Intracranial Transplantation Research Articles

Multi-omics and pharmacological characterization of patient-derived glioma cell lines

Glioblastoma (GBM) is the most common brain tumor and remains incurable. Primary GBM cultures are widely used tools for drug screening, but there is a lack of genomic and pharmacological characterization for these primary GBM cultures. Here, we collect 50 patient-derived glioma cell (PDGC) lines and characterize them by whole genome sequencing, RNA sequencing, and drug response screening. We identify three molecular subtypes among PDGCs: mesenchymal (MES), proneural (PN), and oxidative phosphorylation (OXPHOS). Drug response profiling reveals that PN subtype PDGCs are sensitive to tyrosine kinase inhibitors, whereas OXPHOS subtype PDGCs are sensitive to histone deacetylase inhibitors, oxidative phosphorylation inhibitors, and HMG-CoA reductase inhibitors. PN and OXPHOS subtype PDGCs stably form tumors in vivo upon intracranial transplantation into immunodeficient mice, whereas most MES subtype PDGCs fail to form tumors in vivo. In addition, PDGCs cultured by serum-free medium, especially long-passage PDGCs, carry MYC/MYCN amplification, which is rare in GBM patients. Our study provides a valuable resource for understanding primary glioma cell cultures and clinical translation and highlights the problems of serum-free PDGC culture systems that cannot be ignored.

P53/E2F7 axis promotes temozolomide chemoresistance in glioblastoma multiforme

BackgroundGlioblastoma multiforme (GBM) is the most aggressive form of brain cancer, and chemoresistance poses a significant challenge to the survival and prognosis of GBM. Although numerous regulatory mechanisms that contribute to chemoresistance have been identified, many questions remain unanswered. This study aims to identify the mechanism of temozolomide (TMZ) resistance in GBM.MethodsBioinformatics and antibody-based protein detection were used to examine the expression of E2F7 in gliomas and its correlation with prognosis. Additionally, IC50, cell viability, colony formation, apoptosis, doxorubicin (Dox) uptake, and intracranial transplantation were used to confirm the role of E2F7 in TMZ resistance, using our established TMZ-resistance (TMZ-R) model. Western blot and ChIP experiments provided confirmation of p53-driven regulation of E2F7.ResultsElevated levels of E2F7 were detected in GBM tissue and were correlated with a poor prognosis for patients. E2F7 was found to be upregulated in TMZ-R tumors, and its high levels were linked to increased chemotherapy resistance by limiting drug uptake and decreasing DNA damage. The expression of E2F7 was also found to be regulated by the activation of p53.ConclusionsThe high expression of E2F7, regulated by activated p53, confers chemoresistance to GBM cells by inhibiting drug uptake and DNA damage. These findings highlight the significant connection between sustained p53 activation and GBM chemoresistance, offering the potential for new strategies to overcome this resistance.

Identifying Gene-Treatment Interactions and Targetable Radiation Vulnerabilities in Glioblastoma through Coupling of In Vivo CRISPR Perturbation and Single Cell Transcriptomics

Glioblastoma (GBM) is an incurable brain tumor comprised of dynamic malignant cell states and microenvironment components that underlie treatment resistance. Here we use genome-wide CRISPR/Cas9 functional genomics to define biological drivers and therapeutic vulnerabilities across human and mouse GBM models. To interrogate these mechanisms in the context of the tumor microenvironment and in vivo physiology, we established in vivo Perturb-seq intracranially, a technique coupling functional genomics with single cell transcriptomics, where each cell is an individual experiment. Orthotopic intracranial tumor models were established using human (GBM6, GBM43) or mouse (GL261, SB28) GBM cells stably expressing CRISPR interference (CRISPRi) machinery. Perturb-seq target selection for phenotyping of gene-treatment interactions was performed using genome-wide CRISPRi screens ± radiotherapy in cell cultures. Dual sgRNA lentivirus libraries were transduced either ex vivo prior to intracranial GBM cell transplantation or in vivo using intratumor convection enhanced delivery (CED). Transcriptional phenotyping was performed using single-cell RNA-seq with CRISPR direct capture following focal brain radiotherapy (2 Gy x 5) or mock treatment. GBM cell states were validated using single-nucleus RNA-seq data from 86 primary-recurrent patient-matched GBMs. Mechanistic and functional validation was performed using small molecule inhibitors, immunohistochemistry, clonogenic assays, and in vivo survival experiments. In vivo Perturb-seq ± radiotherapy of 48 genes underlying GBM radiotherapy responses, which were enriched for DNA damage response and metabolic pathways, was performed in > 425,000 single cells. Radiotherapy induced 16 distinct GBM cell states, and genetic perturbations reprogrammed these cell states in a treatment-dependent fashion. Quantitative modeling of gene/radiotherapy interactions using high dimensional manifolds revealed in vivo-specific genetic dependencies. We revealed a critical role for Prkdc, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), as a radiotherapy sensitizer through regulation of cell intrinsic growth and oxidative stress pathways, and cell extrinsic interferon and signaling pathways that altered cell-cell interactions in vivo. These pathways were also disrupted in single-nucleus RNA-seq analysis of post-radiotherapy human GBM tumors. Inhibition of Prkdc using a Food and Drug Administration approved small molecule sensitized GBM cells to radiotherapy and extended survival in mice harboring human intracranial xenografts. We establish in vivo Perturb-seq in orthotopic GBM models as a platform for simultaneous functional genomic discovery and characterization of therapeutic targets, revealing an underappreciated role for Prkdc in GBM tumors in vivo that is targetable using small molecules. These tools are adaptable for a wide range of disease models and treatment modalities.

