for effective treatments. Here, we evaluated the anti-DIPG effect of the oncolytic adenovirus Delta-24-ACT, which was engineered to express the costimulatory ligand 4-1BBL to potentiate the antitumor immune response of the virus. Delta-24-ACT induced the expression of functional 4-1BBL on the membranes of infected DIPG cells, which enhanced the costimulation of CD8+ T lymphocytes. In vivo, Delta-24-ACT treatment of murine DIPG orthotopic tumors significantly improved the survival of treated mice, leading to long-term survivors that developed immunological memory against these tumors. In addition, Delta-24-ACT was safe and caused no local or systemic toxicity. Mechanistic studies showed that Delta-24-ACT modulated the tumor-immune content, not only increasing the number, but also improving the functionality of immune cells. All of these data highlight the safety and potential therapeutic benefit of Delta-24-ACT the treatment of patients with DIPG.

Glioblastoma is the most common primary malignant brain neoplasm and it remains one of the most difficult-to-treat human cancers despite decades of discovery and translational and clinical research. Many advances have been made in our understanding of the genetics and epigenetics of gliomas in general; yet, there remains an urgent need to develop novel agents that will improve the survival of patients with this deadly disease. What sets glioblastoma apart from all other cancers is that it develops and spreads within an organ that renders tumor cells inaccessible to most systemically administered agents because of the presence of the blood-brain barrier. Inadequate drug penetration into the central nervous system is often cited as the most common cause of trial failure in neuro-oncology, and even so-called brain-penetrant therapeutics may not reach biologically relevant concentrations in tumor cells. Evaluation of the pharmacokinetics and pharmacodynamics of a novel therapy is a cornerstone of drug development, but few trials for glioma therapeutics have incorporated these basic elements in an organ-specific manner. Window-of-opportunity clinical trial designs can provide early insight into the biological plausibility of a novel therapeutic strategy in the clinical setting. A variety of window-of-opportunity trial designs, which take into account the limited access to treated tissue and the challenges with obtaining pretreatment control tissues, have been used for the initial development of traditional and targeted small-molecule drugs and biologic therapies, including immunotherapies and oncolytic viral therapies. Early-stage development of glioma therapeutics should include a window-of-opportunity component whenever feasible.

