Sridevi Yadavilli

Insights into pediatric diffuse intrinsic pontine glioma through proteomic analysis of cerebrospinal fluid

Diffuse intrinsic pontine glioma (DIPG) is a leading cause of brain tumor-related death in children. DIPG is not surgically resectable, resulting in a paucity of tissue available for molecular studies. As such, tumor biology is poorly understood, and, currently, there are no effective treatments. In the absence of frozen tumor specimens, body fluids--such as cerebrospinal fluid (CSF), serum, and urine--can serve as more readily accessible vehicles for detecting tumor-secreted proteins. We analyzed a total of 76 specimens, including CSF, serum, urine, and normal and tumor brainstem tissue. Protein profiling of CSF from patients with DIPG was generated by mass spectrometry using an LTQ-Orbitrap-XL and database search using the Sequest algorithm. Quantitative and statistical analyses were performed with ProteoIQ and Partek Genomics Suite. A total of 528 unique proteins were identified, 71% of which are known secreted proteins. CSF proteomic analysis revealed selective upregulation of Cyclophillin A (CypA) and dimethylarginase 1 (DDAH1) in DIPG (n = 10), compared with controls (n = 4). Protein expression was further validated with Western blot analysis and immunohistochemical assays using CSF, brain tissue, serum, and urine from DIPG and control specimens. Immunohistochemical staining showed selective upregulation of secreted but not cytosolic CypA and DDAH1 in patients with DIPG. In this study, we present the first comprehensive protein profile of CSF specimens from patients with DIPG to demonstrate selective expression of tumor proteins potentially involved in brainstem gliomagenesis. Detection of secreted CypA and DDAH1 in serum and urine has potential clinical application, with implications for assessing treatment response and detecting tumor recurrence in patients with DIPG.

Molecular Imaging of Biological Samples on Nanophotonic Laser Desorption Ionization Platforms

Mass spectrometry imaging (MSI) is a comprehensive tool for the analysis of a wide range of biomolecules. The mainstream method for molecular MSI is matrix-assisted laser desorption ionization, however, the presence of a matrix results in spectral interferences and the suppression of some analyte ions. Herein we demonstrate a new matrix-free MSI technique using nanophotonic ionization based on laser desorption ionization (LDI) from a highly uniform silicon nanopost array (NAPA). In mouse brain and kidney tissue sections, the distributions of over 80 putatively annotated molecular species are determined with 40 μm spatial resolution. Furthermore, NAPA-LDI-MS is used to selectively analyze metabolites and lipids from sparsely distributed algal cells and the lamellipodia of human hepatocytes. Our results open the door for matrix-free MSI of tissue sections and small cell populations by nanophotonic ionization.

Manganese-containing Prussian blue nanoparticles for imaging of pediatric brain tumors

Pediatric brain tumors (PBTs) are a leading cause of death in children. For an improved prognosis in patients with PBTs, there is a critical need to develop molecularly-specific imaging agents to monitor disease progression and response to treatment. In this paper, we describe manganese-containing Prussian blue nanoparticles as agents for molecular magnetic resonance imaging (MRI) and fluorescence-based imaging of PBTs. Our core-shell nanoparticles consist of a core lattice structure that incorporates and retains paramagnetic Mn2+ ions, and generates MRI contrast (both negative and positive). The biofunctionalized shell is comprised of fluorescent avidin, which serves the dual purpose of enabling fluorescence imaging and functioning as a platform for the attachment of biotinylated ligands that target PBTs. The surfaces of our nanoparticles are modified with biotinylated antibodies targeting neuron-glial antigen 2 or biotinylated transferrin. Both neuron-glial antigen 2 and the transferrin receptor are protein markers overexpressed in PBTs. We describe the synthesis, biofunctionalization, and characterization of these multimodal nanoparticles. Further, we demonstrate the MRI and fluorescence imaging capabilities of manganese-containing Prussian blue nanoparticles in vitro. Finally, we demonstrate the potential of these nanoparticles as PBT imaging agents by measuring their organ and brain biodistribution in an orthotopic mouse model of PBTs using ex vivo fluorescence imaging.

