Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model.

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

Safety and Efficacy of Biopsy in Patients with Diffuse Intrinsic Pontine Gliomas.

BACKGROUND: Diffuse intrinsic pontine gliomas are aggressive tumors that carry a poor prognosis with a 2-year survival rate of <10%. The imaging appearance is often pathognomonic, and surgical biopsy is not mandatory to initiate treatment in children. Studies of biopsy samples provide insight into the disease's molecular pathobiology and open prospects for targeted therapy. This study was conducted to determine the diagnostic yield and safety of stereotactic biopsies. METHODS: This is a prospective observational study from a single tertiary health care center. All patients with clinical and radiological features diagnostic of diffuse intrinsic pontine gliomas (DIPGs) who underwent biopsy from July 2018 to June 2023 were included. Biopsies were performed using either stereotactic frame-based, frameless, or endoscopic techniques. RESULTS: A total of 165 patients with DIPGs were evaluated in the study period. The option of biopsy with its associated risks and benefits was offered to all patients. A total of 76 biopsies were performed in 74 patients (40 children and 34 adults, including 2 repeat biopsies). The median age was 15 years. Diffuse midline gliomas, H3K27M altered, was the most common histopathological diagnosis (85% pediatric and 55.9% adults). The diagnostic efficacy of the procedure was 94.7%. The complication rate was 10.8%, with no permanent neurological deficits due to surgery. There was no procedure-related mortality. CONCLUSIONS: Establishing the safety of the procedure could be an important step toward popularizing the concept, which might offer a better understanding of the disease. Brainstem eloquence and a lack of direct benefit to patients are the primary obstacles to brainstem biopsy.

High Level Immunoglobulin Gene Expression and Reduced Intra-tumoral Bacteria Associated With the Transition from Initial to Progressive Diffuse Intrinsic Pontine Glioma.

BACKGROUND/AIM: In the past decade, diffuse intrinsic pontine glioma (DIPG), the most common childhood brainstem glioma, has benefitted from an increase in tissue-based research because of improved biopsy collection techniques. However, the adaptive immune receptor (IR) features represented by tumor material and tumor infiltrating lymphocytes have remained poorly understood. MATERIALS AND METHODS: Herein, we characterized the adaptive immune parameters of DIPG through the recovery of IR recombination reads from RNAseq files representing initial and progressive DIPG samples. RESULTS: An elevated level of immunoglobulin gene expression in the progressive DIPG sample files and a reduced number of bacterial sequencing read recoveries in comparison to RNAseq files representing the initial form of DIPG, was found. Furthermore, the RNAseq files representing both initial and progressive DIPG samples had significant numbers of reads representing Cutibacterium acnes, a bacterium previously linked to prostate cancer development. Results also indicated an opportunity to distinguish overall survival probabilities based on IGL complementarity determining region-3 amino acid sequence physicochemical parameters. CONCLUSION: Genomics analyses allow for a better understanding of adaptive IR features and bacterial infections in the DIPG setting.

Diffuse intrinsic pontine glioma (DIPG): A review of current and emerging treatment strategies.

Diffuse intrinsic pontine glioma (DIPG) is a childhood malignancy of the brainstem with a dismal prognosis. Despite recent advances in its understanding at the molecular level, the prognosis of DIPG has remained unchanged. This article aims to review the current understanding of the genetic pathophysiology of DIPG and to highlight promising therapeutic targets. Various DIPG treatment strategies have been investigated in pre-clinical studies, several of which have shown promise and have been subsequently translated into ongoing clinical trials. Ultimately, a multifaceted therapeutic approach that targets cell-intrinsic alterations, the micro-environment, and augments the immune system will likely be necessary to eradicate DIPG.

Very Long-term Survivorship in Pediatric DIPG: Case Report and Review of the Literature.

Diffuse intrinsic pontine gliomas are lethal tumors with a prognosis generally less than 1 year. Few cases of survivors of 5 years or more have been reported. This case report highlights the journey of a 9.5-year survivor who underwent 3 rounds of focal radiotherapy; she experienced 6 years of progression-free survival following the first round but ultimately succumbed to her disease. An autopsy revealed a favorable IDH1 mutation and the absence of H3K27M. This case reiterates the importance of extensive molecular analyses in diffuse intrinsic pontine gliomas and explores the potential benefit of re-irradiation in patients with positive responses and long periods of remission.

Bone Morphogenic Proteins in Pediatric Diffuse Midline Gliomas: How to Make New Out of Old?