Osteosarcoma is an aggressive bone tumor occurring primarily in pediatric patients. Despite years of intensive research, the outcomes of patients with metastatic disease or those who do not respond to therapy have remained poor and have not changed in the last 30 years. Oncolytic virotherapy is becoming a reality to treat local and metastatic tumors while maintaining a favorable safety profile. Delta-24-ACT is a replicative oncolytic adenovirus engineered to selectively target cancer cells and to potentiate immune responses through expression of the immune costimulatory ligand 4-1BB. This work aimed to assess the antisarcoma effect of Delta-24-ACT. MTS and replication assays were used to quantify the antitumor effects of Delta-24-ACT in vitro in osteosarcoma human and murine cell lines. Evaluation of the in vivo antitumor effect and immune response to Delta-24-ACT was performed in immunocompetent mice bearing the orthotopic K7M2 cell line. Immunophenotyping of the tumor microenvironment was characterized by immunohistochemistry and flow cytometry. In vitro, Delta-24-ACT killed osteosarcoma cells and triggered the production of danger signals. In vivo, local treatment with Delta-24-ACT led to antitumor effects against both the primary tumor and spontaneous metastases in a murine osteosarcoma model. Viral treatment was safe, with no noted toxicity. Delta-24-ACT significantly increased the median survival time of treated mice. Collectively, our data identify Delta-24-ACT administration as an effective and safe therapeutic strategy for patients with local and metastatic osteosarcoma. These results support clinical translation of this viral immunotherapy approach., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Oncolytic adenoviruses are promising new treatments against solid tumors, particularly for glioblastoma (GBM), and preclinical models are required to evaluate the mechanisms of efficacy. However, due to the species selectivity of adenovirus, there is currently no single animal model that supports viral replication, tumor oncolysis, and a virus-mediated immune response. To address this gap, we took advantage of the Syrian hamster to develop the first intracranial glioma model that is both adenovirus replication-permissive and immunocompetent., Methods: We generated hamster glioma stem-like cells (hamGSCs) by transforming hamster neural stem cells with hTERT, simian virus 40 large T antigen, and h-RasV12. Using a guide-screw system, we generated an intracranial tumor model in the hamster. The efficacy of the oncolytic adenovirus Delta-24-RGD was assessed by survival studies, and tumor-infiltrating lymphocytes (TILs) were evaluated by flow cytometry., Results: In vitro, hamGSCs supported viral replication and were susceptible to Delta-24-RGD mediated cell death. In vivo, hamGSCs consistently developed into highly proliferative tumors resembling high-grade glioma. Flow cytometric analysis of hamster gliomas revealed significantly increased T-cell infiltration in Delta-24-RGD infected tumors, indicative of immune activation. Treating tumor-bearing hamsters with Delta-24-RGD led to significantly increased survival compared to hamsters treated with phosphate buffered saline (PBS)., Conclusions: This adenovirus-permissive, immunocompetent hamster glioma model overcomes the limitations of previous model systems and provides a novel platform to study the interactions between tumor cells, the host immune system, and oncolytic adenoviral therapy; understanding of which will be critical to implementing oncolytic adenovirus in the clinic., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Delta-24-RGD is an oncolytic adenovirus that is capable of replicating in and killing human glioma cells. Although intratumoral delivery of Delta-24-RGD can be effective, systemic delivery would improve its clinical application. Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) obtained from healthy donors have been investigated as virus carriers. However, it is unclear whether BM-hMSCs can be derived from glioma patients previously treated with marrow-toxic chemotherapy or whether such BM-hMSCs can deliver oncolytic viruses effectively. Herein, the authors undertook a prospective clinical trial to determine the feasibility of obtaining BM-hMSCs from patients with recurrent malignant glioma who were previously exposed to marrow-toxic chemotherapy., Methods: The authors enrolled 5 consecutive patients who had been treated with radiation therapy and chemotherapy. BM aspirates were obtained from the iliac crest and were cultured to obtain BM-hMSCs., Results: The patient-derived BM-hMSCs (PD-BM-hMSCs) had a morphology similar to that of healthy donor-derived BM-hMSCs (HD-BM-hMSCs). Flow cytometry revealed that all 5 cell lines expressed canonical MSC surface markers. Importantly, these cultures could be made to differentiate into osteocytes, adipocytes, and chondrocytes. In all cases, the PD-BM-hMSCs homed to intracranial glioma xenografts in mice after intracarotid delivery as effectively as HD-BM-hMSCs. The PD-BM-hMSCs loaded with Delta-24-RGD (PD-BM-MSC-D24) effectively eradicated human gliomas in vitro. In in vivo studies, intravascular administration of PD-BM-MSC-D24 increased the survival of mice harboring U87MG gliomas., Conclusions: The authors conclude that BM-hMSCs can be acquired from patients previously treated with marrow-toxic chemotherapy and that these PD-BM-hMSCs are effective carriers for oncolytic viruses.

Competing Interests: MMA has a research grant from DNATrix not associated with this work. CG-M and JF have intellectual property licenced to DNATrix. FFL declares no competing interests.

Background: Glioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma., Methods: The in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF., Results: Delta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge., Conclusions: In summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors., Competing Interests: Competing interests: CG-M and JF report ownership interest (including patents) in DNATrix. MMA has a research grant from DNAtrix not related to this work., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Cancer virotherapy is a paradigm-shifting treatment modality based on virus-mediated oncolysis and subsequent antitumor immune responses. Clinical trials of currently available virotherapies showed that robust antitumor immunity characterizes the remarkable and long-term responses observed in a subset of patients. These data suggest that future therapies should incorporate strategies to maximize the immunotherapeutic potential of oncolytic viruses. In this review, we highlight the recent evidence that the antiviral immunity of the patients may limit the immunotherapeutic potential of oncolytic viruses and summarize the most relevant approaches to strategically redirect the immune response away from the viruses and toward tumors to heighten the clinical impact of viro-immunotherapy platforms., Competing Interests: Competing interests: FL, JF and CG-M are reporting ownership interest (including patents). CG-M and JF are consultants and shareholders of DNATrix., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Atypical teratoid/rhabdoid tumors (AT/RT) and central nervous system primitive neuroectodermal tumors (CNS-PNET) are pediatric brain tumors with poor survival and life-long negative side effects. Here, the aim was to characterize the efficacy and safety of the oncolytic adenovirus, Delta-24-RGD, which selectively replicates in and kills tumor cells., Experimental Design: Delta-24-RGD determinants for infection and replication were evaluated in patient expression datasets. Viral replication and cytotoxicity were assessed in vitro in a battery of CNS-PNET and AT/RT cell lines. In vivo , efficacy was determined in different orthotopic mouse models, including early and established tumor models, a disseminated AT/RT lesion model, and immunocompetent humanized mouse models (hCD34 + -NSG-SGM3)., Results: Delta-24-RGD infected and replicated efficiently in all the cell lines tested. In addition, the virus induced dose-dependent cytotoxicity [IC 50 value below 1 plaque-forming unit (PFU)/cell] and the release of immunogenic markers. In vivo , a single intratumoral Delta-24-RGD injection (10 7 or 10 8 PFU) significantly increased survival and led to long-term survival in AT/RT and PNET models. Delta-24-RGD hindered the dissemination of AT/RTs and increased survival, leading to 70% of long-term survivors. Of relevance, viral administration to established tumor masses (30 days after engraftment) showed therapeutic benefit. In humanized immunocompetent models, Delta-24-RGD significantly extended the survival of mice bearing AT/RTs or PNETs (ranging from 11 to 27 days) and did not display any toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD-treated tumors revealed increased CD8 + T-cell infiltration., Conclusions: Delta-24-RGD is a feasible therapeutic option for AT/RTs and CNS-PNETs. This work constitutes the basis for potential translation to the clinical setting., (©2020 American Association for Cancer Research.)

