技术资料

Metabolic imaging of brain tumors using (13)C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-(13)C] pyruvate is a promising neuroimaging strategy which,after a decade of preclinical success in glioblastoma (GBM) models,is now entering clinical trials in multiple centers. Typically,the presence of GBM has been associated with elevated hyperpolarized [1-(13)C] lactate produced from [1-(13)C] pyruvate,and response to therapy has been associated with a drop in hyperpolarized [1-(13)C] lactate. However,to date,lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation,which,in addition to initiating tumor development,also induces metabolic reprogramming. In particular,mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1,MCT4),three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of (13)C MRS of hyperpolarized [1-(13)C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA,MCT1 and MCT4,and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-(13)C] lactate compared to GBM,consistent with their metabolic reprogramming. Furthermore,hyperpolarized [1-(13)C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors,in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas,which,when combined with other clinically available imaging methods,could be used to detect the presence of the IDH1 mutation in vivo. View Publication

Malignant gliomas are the most common and deadly primary malignant brain tumors. In vivo orthotopic models could doubtless represent an appropriate tool to test novel treatment for gliomas. However,methods commonly used to monitor the growth of glioma inside the mouse brain are time consuming and invasive. We tested the reliability of a minimally invasive procedure,based on a secreted luciferase (Gaussia luciferase),to frequently monitor the changes of glioma size. Gluc activity was evaluated from blood samples collected from the tail tip of mice twice a week,allowing to make a growth curve for the tumors. We validated the correlation between Gluc activity and tumor size by analysing the tumor after brain dissection. We found that this method is reliable for monitoring human glioma transplanted in immunodeficient mice,but it has strong limitation in immunocompetent models,where an immune response against the luciferase is developed during the first weeks after transplant. View Publication

Pituitary somatotroph adenomas result in dysregulated growth hormone (GH) hypersecretion and acromegaly; however,regulatory mechanisms that promote GH hypersecretion remain elusive. Here,we provide evidence that STAT3 directly induces somatotroph tumor cell GH. Evaluation of pituitary tumors revealed that STAT3 expression was enhanced in human GH-secreting adenomas compared with that in nonsecreting pituitary tumors. Moreover,STAT3 and GH expression were concordant in a somatotroph adenoma tissue array. Promoter and expression analysis in a GH-secreting rat cell line (GH3) revealed that STAT3 specifically binds the Gh promoter and induces transcription. Stable expression of STAT3 in GH3 cells induced expression of endogenous GH,and expression of a constitutively active STAT3 further enhanced GH production. Conversely,expression of dominant-negative STAT3 abrogated GH expression. In primary human somatotroph adenoma-derived cell cultures,STAT3 suppression with the specific inhibitor S3I-201 attenuated GH transcription and reduced GH secretion in the majority of derivative cultures. In addition,S3I-201 attenuated somatotroph tumor growth and GH secretion in a rat xenograft model. GH induced STAT3 phosphorylation and nuclear translocation,indicating a positive feedback loop between STAT3 and GH in somatotroph tumor cells. Together,these results indicate that adenoma GH hypersecretion is the result of STAT3-dependent GH induction,which in turn promotes STAT3 expression,and suggest STAT3 as a potential therapeutic target for pituitary somatotroph adenomas. View Publication

Glioblastoma growth is driven by cancer cells that have stem cell properties,but molecular determinants of their tumorigenic behavior are poorly defined. In cancer,altered activity of the epigenetic modifiers Polycomb and Trithorax complexes may contribute to the neoplastic phenotype. Here,we provide the first mechanistic insights into the role of the Trithorax protein mixed lineage leukemia (MLL) in maintaining cancer stem cell characteristics in human glioblastoma. We found that MLL directly activates the Homeobox gene HOXA10. In turn,HOXA10 activates a downstream Homeobox network and other genes previously characterized for their role in tumorigenesis. The MLL-Homeobox axis we identified significantly contributes to the tumorigenic potential of glioblastoma stem cells. Our studies suggest a role for MLL in contributing to the epigenetic heterogeneity between tumor-initiating and non-tumor-initiating cells in glioblastoma. View Publication