The Role of NG2 Proteoglycan in Glioma

Neuron glia antigen-2 ((NG2), also known as chondroitin sulphate proteoglycan 4, or melanoma-associated chondroitin sulfate proteoglycan) is a type-1 membrane protein expressed by many central nervous system (CNS) cells during development and differentiation and plays a critical role in proliferation and angiogenesis. ‘NG2’ often references either the protein itself or the highly proliferative and undifferentiated glial cells expressing high levels of NG2 protein. NG2 glia represent the fourth major type of neuroglia in the mammalian nervous system and are classified as oligodendrocyte progenitor cells by virtue of their committed oligodendrocyte generation in developing and adult brain. Here, we discuss NG2 glial cells as well as NG2 protein and its expression and role with regards to CNS neoplasms as well as its potential as a therapeutic target for treating childhood CNS cancers.

Vamorolone, a dissociative steroidal compound, reduces pro-inflammatory cytokine expression in glioma cells and increases activity and survival in a murine model of cortical tumor

Corticosteroids, such as dexamethasone, are routinely used as palliative care in neuro-oncology for their anti-inflammatory benefits, however many patients experience dose limiting side effects caused by glucocorticoid response element (GRE)-mediated transcription. The purpose of this study was to use a murine model to investigate a new steroid alternative, vamorolone, which promises to reduce side effects through dissociating GRE-mediated transcription and NF-κB -mediated anti-inflammatory actions. To compare vamorolone to dexamethasone in reducing pro-inflammatory signals in vitro, murine glioma cells were treated with dexamethasone, vamorolone or vehicle control. Changes in mRNA expression were assessed using the nanostring inflammatory platform. Furthermore, drug efficacy, post-treatment behavioral activity and side effects were assessed by treating two cohorts of brain tumor bearing mice with dexamethasone, vamorolone, or vehicle control. Our investigation showed that treatment with vamorolone resulted in a reduction of pro-inflammatory signals in tumor cells in vitro similar to treatment with dexamethasone. Treatment with vamorolone resulted in a better safety profile in comparison to dexamethasone treatment. Vamorolone- treated mice showed similar or better activity and survival when compared to dexamethasone-treated mice. Our data indicate vamorolone is a potential steroid-sparing alternative for treating patients with brain tumors.

Matrix-free Mass Spectrometry Imaging of Mouse Brain Tissue Sections on Silicon Nanopost Arrays

Mass spectrometry imaging (MSI) is capable of detection and identification of diverse classes of compounds in brain tissue sections, whereas simultaneously mapping their spatial distributions. Given the vast array of chemical components present in neurological systems, as well as the innate diversity within molecular classes, MSI platforms capable of detecting a wide array of species are useful for achieving a more comprehensive understanding of their biological roles and significance. Currently, matrix‐assisted laser desorption ionization (MALDI) is the method of choice for the molecular imaging of brain samples by mass spectrometry. However, nanostructured laser desorption ionization platforms, such as silicon nanopost arrays (NAPA), are emerging as alternative MSI techniques that can provide complementary insight into molecular distributions in the central nervous system. In this work, the molecular coverage of mouse brain lipids afforded by NAPA‐MSI is compared to that of MALDI‐MSI using two common MALDI matrices. In positive ion mode, MALDI spectra were dominated by phosphatidylcholines and phosphatidic acids. NAPA favored the ionization of phosphatidylethanolamines and glycosylated ceramides, which were poorly detected in MALDI‐MSI. In negative ion mode, MALDI favored sulfatides and free fatty acids, whereas NAPA spectra were dominated by signal from phosphatidylethanolamines. The complementarity in lipid coverages between the NAPA‐ and MALDI‐MSI platforms presents the possibility of selective lipid analysis and imaging dependent upon which platform is used. Nanofabrication of the NAPA platform offers better uniformity compared to MALDI, and the wider dynamic range offered by NAPA promises improved quantitation in imaging.

