Publications by authors named "Maria G Castro"

155 Publications

Current Approaches for Glioma Gene Therapy and Virotherapy.

Front Mol Neurosci 2021 11;14:621831. Epub 2021 Mar 11.

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in the adult population and it carries a dismal prognosis. Inefficient drug delivery across the blood brain barrier (BBB), an immunosuppressive tumor microenvironment (TME) and development of drug resistance are key barriers to successful glioma treatment. Since gliomas occur through sequential acquisition of genetic alterations, gene therapy, which enables to modification of the genetic make-up of target cells, appears to be a promising approach to overcome the obstacles encountered by current therapeutic strategies. Gene therapy is a rapidly evolving field with the ultimate goal of achieving specific delivery of therapeutic molecules using either viral or non-viral delivery vehicles. Gene therapy can also be used to enhance immune responses to tumor antigens, reprogram the TME aiming at blocking glioma-mediated immunosuppression and normalize angiogenesis. Nano-particles-mediated gene therapy is currently being developed to overcome the BBB for glioma treatment. Another approach to enhance the anti-glioma efficacy is the implementation of viro-immunotherapy using oncolytic viruses, which are immunogenic. Oncolytic viruses kill tumor cells due to cancer cell-specific viral replication, and can also initiate an anti-tumor immunity. However, concerns still remain related to off target effects, and therapeutic and transduction efficiency. In this review, we describe the rationale and strategies as well as advantages and disadvantages of current gene therapy approaches against gliomas in clinical and preclinical studies. This includes different delivery systems comprising of viral, and non-viral delivery platforms along with suicide/prodrug, oncolytic, cytokine, and tumor suppressor-mediated gene therapy approaches. In addition, advances in glioma treatment through BBB-disruptive gene therapy and anti-EGFRvIII/VEGFR gene therapy are also discussed. Finally, we discuss the results of gene therapy-mediated human clinical trials for gliomas. In summary, we highlight the progress, prospects and remaining challenges of gene therapies aiming at broadening our understanding and highlighting the therapeutic arsenal for GBM.
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http://dx.doi.org/10.3389/fnmol.2021.621831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006286PMC
March 2021

Immune-stimulatory (TK/Flt3L) gene therapy opens the door to a promising new treatment strategy against brainstem gliomas.

Oncotarget 2020 Dec 15;11(50):4607-4612. Epub 2020 Dec 15.

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Diffuse intrinsic pontine glioma (DIPG) is a rare brainstem tumor which carries a dismal prognosis. To date. there are no effective treatments for DIPG. Transcriptomic studies have shown that DIPGs have a distinct profile compared to hemispheric high-grade pediatric gliomas. These specific genomic features coupled with the younger median age group suggest that DIPG is of developmental origin. There is a major unmet need for novel effective therapeutic approaches for DIPG. Clinical and preclinical studies have expanded our understanding of the molecular pathways in this deadly disease. We have developed a genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, activin A receptor type I (ACVR1)-G328V (mACVR1) using the sleeping beauty transposon system. DIPG neurospheres isolated from the genetically engineered mouse model were implanted into the pons of immune-competent mice to assess the therapeutic efficacy and toxicity of immunostimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). Immunostimulatory adenoviral-mediated delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and improved median survival by stimulating the host antitumor immune response. Therapeutic efficacy of the immunostimulatory gene therapy strategy will be tested in the clinical arena in a Phase I clinical trial. We also discuss immunotherapeutic interventions currently being implemented in DIPG patients and discuss the profound therapeutic implications of immunotherapy for this patient populations.
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http://dx.doi.org/10.18632/oncotarget.27834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7747859PMC
December 2020

Blocking NHE1 stimulates glioma tumor immunity by restoring OXPHOS function of myeloid cells.

Theranostics 2021 1;11(3):1295-1309. Epub 2021 Jan 1.

Department of Neurology and Pittsburgh Institute for Neurodegenerative Disease, University of Pittsburgh, Pittsburgh, PA 15213.

Immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) is one of the contributing factors for failed immunotherapies. Therefore, there is an urgent need to better understand TME and to identify novel modulators of TME for more effective GBM therapies. We hypothesized that H extrusion protein Na/H exchanger 1 (NHE1) plays a role in dysregulation of glucose metabolism and immunosuppression of GBM. We investigated the efficacy of blockade of NHE1 activity in combination with temozolomide (TMZ) therapy in increasing anti-tumor immunity. Mouse syngeneic intracranial glioma model was used to test four treatment regimens: DMSO (Vehicle-control), TMZ, NHE1 specific inhibitor HOE642, or TMZ+HOE642 (T+H) combination. H/Fluorine magnetic resonance imaging (MRI) with cell tracking agent Vsense was performed to monitor the infiltration of glioma-associated microglia/myeloid cells (GAMs). Glucose metabolism and transcriptome profiles were analyzed by Seahorse analyzer and bulk RNA-sequencing. The impact of selective deletion in GAMs on sensitivity to anti-PD-1 therapy was evaluated in transgenic knockout () mice. Among the tested treatment regimens, the T+H combination therapy significantly stimulated the infiltration of GAMs and T-cells; up-regulated Th1 activation, and mitochondrial oxidative phosphorylation (OXPHOS) pathway genes, increased glucose uptake and mitochondrial mass, and decreased aerobic glycolysis in GAMs. Selective deletion of in Cx3cr1 mice increased anti-tumor immunity and sensitivity to TMZ plus anti-PD-1 combinatorial therapy. NHE1 plays a role in developing glioma immunosuppressive TME in part by dysregulating glucose metabolism of GAMs and emerges as a therapeutic target for improving glioma immunity.
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http://dx.doi.org/10.7150/thno.50150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738877PMC
January 2021

Genetically Engineered Mouse Model of Brainstem High-Grade Glioma.

STAR Protoc 2020 Dec 25;1(3):100165. Epub 2020 Nov 25.

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Brainstem gliomas are aggressive tumors that are more prevalent in pediatric patients. The location of these tumors makes them inoperable, and currently there is no effective treatment. Recent genomic data revealed the unique biology of these tumors. The following protocol provides a method to incorporate these specific genetic lesions in a mouse glioma model. Using this model, the effects of these mutations in tumor progression and response to treatments can be studied within a relevant context. For complete details on the use and execution of this protocol, please refer to Mendez et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757359PMC
December 2020

Hemispherical Pediatric High-Grade Glioma: Molecular Basis and Therapeutic Opportunities.

Int J Mol Sci 2020 Dec 17;21(24). Epub 2020 Dec 17.

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

In this review, we discuss the molecular characteristics, development, evolution, and therapeutic perspectives for pediatric high-grade glioma (pHGG) arising in cerebral hemispheres. Recently, the understanding of biology of pHGG experienced a revolution with discoveries arising from genomic and epigenomic high-throughput profiling techniques. These findings led to identification of prevalent molecular alterations in pHGG and revealed a strong connection between epigenetic dysregulation and pHGG development. Although we are only beginning to unravel the molecular biology underlying pHGG, there is a desperate need to develop therapies that would improve the outcome of pHGG patients, as current therapies do not elicit significant improvement in median survival for this patient population. We explore the molecular and cell biology and clinical state-of-the-art of pediatric high-grade gliomas (pHGGs) arising in cerebral hemispheres. We discuss the role of driving mutations, with a special consideration of the role of epigenetic-disrupting mutations. We will also discuss the possibilities of targeting unique molecular vulnerabilities of hemispherical pHGG to design innovative tailored therapies.
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http://dx.doi.org/10.3390/ijms21249654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766684PMC
December 2020

Inhibition of 2-hydroxyglutarate elicits metabolic reprogramming and mutant IDH1 glioma immunity in mice.

J Clin Invest 2021 Feb;131(4)

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Mutant isocitrate dehydrogenase 1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into 2 molecular subgroups: 1p/19q codeletion/TERT-promoter mutations or inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work focuses on glioma subtypes harboring mIDH1, TP53, and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts α-ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of D-2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma-bearing mice. Also, D-2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to D-2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in WT-IDH gliomas. Thus, we combined D-2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint blockade and observed complete tumor regression in 60% of mIDH1 glioma-bearing mice. This combination strategy reduced T cell exhaustion and favored the generation of memory CD8+ T cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data support the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients.
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http://dx.doi.org/10.1172/JCI139542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880418PMC
February 2021

Systemic brain tumor delivery of synthetic protein nanoparticles for glioblastoma therapy.

Nat Commun 2020 11 10;11(1):5687. Epub 2020 Nov 10.

Biointerfaces Institute, University of Michigan, 2800 Plymouth Road, Ann Arbor, MI, 48109, USA.

Glioblastoma (GBM), the most aggressive form of brain cancer, has witnessed very little clinical progress over the last decades, in part, due to the absence of effective drug delivery strategies. Intravenous injection is the least invasive drug delivery route to the brain, but has been severely limited by the blood-brain barrier (BBB). Inspired by the capacity of natural proteins and viral particulates to cross the BBB, we engineered a synthetic protein nanoparticle (SPNP) based on polymerized human serum albumin (HSA) equipped with the cell-penetrating peptide iRGD. SPNPs containing siRNA against Signal Transducer and Activation of Transcription 3 factor (STAT3i) result in in vitro and in vivo downregulation of STAT3, a central hub associated with GBM progression. When combined with the standard of care, ionized radiation, STAT3i SPNPs result in tumor regression and long-term survival in 87.5% of GBM-bearing mice and prime the immune system to develop anti-GBM immunological memory.
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http://dx.doi.org/10.1038/s41467-020-19225-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655867PMC
November 2020

An Optimized Protocol for In Vivo Analysis of Tumor Cell Division in a Sleeping Beauty-Mediated Mouse Glioma Model.