Flow Cytometry Identification of Cell Compartments in the Murine Brain.

Glioma can be modelled in the murine brain through the induction of genetically engineered mouse models or intracranial transplantation. Gliomas (oligodendroglioma and astrocytoma) are thought to arise from neuronal and glial progenitor populations in the brain and are poorly infiltrated by immune cells. An improved understanding of oligodendrocytes, astrocytes, and the immune environment throughout tumor development will enhance the analysis and development of brain cancer models. Here, we describe the isolation and analysis of murine brain cell types using a combination of flow cytometry and quantitative RT-PCR strategies to analyze these individual cell populations in vivo.

Activity of 2-[3-(2-chloroethyl)-3-nitrosoureido]-1,3-propandyоl (chlonisol) versus lomustine in intracranial transplanted tumor model in mice

Background. Alkylating drugs have been used in tumor chemo therapy for many decades, and the search for effective compounds continues.The aim of the study was to study the activity of the developed new compound – 2-[3-(2-chloroethyl)-3-nitrosoureido]-1,3-propanediol (chlonisol) in comparison with lomustine (CCNU) from the nitrosoalkylurea group, which is similar in chemical structure, in the model of intracranially transplanted Ehrlich's tumor and sarcoma 180 in mice.Methods. According to the developed technique, 64 female mice of the BALB/c line were punctured in the skull under anesthesia and inoculated with tumor cells of Ehrlich's carcinoma or sarcoma 180 in 0.9 % sodium chloride solution. After 24 hours, the test substances were administered at the maximum tolerated doses – chlonisol (20 mg/kg, i.p.) and lomustine (50 mg/kg, orally), once. The effect was compared with the control (solvent injection).Results. Chlonisol significantly increased the median overall survival (MOS) of animals after intracranial transplantation of both Ehrlich's tumor (by 39 %) and sarcoma 180 (by 84 %) compared with control (p<0.0001). Chlonisol reduced the risk of death in mice by 73 % compared to control in Ehrlich tumor transplantation and by 83 % in sarcoma 180 (p<0.0001). In contrast, lomustine did not show a significant therapeutic effect in intracranial transplantation of both tumors.Conclusion. The high activity of chlonisol in comparison with lomustine gives reason to consider it as a potential cytostatic agent in the treatment of nervous system tumors.

Comparative Activity of Chlonisol and Temozolomide in Mice After Intracranial Transplantation of Ehrlich Carcinoma and HER2-Positive Breast Cancer

Comparative Activity of Chlonisol and Temozolomide in Mice After Intracranial Transplantation of Ehrlich Carcinoma and HER2-Positive Breast Cancer

TMIC-85. MICROGLIA REPLACEMENT CHANGES THE TRANSCRIPTIONAL PROFILE OF TUMOR ASSOCIATED MYELOID CELLS IN MURINE MODELS OF BRAIN MALIGNANCIES