The BMP pathway is one of the major signaling pathways in embryonic development, ontogeny and homeostasis, identified many years ago by pioneers in developmental biology. Evidence of the deregulation of its activity has also emerged in many cancers, with complex and sometimes opposing effects. Recently, its role has been suspected in Diffuse Midline Gliomas (DMG), among which Diffuse Intrinsic Pontine Gliomas (DIPG) are one of the most complex challenges in pediatric oncology. Genomic sequencing has led to understanding part of their molecular etiology, with the identification of histone H3 mutations in a large proportion of patients. The epigenetic remodeling associated with these genetic alterations has also been precisely described, creating a permissive context for oncogenic transcriptional program activation. This review aims to describe the new findings about the involvement of BMP pathway activation in these tumors, placing their appearance in a developmental context. Targeting the oncogenic synergy resulting from this pathway activation in an H3K27M context could offer new therapeutic perspectives based on targeting treatment-resistant cell states.

H3 K27M-altered glioma and diffuse intrinsic pontine glioma: Semi-systematic review of treatment landscape and future directions.

H3 K27M-mutant diffuse glioma is a recently identified brain tumor associated with poor prognosis. As of 2016, it is classified by the World Health Organization as a distinct form of grade IV glioma. Despite recognition as an important prognostic and diagnostic feature in diffuse glioma, radiation remains the sole standard of care and no effective systemic therapies are available for H3K27M mutant tumors. This review will detail treatment interventions applied to diffuse midline glioma and diffuse intrinsic pontine glioma (DIPG) prior to the identification of the H3 K27M mutation, the current standard-of-care for H3 K27M-mutant diffuse glioma treatment, and ongoing clinical trials listed on www.clinicaltrials.gov evaluating novel therapeutics in this population. Current clinical trials were identified using clinicaltrials.gov, and studies qualifying for this analysis were active or ongoing interventional trials that evaluated a therapy in at least 1 treatment arm or cohort comprised exclusively of patients with DIPG and H3 K27M-mutant glioma. Forty-one studies met these criteria, including trials evaluating H3 K27M vaccination, chimeric antigen receptor T-cell therapy, and small molecule inhibitors. Ongoing evaluation of novel therapeutics is necessary to identify safe and effective interventions in this underserved patient population.

Microtubule-Targeting Combined with HDAC Inhibition Is a Novel Therapeutic Strategy for Diffuse Intrinsic Pontine Gliomas.

Diffuse intrinsic pontine gliomas (DIPG) are an incurable childhood brain cancer for which novel treatments are needed. DIPGs are characterized by a mutation in the H3 histone (H3K27M), resulting in loss of H3K27 methylation and global gene dysregulation. TRX-E-009-1 is a novel anticancer agent with preclinical activity demonstrated against a range of cancers. We examined the antitumor activity of TRX-E-009-1 against DIPG neurosphere cultures and observed tumor-specific activity with IC50s ranging from 20 to 100 nmol/L, whereas no activity was observed against normal human astrocyte cells. TRX-E-009-1 exerted its anti-proliferative effect through the induction of apoptotic pathways, with marked increases in cleaved caspase 3 and cleaved PARP levels, while also restoring histone H3K27me3 methylation. Co-administration of TRX-E-009-1 and the histone deacetylase (HDAC) inhibitor SAHA extended survival in DIPG orthotopic animal models. This antitumor effect was further enhanced with irradiation. Our findings indicate that TRX-E-009-1, combined with HDAC inhibition, represents a novel, potent therapy for children with DIPG.

Rapid Peroxide Removal Limits the Radiosensitization of Diffuse Intrinsic Pontine Glioma (DIPG) Cells by Pharmacologic Ascorbate.

Diffuse intrinsic pontine gliomas (DIPG) are an aggressive type of pediatric brain tumor with a very high mortality rate. Surgery has a limited role given the tumor's location. Palliative radiation therapy alleviates symptoms and prolongs survival, but median survival remains less than 1 year. There is no clear role for chemotherapy in DIPGs as trials adding chemotherapy to palliative radiation therapy have failed to improve survival compared to radiation alone. Thus, there is a critical need to identify tissue-specific radiosensitizers to improve clinical outcomes for patients with DIPGs. Pharmacologic (high dose) ascorbate (P-AscH-) is a promising anticancer therapy that sensitizes human tumors, including adult high-grade gliomas, to radiation by acting selectively as a generator of hydrogen peroxide (H2O2) in cancer cells. In this study we demonstrate that in contrast to adult glioma models, P-AscH- does not radiosensitize DIPG. DIPG cells were sensitive to bolus of H2O2 but have faster H2O2 removal rates than GBM models which are radiosensitized by P-AscH-. These data support the hypothesis that P-AscH- does not enhance DIPG radiosensitivity, likely due to a robust capacity to detoxify and remove hydroperoxides.