STAR Protoc 2020 Sep 6;1(2). Epub 2020 Jun 6.

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Malignant gliomas are the most common and aggressive primary brain tumor in adults, and high mitotic rates are associated with their malignancy. Gliomas were modeled in mice using the Sleeping Beauty system to encode genetic lesions recapitulating the human disease. The presented workflow allows the study of the proliferation of glioma cells , enabling the identification of different phases of the cell cycle, with the advantage that 5-ethynyl-2'-deoxyuridine staining does not involve denaturation steps and samples do not require histological processing. For complete details on the use and execution of this protocol, please refer to Núñez et al. (2019).
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http://dx.doi.org/10.1016/j.xpro.2020.100044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518519PMC
September 2020

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography.

J Vis Exp 2020 08 16(162). Epub 2020 Aug 16.

Department of Internal Medicine, University of Michigan Ann Arbor; Rogel Cancer Center, University of Michigan Ann Arbor;

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (µmBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model µmBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.
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http://dx.doi.org/10.3791/61654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831113PMC
August 2020

Glioblastoma Utilizes Fatty Acids and Ketone Bodies for Growth Allowing Progression during Ketogenic Diet Therapy.

iScience 2020 Aug 13;23(9):101453. Epub 2020 Aug 13.

Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA; Department of Psychiatry and Biobehavioral Sciences and Semel Institute for Neuroscience & Human Behavior, UCLA, Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA, USA. Electronic address:

Glioblastoma (GBM) metabolism has traditionally been characterized by a primary dependence on aerobic glycolysis, prompting the use of the ketogenic diet (KD) as a potential therapy. In this study we evaluated the effectiveness of the KD in GBM and assessed the role of fatty acid oxidation (FAO) in promoting GBM propagation. In vitro assays revealed FA utilization throughout the GBM metabolome and growth inhibition in nearly every cell line in a broad spectrum of patient-derived glioma cells treated with FAO inhibitors. In vivo assessments revealed that knockdown of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme for FAO, reduced the rate of tumor growth and increased survival. However, the unrestricted ketogenic diet did not reduce tumor growth and for some models significantly reduced survival. Altogether, these data highlight important roles for FA and ketone body metabolism that could serve to improve targeted therapies in GBM.
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http://dx.doi.org/10.1016/j.isci.2020.101453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471621PMC
August 2020

Purine metabolism regulates DNA repair and therapy resistance in glioblastoma.

Nat Commun 2020 07 30;11(1):3811. Epub 2020 Jul 30.

Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, 48109, USA.

Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming therapy resistance. Treatments that are effective independent of genotype are urgently needed. By correlating intracellular metabolite levels with radiation resistance across dozens of genomically-distinct models of GBM, we find that purine metabolites, especially guanylates, strongly correlate with radiation resistance. Inhibiting GTP synthesis radiosensitizes GBM cells and patient-derived neurospheres by impairing DNA repair. Likewise, administration of exogenous purine nucleosides protects sensitive GBM models from radiation by promoting DNA repair. Neither modulating pyrimidine metabolism nor purine salvage has similar effects. An FDA-approved inhibitor of GTP synthesis potentiates the effects of radiation in flank and orthotopic patient-derived xenograft models of GBM. High expression of the rate-limiting enzyme of de novo GTP synthesis is associated with shorter survival in GBM patients. These findings indicate that inhibiting purine synthesis may be a promising strategy to overcome therapy resistance in this genomically heterogeneous disease.
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http://dx.doi.org/10.1038/s41467-020-17512-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393131PMC
July 2020

Tumor mutational burden predicts survival in patients with low-grade gliomas expressing mutated IDH1.

Neurooncol Adv 2020 Jan-Dec;2(1):vdaa042. Epub 2020 Mar 27.

Department of Neurosurgery, University of Michigan Medical School, MSRB II, Ann Arbor, Michigan, USA.

Background: Gliomas are the most common primary brain tumors. High-Grade Gliomas have a median survival (MS) of 18 months, while Low-Grade Gliomas (LGGs) have an MS of approximately 7.3 years. Seventy-six percent of patients with LGG express mutated isocitrate dehydrogenase (mIDH) enzyme. Survival of these patients ranges from 1 to 15 years, and tumor mutational burden ranges from 0.28 to 3.85 somatic mutations/megabase per tumor. We tested the hypothesis that the tumor mutational burden would predict the survival of patients with tumors bearing mIDH.