Abstract Tumor associated microglia and macrophages (TAMs) represent a main cell type of brain malignancies and demonstrate complex interactions with cancerous cells and the tumor microenvironment. These interactions have important implications for the progression and treatment of malignant central nervous system (CNS) tumors. In a non-oncological context, it has been shown that endogenous microglia can be replaced by peripheral hematopoietic cells by genetic or pharmacological intervention. A highly efficient replacement protocol (Shibuya et al, Sci Transl Med 2022, PMID:35294256) utilizing myeloablative bone marrow transplantation (BMT) followed by microglial depletion via pharmacological inhibition of the Colony-stimulating factor-1 receptor (CSF-1R) leads to almost complete repopulation of the CNS myeloid niche by circulation-derived myeloid cells (CDMCs). We investigated if this approach could be utilized to integrate CDMCs into brain tumors and study associated changes in the TAM signature. Microglia replacement in immune-competent mice was followed by intracranial transplantation of syngeneic cell lines of either high-grade glioma (GL261) or breast cancer metastasis (E0771). Lineage tracing proved high intra- and peri-tumoral chimerism of BMT-graft derived cells among CNS myeloid cells in both models two weeks after tumor cell injection. Tumor associated CDMCs (TA-CDMCs) showed a similarly activated morphology as naïve TAMs. We used single cell RNA sequencing of CD45-positive cells to further profile the transcriptomic identity of glioma TA-CDMCs. Pathway enrichment analysis revealed an upregulation of immune response pathways in TA-CDMCs compared to regular TAMs, including the overexpression of genes relevant for the regulation of T cell activation, suggesting possible modification of the glioma immune-environment. Strategies to manipulate the myeloid niche have the potential to provide further understanding of the pathobiology of TAMs and to build the basis for novel cell therapeutic approaches in the neuro-oncological domain.

Dental pulp stem cell transplantation facilitates neuronal neuroprotection following cerebral ischemic stroke

This study aimed to identify and evaluate the intracranial transplantation of dental pulp stem cells (DPSCs) as a possible ischemic stroke therapy that mitigates neuronal death/apoptosis. DPSCs were isolated from the impacted third molars of healthy volunteers and then intracranially injected at 24h post-ischemic stroke to Sprague Dawley rats that had been subjected to 2h of middle cerebral artery occlusion. Neurological functional deficits were assessed using the modified neurological severity score (mNSS), and cerebral edema was quantified using brain water content. Neuronal death/apoptosis was indicated by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, NeuN immunofluorescence and immunohistochemistry, and Western blot analysis of the protein expression of anti-apoptotic indicator of Bcl-2 and apoptotic indicators of Bax and caspase 3. DPSC transplantation could ameliorate neurological dysfunction and brain edema, reduce infarct volume, decrease the percentage of TUNEL-positive nuclei, increase the number and percentage of NeuN-positive cells in ischemic penumbra, increase the ratio of Bcl-2 and Bax and down-regulate the production of caspase 3 in the cortical infarct zone. DPSC therapy via intracranial injection exerted remarkably neuroprotection mainly by inhibiting neuronal death/apoptosis.

Assessing fetal human neural stem cells tumorigenicity potential in athymic rats with penetrating traumatic brain injury (pTBI)

Traumatic brain injuries (TBI) often produce disability in survivors due to unresolved inflammation and progressive neurodegeneration. The central nervous system in mammals is incapable of self-repair. Two decades of preclinical studies and clinical trials have provided insights into TBI pathophysiology that could be utilized to develop clinically relevant therapy. Our laboratory recently reported efficacy of clinical trial grade fetal human neural stem cells (hNSCs) in immunosuppressed rats with penetrating traumatic brain injury (pTBI). Next, in compliance with the United States Food and Drug Administration (USFDA) guidance, this study explores safety by assessing the tumorigenicity potential of intracranial hNSC transplants in athymic rats with pTBI. First, the maximum tolerated dose (MTD) was determined. Then, forty athymic pTBI rats were randomized to either: Group A. pTBI + vehicle or Group B. pTBI + hNSCs at MTD one week after injury with 6-months survival, sufficient time to uncover transplant associated tumorigenicity. A board-certified Pathologist examined hematoxylin-eosin (H&E), Ki67 immunostained brain and spinal cord, serial sections along with several abnormal peripheral masses for evidence of lesion, transplant, and oncogenesis. There was no evidence of transplant derived tumors or oncogenic tissue necrosis. Consistent with athymic literature, the lesion remained unchanged even after robust hNSC engraftment. This safety study supports the conclusion that hNSCs are safe for transplantation in pTBI. The differences in lesion expansion between immunosuppressed and athymic rats in the presence of hNSCs suggests an unexpected role for thymus derived cells in resolution of trauma induced inflammation.

