技术资料

OBJECTIVE In this work we set to develop and to validate a new in vivo frameless orthotopic Diffuse Intrinsic Pontine Glioma (DIPG) model based in the implantation of a guide-screw system. METHODS It consisted of a guide-screw also called bolt,a Hamilton syringe with a 26-gauge needle and an insulin-like 15-gauge needle. The guide screw is 2.6 mm in length and harbors a 0.5 mm central hole which accepts the needle of the Hamilton syringe avoiding a theoretical displacement during insertion. The guide-screw is fixed on the mouse skull according to the coordinates: 1mm right to and 0.8 mm posterior to lambda. To reach the pons the Hamilton syringe is adjusted to a 6.5 mm depth using a cuff that serves as a stopper. This system allows delivering not only cells but also any kind of intratumoral chemotherapy,antibodies or gene/viral therapies. RESULTS The guide-screw was successfully implanted in 10 immunodeficient mice and the animals were inoculated with DIPG human cell lines during the same anesthetic period. All the mice developed severe neurologic symptoms and had a median overall survival of 95 days ranging the time of death from 81 to 116 days. Histopathological analysis confirmed tumor into the pons in all animals confirming the validity of this model. CONCLUSION Here we presented a reproducible and frameless DIPG model that allows for rapid evaluation of tumorigenicity and efficacy of chemotherapeutic or gene therapy products delivered intratumorally to the pons. View Publication

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation,migration,and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme,a highly aggressive brain cancer,suggesting that ion channel expression may be perturbed in this population. However,little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing,we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance,expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally,genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes,gene mutations,survival outcomes,regional tumor expression,and experimental responses to loss-of-function. Together,the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance. View Publication

BACKGROUND Glioblastomas (GBMs) are a heterogeneous group of primary brain tumors. These tumors are resistant to therapeutic interventions and invariably recur after surgical resection. The multifunctional protein transglutaminase 2 (TG2) has been shown to promote cell survival in a number of different tumors. There is also evidence that TG2 may be a pro-survival factor in GBMs. However,the roles that TG2 plays in facilitating GBM survival and proliferation have not yet been clearly delineated . METHODS The functions of TG2 are often cell- and context-specific. Therefore,in this study we examined the ability of TG2 to facilitate GBM proliferation using colony formation assays and 5-ethynyl-2'-deoxyuridine (EdU) incorporation in several different GBM cell lines as well as neurospheres derived from patient tumors representing the 3 major subtypes of GBM tumors (mesenchymal,proneural,and classical) and maintained in the absence of serum. TG2 knockdown or selective TG2 inhibitors were used to modulate TG2 expression and activity. RESULTS We show that TG2 plays differential roles in the proliferative process depending on the cell type. In most,but not all,GBM models TG2 plays a crucial role in the proliferative process,and some but not all TG2 inhibitors were highly effective at reducing proliferation in a large subset of the GBM models. CONCLUSION Our results show that TG2 plays an important-but notoriously context-specific-role in GBM cell biology. Nonetheless,as future studies unravel the genetic fingerprints" that make TG2 inhibitors effective this information could be exploited to develop TG2 inhibitors into personalized GBM therapies. View Publication

Surgery is an essential component in the treatment of brain tumors. However,delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology,SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally,we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue,SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct. View Publication

BIX01294 (Bix) is known to be a euchromatic histone-lysine N-methyltransferase 2 inhibitor and treatment with Bix suppresses cancer cell survival and proliferation. In the present study,it was observed that sequential treatment with low-dose Bix notably increases glioblastoma cell migration and metastasis. It was demonstrated that U251 cells sequentially treated with low-dose Bix exhibited induced characteristic changes in critical epithelial-mesenchymal transition (EMT) markers,including E-cadherin,N-cadherin,β-catenin and zinc finger protein SNAI2. Notably,sequential treatment with Bix also increased the expression of cancer stem cell-associated markers,including sex determining region Y-box 2,octamer-binding transcription factor 4 and cluster of differentiation 133. Neurosphere formation was significantly enhanced in cells sequentially treated with Bix,compared with control cells (control: P=0.011; single treatment of Bix,P=0.045). The results of the present study suggest that accumulation of low-dose Bix enhanced the migration and metastatic potential of glioblastoma cells by regulating EMT-associated gene expression,which may be the cause of the altered properties of glioblastoma stem cells. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM However,clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy,in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression,we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells,we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs,modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type,expressing pro-angiogenic factors. In contrast,phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data. View Publication

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here,we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes,suggesting that these cells could be committed to the neural lineage. After neural induction,NeuroD1,Sox1,Double Cortin,and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties,indicating the inability of these cells to differentiate into mature neurons. In contrast,the differentiated cells expressed a high level of CLDN11,as demonstrated using molecular method,and stained positively for the glial cell markers CLDN11 and GFAP,as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production,which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion,our data demonstrate morphological,molecular,and immunocytochemical evidence of initial neural differentiation of mature adipocytes,committing to a glial lineage. View Publication

We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells,and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse,we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality. View Publication

MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes,while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells,including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously,the common mechanism conferring the miR-10b-sustained viability of GSC is unknown. Here,we demonstrate that in heterogeneous GSC,miR-10b regulates cell cycle and alternative splicing,often through the non-canonical targeting via 5'UTRs of its target genes,including MBNL1-3,SART3,and RSRC1. We have further assessed the inhibition of miR-10b in intracranial human GSC-derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR-10b antisense oligonucleotide inhibitors (ASO),direct intratumoral injections,continuous osmotic delivery,and systemic intravenous injections,have been explored. In all cases,the treatment with miR-10b ASO led to targets' derepression,and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR-10b is a promising candidate for the development of targeted therapies against all GBM subtypes. View Publication

BACKGROUND In glioblastoma (GBM),Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance. METHODS Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2),a stabilized form of PGE2,to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1,a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting,quantitative real-time (qRT)-PCR,and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM. RESULTS In GBM cells,dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads,in turn,to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage,which are dependent on Id1. CONCLUSIONS In GBM,Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively,these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients. View Publication