技术资料

Fetal-onset hydrocephalus affects 1 to 3 per 1,000 live births. It is not only a disorder of cerebrospinal fluid dynamics but also a brain disorder that corrective surgery does not ameliorate. We hypothesized that cell junction abnormalities of neural stem cells (NSCs) lead to the inseparable phenomena of fetal-onset hydrocephalus and abnormal neurogenesis. We used bromodeoxyuridine labeling,immunocytochemistry,electron microscopy,and cell culture to study the telencephalon of hydrocephalic HTx rats and correlated our findings with those in human hydrocephalic and nonhydrocephalic human fetal brains (n = 12 each). Our results suggest that abnormal expression of the intercellular junction proteins N-cadherin and connexin-43 in NSC leads to 1) disruption of the ventricular and subventricular zones,loss of NSCs and neural progenitor cells; and 2) abnormalities in neurogenesis such as periventricular heterotopias and abnormal neuroblast migration. In HTx rats,the disrupted NSC and progenitor cells are shed into the cerebrospinal fluid and can be grown into neurospheres that display intercellular junction abnormalities similar to those of NSC of the disrupted ventricular zone; nevertheless,they maintain their potential for differentiating into neurons and glia. These NSCs can be used to investigate cellular and molecular mechanisms underlying this condition,thereby opening the avenue for stem cell therapy. View Publication

UNLABELLED A noninvasive technology that indiscriminately detects tumor tissue in the brain could substantially enhance the management of primary or metastatic brain tumors. Although the documented molecular heterogeneity of diseases that initiate or eventually deposit in the brain may preclude identifying a single smoking-gun molecular biomarker,many classes of brain tumors are generally avid for transferrin. Therefore,we reasoned that applying a radiolabeled derivative of transferrin ((89)Zr-labeled transferrin) may be an effective strategy to more thoroughly identify tumor tissue in the brain,regardless of the tumor's genetic background. METHODS Transferrin was radiolabeled with (89)Zr,and its properties with respect to human models of glioblastoma multiforme were studied in vivo. RESULTS In this report,we show proof of concept that (89)Zr-labeled transferrin ((89)Zr-transferrin) localizes to genetically diverse models of glioblastoma multiforme in vivo. Moreover,we demonstrate that (89)Zr-transferrin can detect an orthotopic lesion with exceptional contrast. Finally,the tumor-to-brain contrast conferred by (89)Zr-transferrin vastly exceeded that observed with (18)F-FDG,currently the most widely used radiotracer to assess tumor burden in the brain. CONCLUSION The results from this study suggest that (89)Zr-transferrin could be a broadly applicable tool for identifying and monitoring tumors in the brain,with realistic potential for near-term clinical translation. View Publication

Neurotrophins and their receptors are frequently expressed in malignant gliomas,yet their functions are largely unknown. Previously,we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However,the role of Trk receptors has not been examined. In this study,we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here,we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC,not TrkA,and they also express neurotrophins NGF,BDNF,and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely,TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further,pharmacological inhibition of both ERK and Akt pathways blocked BDNF,and NT3 stimulated BTIC survival. Importantly,attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling,and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma. View Publication

OBJECTIVE To develop in vitro adenoid cystic carcinoma cell line as a surrogate for functional studies. MATERIALS AND METHODS Cells obtained from a primary ACC of the base of tongue were cultivated in vitro and immortalized with h-TERT. Morphologic,cytogenetic and functional studies were performed. RESULTS Tumor cells were verified by positive reactions to keratin and smooth muscle actin and phenotypic cellular and nuclear features. In-vitro cell growth and colony formation assay supported their tumor nature. CONCLUSION We authenticated an ACC cell line with hybrid epithelial-myoepithelial feature as a resource for functional experimentation. View Publication

BACKGROUND Glioma is the most common form of primary malignant brain tumor in adults,with approximately 4 cases per 100 000 people each year. Gliomas,like many tumors,are thought to primarily metabolize glucose for energy production; however,the reliance upon glycolysis has recently been called into question. In this study,we aimed to identify the metabolic fuel requirements of human glioma cells. METHODS We used database searches and tissue culture resources to evaluate genotype and protein expression,tracked oxygen consumption rates to study metabolic responses to various substrates,performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates,and employed an animal model of malignant glioma to evaluate a new therapeutic intervention. RESULTS We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition,we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover,inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma. CONCLUSIONS Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition,the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice. View Publication

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress,however,results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts,implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings,we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ˜425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR,including a compound with a 2,9-diazaspiro[5.5]undecane core,which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines,including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models. View Publication

Glioblastoma multiforme (GBM),the most common and lethal tumor of the adult brain,generally shows chemo- and radioresistance. MicroRNAs (miRs) regulate physiological processes,such as resistance of GBM cells to temozolomide (TMZ). Although miRs are attractive targets for cancer therapeutics,the effectiveness of this approach requires targeted delivery. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers,including GBM. We report on an increase in miR-9 in TMZ-resistant GBM cells. miR-9 was involved in the expression of the drug efflux transporter,P-glycoprotein. To block miR-9,methods were developed with Cy5-tagged anti-miR-9. Dye-transfer studies indicated intracellular communication between GBM cells and MSCs. This occurred by gap junctional intercellular communication and the release of microvesicles. In both cases,anti-miR-9 was transferred from MSCs to GBM cells. However,the major form of transfer occurred with the microvesicles. The delivery of anti-miR-9 to the resistant GBM cells reversed the expression of the multidrug transporter and sensitized the GBM cells to TMZ,as shown by increased cell death and caspase activity. The data showed a potential role for MSCs in the functional delivery of synthetic anti-miR-9 to reverse the chemoresistance of GBM cells.Molecular Therapy-Nucleic Acids (2013) 2,e126; doi:10.1038/mtna.2013.60; published online 1 October 2013. 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