Mouse Model Of Glioma Research Articles

Interferon-γ resistance and immune evasion in glioma develop via Notch-regulated co-evolution of malignant and immune cells.

Immune surveillance is critical to prevent tumorigenesis. Gliomas evade immune attack, but the underlying mechanisms remain poorly understood. We show that glioma cells can sustain growth independent of immune system constraint by reducing Notch signaling. Loss of Notch activity in a mouse model of glioma impairs MHC-I and cytokine expression and curtails the recruitment of anti-tumor immune cell populations in favor of immunosuppressive tumor-associated microglia/macrophages (TAMs). Depletion of Tcells simulates Notch inhibition and facilitates tumor initiation. Furthermore, Notch-depleted glioma cells acquire resistance to interferon-γ and TAMs re-educating therapy. Decreased interferon response and cytokine expression by human and mouse glioma cells correlate with low Notch activity. These effects are paralleled byupregulation of oncogenes anddownregulation of quiescence genes. Hence, suppression of Notch signaling enables gliomas to evade immune surveillance and increases aggressiveness. Our findings provide insights into how brain tumor cells shape their microenvironment to evade immune niche control.

Exploratory Analysis of Serial 18F-fluciclovine PET-CT and Multiparametric MRI during Chemoradiation for Glioblastoma.

Simple SummaryGlioblastoma (GBM), the most common malignant adult primary brain tumour, has a prognosis of ~12–15 months. Poor prognosis is partly due to the inability to accurately define the extent of tumour infiltration; currently demarcated using magnetic resonance imaging (MRI) sequences (e.g., post-contrast T1-weighted (Gd-T1) and dynamic contrast-enhanced (DCE-MRI)). Anti-1-amino-3-18fluorine-fluorocyclobutane-1-carboxylic acid (18F-fluciclovine) positron emission tomography (PET) may depict GBM better than MRI. This prospective pilot study aimed to explore the relationship of 18F-fluciclovine PET, DCE-MRI and Gd-T1 in patients with GBM undergoing standard-of-care adjuvant chemoradiotherapy. A parallel mouse glioma model was used to investigate the relationship between 18F-fluciclovine PET, MRI and tumour biology. Clinical results showed that GBM volume on 18F-fluciclovine PET tended to be larger than Gd-T1 and DCE-MRI in patients with shorter overall survival (OS) but smaller in patients with longer OS. The preclinical study confirmed that 18F-fluciclovine uptake reflected biologically active tumour. Results suggest that 18F-fluciclovine PET may better define GBM infiltration than MRI.Anti-1-amino-3-18fluorine-fluorocyclobutane-1-carboxylic acid (18F-fluciclovine) positron emission tomography (PET) shows preferential glioma uptake but there is little data on how uptake correlates with post-contrast T1-weighted (Gd-T1) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) activity during adjuvant treatment. This pilot study aimed to compare 18F-fluciclovine PET, DCE-MRI and Gd-T1 in patients undergoing chemoradiotherapy for glioblastoma (GBM), and in a parallel pre-clinical GBM model, to investigate correlation between 18F-fluciclovine uptake, MRI findings, and tumour biology. 18F-fluciclovine-PET-computed tomography (PET-CT) and MRI including DCE-MRI were acquired before, during and after adjuvant chemoradiotherapy (60 Gy in 30 fractions with temozolomide) in GBM patients. MRI volumes were manually contoured; PET volumes were defined using semi-automatic thresholding. The similarity of the PET and DCE-MRI volumes outside the Gd-T1 volume boundary was measured using the Dice similarity coefficient (DSC). CT-2A tumour-bearing mice underwent MRI and 18F-fluciclovine PET-CT. Post-mortem mice brains underwent immunohistochemistry staining for ASCT2 (amino acid transporter), nestin (stemness) and Ki-67 (proliferation) to assess for biologically active tumour. 6 patients were recruited (GBM 1–6) and grouped according to overall survival (OS)—short survival (GBM-SS, median OS 249 days) and long survival (GBM-LS, median 903 days). For GBM-SS, PET tumour volumes were greater than DCE-MRI, in turn greater than Gd-T1. For GBM-LS, Gd-T1 and DCE-MRI were greater than PET. Tumour-specific 18F-fluciclovine uptake on pre-clinical PET-CT corresponded to immunostaining for Ki-67, nestin and ASCT2. Results suggest volumes of 18F-fluciclovine-PET activity beyond that depicted by DCE-MRI and Gd-T1 are associated with poorer prognosis in patients undergoing chemoradiotherapy for GBM. The pre-clinical model confirmed 18F-fluciclovine uptake reflected biologically active tumour.