Methods: We analyzed the effect of tumor mutational burden on patients' survival using clinical and genomic data of 1199 glioma patients from The Cancer Genome Atlas and validated our results using the Glioma Longitudinal AnalySiS consortium.

Results: High tumor mutational burden negatively correlates with the survival of patients with LGG harboring mIDH ( = .005). This effect was significant for both Oligodendroglioma (LGG-IDH-; MS = 2379 vs 4459 days in high vs low, respectively; = .005) and Astrocytoma (LGG-IDH-; MS = 2286 vs 4412 days in high vs low respectively; = .005). There was no differential representation of frequently mutated genes (eg, , , , and ) in either group. Gene set enrichment analysis revealed an enrichment in Gene Ontologies related to cell cycle, DNA-damage response in high versus low tumor mutational burden. Finally, we identified 6 gene sets that predict survival for LGG-IDH- and LGG-IDH-.

Conclusions: we demonstrate that tumor mutational burden is a powerful, robust, and clinically relevant prognostic factor of MS in mIDH patients.
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http://dx.doi.org/10.1093/noajnl/vdaa042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212865PMC
March 2020

Synthetic High-density Lipoprotein Nanodiscs for Personalized Immunotherapy Against Gliomas.

Clin Cancer Res 2020 08 21;26(16):4369-4380. Epub 2020 May 21.

Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan.

Purpose: Gliomas are brain tumors with dismal prognoses. The standard-of-care treatments for gliomas include surgical resection, radiation, and temozolomide administration; however, they have been ineffective in providing significant increases in median survival. Antigen-specific cancer vaccines and immune checkpoint blockade may provide promising immunotherapeutic approaches for gliomas.

Experimental Design: We have developed immunotherapy delivery vehicles based on synthetic high-density lipoprotein (sHDL) loaded with CpG, a Toll-like receptor-9 agonist, and tumor-specific neoantigens to target gliomas and elicit immune-mediated tumor regression.

Results: We demonstrate that vaccination with neoantigen peptide-sHDL/CpG cocktail in combination with anti-PD-L1 immune checkpoint blocker elicits robust neoantigen-specific T-cell responses against GL261 cells and eliminated established orthotopic GL261 glioma in 33% of mice. Mice remained tumor free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of immunologic memory. Moreover, in a genetically engineered murine model of orthotopic mutant IDH1 (mIDH1) glioma, sHDL vaccination with mIDH1 neoantigen eliminated glioma in 30% of animals and significantly extended the animal survival, demonstrating the versatility of our approach in multiple glioma models.

Conclusions: Overall, our strategy provides a general roadmap for combination immunotherapy against gliomas and other cancer types.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-0341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442596PMC
August 2020

Immunotherapy for gliomas: shedding light on progress in preclinical and clinical development.

Expert Opin Investig Drugs 2020 Jul 4;29(7):659-684. Epub 2020 Jun 4.

Department of Neurosurgery, University of Michigan Medical School , Ann Arbor, MI, USA.

Introduction: Gliomas are infiltrating brain tumors associated with high morbidity and mortality. Current standard of care includes radiation, chemotherapy, and surgical resection. Today, survival rates for malignant glioma patients remain dismal and unchanged for decades. The glioma microenvironment is highly immunosuppressive and consequently this has motivated the development of immunotherapies for counteracting this condition, enabling the immune cells within the tumor microenvironment to react against this tumor.

Areas Covered: The authors discuss immunotherapeutic strategies for glioma in phase-I/II clinical trials and illuminate their mechanisms of action, limitations, and key challenges. They also examine promising approaches under preclinical development.

Expert Opinion: In the last decade there has been an expansion in immune-mediated anti-cancer therapies. In the glioma field, sophisticated strategies have been successfully implemented in preclinical models. Unfortunately, clinical trials have not yet yielded consistent results for glioma patients. This could be attributed to our limited understanding of the complex immune cell infiltration and its interaction with the tumor cells, the selected time for treatment, the combination with other therapies and the route of administration of the agent. Applying these modalities to treat malignant glioma is challenging, but many new alternatives are emerging to by-pass these hurdles.
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http://dx.doi.org/10.1080/13543784.2020.1768528DOI Listing
July 2020

Blockade of Cell Volume Regulatory Protein NKCC1 Increases TMZ-Induced Glioma Apoptosis and Reduces Astrogliosis.

Mol Cancer Ther 2020 07 11;19(7):1550-1561. Epub 2020 May 11.

Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania.

Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that gene and its encoded Na-K-2Cl cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II-IV gliomas. NKCC1 regulates cell volume and intracellular Cl concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0910DOI Listing
July 2020

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion.

J Vis Exp 2020 04 12(158). Epub 2020 Apr 12.