Dimethyl alpha-ketoglutarate inhibits proliferation in diffuse intrinsic pontine glioma by reprogramming epigenetic and transcriptional networks.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with limited therapeutic options. Here, we investigated the potential of dimethyl alpha-ketoglutarate (DMKG) as an anti-proliferative agent against DIPG and unraveled its underlying molecular mechanisms. DMKG exhibited robust inhibition of DIPG cell proliferation, colony formation, and neurosphere growth. Transcriptomic analysis revealed substantial alterations in gene expression, with upregulated genes enriched in hypoxia-related pathways and downregulated genes associated with cell division and the mitotic cell cycle. Notably, DMKG induced G1/S phase cell cycle arrest and downregulated histone H3 lysine 27 acetylation (H3K27ac) without affecting H3 methylation levels. The inhibition of AKT and ERK signaling pathways by DMKG coincided with decreased expression of the CBP/p300 coactivator. Importantly, we identified the c-MYC-p300/ATF1-p300 axis as a key mediator of DMKG's effects, demonstrating reduced binding to target gene promoters and decreased H3K27ac levels. Depletion of c-MYC or ATF1 effectively inhibited DIPG cell growth. These findings highlight the potent anti-proliferative properties of DMKG, its impact on epigenetic modifications, and the involvement of the c-MYC-p300/ATF1-p300 axis in DIPG, shedding light on potential therapeutic strategies for this devastating disease.

Advances and Clinical Trials Update in the Treatment of Diffuse Intrinsic Pontine Gliomas.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) are high-grade gliomas (HGGs) that occur primarily in children, and represent a leading cause of death in pediatric patients with brain tumors with a median overall survival of only 8-11 months. SUMMARY: While these lesions were previously thought to behave similarly to adult HGG, emerging data have demonstrated that DIPG is a biologically distinct entity from adult HGG frequently driven by mutations in the histone genes H3.3 and H3.1 not found in adult glioma. While biopsy of DIPG was historically felt to confer unacceptable risk of morbidity and mortality, multiple studies have demonstrated that stereotactic biopsy of DIPG is safe, allowing not only for improved understanding of DIPG but also forming the basis for protocols for personalized medicine in DIPG. However, current options for personalized medicine in DIPG are limited by the lack of efficacious targeted therapies for the mutations commonly found in DIPG. Multiple treatment modalities including targeted therapies, immunotherapy, convection-enhanced delivery, and focused ultrasound are in various stages of investigation. KEY MESSAGE: Increasing frequency of biopsy for DIPG has identified distinct driving mutations that may serve as therapeutic targets. Novel treatment modalities are under investigation.

Chorioallantoic membrane (CAM) assay to study treatment effects in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor. While there are a number of in vivo rodent models for evaluating tumor biology and response to therapy, these models require significant time and resources. Here, we established the chick-embryo chorioallantoic (CAM) assay as an affordable and time efficient xenograft model for testing a variety of treatment approaches for DIPG. We found that patient-derived DIPG tumors develop in the CAM and maintain the same genetic and epigenetic characteristics of native DIPG tumors. We monitored tumor response to pharmaco- and radiation therapy by 3-D ultrasound volumetric and vasculature analysis. In this study, we established and validated the CAM model as a potential intermediate xenograft model for DIPG and its use for testing novel treatment approaches that include pharmacotherapy or radiation.

TP53 wild-type/PPM1D mutant diffuse intrinsic pontine gliomas are sensitive to a MDM2 antagonist.

Diffuse intrinsic pontine gliomas (DIPGs) are high-grade tumors of the brainstem that often occur in children, with a median overall survival of less than one year. Given the fact that DIPGs are resistant to chemotherapy and are not amenable to surgical resection, it is imperative to develop new therapeutic strategies for this deadly disease. The p53 pathway is dysregulated by TP53 (~ 60%) or PPM1D gain-of-function mutations (~ 30%) in DIPG cases. PPM1D gain-of-function mutations suppress p53 activity and result in DIPG tumorigenesis. While MDM2 is a major negative regulator of p53, the efficacy of MDM2 inhibitor has not been tested in DIPG preclinical models. In this study, we performed a comprehensive validation of MDM2 inhibitor RG7388 in patient-derived DIPG cell lines established from both TP53 wild-type/PPM1D-mutant and TP53 mutant/PPM1D wild-type tumors, as well in TP53 knockout isogenic DIPG cell line models. RG7388 selectively inhibited the proliferation of the TP53 wild-type/PPM1D mutant DIPG cell lines in a dose- and time-dependent manner. The anti-proliferative effects were p53-dependent. RNA-Seq data showed that differential gene expression induced by RG7388 treatment was enriched in the p53 pathways. RG7388 reactivated the p53 pathway and induced apoptosis as well as G1 arrest. In vivo, RG7388 was able to reach the brainstem and exerted therapeutic efficacy in an orthotopic DIPG xenograft model. Hence, this study demonstrates the pre-clinical efficacy potential of RG7388 in the TP53 wild-type/PPM1D mutant DIPG subgroup and may provide critical insight on the design of future clinical trials applying this drug in DIPG patients.