Neutrophils Promote Glioblastoma Tumor Cell Migration after Biopsy.

Glioblastoma is diagnosed by biopsy or, if clinically feasible, tumor resection. However, emerging evidence suggests that this surgical intervention may increase the risk of tumor cell spread. It has been hypothesized that the damage to the tumor leads to infiltration of immune cells that consequently form an environment that favors tumor cell motility. In mouse glioma models, it was previously found that biopsy induced migration of tumor cells in vivo and that recruitment of monocytes from the blood was involved in this effect. However, the role of neutrophils in this process is still unclear. Here, we study the contribution of neutrophils on the pro-migratory effect of surgical interventions in glioma. Using repetitive intravital microscopy, in vivo migration of glioma tumor cells before and after biopsy was compared in mice systemically depleted of neutrophils. Interestingly, macrophages/microglia were almost completely absent from neutrophil-depleted tumors, indicating that neutrophils may be indirectly involved in biopsy-induced migration of glioma tumor cells through the recruitment of macrophages to the tumor. To further investigate whether neutrophils have the potential to also directly promote glioblastoma tumor cell migration, we performed in vitro migration assays using human neutrophils. Indeed, wound-healing of human primary glioblastoma tumor cell lines was promoted by human neutrophils. The pro-migratory effects of human neutrophils on glioblastoma tumor cells could also be recapitulated in transwell migration assays, indicating that soluble factor(s) are involved. We therefore provide evidence for both an indirect and direct involvement of neutrophils in tumor spread following biopsy of glioblastoma tumors.

Spatiotemporal analysis of glioma heterogeneity reveals COL1A1 as an actionable target to disrupt tumor progression

Intra-tumoral heterogeneity is a hallmark of glioblastoma that challenges treatment efficacy. However, the mechanisms that set up tumor heterogeneity and tumor cell migration remain poorly understood. Herein, we present a comprehensive spatiotemporal study that aligns distinctive intra-tumoral histopathological structures, oncostreams, with dynamic properties and a specific, actionable, spatial transcriptomic signature. Oncostreams are dynamic multicellular fascicles of spindle-like and aligned cells with mesenchymal properties, detected using ex vivo explants and in vivo intravital imaging. Their density correlates with tumor aggressiveness in genetically engineered mouse glioma models, and high grade human gliomas. Oncostreams facilitate the intra-tumoral distribution of tumoral and non-tumoral cells, and potentially the collective invasion of the normal brain. These fascicles are defined by a specific molecular signature that regulates their organization and function. Oncostreams structure and function depend on overexpression of COL1A1. Col1a1 is a central gene in the dynamic organization of glioma mesenchymal transformation, and a powerful regulator of glioma malignant behavior. Inhibition of Col1a1 eliminates oncostreams, reprograms the malignant histopathological phenotype, reduces expression of the mesenchymal associated genes, induces changes in the tumor microenvironment and prolongs animal survival. Oncostreams represent a pathological marker of potential value for diagnosis, prognosis, and treatment.

Abstract 705: Inhibition of H3.3 S31 phosphorylation by the pediatric glioma driver mutation, H3.3 K27M, results in chromosomal instability, loss of p53 regulation, and tumorigenesis