Dept. of Neurosurgery, University of Michigan Medical School; Dept. of Cell and Developmental Biology, University of Michigan Medical School; Rogel Cancer Center, University of Michigan Medical School;

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma's growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.
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http://dx.doi.org/10.3791/60939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253033PMC
April 2020

Therapeutic Efficacy of Immune Stimulatory Thymidine Kinase and fms-like Tyrosine Kinase 3 Ligand (TK/Flt3L) Gene Therapy in a Mouse Model of High-Grade Brainstem Glioma.

Clin Cancer Res 2020 08 24;26(15):4080-4092. Epub 2020 Apr 24.

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan.

Purpose: Diffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, Activin A receptor type I (ACVR1)-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy.

Experimental Design: We utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNA-sequencing analysis on neurospheres harboring mACVR1. mACVR1 neurospheres were implanted into the pons of immune-competent mice to test the therapeutic efficacy and toxicity of immune-stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 neurospheres expressing the surrogate tumor antigen ovalbumin were generated to investigate whether TK/Flt3L treatment induces the recruitment of tumor antigen-specific T cells.

Results: Histologic analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 neurosphere identified an increase in the TGFβ signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in antitumor immunity, recruitment of antitumor-specific T cells, and increased median survival (MS).

Conclusions: This study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune-stimulatory gene therapy targeting the hosts' antitumor immune response inhibits tumor progression and increases MS of mice bearing mACVR1 tumors.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415674PMC
August 2020

Synthetic HDL Nanoparticles Delivering Docetaxel and CpG for Chemoimmunotherapy of Colon Adenocarcinoma.

Int J Mol Sci 2020 Mar 5;21(5). Epub 2020 Mar 5.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA.

Colon carcinomas comprise over two-thirds of all colorectal cancers with an overall 5-year survival rate of 64%, which rapidly decreases to 14% when the cancer becomes metastatic. Depending on the stage of colon carcinoma at diagnosis, patients can undergo surgery to attempt complete tumor resection or move directly to chemotherapy with one or a combination of drugs. As with most cancers, colon carcinomas do not always respond to chemotherapies, so targeted therapies and immunotherapies have been developed to aid chemotherapy. We report the development of a local combination therapy for colon carcinoma whereby chemo- and immunotherapeutic entities are delivered intratumorally to maximize efficacy and minimize off-target side effects. A hydrophobic chemotherapeutic agent, docetaxel (DTX), and cholesterol-modified Toll-like receptor 9 (TLR9) agonist CpG (cho-CpG) oligonucleotide are co-loaded in synthetic HDL (sHDL) nanodiscs. In vivo survival analysis of MC-38 tumor-bearing mice treated intratumorally with DTX-sHDL/CpG (median survival; MS = 43 days) showed significant improvement in overall survival compared to mice treated with single agents, free DTX (MS = 23 days, < 0.0001) or DTX-sHDL (MS = 28 days, < 0.0001). Two of seven mice treated with DTX-sHDL/CpG experienced complete tumor regression. None of the mice experienced any systemic toxicity as indicated by body weight maintenance and normal serum enzyme and protein levels. In summary, we have demonstrated that chemo- and immunotherapies can be co-loaded into sHDLs, delivered locally to the tumor, and can be used to improve survival outcomes significantly compared to chemotherapy alone.
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http://dx.doi.org/10.3390/ijms21051777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084365PMC
March 2020

Epigenetic reprogramming and chromatin accessibility in pediatric diffuse intrinsic pontine gliomas: a neural developmental disease.

Neuro Oncol 2020 02;22(2):195-206

Department of Cell and Developmental Biology and Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, Michigan.

Diffuse intrinsic pontine glioma (DIPG) is a rare but deadly pediatric brainstem tumor. To date, there is no effective therapy for DIPG. Transcriptomic analyses have revealed DIPGs have a distinct profile from other pediatric high-grade gliomas occurring in the cerebral hemispheres. These unique genomic characteristics coupled with the younger median age group suggest that DIPG has a developmental origin. The most frequent mutation in DIPG is a lysine to methionine (K27M) mutation that occurs on H3F3A and HIST1H3B/C, genes encoding histone variants. The K27M mutation disrupts methylation by polycomb repressive complex 2 on histone H3 at lysine 27, leading to global hypomethylation. Histone 3 lysine 27 trimethylation is an important developmental regulator controlling gene expression. This review discusses the developmental and epigenetic mechanisms driving disease progression in DIPG, as well as the profound therapeutic implications of epigenetic programming.
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http://dx.doi.org/10.1093/neuonc/noz218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032633PMC
February 2020

The IDH-TAU-EGFR triad defines the neovascular landscape of diffuse gliomas.