Presenilin1 inhibits glioblastoma cell invasiveness via promoting Sortilin cleavage

BackgroundAlzheimer’s disease (AD) and glioblastoma are the most common and devastating diseases in the neurology and neurosurgery departments, respectively. Our previous research reports that the AD-related protein Presenilin1 represses cell proliferation by inhibiting the Wnt/β-catenin pathway in glioblastoma. However, the function of Presenilin1 and the underlying mechanism need to be further investigated.MethodsThe correlations of two genes were conducted on the R2 microarray platform and CGGA. Wound healing, Transwell assays and glioblastoma transplantation were performed to detect invasion ability. Phalloidin staining was employed to show cell morphology. Proximity ligation assays and protein docking assays were employed to detect two protein locations. We also employed western blotting to detect protein expression.ResultsWe found that Presenilin1 clearly repressed the migration, invasion and mesenchymal transition of glioblastoma cells. Intriguingly, we observed that the expression of Presenilin1 was positively correlated with Sortilin, which is identified as a pro-invasion molecule in glioma. Furthermore, Presenilin1 interacted with Sortilin at the transmembrane domain and repressed Sortilin expression by cleaving it in glioblastoma cells. First, we found that Sortilin introduced the function of Presenilin1 in phosphorylating β-catenin and repressing invasion in glioblastoma cells. Last, Presenilin1 stimulation sharply suppressed the invasion and mesenchymal transition of glioblastoma in mouse subcutaneous and intracranial transplantation models.ConclusionsOur study reveals that Sortilin mediates the regulation of β-catenin by Presenilin1 and transduces the anti-invasive function of Presenilin1, which may provide novel therapeutic targets for glioblastoma treatment.EaRYnAMsaFgrDcEnmWscyLVideo

Evaluation of the antitumor activity of 2-[3-(2-chloroethyl)-3-nitrosoureido]-1,3-propanediol (chlonisol) in C57BL/6 mice with intracranially transplanted B16 melanoma

Background. The arsenal of antitumor drug therapy for melanoma brain metastases is limited. The search and study of new agents capable to penetrate the blood-brain barrier and provide a therapeutic effect against intracranial tumors remains an unmet clinical need. The aim is to evaluate the antitumor activity of the domestic derivative of nitrosoalkylureas, chlonisol, in mice with intracranially transplanted syngeneic B16 melanoma. Methods. The experiment was carried out in 18 female inbred C57BL/6 mice. After intracranial tumor transplantation, performed according to modified technique, the animals were randomized into two groups: I. Control (n = 10) – the animals were injected with normal saline 10 ml/kg intraperitoneally; II. Chlonisol (n = 8) – the animals were treated with the test compound at a dose of 15 mg/kg in normal saline intraperitoneally. The single administration of normal saline and chlonisol was performed 24 hours after tumor transplantation. The end point of the study was overall survival (OS) of the animals. Results. Compared with the control group, administration of chlonisol significantly increased the median OS of mice from 13 to 18 days (log rank test, p = 0.0005). Chlonisol significantly decreased the risk of death by 71 % compared with the control group (HR = 0.29; 95 %CI 0.10–0.82). By the 15th day after intracranial transplantation of B16 melanoma, all 10 mice in the control group died from intracerebral tumors (100 %), whereas in the chlonisol group only 2 out of 8 (25 %) mice died (Fisher's exact test, p = 0.0015). Conclusion. Despite the exploratory nature of the present study, it provides a good starting point for further research of chlonisol in brain tumors.

Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia.

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

Mannan-BAM, TLR ligands, and anti-CD40 immunotherapy in established murine pancreatic adenocarcinoma: understanding therapeutic potentials and limitations.

Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths, and its therapy remains a challenge. Our proposed therapeutic approach is based on the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 antibody (thus termed MBTA therapy), and has shown promising results in the elimination of subcutaneous murine melanoma, pheochromocytoma, colon carcinoma, and smaller pancreatic adenocarcinoma (Panc02). Here, we tested the short- and long-term effects of MBTA therapy in established subcutaneous Panc02 tumors two times larger than in previous study and bilateral Panc02 models as well as the roles of CD4+ and CD8+ T lymphocytes in this therapy. The MBTA therapy resulted in eradication of 67% of Panc02 tumors with the development of long-term memory as evidenced by the rejection of Panc02 cells after subcutaneous and intracranial transplantations. The initial Panc02 tumor elimination is not dependent on the presence of CD4+ T lymphocytes, although these cells seem to be important in long-term survival and resistance against tumor retransplantation. The resistance was revealed to be antigen-specific due to its inability to reject B16-F10 melanoma cells. In the bilateral Panc02 model, MBTA therapy manifested a lower therapeutic response. Despite numerous combinations of MBTA therapy with other therapeutic approaches, our results show that only simultaneous application of MBTA therapy into both tumors has potential for the treatment of the bilateral Panc02 model.