Population pharmacokinetic analysis of crizotinib in children with progressive/recurrent high-grade and diffuse intrinsic pontine gliomas.

PURPOSE: Crizotinib, a potent oral tyrosine kinase inhibitor, was evaluated in combination with dasatinib in a phase 1 trial (NCT01644773) in children with progressive or recurrent high-grade and diffuse intrinsic pontine gliomas (HGG and DIPG). This study aimed to characterize the pharmacokinetics of crizotinib in this population and identify significant covariates. METHODS: Patients (N = 36, age range 2.9-21.3 years) were treated orally once or twice-daily with 100-215 mg/m2 crizotinib and 50-65 mg/m2 dasatinib. Pharmacokinetic studies were performed for crizotinib alone after the first dose and at steady state, and for the drug combination at steady state. Crizotinib plasma concentrations were measured using a validated LC-MS/MS method. Population modeling was performed (Monolix) and the impact of factors including patient demographics and co-medications were investigated on crizotinib pharmacokinetics. RESULTS: Crizotinib concentrations were described with a linear two-compartment model and absorption lag time. Concomitant dasatinib and overweight/obese status significantly influenced crizotinib pharmacokinetics, resulting in clinically relevant impact (> 20%) on drug exposure. Crizotinib mean apparent clearance (CL/F) was 66.7 L/h/m2 after single-dose and decreased to 26.5 L/h/m2 at steady state when given alone, but not when combined with dasatinib (mean 60.8 L/h/m2). Overweight/obese patients exhibited lower crizotinib CL/F and apparent volume V1/F (mean 46.2 L/h/m2 and 73.3 L/m2) compared to other patients (mean 75.5 L/h/m2 and 119.3 L/m2, p < 0.001). CONCLUSION: A potential pharmacokinetic interaction was observed between crizotinib and dasatinib in children with HGG and DIPG. Further, crizotinib exposure was significantly higher in overweight/obese patients, who may require a dosing adjustment.

Ketogenic diet treatment in diffuse intrinsic pontine glioma in children: Retrospective analysis of feasibility, safety, and survival data.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is one of the most devastating diseases among children with cancer, thus novel strategies are urgently needed. AIMS: We retrospectively evaluated DIPG patients exposed to the carbohydrate restricted ketogenic diet (KD) with regard of feasibility, safety, and overall survival (OS). METHODS AND RESULTS: Searches of MEDLINE and Embase identified five hits meeting the search criteria (diagnosis of DIPG and exposure to KD). One additional case was identified by contact with experts. Individual patient data were extracted from publications or obtained from investigators. The inclusion criteria for analysis of the data were defined as DIPG patients who were exposed to the KD for ≥3 months. Feasibility, as described in the literature, was the number of patients able to follow the KD for 3 months out of all DIPG patients identified. OS was estimated by the Kaplan-Meier method. Five DIPG patients (males, n = 3; median age 4.4 years; range, 2.5-15 years) meeting the inclusion criteria were identified. Analysis of the available data suggested that the KD is generally relatively well tolerated. Only mild gastro-intestinal complaints, one borderline hypoglycemia (2.4 mmol/L) and one hyperketosis (max 7.2 mmol/L) were observed. Five out of six DIPG patients identified adhered for ≥3 months (median KD duration, 6.5 months; range, 0.25-2 years) to the diet. The median OS was 18.7 months. CONCLUSION: Our study provides evidence that it may be feasible for pediatric DIPG patients to adhere for at least 3 months to KD. In particular cases, diet modifications were done. The clinical outcome and OS appear not to be impacted in a negative way. KD might be proposed as adjuvant therapy when large prospective studies have shown feasibility and safety. Future studies might ideally assess the impact of KD on clinical outcome, quality of life, and efficacy.