Sci Transl Med 2020 01;12(527)

Neurooncology Unit, Instituto de Salud Carlos III-UFIEC, Madrid 28220, Spain.

Gliomas that express the mutated isoforms of isocitrate dehydrogenase 1/2 (IDH1/2) have better prognosis than wild-type (wt) IDH1/2 gliomas. However, how these mutant (mut) proteins affect the tumor microenvironment is still a pending question. Here, we describe that the transcription of microtubule-associated protein TAU (), a gene that has been classically associated with neurodegenerative diseases, is epigenetically controlled by the balance between wt and mut IDH1/2 in mouse and human gliomas. In IDH1/2 mut tumors, we found high expression of TAU that decreased with tumor progression. Furthermore, was almost absent from tumors with epidermal growth factor receptor () mutations, whereas its trancription negatively correlated with overall survival in gliomas carrying wt or amplified (amp) We demonstrated that the overexpression of TAU, through the stabilization of microtubules, impaired the mesenchymal/pericyte-like transformation of glioma cells by blocking EGFR, nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and the transcriptional coactivator with PDZ-binding motif (TAZ). Our data also showed that mut EGFR induced a constitutive activation of this pathway, which was no longer sensitive to TAU. By inhibiting the transdifferentiation capacity of EGFRamp/wt tumor cells, TAU protein inhibited angiogenesis and favored vascular normalization, decreasing glioma aggressiveness and increasing their sensitivity to chemotherapy.
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http://dx.doi.org/10.1126/scitranslmed.aax1501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055928PMC
January 2020

Prospects of biological and synthetic pharmacotherapies for glioblastoma.

Expert Opin Biol Ther 2020 03 20;20(3):305-317. Epub 2020 Jan 20.

Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA.

: The field of neuro-oncology has experienced significant advances in recent years. More is known now about the molecular and genetic characteristics of glioma than ever before. This knowledge leads to the understanding of glioma biology and pathogenesis, guiding the development of targeted therapeutics and clinical trials. The goal of this review is to describe the state of basic, translational, and clinical research as it pertains to biological and synthetic pharmacotherapy for gliomas.: Challenges remain in designing accurate preclinical models and identifying patients that are likely to respond to a particular targeted therapy. Preclinical models for therapeutic assessment are critical to identify the most promising treatment approaches.: Despite promising new therapeutics, there have been no significant breakthroughs in glioma treatment and patient outcomes. Thus, there is an urgent need to better understand the mechanisms of treatment resistance and to design effective clinical trials.
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http://dx.doi.org/10.1080/14712598.2020.1713085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059118PMC
March 2020

Fyn tyrosine kinase, a downstream target of receptor tyrosine kinases, modulates antiglioma immune responses.

Neuro Oncol 2020 06;22(6):806-818

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, Michigan.

Background: High-grade gliomas are aggressive and immunosuppressive brain tumors. Molecular mechanisms that regulate the inhibitory immune tumor microenvironment (TME) and glioma progression remain poorly understood. Fyn tyrosine kinase is a downstream target of the oncogenic receptor tyrosine kinase pathway and is overexpressed in human gliomas. Fyn's role in vivo in glioma growth remains unknown. We investigated whether Fyn regulates glioma initiation, growth and invasion.

Methods: We evaluated the role of Fyn using genetically engineered mouse glioma models (GEMMs). We also generated Fyn knockdown stem cells to induce gliomas in immune-competent and immune-deficient mice (nonobese diabetic severe combined immunodeficient gamma mice [NSG], CD8-/-, CD4-/-). We analyzed molecular mechanism by RNA sequencing and bioinformatics analysis. Flow cytometry was used to characterize immune cellular infiltrates in the Fyn knockdown glioma TME.

Results: We demonstrate that Fyn knockdown in diverse immune-competent GEMMs of glioma reduced tumor progression and significantly increased survival. Gene ontology (GO) analysis of differentially expressed genes in wild-type versus Fyn knockdown gliomas showed enrichment of GOs related to immune reactivity. However, in NSG and CD8-/- and CD4-/- immune-deficient mice, Fyn knockdown gliomas failed to show differences in survival. These data suggest that the expression of Fyn in glioma cells reduces antiglioma immune activation. Examination of glioma immune infiltrates by flow cytometry displayed reduction in the amount and activity of immune suppressive myeloid derived cells in the Fyn glioma TME.

Conclusions: Gliomas employ Fyn mediated mechanisms to enhance immune suppression and promote tumor progression. We propose that Fyn inhibition within glioma cells could improve the efficacy of antiglioma immunotherapies.
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http://dx.doi.org/10.1093/neuonc/noaa006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283034PMC
June 2020

Functional characterization of tumor antigen-specific T-cells isolated from the tumor microenvironment of sleeping beauty induced murine glioma models.