Abstract The heterozygous H3.3 K27M mutation is found in the majority of pediatric High-Grade Glioma’s (pHGG). H3.3 K27M is thought to promote tumor formation through altered epigenetic gene regulation. However, it is unclear if epigenetic reprogramming alone is sufficient for glioma formation. H3.3 contains a unique Serine at position 31 which is phosphorylated in early mitosis (where it functions in chromosome segregation) and again in late M/early G1 when a mitotic error has occurred (triggering p53 expression). We found that pHGG cell lines harboring H3.3 K27M have significantly reduced S31 phosphorylation as compared to both pHGG and non-transformed H3.3 WT cells. When compared to WT H3.3 in an in vitro kinase assay, H3.3 K27M exhibits an ~60% reduction in S31 phosphorylation by Chk1, the mitotic S31 kinase. In normal diploid cells, expression of K27M or non-phosphorylatable S31A mutant significantly increased chromosome missegregation. Yet expressing a phosphomimetic double mutant (K27M/S31E) did not significantly alter the rate of chromosome mis-segregation compared to WT. Furthermore, patient-derived pHGG lines harboring K27M have significantly higher rates of chromosome mis-segregation compared to an H3.3 WT pHGG line or H3.3 WT non-transformed human cells. We used CRISPR gene editing to remove the H3.3 K27M allele or replace it with WT H3.3. In both cases, loss of K27M elevated pS31 levels and reduced chromosome mis-segregations. Yet, when K27M was replaced with S31A, the mis-segregation rate remained similar to the parental K27M cells. In normal cells, chromosome missegregation stimulates p53-dependent cell cycle arrest in G1 to prevent the proliferation of aneuploid daughters. However, cells expressing H3.3 K27M or S31A failed to arrest following missegregation - despite having WT p53. To determine if the induction of chromosomal instability would alter tumor formation, we expressed H3.3 WT, K27M or S31A in combination with PDGFβ in a glioma mouse model. Expression of WT H3.3 failed to generate high-grade tumors. But 66% of mice expressing K27M and 93% of those expressing H3.3 S31A developed diffuse high-grade brain tumors by 100 days. Our results suggest that loss of phospho-S31 alone is oncogenic because H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that H3.3 K27M inhibits Ser31 phosphorylation both in vitro and in vivo, leading to both chromosome missegregation and loss of subsequent G1 arrest - thus creating diffuse midline gliomas with both dynamic, complex karyotypes and epigenetic reprogramming. Citation Format: Charles Day, Florina Grigore, Alyssa Langfald, David J. Daniels, James Robinson, Edward Hinchcliffe. Inhibition of H3.3 S31 phosphorylation by the pediatric glioma driver mutation, H3.3 K27M, results in chromosomal instability, loss of p53 regulation, and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 705.

Abstract 2476: Spatiotemporal analyses of preclinical glioma models reveal ‘oncostreams’ as dynamic fascicles regulating tumor mesenchymal transformation, invasion, and malignancy

Abstract Glioblastomas multiforme (GBMs) are the most lethal tumors of the brain. Tumoral mesenchymal transformation is a hallmark of GBMs associated with alterations in cellular morphology and dynamic organization. However, little is known about the mechanisms that control this pathological process. Here, we report a comprehensive spatiotemporal study integrating novel intra-tumoral histopathological structures, ‘oncostreams’, with tumor dynamic properties, microenvironment assets and spatial molecular features. Cellular analyses of genetic engineered mouse models of glioma identified that oncostreams are heterogenous structures formed by elongated and aligned neoplastic cells enriched in non-neoplastic cells such as ACTA2+ mesenchymal like cells and CD68+ tumor associated microglia/macrophages (TAM). Deep learning analysis of H&E glioma histological samples from mouse and human gliomas identified that oncostream density correlates with tumor aggressiveness. To determine whether oncostreams fascicles are characterized by a specific gene expression profile, we performed transcriptomic analysis using laser capture microdissection coupled to RNA-sequencing. We found that oncostreams are defined by a transcriptomic signature enriched in mesenchymal genes. Network analyses identified that COL1A1 is a critical gene that regulates oncostream organization and function. Correspondingly, human and mouse high-grade gliomas with high oncostream densities showed prominent alignment of collagen fibers along these fascicles and higher COL1A1 expression compared to low-grade gliomas. To evaluate the functional role of COL1A1 in oncostream formation we generated a COL1A1-deficient GEMM of glioma. We observed that COL1A1 inhibition decreased oncostream formation, impaired tumor cell proliferation and remodeled the tumor microenvironment by diminishing CD68+ TAM cells, CD31+ endothelial vascular proliferation and ACTA2+ perivascular mesenchymal cells, thus increasing animal survival. Further studies, using time lapse confocal imaging in ex vivo glioma explants, and intravital imaging in vivo demonstrated that oncostreams are organized collective dynamic structures present at the tumor core and the invasive tumor border. Oncostreams dynamics increased the intra-tumoral spread of cells within the tumor and foster glioma aggressiveness through collective invasion of the normal brain parenchyma. The analysis of glioma invasion in COL1A1 knockdown tumors exhibited a reduction in collective migration patterns, strongly supporting its importance in tumor progression. We propose that oncostreams represent a novel pathological marker of potential value for diagnosis and COL1A1 depletion within oncostreams is a promising approach and reprogram mesenchymal transformation to reduce the tumor malignancy. Citation Format: Andrea Comba, Syed Faisal, Patrick J. Dunn, Anna E. Argento, Todd C. Hollon, Wajd N. Al-Holou, Maria L. Varela, Daniel B. Zamler, Gunnar L. Quass, Pierre F. Apostolides, Christine E. Brown, Phillip E. E. Kish, Alon Kahana, Celina G. Kleer, Sebastien Motsch, Maria G. Castro, Pedro R. Lowenstein. Spatiotemporal analyses of preclinical glioma models reveal ‘oncostreams’ as dynamic fascicles regulating tumor mesenchymal transformation, invasion, and malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2476.