TMOD-22. AKALUC BIOLUMINESCENCE OFFERS SUPERIOR SENSITIVITY TO TRACK IN VIVO GBM EXPANSION

Abstract Longitudinal tracking of tumor growth using non-invasive bioluminescence imaging (BLI) is a key approach for in vivo cancer studies, but the current method of firefly luciferase (Fluc) BLI has quantitative limitations, as it is only suited for detection of tumors of considerable sizes at advanced stage, typically in the order of >105 cells. Recently, Akaluciferase (Akaluc) has been developed as an alternative BLI system that offers higher signal strength and better light penetration of tissue due to its red-shifted emission. Here, we established Akaluc BLI as a new sensitive method for in vivo tracking of glioblastoma (GBM) expansion in intracranial transplant models. In multiple GBM cell lines, including the frequently used U87MG and GL261, as well as patient-derived glioma stem cells (GSC), we demonstrate that Akaluc-expressing GBM cells produced more than 50-times brighter BLI signals in vitro and up to 100-fold higher signal intensities in vivo over Fluc-expressing counterparts. The higher sensitivity of Akaluc BLI permits early in vivo detection of intracranial GBM transplants starting as early as 4 hours after implantation and with as little as 5,000 transplanted GSC. We also reveal a prolonged engraftment period in intracranial GSC transplants before wide dissemination into host brain parenchyma. Akaluc BLI is also advantageous for longitudinal monitoring of therapeutic effects of chemoradiation for GBM and detection of early phase of tumor relapse. Thus, Akaluc BLI offers an important addition to the tool box for cancer research. SIGNIFICANCE: The high sensitivity of Akaluc bioluminescence is a significant improvement for the non-invasive tracking of tumors in preclinical cancer studies, including detection of small incipient tumors and micro-metastasis.

TAMI-18. SINGLE-CELL SEQUENCING AND GENETIC LABELING TO CHARACTERIZE HYPOXIC GBM CELLS IN THEIR MICROENVIRONMENT

Abstract Glioblastoma (GBM) recurrence, arising from treatment-resistant hypoxic cells, is a major contributor to patient mortality. We have engineered GBM cell lines with a novel genetic reporter system (“HRE-UnaG”), which sensitively labels hypoxic cells with green fluorescence. Using murine GL261 HRE-UnaG GBM cells in intracranial transplants in immunocompetent hosts, we detected hypoxic cells primarily around pseudopalisading structures. Here, we report our new results of single cell RNA-seq sequencing of intracranial GL261-HRE-dUnaG GBM tumors, which revealed in hypoxic UnaG+ GBM cells upregulation of more than 60 genes, including canonical hypoxia response genes such as Slc2a1, Bnip3, Ldha and Vegfa, and downregulation of a smaller number of genes, such as Rgcc, Cdc20 and Hist1h2ap, which regulate cell cycle progression. This novel GBM hypoxia signature was in TCGA patient samples mostly upregulated in recurrent GBM and associated with worse prognosis, especially within proneural and mesenchymal subtypes. Gene set enrichment analysis revealed that the GBM hypoxia signature was positively correlated with hypoxia, glycolysis, TNFα and MTORC1 signaling, and negatively correlated with oxidative phosphorylation, Myc and DNA repair pathways. Subclustering revealed four distinct subpopulations of hypoxic GBM cells, which were distinguished by differential expression of pathway genes for proliferation, angiogenesis, stress response, and type I/II interferon signaling. Intercellular communication between hypoxic GBM cells and the stromal cells were predicted using NicheNET package. To spatio-temporally label in future studies hypoxic GBM cells and their progeny, we have engineered GBM cells with an inducible, hypoxia-sensitive lineage-tracing reporter (HRE-Cre; floxed-tdTomato). In combination with the HRE-UnaG hypoxia reporter, we will be able to reveal the role of “hypoxia memory” in GBM expansion and recurrence after treatment. Identifying key markers of hypoxic cells in respect to treatment resistance and relapse will lead to improvements in GBM detection and new options for therapeutic treatments.