Methods Enzymol 2020 8;631:91-106. Epub 2019 Jun 8.

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States; Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States. Electronic address:

Diffuse Gliomas represent 80% of brain tumors with an average survival of the most aggressive form glioblastoma (GBM) 15-22 months from the time of diagnosis. The current standard of care includes tumor resection, chemotherapy and radiation, nevertheless, the incidence of recurrence remains high and there is a critical need for developing new therapeutic strategies. T-cell mediated immunotherapy that triggers an anti-tumor T cell-mediated memory response is a promising approach since it will not only attack the primary tumor but also prevent recurrence. Multiple immunotherapeutic strategies against glioma are currently being tested in clinical trials. We have developed an immune-mediated gene therapy (Thymidine kinase plus Fms-like tyrosine kinase 3 ligand: TK/Flt3L) which induces a robust anti-tumor T cell response leading to tumor regression, long-term survival and immunological memory in GBM models. Efficacy of the anti-glioma T cell therapy is determined by anti-tumor specific effector T cells. Therefore, assessing effector T cell activation status and function are critical readouts for determining the effectiveness of the therapy. Here, we detail methodologies to evaluate tumor specific T-cell responses using a genetically engineered Sleeping Beauty transposase-mediated glioma model. We first describe the glioma model and the generation of neurospheres (NS) that express the surrogate antigen cOVA. Then, we describe functional assays to determine anti-tumor T-cell response.
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http://dx.doi.org/10.1016/bs.mie.2019.05.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021207PMC
January 2021

Engineering patient-specific cancer immunotherapies.

Nat Biomed Eng 2019 10 12;3(10):768-782. Epub 2019 Aug 12.

Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA.

Research into the immunological processes implicated in cancer has yielded a basis for the range of immunotherapies that are now considered the fourth pillar of cancer treatment (alongside surgery, radiotherapy and chemotherapy). For some aggressive cancers, such as advanced non-small-cell lung carcinoma, combination immunotherapies have resulted in unprecedented treatment efficacy for responding patients, and have become frontline therapies. Individualized immunotherapy, enabled by the identification of patient-specific mutations, neoantigens and biomarkers, and facilitated by advances in genomics and proteomics, promises to broaden the responder patient population. In this Perspective, we give an overview of immunotherapies leveraging engineering approaches, including the design of biomaterials, delivery strategies and nanotechnology solutions, for the realization of individualized cancer treatments such as nanoparticle vaccines customized with neoantigens, cell therapies based on patient-derived dendritic cells and T cells, and combinations of theranostic strategies. Developments in precision cancer immunotherapy will increasingly rely on the adoption of engineering principles.
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http://dx.doi.org/10.1038/s41551-019-0436-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783331PMC
October 2019

Effect of caveolin-1 on Stat3-ptyr705 levels in breast and lung carcinoma cells.

Biochem Cell Biol 2019 10 15;97(5):638-646. Epub 2019 Apr 15.

Department of Biomedical and Molecular Sciences, Pathology and Molecular Medicine, and Queen's University Cancer Research Institute, Queen's University, Kingston, ON K7L 3N6, Canada.

We recently demonstrated that Cav1 (caveolin-1) is a negative regulator of Stat3 (signal transducer and activator of transcription-3) activity in mouse fibroblasts and human lung carcinoma SHP77 cells. We now examined whether the cellular context may affect their levels as well as the relationship between them, by assessing Cav1 and Stat3-ptyr705 amounts in different cell lines. In MDA-MB-231, A549, and HaCat cells, Cav1 levels were high and Stat3-ptyr705 levels were low, consistent with the notion of a negative effect of endogenous Cav1 on Stat3-ptyr705 levels in these lines. In addition, manipulation of Cav1 levels revealed a negative effect in MCF7 and mouse fibroblast cells, while Cav1 upregulation induced apoptosis in MCF7 cells. In contrast, however, line MRC9 had high Cav1 and high Stat3-ptyr705 levels, indicating that high Cav1 is insufficient to reduce Stat3-ptyr705 levels in this line. MCF7 and LuCi6 cells had very low Cav1 and Stat3-ptyr705 levels, indicating that the low Stat3-ptyr705 can be independent from Cav1 levels altogether. Our results reveal a further level of complexity in the relationship between Cav1 and Stat3-ptyr705 than previously thought. In addition, we demonstrate that in a feedback loop, Stat3 inhibition upregulates Cav1 in HeLa cells but not in other lines tested.
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http://dx.doi.org/10.1139/bcb-2018-0367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038897PMC
October 2019

A platform for artificial intelligence based identification of the extravasation potential of cancer cells into the brain metastatic niche.