Abstract 1331: Elucidating cell-to-cell communication and immunotherapy responses in deeply characterized mouse models of human glioma subtypes

Abstract Immunotherapy is a promising treatment modality for highly invasive gliomas; however, clinical trials thus far have failed to provide significant clinical benefit to most GBM patients. GBM is one of the “immune cold” tumors characterized by poor infiltration of T cells even though greater than 40% of glioma cells are composed of immune cells in some patients. The majority of immune cells in GBM are immune-suppressive myeloid cells that block T cell infiltration and/or activation. To elucidate the mechanisms of immune evasion and to understand how the immune system interacts with glioma and stromal cells that shape the immune-suppressive landscape in GBM, there is an urgent need for immune-competent preclinical models that recapitulate the human disease. Human GBM is divided into three molecular subtypes (proneural:PR, classical:CL, and mesenchymal:MES) based on specific gene expression patterns and signature mutational profiles. Here, we report multi-dimensional analyses of six different transplantable mouse glioma models in the C57BL6/J background that represent all three human GBM molecular subtypes. We performed whole-exome sequencing as well as STR fingerprinting of each primary tumorsphere line and also performed immune phenotyping of each glioma model with flow cytometry. To gain molecular insights and determine cellular heterogeneity, we also performed single-cell RNA sequencing from the six models. Glioma cell analysis at the single-cell level revealed that cell-of-origin rather than the oncogenic driver (such as EGFRviii) plays a dominant role in determining the molecular phenotypes of glioma cells, driving their classification into a molecular subtype defined by human studies. In addition, we identified eight molecular subtypes of glioma-associated myeloid (GAMs), seven different subtypes of T cells, and provide molecular definitions of glioma-associated pericytes and endothelial cells in murine gliomas. In addition, we performed cross-species comparisons of glioma and immune cell subtypes between humans and mice at the single-cell level. Furthermore, we report qualitative and quantitative differences in the cell-to-cell communication among different stromal cells and glioma cells in each model, and propose that these interactions shape the local niche and functional neighborhoods. Finally, we leverage these preclinical models to elucidate underlying molecular mechanisms that drive differential sensitivities of each model to immunotherapies: anti-PD1, CTLA4, and 4-1BB in vivo. In summary, we report deeply characterized mouse models of human GBM subtypes and highlight their utility as preclinical models for immunotherapy evaluation and foundational tumor immunology studies. Citation Format: Caiyi Wang, Jose Maldonado, Thomas D. Gallup, Nourhan Abdelfattah, Jia-Shiun Leu, Nithin Joshy, joshy George, Jihye Paik, Massimo Squatrito, Kyuson Yun. Elucidating cell-to-cell communication and immunotherapy responses in deeply characterized mouse models of human glioma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1331.