TMOD-08. PRIMARY AND RECURRENT GLIOMA PATIENT-DERIVED ORTHOTOPIC XENOGRAFTS (PDOX) REPRESENT RELEVANT PATIENT AVATARS FOR PRECISION MEDICINE

Abstract Patient-derived cancer models are essential tools for studying tumor biology and for preclinical interventions. Although numerous clinical cancer trials are being conducted, many fail due to inappropriate selection of compounds at the preclinical stage. Therefore, better preclinical models are crucial for predicting successful clinical impact. Orthotropic patient-derived xenograft (PDOX) models are of particular importance for brain cancers, as they allow to better recapitulate the brain tumor environment and the blood brain barrier. We created a large collection of PDOXs from primary and recurrent gliomas with and without mutations in IDH1. PDOX models were based on 3D organoids, derived from mechanically minced patient material. Organoids were implanted in the brain of immunodeficient mice and further propagated by serial intracranial transplantations. High grade glioma PDOX models, starting with viable patient-derived organoids, have generally a high tumor take rate, a reproducible phenotype and tumor development time. PDOXs retain histopathological, genetic, epigenetic and transcriptomic features of patient tumors with no mouse-specific clonal evolution. Longitudinal PDOX models confirmed limited evolution of gliomas upon treatment observed in patient tumors. PDOX-derived standardized tumor organoid cultures enabled assessment of drug responses, which were validated in mice. PDOXs showed clinically relevant responses to Temozolomide and to targeted treatments such as EGFR and CDK4/6 inhibitors in (epi)genetically defined groups, according to MGMT promoter and EGFR/CDK status respectively. These data indicate that glioma PDOXs represent clinically relevant avatars for personalized treatment. The use of these models should lead to a more realistic evaluation of the efficacy of novel drugs, thereby increasing the success of clinical studies.

Glucose transporter Glut1 controls diffuse invasion phenotype with perineuronal satellitosis in diffuse glioma microenvironment.

BackgroundGliomas typically escape surgical resection and recur due to their “diffuse invasion” phenotype, enabling them to infiltrate diffusely into the normal brain parenchyma. Over the past 80 years, studies have revealed 2 key features of the “diffuse invasion” phenotype, designated the Scherer’s secondary structure, and include perineuronal satellitosis (PS) and perivascular satellitosis (PVS). However, the mechanisms are still unknown.MethodsWe established a mouse glioma cell line (IG27) by manipulating the histone H3K27M mutation, frequently harboring in diffuse intrinsic pontine gliomas, that reproduced the diffuse invasion phenotype, PS and PVS, following intracranial transplantation in the mouse brain. Further, to broadly apply the results in this mouse model to human gliomas, we analyzed data from 66 glioma patients.ResultsIncreased H3K27 acetylation in IG27 cells activated glucose transporter 1 (Glut1) expression and induced aerobic glycolysis and TCA cycle activation, leading to lactate, acetyl-CoA, and oncometabolite production irrespective of oxygen and glucose levels. Gain- and loss-of-function in vivo experiments demonstrated that Glut1 controls the PS of glioma cells, that is, attachment to and contact with neurons. GLUT1 is also associated with early progression in glioma patients.ConclusionsTargeting the transporter Glut1 suppresses the unique phenotype, “diffuse invasion” in the diffuse glioma mouse model. This work leads to promising therapeutic and potential useful imaging targets for anti-invasion in human gliomas widely.

Extracellular Vesicle-Derived miR-124 Resolves Radiation-Induced Brain Injury.