Abstract 5004: NG2 upregulation and its defective asymmetric distribution in pediatric brainstem glioma and diffuse intrinsic pontine glioma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Pediatric brainstem glioma (BSG) is one of the most difficult cancers to treat accounting for 10-20% of all pediatric central nervous system (CNS) tumors. BSGs may occur throughout the brainstem and are categorized into two main groups: diffuse intrinsic pontine gliomas (DIPGs) and focal brainstem gliomas. DIPGs represent about 80% of BSG and have a peak onset of six to nine years of age. DIPGs invade throughout the pons and may spread to other portions of brainstem. To better understand the pathophysiology of the disease, a genetically engineered (PDGFα-expressing) BSG mouse and a xenograft model have been established. Results: We recently reported proteome profiling of DIPG CSF and formalin fixed specimens. To expand our molecular studies of the disease, we generated complete protein profiles of specimens obtained from BSG mouse tumor and aged-matched healthy controls. Neuroglia 2 (NG2), also known as chondroitine sulfate proteoglycan 4 (CSPG4) was selected for further analysis based on its exclusive expression in BSG specimens. Although NG2 has been previously implicated in adult gliomas, its role in pediatric gliomas has not been investigated and currently there are no publications on the role of NG2 in DIPGs. Recently, NG2 expression has been shown to play a significant role in neoplastic transformation of glioma precursor cells. Gliomas are thought to originate from cells including astrocytes, stem cells, and glioma progenitor cells. Glial progenitor cells are specifically important since several groups have reported detection of oligodendrocyte markers including NG2, PDGFRα, and Olig-2 in gliomas. Our preliminary data shows high expression of NG2 in murine model of brainstem glioma as well as 80% of pediatric DIPG specimens tested. We show that shRNA-mediated knockdown of NG2 reduces cellular migration in vitro. NG2 expression is defective (symmetric) in dividing cells in vitro and in vivo. The defective NG2 expression is consistent with a recent observation in adult high grade gliomas. Injection of NG2 expressing neurospheres (NS) into brainstems of 2 day old mice (P2) results in highly aggressive brainstem tumors resulting in death within 3-7 weeks post-injection. Therefore the NS injected mouse model of brainstem glioma provides a solid model for testing therapeutics and evaluating interventions. Furthermore, we show selective delivery of liposomal nanoparticles to brainstem of our robust BSG mouse model. We also show that nanoparticle-mediated delivery of doxorubicin will induce apoptosis in tumor and not the adjacent normal region. Conclusion: We introduce a robust murine model of brainstem glioma that is developed using NG2 expressing cells. High expression of NG2 in a subset of DIPGs and its defective expression may provide novel approaches for treating DIPGs and BSGs. Citation Format: Sridevi Yadavilli, Madhuri Kambhampati, Oren J. Becher, Tobey MacDonald, Ravi Bellamkonda, Roger J. Paker, Javad Nazarian. NG2 upregulation and its defective asymmetric distribution in pediatric brainstem glioma and diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5004. doi:10.1158/1538-7445.AM2013-5004

Abstract C30: Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma

Introduction: Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Molecular characterization is limited due to lack of tissue. Recent investigations suggest possible molecular subtypes may account for the historical poor response to therapy. We previously generated protein profiles of CSF and formalin fixed DIPG tumor specimens to characterize patterns of protein expression. Here, we present the first comprehensive tissue proteome of fresh frozen DIPG tumor specimens (n=16) and normal brain tissue (n=10). We characterize differential protein expression in DIPG tumor specimens, and compare these to gene expression and DNA methylation profiles of the same tissue. Methods: Normal brain and tumor tissue was collected intraoperatively or post-mortem. Extracted total tissue protein was quantified by mass spectrometry (MS/MS) via LTQ-Orbitrap-XL and database search using the Sequest algorithm. Gene expression profiles were detected using whole-genome Human HT-4 v12 Gene Expression Bead Chips. DNA methylation profiles were characterized after bisulphite conversion using Infinium HumanMethylation 450K BeadChip arrays. Quantitative and statistical analysis was performed with Genome Studio, ProteoIQ, and Partek Genomics Suite. Functional analysis was performed using Ingenuity Pathways Analysis. Gene and protein expression was validated via western blot and immunohistochemical staining of tumor and normal brain tissue. Results: 1,918 differentially expressed genes were detected in DIPG tumor tissue (ANOVA, p 2 or 2), with differential ShH pathway (GLI1 z-score -0.626 vs. 2.254) and protein expression COL1A2, LMNA, MAP4, NES, NRCAM, STMN1, and TNC between subgroups (ANOVA, p 2 or 2). Conclusions: We present the first comprehensive protein profile of DIPG fresh frozen tumor tissue and correlate to tissue gene expression profiles, which suggest differential activity of SHh pathway. This may in part be explained by differential methylation patterns of key genes. Proteomic analysis of DIPG tumor tissue reveals protein expression profiles reflective of this differential pathway activation, and hence may be useful tool for elucidating mechanisms of brainstem gliomagenesis in what likely is a heterogeneous tumor population. Biomarkers identified through proteomic analysis may in turn serve to more accurately diagnose patients with DIPG and measure response to therapy. Citation Format: Amanda Saratsis, Sridevi Yadavilli, Madhuri Kambhampati, Eric Raabe, Suresh Magge, Javad Nazarian. Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C30.