IMMU-12. Exploring and modulating the tumour immune microenvironment to facilitate the selection of immunotherapies for paediatric-type diffuse high-grade glioma

Abstract Immune cells have the potential to selectively eradicate high-risk brain tumours such as paediatric-type diffuse high-grade glioma (PDHGG). We aim to characterize the tumour immune microenvironment (TIME) of intra-cranial syngeneic mouse models of diffuse hemispheric glioma, H3G34 (DHG-H3G34) and diffuse midline glioma, H3K27 (DMG-H3K27). We also demonstrate how an oncolytic reovirus (Reolysin) can “heat-up” the TIME of our syngeneic models. Orthotopic immunocompetent mouse models of DHG-H3G34 (C57BL/6, NRASG12V + shp53 + shATRX +/- H3.3G34R) and DMG-H3K27 (Nestin-Tv-a/p53fl/fl, RCAS-ACVR1R206H + RCAS-H3.1K27M) were profiled using single-cell RNA-sequencing (scRNA-seq) (10x genomics), a 22-colour custom flow cytometry immune panel and spatial transcriptomics. Differential marker expression was validated with immunohistochemistry and immunofluorescence in tissue sections. Syngeneic mouse tumours treated systemically with Reolysin were also profiled to evaluate the effects of the oncolytic virus on the TIME. Cell type predictions in scRNA-seq using singleR, ssGSEA and expression of individual marker genes suggested that the predominant immune cell types within hemispheric tumours were monocytes (11-21%) and macrophages (10-19%) with much smaller proportions of CD4+ and CD8+ T-cells (4-10%). By contrast, much smaller proportions of monocytes (2%) and macrophages (3%) were observed in the H3.1K27M pontine model. Flow cytometry, immunohistochemistry and immunofluorescence validated scRNA-seq immune profiles and characterised signalling of the PD-1/PD-L1 checkpoint pathway. Spatial transcriptomics allowed immune cell populations to be positioned within tumour sections and showed significant co-localization of CD4+ and CD8+ lymphocytes at tumour margins. Treatment of syngeneic mouse tumours with Reolysin resulted in reduced tumour volumes and altered the TIME, in particular increasing cytotoxic T-cell tumour infiltration. Our results highlight immunological heterogeneity within molecular subgroups of PDHGG and demonstrate ability of a systemically delivered oncolytic virus, Reolysin, to “heat-up” the TIME, contributing to a more immune actionable profile. Future work will help to identify optimal combinations for the next generation of immunotherapies in PDHGG.

Locally secreted BiTEs complement CAR T cells by enhancing killing of antigen heterogeneous solid tumors

Bispecific Tcell engagers (BiTEs) are bispecific antibodies that redirect Tcells to target antigen-expressing tumors. We hypothesized that BiTE-secreting Tcells could be a valuable therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) Tcells. Glioblastomas represent a good model for solidtumor heterogeneity, representing a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III, and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the single chain variable fragments to generate new BiTE molecules. Compared with CAR Tcells, BiTE Tcells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE-secreting bivalent Tcells compared with bivalent CAR Tcells in a glioma mouse model at early phase, but not in the long term. In summary, BiTEs secreted by mono- or multi-valent Tcells have potent anti-tumor activity invitro and invivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

A novel mouse model of diffuse midline glioma initiated in neonatal oligodendrocyte progenitor cells highlights cell-of-origin dependent effects of H3K27M.

Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells‐of‐origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs. Here, we used the RCAS/Tv‐a avian retroviral system to generate DMG in Olig2‐expressing progenitors and Nestin‐expressing progenitors in the neonatal mouse brainstem. PDGF‐A or PDGF‐B overexpression, along with p53 deletion, resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M‐positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF‐A and PDGF‐B‐driven models, suggesting that the requirement for H3.3K27M is reduced when tumorigenesis is initiated in Olig2+ cells. RNA‐sequencing analysis revealed that the differentially expressed genes in H3.3K27M tumors were non‐overlapping between Olig2;PDGF‐B, Olig2;PDGF‐A, and Nestin;PDGF‐A models. GSEA analysis of PDGFA tumors confirmed that the transcriptomal effects of H3.3K27M are cell‐of‐origin dependent with H3.3K27M promoting epithelial‐to‐mesenchymal transition (EMT) and angiogenesis when Olig2 marks the cell‐of‐origin and inhibiting EMT and angiogenesis when Nestin marks the cell‐of‐origin. We did observe some overlap with H3.3K27M promoting negative enrichment of TNFA_Signaling_Via_NFKB in both models. Our study suggests that the tumorigenic effects of H3.3K27M are cell‐of‐origin dependent, with H3.3K27M being more oncogenic in Nestin+ cells than Olig2+ cells.

Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma

BackgroundLong-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive...

