Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Calinescu, Anda-Alexandra; Núñez, Felipe J.; Koschmann, Carl; Kolb, Bradley; Löwenstein, Pedro R.; Castro, Maria G.

doi:10.3791/52443

Cited by 34 publications

(81 citation statements)

References 26 publications

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“…M7 and OL61 neurospheres were derived from sleeping beauty transposase system-induced tumors in wild-type mice. 44,45 While M7 tumors were generated using NRASG12V and SV40-large T antigen plasmids, OL61 tumors contained NRASG12V, short hairpin (sh)-targeting p53, and platelet-derived growth factor b (PDGFb) overexpression. 44 Almost 80% of the CD45 high compartment was comprised of MDSCs ( Figure S1A).…”

Section: Malignant Brain Tumors (Gliomasmentioning

confidence: 99%

“…44 Neurospheres were derived from tumors generated in C57BL/6 mice using the Sleeping Beauty (SB) transposase system. 44,45 Tumor tissue was dissociated using HyQTase (HyClone), passed through a 70-mm cell strainer, and cultured in media containing DMEM-F12, N2, and B27 supplements, 1% penicillin-streptomycin (Invitrogen), Normocin (Invivogen), and supplemented with 20 ng/mL EGF and 20 ng/mL FGF (Peprotech). Growth factors were added every 2 to 3 days, and cells were passaged once to twice each week, depending on density.…”

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confidence: 99%

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Immunosuppressive Myeloid Cells’ Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy

et al. 2017

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Survival of glioma (GBM) patients treated with the current standard of care remains dismal. Immunotherapeutic approaches that harness the cytotoxic and memory potential of the host immune system have shown great benefit in other cancers. GBMs have developed multiple strategies, including the accumulation of myeloid-derived suppressor cells (MDSCs) to induce immunosuppression. It is therefore imperative to develop multipronged approaches when aiming to generate a robust anti-tumor immune response. Herein, we tested whether combining MDSC depletion or checkpoint blockade would augment the efficacy of immune-stimulatory herpes simplex type-I thymidine kinase (TK) plus Fms-like tyrosine kinase ligand (Flt3L)-mediated immune stimulatory gene therapy. Our results show that MDSCs constitute >40% of the tumorinfiltrating immune cells. These cells express IL-4Ra, inducible nitric oxide synthase (iNOS), arginase, programmed death ligand 1 (PDL1), and CD80, molecules that are critically involved in antigen-specific T cell suppression. Depletion of MDSCs strongly enhanced the TK/Flt3L gene therapy-induced tumor-specific CD8 T cell response, which lead to increased median survival and percentage of long-term survivors. Also, combining PDL1 or CTLA-4 immune checkpoint blockade greatly improved the efficacy of TK/Flt3L gene therapy. Our results, therefore, indicate that blocking MDSC-mediated immunosuppression holds great promise for increasing the efficacy of gene therapy-mediated immunotherapies for GBM.

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Section: Malignant Brain Tumors (Gliomasmentioning

confidence: 99%

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confidence: 99%

Immunosuppressive Myeloid Cells’ Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy

et al. 2017

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“…NPC-derived tumors were generated and characterized by histology and immunohistochemistry as previously described (31) and elaborated in the Supplementary Methods. In vivo treatment with AMD3100 is detailed in the Supplementary Methods.…”

Section: Methodsmentioning

confidence: 99%

Survival and Proliferation of Neural Progenitor–Derived Glioblastomas Under Hypoxic Stress is Controlled by a CXCL12/CXCR4 Autocrine-Positive Feedback Mechanism

Calinescu

Yadav

Carballo

et al. 2017

Clinical Cancer Research

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Purpose One likely cause of treatment failure in glioblastoma is the persistence of glioma stem-like cells (GSLCs) which are highly resistant to therapies currently employed. We found that CXCL12 has highest expression in glioma cells derived from neural progenitor cells (NPCs). The development and molecular signature of NPC derived GBMs were analyzed and the therapeutic effect of blocking CXCL12 was tested. Experimental Design Tumors were induced by injecting DNA into the lateral ventricle of neonatal mice, using the Sleeping Beauty transposase method. Histology and expression of GSLC markers were analyzed during disease progression. Survival upon treatment with pharmacologic (Plerixafor) or genetic inhibition of CXCR4 was analyzed. Primary neurospheres were generated and analyzed for proliferation, apoptosis and expression of proteins regulating survival and cell cycle progression. Results Tumors induced from NPCs display histological features of human GBM and express markers of GSLC. In vivo, inhibiting the CXCL12/CXCR4 signaling axis results in increased survival of tumor-bearing animals. In vitro, CXCR4 blockade induces apoptosis and inhibits cell cycle progression, downregulates molecules regulating survival and proliferation and also blocks the hypoxic-induction of HIF-1α and CXCL12. Exogenous administration of CXCL12 rescues the drug-induced decrease in proliferation. Conclusions This study demonstrates that the CXCL12/CXCR4 axis operates in GBM cells under hypoxic stress via an autocrine positive feedback mechanism, which promotes survival and cell cycle progression. Our study brings new mechanistic insight and encourages further exploration of the use of drugs blocking CXCL12 as adjuvant agents to target hypoxia-induced GBM progression, prevent resistance to treatment and recurrence of the disease.

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“…Induce the brain tumor in mouse using the Sleeping Beauty transposon system in which plasmids encoding oncogenes or shRNA targeting tumor suppressors is injected into the lateral ventricles of neonates to generate spontaneous brain tumors 9 . Select a mouse with a large tumor, which is confirmed with bioluminescence imaging.…”

Section: Protocolmentioning

confidence: 99%

“…NOTE: More detailed information about the Sleeping Beauty Transposon System and plasmid constructs used can be found in Calinescu et al 9 .…”

Section: Protocolmentioning

confidence: 99%

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Mendez

Núñez

Zorrilla-Veloz

et al. 2018

JoVE

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Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

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Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Cited by 34 publications

References 26 publications

Immunosuppressive Myeloid Cells’ Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy

Immunosuppressive Myeloid Cells’ Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy

Survival and Proliferation of Neural Progenitor–Derived Glioblastomas Under Hypoxic Stress is Controlled by a CXCL12/CXCR4 Autocrine-Positive Feedback Mechanism

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

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