Lab Chip 2019 03;19(7):1162-1173

Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

Brain metastases are the most lethal complication of advanced cancer; therefore, it is critical to identify when a tumor has the potential to metastasize to the brain. There are currently no interventions that shed light on the potential of primary tumors to metastasize to the brain. We constructed and tested a platform to quantitatively profile the dynamic phenotypes of cancer cells from aggressive triple negative breast cancer cell lines and patient derived xenografts (PDXs), generated from a primary tumor and brain metastases from tumors of diverse organs of origin. Combining an advanced live cell imaging algorithm and artificial intelligence, we profile cancer cell extravasation within a microfluidic blood-brain niche (μBBN) chip, to detect the minute differences between cells with brain metastatic potential and those without with a PPV of 0.91 in the context of this study. The results show remarkably sharp and reproducible distinction between cells that do and those which do not metastasize inside of the device.
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http://dx.doi.org/10.1039/c8lc01387jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510031PMC
March 2019

IDH1-R132H acts as a tumor suppressor in glioma via epigenetic up-regulation of the DNA damage response.

Sci Transl Med 2019 02;11(479)

Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Patients with glioma whose tumors carry a mutation in isocitrate dehydrogenase 1 (IDH1) are younger at diagnosis and live longer. mutations co-occur with other molecular lesions, such as 1p/19q codeletion, inactivating mutations in the tumor suppressor protein 53 ) gene, and loss-of-function mutations in alpha thalassemia/mental retardation syndrome X-linked gene (). All adult low-grade gliomas (LGGs) harboring ATRX loss also express the IDH1 mutation. The current molecular classification of LGGs is based, partly, on the distribution of these mutations. We developed a genetically engineered mouse model harboring IDH1, and inactivating mutations, and activated NRAS G12V. Previously, we established that ATRX deficiency, in the context of wild-type IDH1, induces genomic instability, impairs nonhomologous end-joining DNA repair, and increases sensitivity to DNA-damaging therapies. In this study, using our mouse model and primary patient-derived glioma cultures with IDH1 mutations, we investigated the function of IDH1 in the context of TP53 and ATRX loss. We discovered that IDH1 expression in the genetic context of and gene inactivation (i) increases median survival in the absence of treatment, (ii) enhances DNA damage response (DDR) via epigenetic up-regulation of the ataxia-telangiectasia-mutated (ATM) signaling pathway, and (iii) elicits tumor radioresistance. Accordingly, pharmacological inhibition of ATM or checkpoint kinases 1 and 2, essential kinases in the DDR, restored the tumors' radiosensitivity. Translation of these findings to patients with IDH1 glioma harboring TP53 and ATRX loss could improve the therapeutic efficacy of radiotherapy and, consequently, patient survival.
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http://dx.doi.org/10.1126/scitranslmed.aaq1427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400220PMC
February 2019

High-Density Lipoprotein-Mimicking Nanodiscs for Chemo-immunotherapy against Glioblastoma Multiforme.

ACS Nano 2019 02 11;13(2):1365-1384. Epub 2019 Feb 11.

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor, for which there is no cure. Treatment effectiveness for GBM has been limited due to tumor heterogeneity, an immunosuppressive tumor microenvironment (TME), and the presence of the blood-brain barrier, which hampers the transport of chemotherapeutic compounds to the central nervous system (CNS). High-density lipoprotein (HDL)-mimicking nanodiscs hold considerable promise to achieve delivery of bioactive compounds into tumors. Herein, we tested the ability of synthetic HDL nanodiscs to deliver chemotherapeutic agents to the GBM microenvironment and elicit tumor regression. To this end, we developed chemo-immunotherapy delivery vehicles based on sHDL nanodiscs loaded with CpG, a Toll-like receptor 9 (TLR9) agonist, together with docetaxel (DTX), a chemotherapeutic agent, for targeting GBM. Our data show that delivery of DTX-sHDL-CpG nanodiscs into the tumor mass elicited tumor regression and antitumor CD8 T cell responses in the brain TME. We did not observe any overt off-target side effects. Furthermore, the combination of DTX-sHDL-CpG treatment with radiation (IR), which is the standard of care for GBM, resulted in tumor regression and long-term survival in 80% of GBM-bearing animals. Mice remained tumor-free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of anti-GBM immunological memory. Collectively, these data indicate that sHDL nanodiscs constitute an effective drug delivery platform for the treatment of GBM, resulting in tumor regression, long-term survival, and immunological memory when used in combination with IR. The proposed delivery platform has significant potential for clinical translation.
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http://dx.doi.org/10.1021/acsnano.8b06842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484828PMC
February 2019

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures.

J Vis Exp 2019 01 9(143). Epub 2019 Jan 9.

Department of Neurosurgery, University of Michigan Medical School; Department of Cell & Developmental Biology, University of Michigan;

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.
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http://dx.doi.org/10.3791/58931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6352905PMC
January 2019