Tryptophan hydroxylase 1 drives glioma progression by modulating the serotonin/L1CAM/NF-\u03baB signaling pathway

BackgroundGlioma is one of the main causes of cancer-related mortality worldwide and is associated with high heterogeneity. However, the key players determining the fate of glioma remain obscure. In the present study, we shed light on tumor metabolism and aimed to investigate the role of tryptophan hydroxylase 1 (TPH-1) in the advancement of glioma.MethodHerein, the levels of TPH-1 expression in glioma tissues were evaluated using The Cancer Genome Atlas (TCGA) database. Further, the proliferative characteristics and migration ability of TPH-1 overexpressing LN229/T98G cells were evaluated. Additionally, we performed a cytotoxicity analysis using temozolomide (TMZ) in these cells. We also examined the tumor growth and survival time in a mouse model of glioma treated with chemotherapeutic agents and a TPH-1 inhibitor.ResultsThe results of both clinical and experimental data showed that excess TPH-1 expression resulted in sustained glioma progression and a dismal overall survival in these patients. Mechanistically, TPH-1 increased the production of serotonin in glioma cells. The elevated serotonin levels then augmented the NF-κB signaling pathway through the upregulation of the L1-cell adhesion molecule (L1CAM), thereby contributing to cellular proliferation, invasive migration, and drug resistance. In vivo experiments demonstrated potent antitumor effects, which benefited further from the synergistic combination of TMZ and LX-1031.ConclusionTaken together, these data suggested that TPH-1 facilitated cellular proliferation, migration, and chemoresistance in glioma through the serotonin/L1CAM/NF-κB pathway. By demonstrating the link of amino acid metabolic enzymes with tumor development, our findings may provide a potentially viable target for therapeutic manipulation aimed at eradicating glioma.

Sevoflurane inhibits the malignant phenotypes of glioma through regulating miR-146b-5p/NFIB axis.

Sevoflurane is a common used inhaled anesthetic that was reported to regulate the progression of multiple cancers. Here, we aimed to investigate the function and regulatory mechanism underlying sevoflurane in glioma cells. A172 and U251 cells were treated with different concentrations of sevoflurane. Colony formation, EdU satining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and transwell assays were performed to evaluate cell proliferation, apoptosis, migration and invasion, respectively. Circ_VCAN, microRNA-146b-5p (miR-146b-5p) and nuclear factor I B (NFIB) expression levels were assessed by real-time quantitative PCR (RT-qPCR) or western blot. Bioinformatics analysis and dual-luciferase reporter assay were applied to evaluate the correlation between miR-146b-5p and circ_VCAN or NFIB. A xenograft glioma mice model was established to verify the effect of sevoflurane on tumor growth in vivo. Sevoflurane (Sev) inhibited proliferation, migration, invasion, and elevated apoptosis of A172 and U251 cells. Sevoflurane treatment inhibited the expression of circ_VCAN and NFIB, but elevated the expression of miR-146b-5p in glioma cells. Overexpression of circ_VCAN alleviated the inhibition effects of sevoflurane on the malignant phenotypes of glioma in vitro and in vivo. Besides, miR-146b-5p is a target of circ_VCAN and negatively regulated NFIB expression. Overexpression of miR-146b-5p partly reversed the effects of circ_VCAN in Sev-treated glioma cells. Furthermore, miR-146b-5p deletion enhanced glioma progression in sevoflurane treated glioma cells by targeting NFIB. Moreover, circ_VCAN could upregulate NFIB expression by sponging miR-146b-5p in Sev-treated glioma cells. Sevoflurane alleviated proliferation, migration and invasion, but enhanced apoptosis of glioma cells through regulating circ_VCAN/miR-146b-5p/NFIB axis.

Microglia/macrophage-derived human CCL18 promotes glioma progression via CCR8-ACP5 axis analyzed in humanized slice model.

Factors released from glioma-associated microglia/macrophages (GAMs) play a crucial role in glioblastoma multiforme (GBM) progression. Here, we study the importance of CCL18, a cytokine expressed in human but not in rodent GAMs, as a modulator of glioma growth. Since CCL18 signaling could not be studied in classical mouse glioma models, we developed an approach by transplanting induced pluripotent stem cell-derived human microglia and human glioma cells into mouse brain slices depleted of their intrinsic microglia. We observe that CCL18 promotes glioma cell growth and invasion. Chemokine (C-C motif) receptor 8 (CCR8) is identified as a functional receptor for CCL18 on glioma cells, and ACP5 (acid phosphatase 5) is revealed as an important part of the downstream signaling cascade for mediating glioma growth. We conclude, based on the results from an invitro, exvivo humanized glioma model and an invivo GBM model that microglia/macrophage-derived CCL18 promotes glioma growth.