Background: Delta-24-RGD, an oncolytic adenovirus, shows promise against glioblastoma. To enhance virus delivery, we recently demonstrated that human bone marrow-derived mesenchymal stem cells loaded with Delta-24-RGD (hMSC-D24) can eradicate glioblastomas in mouse models. There are no studies examining the safety of endovascular selective intra-arterial (ESIA) infusions of MSC-D24 in large animals simulating human clinical situations., Objective: To perform canine preclinical studies testing the feasibility and safety of delivering increasing doses of hMSCs-D24 via ESIA infusions., Methods: ESIA infusions of hMSC-D24 were performed in the cerebral circulation of 10 normal canines in the target vessels (internal carotid artery [ICA]/P1) via transfemoral approach using commercially available microcatheters. Increasing concentrations of hMSC-D24 or particles (as a positive control) were injected into 1 hemisphere; saline (negative control) was infused contralaterally. Toxicity (particularly embolic stroke) was assessed on postinfusion angiography, diffusion-weighted magnetic resonance imaging, clinical exam, and necropsy., Results: ESIA injections were performed in the ICA (n = 7) or P1 (n = 3). In 2 animals injected with particles (positive control), strokes were detected by all assays. Of 6 canines injected with hMSC-D24 through the anterior circulation, escalating dose from 2 × 106 cells/20 mL to 1 × 108 cells/10 mL resulted in no strokes. Two animals had ischemic and hemorrhagic strokes after posterior cerebral artery catheterization. A survival experiment of 2 subjects resulted in no complications detected for 24-h before euthanization., Conclusion: This novel study simulating ESIA infusion demonstrates that MSCs-D24 can be infused safely at least up to doses of 1 × 108 cells/10 mL (107 cells/ml) in the canine anterior circulation using commercially available microcatheters. These findings support a clinical trial of ESIA infusion of hMSCs-D24., (Copyright © 2020 by the Congress of Neurological Surgeons.)

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults and prognosis is poor despite maximum therapeutic efforts. GBM is composed of heterogeneous cell populations, among which the glioma stem-like cells (GSCs) play an important role in tumor cell self-renewal and the ability to initiate and drive tumor growth and recurrence. The transcription factor SOX2 is enriched in GSCs where it controls the stem cell phenotype, invasion and maintenance of tumorigenicity. Therefore, understanding the molecular mechanisms governed by SOX2 in GSCs is crucial to developing targeted therapies against this resistant cell population. In this study, we identified and validated a miRNA profile regulated by SOX2 in GSCs. Among these miRNAs, miR-425-5p emerged as a significant robust candidate for further study. The expression of miR-425-5p was significantly enriched in clinical GBM specimens compared with a human brain reference sample and showed a positive correlation with SOX2 expression. Using a combination of in silico analyses and molecular approaches, we show that SOX2 binds to the promoter of miR-425-5p. Loss of function studies show that repressing miR-425-5p expression in multiple GSCs inhibited neurosphere renewal and induced cell death. More importantly, miR-425-5p inhibition extended survival in an orthotopic GBM mouse model. Finally, combining several bioinformatics platforms with biological endpoints in multiple GSC lines, we identified FOXJ3 and RAB31 as high confidence miR-425-5p target genes. Our findings show that miR-425-5p is a GBM stem cell survival factor and that miR-425-5p inhibition function is a potential strategy for treating GBM., Competing Interests: Conflict of Interest Disclosure Statement The authors declare that they have no conflict of interest.

Purpose: Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas., Experimental Design: We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6 mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry., Results: In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells and their migration to tumors expressing the target antigen., Conclusions: Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in systemic immunity against disseminated s.c. and i.c. tumors., (©2019 American Association for Cancer Research.)