BACKGROUND Mechanisms of glioma invasion remain to be fully elucidated. Glioma cells within glioblastoma multiforme (GBM) range from well-differentiated tumor cells to less-differentiated brain tumor-initiating cells (BTICs). The β2-subunit of Na(+)/K(+)-ATPase,called the adhesion molecule on glia (AMOG),is highly expressed in normal glia but is thought to be universally downregulated in GBM. To test our hypothesis that expression of AMOG is heterogeneous in GBM and confers a less invasive phenotype,we compared it between BTICs and differentiated cells from patient-matched GBM and then tested GBM invasion in vitro after AMOG overexpression. METHODS Immunohistochemistry,immunoblotting,and real-time PCR were used to characterize AMOG protein and mRNA expression in tumor samples,BTICs,and differentiated cells. Matrigel invasion assay,scratch assay,and direct cell counting were used for testing in vitro invasion,migration,and proliferation,respectively. RESULTS While AMOG expression is heterogeneous in astrocytomas of grades II-IV,it is lost in most GBM. BTICs express higher levels of AMOG mRNA and protein compared with patient-matched differentiated tumor cells. Overexpression of AMOG decreased GBM cell and BTIC invasion without affecting migration or proliferation. Knockdown of AMOG expression in normal human astrocytes increased invasion. CONCLUSIONS AMOG expression inhibits GBM invasion. Its downregulation increases invasion in glial cells and may also represent an important step in BTIC differentiation. These data provide compelling evidence implicating the role of AMOG in glioma invasion and provide impetus for further investigation. View Publication

Glioblastoma is the most common form of primary malignant brain tumour. These tumours are highly proliferative and infiltrative resulting in a median patient survival of only 14 months from diagnosis. The current treatment regimens are ineffective against the small population of cancer stem cells residing in the tumourigenic niche; however,a new therapeutic approach could involve the removal of these cells from the microenvironment that maintains the cancer stem cell phenotype. We have isolated multipotent sphere-forming cells from human high grade glioma (glioma sphere-forming cells (GSCs)) to investigate the adhesive and migratory properties of these cells in vitro. We have focused on the role of two closely related metalloproteinases ADAM10 and ADAM17 due to their high expression in glioblastoma and GSCs and their ability to activate cytokines and growth factors. Here,we report that ADAM10 and ADAM17 inhibition selectively increases GSC,but not neural stem cell,migration and that the migrated GSCs exhibit a differentiated phenotype. We also observed a correlation between nestin,a stem/progenitor marker,and fibronectin,an extracellular matrix protein,expression in high grade glioma tissues. GSCs adherence on fibronectin is mediated by α5β1 integrin,where fibronectin further promotes GSC migration and is an effective candidate for in vivo cancer stem cell migration out of the tumourigenic niche. Our results suggest that therapies against ADAM10 and ADAM17 may promote cancer stem cell migration away from the tumourigenic niche resulting in a differentiated phenotype that is more susceptible to treatment. View Publication

The processes involved in malignant gliomas damage were quantitatively evaluated by microscopy. The near-infrared fluorescent dye IR700 that is conjugated to an anti-CD133 antibody (IR700-CD133) specifically targets malignant gliomas (U87MG) and stem cells (BT142) and is endocytosed into the cells. The gliomas are then photodamaged by the release of reactive oxygen species (ROS) and the heat induced by illumination of IR700 by a red laser,and the motility of the vesicles within these cells is altered as a result of cellular damage. To investigate these changes in motility,we developed a new method that measures fluctuations in the intensity of phase-contrast images obtained from small areas within cells. The intensity fluctuation in U87MG cells gradually decreased as cell damage progressed,whereas the fluctuation in BT142 cells increased. The endocytosed IR700 dye was co-localized in acidic organelles such as endosomes and lysosomes. The pH in U87MG cells,as monitored by a pH indicator,was decreased and then gradually increased by the illumination of IR700,while the pH in BT142 cells increased monotonically. In these experiments,the processes of cell damage were quantitatively evaluated according to the motility of vesicles and changes in pH. View Publication

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently,reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture,apoptosis assays,protein expression,limiting dilution clonal frequency assay,genetic affymetrix analysis,and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin,P=0.9) were similar as well. Likewise,markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue,DiIC,caspase-3,and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition,genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally,glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional,protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence,both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. View Publication