Bone Marrow Mesenchymal Stem Cell (BMSC)-Exosomes Overexpressing miR-141 Inhibit the Malignant Biological Behavior of Glioma Cells via Wnt Signaling

Our study explores whether BMSC-exosomes overexpressing miR-141 can regulate Wnt signal to inhibit the malignant biological behavior of glioma cells. Thirty healthy mice were selected to construct a glioma mouse model and assigned randomly into the control group, miR-141 NC group, and miR-141 mimic group followed by analysis of cell proliferation, apoptosis, protein expression and mRNA expression by MTT method, flow cytometry, Western blot and RT-PCR methods. Compared with the other two groups, miR-141 mimic group showed reduced number of cell proliferation at 24 h and 48 h, decreased cell migration and invasion ability, and the increased cell apoptosis rate (P < 0.05). In miR-141 mimic group, the protein expression of miR-141 was the highest, while the protein expression of β-catenin, survivin and c-myc was the lowest (P < 0.05). In conclusion, BMSC-exosomes overexpressing miR-141 can inhibit the malignant biological behavior of GC cells possibly by inhibiting the activation of Wnt signaling pathway.

Brain-targeting biomimetic nanoparticles for codelivery of celastrol and LY2157299 for reversing glioma immunosuppression

The treatment of glioblastoma remains a huge challenge due to the lack of an efficient way to deliver drugs across the blood–brain barrier (BBB), and the pharmacotherapy options are very limited. In this work, a biomimetic BBB-penetrating albumin nanosystem modified by a brain-targeting peptide was designed for co-delivering a TGF-β receptor I (TGFβRI) inhibitor (LY2157299) and an mTOR inhibitor (celastrol). The albumin nanosystem can target nAChRs overexpressed both on the BBB and glioma cells, thereby promoting drug delivery into the glioma. The biomimetic nanoparticles could repolarize tumor-associated macrophages (TAMs) from M2 to M1 phenotype by suppressing the STAT6 pathway, thereby reducing TGF-β1 secretion and inducing cell apoptosis. In addition, the treatment also blocked TGF-β/SMAD2 signaling pathway. The glioma-targeting ability and therapeutic efficacy were confirmed in an orthotopic glioma mouse model. The biomimetic nanoparticles significantly prolonged the survival rate, showing a decrease in the proportion of M2-like TAMs and the levels of TGF-β1 and lactic acid in the glioma tissues. This delivery and treatment strategy provides a new approach for the treatment of gliomas.

Late-Stage Glioma Is Associated with Deleterious Alteration of Gut Bacterial Metabolites in Mice.

Brain-gut axis refers to the bidirectional functional connection between the brain and the gut, which sustains vital functions for vertebrates. This connection also underlies the gastrointestinal (GI) comorbidities associated with brain disorders. Using a mouse model of glioma, based on the orthotopic injection of GL261 cell line in syngeneic C57BL6 mice, we show that late-stage glioma is associated with GI functional alteration and with a shift in the level of some bacterial metabolites in the cecum. By performing cecal content transfer experiments, we further show that cancer-associated alteration in cecal metabolites is involved in end-stage disease progression. Antibiotic treatment results in a slight but significant delay in mice death and a shift in the proportion of myeloid cells in the brain tumor environment. This work rationally considers microbiota modulating strategies in the clinical management of patients with late-stage glioma.

Short TE downfield magnetic resonance spectroscopy in a mouse model of brain glioma.