Radiation-induced cognitive dysfunction (RICD) is a progressive and debilitating health issue facing patients following cranial radiotherapy to control central nervous system cancers. There has been some success treating RICD in rodents using human neural stem cell (hNSC) transplantation, but the procedure is invasive, requires immunosuppression, and could cause other complications such as teratoma formation. Extracellular vesicles (EV) are nanoscale membrane-bound structures that contain biological contents including mRNA, miRNA, proteins, and lipids that can be readily isolated from conditioned culture media. It has been previously shown that hNSC-derived EV resolves RICD following cranial irradiation using an immunocompromised rodent model. Here, we use immunocompetent wild-type mice to show that hNSC-derived EV treatment administered either intravenously via retro-orbital vein injection or via intracranial transplantation can ameliorate cognitive deficits following 9 Gy head-only irradiation. Cognitive function assessed on the novel place recognition, novel object recognition, and temporal order tasks was not only improved at early (5 weeks) but also at delayed (6 months) postirradiation times with just a single EV treatment. Improved behavioral outcomes were also associated with reduced neuroinflammation as measured by a reduction in activated microglia. To identify the mechanism of action, analysis of EV cargo implicated miRNA (miR-124) as a potential candidate in the mitigation of RICD. Furthermore, viral vector-mediated overexpression of miR-124 in the irradiated brain ameliorated RICD and reduced microglial activation. Our findings demonstrate for the first time that systemic administration of hNSC-derived EV abrogates RICD and neuroinflammation in cranially irradiated wild-type rodents through a mechanism involving miR-124. SIGNIFICANCE: Radiation-induced neurocognitive decrements in immunocompetent mice can be resolved by systemic delivery of hNSC-derived EVs involving a mechanism dependent on expression of miR-124.

LINC00470 promotes tumour proliferation and invasion, and attenuates chemosensitivity through the LINC00470/miR-134/Myc/ABCC1 axis in glioma.

Glioma is the most common primary malignant tumour in the brain; temozolomide (TMZ) is the most prevalent chemotherapeutic drug currently used to combat this cancer. We reported previously that the long intergenic non‐protein coding RNA 470 (LINC00470) is a novel prognostic biomarker for glioma and promotes the tumour growth in an intracranial transplantation mouse model. However, the effects of LINC00470 on glioma cell proliferation, invasion and TMZ chemosensitivity, as well as its molecular mechanism, remain unclear. In this study, we found elevated expression levels of LINC00470 and MYC in glioma tissues and cells and decreased expression of microRNA‐134 (miR‐134). Functional studies have shown that LINC00470 promotes proliferation and invasion, and attenuates chemosensitivity of glioma cells, while miR‐134 exerts the opposite effect. In the rescue experiments, the tumorigenic effect of LINC00470 was offset by miR‐134. In the mechanism study, we found that LINC00470 was a competitive endogenous RNA (ceRNA) of miR‐134 and that miR‐134 can directly target MYC and negatively regulate its expression. Furthermore, MYC was positively correlated with ATP‐binding cassette subfamily C member 1 (ABCC1) expression in glioma cells and MYC up‐regulated ABCC1 expression. Further studies found that LINC00470 regulated MYC by sponging miR‐134 to regulate the expression of ABCC1. We concluded that LINC00470 promoted the expression of MYC and ABCC1 by suppressing miR‐134, thus promoting glioma cell proliferation and invasion, and attenuating TMZ chemosensitivity. Moreover, the LINC00470/miR‐134/MYC/ABCC1 axis constitutes a potential therapeutic target.

Mesenchymal stem cells derived extracellular vesicles improve behavioral and biochemical deficits in a phencyclidine model of schizophrenia

Schizophrenia is a debilitating psychiatric disorder with a significant number of patients not adequately responding to treatment. Phencyclidine (PCP) is used as a validated model for schizophrenia, shown to reliably induce positive, negative and cognitive-like behaviors in rodents. It was previously shown in our lab that behavioral phenotypes of PCP-treated mice can be alleviated after intracranial transplantation of mesenchymal stem cells (MSC). Here, we assessed the feasibility of intranasal delivery of MSCs-derived-extracellular vesicles (EVs) to alleviate schizophrenia-like behaviors in a PCP model of schizophrenia. As MSCs-derived EVs were already shown to concentrate at the site of lesion in the brain, we determined that in PCP induced injury the EVs migrate to the prefrontal cortex (PFC) of treated mice, a most involved area of the brain in schizophrenia. We show that intranasal delivery of MSC-EVs improve social interaction and disruption in prepulse inhibition (PPI) seen in PCP-treated mice. In addition, immunohistochemical studies demonstrate that the EVs preserve the number of parvalbumin-positive GABAergic interneurons in the PFC of treated mice. Finally, MSCs-EVs reduced glutamate levels in the CSF of PCP-treated mice, which might explain the reduction of toxicity. In conclusion, we show that MSCs-EVs improve the core schizophrenia-like behavior and biochemical markers of schizophrenia and might be used as a novel treatment for this incurable disorder.