AIM Bevacizumab has been reported to result in increased tumor invasion when used to treat malignant glioma. We hypothesized that BMP4 would prevent diffuse tumor infiltration induced by bevacizumab for malignant glioma in a xenograft model. METHODS Human glioblastoma (GBM) tumor cells were implanted in the striatum of immunocompromised mice. The animals were treated with bevacizumab and BMP4. Tumor growth and invasion were measured. RESULTS The bevacizumab-treated mice had increased survival compared with control animals (p = 0.02). BMP4 alone did not result in improved survival (p = 1.0). The bevacizumab (p = 0.006) and bevacizumab plus BMP4 (p = 0.006) groups demonstrated significantly decreased total tumor size compared with control. Tumor invasion was significantly decreased in the bevacizumab (p = 0.005),BMP4 (p = 0.04) alone and bevacizumab plus BMP4 (p = 0.002) groups compared with control. No synergistic effect between bevacizumab and BMP4 was observed. CONCLUSION Bevacizumab treatment did not result in diffuse infiltration of human GBM in a mouse xenograft model. BMP4 did have an independent favorable effect on GBM that was not synergistic with bevacizumab treatment. View Publication

Glioblastoma multiforme (GBM) comprises several molecular subtypes,including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment,such as tumor-associated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model,which significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly,TAMs were not depleted in treated mice. Instead,glioma-secreted factors,including granulocyte-macrophage CSF (GM-CSF) and interferon-γ (IFN-γ),facilitated TAM survival in the context of CSF-1R inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs,which is consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R inhibition for GBM. View Publication

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies characterized by an intense tumor stroma with hypoperfused regions,a significant inflammatory response and pronounced therapy resistance. New therapeutic agents are urgently needed. The plant-derived agent triptolide also known as thunder god vine" has a long history in traditional Chinese medicine for treatment of rheumatoid arthritis and cancer and is now in a clinical phase II trial for establishing the efficacy against a placebo. The authors mimicked the situation in patient tumors by induction of hypoxia in experimental models of pancreatic cancer stem cells (CSCs) and evaluated the therapeutic effect of triptolide. Hypoxia led to induction of colony and spheroid formation aldehyde dehydrogenase 1 (ALDH1) and NF-κB activity migratory potential and a switch in morphology to a fibroblastoid phenotype as well as stem cell- and epithelial-mesenchymal transition-associated protein expression. Triptolide efficiently inhibited hypoxia-induced transcriptional signaling and downregulated epithelial-mesenchymal transition (EMT) and CSC features in established highly malignant cell lines whereas sensitive cancer cells or nonmalignant cells were less affected. In vivo triptolide inhibited tumor take and tumor growth. In primary CSCs isolated from patient tumors triptolide downregulated markers of CSCs proliferation and mesenchymal cells along with upregulation of markers for apoptosis and epithelial cells. This study is the first to show that triptolide reverses EMT and CSC characteristics and therefore may be superior to current chemotherapeutics for treatment of PDA. View Publication

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with high mortality and has been well known to involve many molecular pathways,including G-protein coupled receptor (GPCR)-mediated signaling (such as epithelial growth factor receptor [EGFR] and platelet derived growth factor receptor [PDGFR]). G protein-coupled receptor kinases (GRK) directly regulate GPCR activity by phosphorylating activated agonist-bound receptors to desensitize signaling and internalize receptors through beta-arrestins. Recent studies in various cancers,including prostate and breast cancer,have highlighted the role of change in GRK expression to oncogenesis and tumor proliferation. In this study,we evaluated the expression of GRK5 in grade II to grade IV glioma specimens using immunohistochemistry and found that GRK5 expression levels are highly correlated with aggressiveness of glioma. We used culture conditions to selectively promote the growth of either glioblastoma cells with stem cell markers (GSC) or differentiated glioblastoma cells (DGC) from fresh GBM specimens. GSC are known to be highly invasive and mobile,and have the capacity to self-renew and are more resistant to chemotherapy and radiation compared to differentiated populations of GBM. We examined the expression of GRK5 in these two sets of culturing conditions for GBM cells and found that GRK5 expression is upregulated in GSC compared to differentiated GBM cells. To better understand the role of GRK5 in GBM-derived stem cells,we created stable GRK5 knockdown and evaluated the proliferation rate. Using an ATP chemiluminescence assay,we show,for the first time,that knocking down the expression of GRK5 decreased the proliferation rate of GSC in contrast to control. View Publication