Enhanced cell proliferation in tumors can be associated with altered metabolic profiles and dramatic microenvironmental changes. Downfield magnetic resonance spectroscopy (MRS) has received increasing attention due to its ability to report on labile resonances of molecules not easily detected in upfield 1 H MRS. Image-selected-in-vivo-spectroscopy-relaxation enhanced MRS (iRE-MRS) was recently introduced for acquiring short echo-time (TE) spectra. Here, iRE-MRS was used to investigate in-vivo downfield spectra in glioma-bearing mice. Experiments were performed in vivo in an immunocompetent glioma mouse model at 9.4T using a cryogenic coil. iRE-MRS spectra were acquired in N=6 glioma-bearing mice (voxel size=2.23 mm3 ) and N=6 control mice. Spectra were modeled by a sum of Lorentzian peaks simulating known downfield resonances, and differences between controls and tumors were quantified using relative peak areas. Short TE tumor spectra exhibited large qualitative differences compared to control spectra. Most peaks appeared modulated, with strong attenuation of NAA (∼7.82, 7.86ppm) and changes in relative peak areas between 6.75 and 8.49ppm. Peak areas tended to be smaller for DF6.83 , DF7.60 , DF8.18 and NAA; and larger for DF7.95 and DF8.24 . Differences were also detected in signals resonating above 8.5ppm, assumed to arise from NAD+. In-vivo downfield 1 H iRE-MRS of mouse glioma revealed differences between controls and tumor bearing mice, including in metabolites which are not easily detectable in the more commonly investigated upfield spectrum. These findings motivate future downfield MRS investigations exploring pH and exchange contributions to these differences.

Hypoxia-induced GLT8D1 promotes glioma stem cell maintenance by inhibiting CD133 degradation through N-linked glycosylation.

Gliomas are the most aggressive primary brain tumors. However, no significant improvement in survival has been achieved with the addition of temozolomide (TMZ) or radiation as initial therapy, although many clinical efforts have been carried out to target various signaling pathways or putative driver mutations. Here, we report that glycosyltransferase 8 domain containing 1 (GLT8D1), induced by HIF-1α under a hypoxic niche, significantly correlates with a higher grade of glioma, and a worse clinical outcome. Depletion of GLT8D1 inhibits self-renewal of glioma stem cell (GSC) in vitro and represses tumor growth in glioma mouse models. GLT8D1 knockdown promotes cell cycle arrest at G2/M phase and cellular apoptosis with or without TMZ treatment. We reveal that GLT8D1 impedes CD133 degradation through the endosomal-lysosomal pathway by N-linked glycosylation and protein-protein interaction. Directly blocking the GLT8D1/CD133 complex formation by CD133N1~108 (referred to as FECD133), or inhibiting GLT8D1 expression by lercanidipine, suppresses Wnt/β-catenin signaling dependent tumorigenesis both in vitro and in patient-derived xenografts mouse model. Collectively, these findings offer mechanistic insights into how hypoxia promotes GLT8D1/CD133/Wnt/β-catenin signaling during glioma progression, and identify GLT8D1 as a potential therapeutic target in the future.

Bone Marrow Mesenchymal Stem Cells (BMSC)-Derived MicroRNA-189 Inhibits Glioma Tumorigenesis Through Suppressing Tumor Necrosis Factor-α (TNF-α)-Mediated Nuclear Factor Kappa Light Chain Enhancer of Activated B Cells (NF-κB) Signaling Pathway

This study aims at investigating the mechanism underlying bone marrow mesenchymal stem cells (BMSC) function in glioma. Glioma cells were administered with plasmids loading NF-κB siRNA, microRNA (miRNA)-189 inhibitor, or miR-189 mimics for transfection followed by analysis of miR-189 expression by RT-qPCR, cell apoptosis by flow cytometry, cell proliferation by MTT assay,invasion and migration by Transwell assay, inflammatory factors secretion by ELISA as well as proteins expression by western blot. A mouse model of glioma was established to detect the in vivo effect of BMSCs. miR-189 was lowly expressed in glioma cell lines but enriched in BMSCs. When miR-189 was silenced, cell proliferation, invasion and migration were potentiated and apoptosis was decreased, along with enhancement of N-cadherin, Vimentin, MMP-2 and and MMP-9, and decline in Bax, cleaved casepase-3 and cleaved PARP. Silencing of NF-κB reversed the effect of miR-189 inhibitor on cell progression, accompanied with reduction of inflammatory factors. BMSCs treatment effectively promoted miR-189 expression in glioma and inactivated TNF-α/NF-κB signaling, thereby suppressing tumor growth. In conclusion, miR-189 derived from BMSC inhibits glioma progression through regulation of TNF-α/NF-κB signaling pathway.