Design considerations of an IL13Rα2 antibody-drug conjugate for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG), a rare pediatric brain tumor, afflicts approximately 350 new patients each year in the United States. DIPG is noted for its lethality, as fewer than 1% of patients survive to five years. Multiple clinical trials involving chemotherapy, radiotherapy, and/or targeted therapy have all failed to improve clinical outcomes. Recently, high-throughput sequencing of a cohort of DIPG samples identified potential therapeutic targets, including interleukin 13 receptor subunit alpha 2 (IL13Rα2) which was expressed in multiple tumor samples and comparably absent in normal brain tissue, identifying IL13Rα2 as a potential therapeutic target in DIPG. In this work, we investigated the role of IL13Rα2 signaling in progression and invasion of DIPG and viability of IL13Rα2 as a therapeutic target through the use of immunoconjugate agents. We discovered that IL13Rα2 stimulation via canonical ligands demonstrates minimal impact on both the cellular proliferation and cellular invasion of DIPG cells, suggesting IL13Rα2 signaling is non-essential for DIPG progression in vitro. However, exposure to an anti-IL13Rα2 antibody-drug conjugate demonstrated potent pharmacological response in DIPG cell models both in vitro and ex ovo in a manner strongly associated with IL13Rα2 expression, supporting the potential use of targeting IL13Rα2 as a DIPG therapy. However, the tested ADC was effective in most but not all cell models, thus selection of the optimal payload will be essential for clinical translation of an anti-IL13Rα2 ADC for DIPG.

Dual targeting of the epigenome via FACT complex and histone deacetylase is a potent treatment strategy for DIPG.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable childhood brain tumor for which new treatments are needed. CBL0137 is an anti-cancer compound developed from quinacrine that targets facilitates chromatin transcription (FACT), a chromatin remodeling complex involved in transcription, replication, and DNA repair. We show that CBL0137 displays profound cytotoxic activity against a panel of patient-derived DIPG cultures by restoring tumor suppressor TP53 and Rb activity. Moreover, in an orthotopic model of DIPG, treatment with CBL0137 significantly extends animal survival. The FACT subunit SPT16 is found to directly interact with H3.3K27M, and treatment with CBL0137 restores both histone H3 acetylation and trimethylation. Combined treatment of CBL0137 with the histone deacetylase inhibitor panobinostat leads to inhibition of the Rb/E2F1 pathway and induction of apoptosis. The combination of CBL0137 and panobinostat significantly prolongs the survival of mice bearing DIPG orthografts, suggesting a potential treatment strategy for DIPG.

A pilot radiogenomic study of DIPG reveals distinct subgroups with unique clinical trajectories and therapeutic targets.

An adequate understanding of the relationships between radiographic and genomic features in diffuse intrinsic pontine glioma (DIPG) is essential, especially in the absence of universal biopsy, to further characterize the molecular heterogeneity of this disease and determine which patients are most likely to respond to biologically-driven therapies. Here, a radiogenomics analytic approach was applied to a cohort of 28 patients with DIPG. Tumor size and imaging characteristics from all available serial MRIs were evaluated by a neuro-radiologist, and patients were divided into three radiographic response groups (partial response [PR], stable disease [SD], progressive disease [PD]) based on MRI within 2 months of radiotherapy (RT) completion. Whole genome and RNA sequencing were performed on autopsy tumor specimens. We report several key, therapeutically-relevant findings: (1) Certain radiologic features on first and subsequent post-RT MRIs are associated with worse overall survival, including PD following irradiation as well as present, new, and/or increasing peripheral ring enhancement, necrosis, and diffusion restriction. (2) Upregulation of EMT-related genes and distant tumor spread at autopsy are observed in a subset of DIPG patients who exhibit poorer radiographic response to irradiation and/or higher likelihood of harboring H3F3A mutations, suggesting possible benefit of upfront craniospinal irradiation. (3) Additional genetic aberrations were identified, including DYNC1LI1 mutations in a subgroup of patients with PR on post-RT MRI; further investigation into potential roles in DIPG tumorigenesis and/or treatment sensitivity is necessary. (4) Whereas most DIPG tumors have an immunologically "cold" microenvironment, there appears to be a subset which harbor a more inflammatory genomic profile and/or higher mutational burden, with a trend toward improved overall survival and more favorable radiographic response to irradiation, in whom immunotherapy should be considered. This study has begun elucidating relationships between post-RT radiographic response with DIPG molecular profiles, revealing radiogenomically distinct subgroups with unique clinical trajectories and therapeutic targets.

Mesenchymal Stem Cells Successfully Deliver Oncolytic Virotherapy to Diffuse Intrinsic Pontine Glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors. EXPERIMENTAL DESIGN: Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation. RESULTS: Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01). CONCLUSIONS: Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.