技术资料

Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind,we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation,of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin,deguelin,patulin and phenethyl caffeate) exhibited high cytotoxicity,with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular,the FDA approved drug for the treatment of alcoholism,disulfiram (DSF),was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu),which markedly increased GSC death. DSF-Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs,consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier,we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM. View Publication

Peripheral nerve injuries and neuropathies lead to profound functional deficits. Here,we have demonstrated that muscle-derived stem/progenitor cells (MDSPCs) isolated from adult human skeletal muscle (hMDSPCs) can adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model. Transplanted hMDSPCs surrounded the axonal growth cone,while hMDSPCs infiltrating the regenerating nerve differentiated into myelinating Schwann cells. Engraftment of hMDSPCs into the area of the damaged nerve promoted axonal regeneration,which led to functional recovery as measured by sustained gait improvement. Furthermore,no adverse effects were observed in these animals up to 18 months after transplantation. Following hMDSPC therapy,gastrocnemius muscles from mice exhibited substantially less muscle atrophy,an increase in muscle mass after denervation,and reorganization of motor endplates at the postsynaptic sites compared with those from PBS-treated mice. Evaluation of nerve defects in animals transplanted with vehicle-only or myoblast-like cells did not reveal histological or functional recovery. These data demonstrate the efficacy of hMDSPC-based therapy for peripheral nerve injury and suggest that hMDSPC transplantation has potential to be translated for use in human neuropathies. View Publication

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

Aldehyde dehydrogenase (ALDH) has been identified in stem cells from both normal and cancerous tissues. This study aimed to evaluate the potential of ALDH as a universal brain tumour initiating cell (BTIC) marker applicable to primary brain tumours and their biological role in maintaining stem cell status. Cells from various primary brain tumours (24paediatric and 6 adult brain tumours) were stained with Aldefluor and sorted by flow cytometry. We investigated the impact of ALDH expression on BTIC characteristics in vitro and on tumourigenic potential in vivo. Primary brain tumours showed universal expression of ALDH,with 0.3-28.9% of the cells in various tumours identified as ALDH(+). The proportion of CD133(+) cells within ALDH(+) is higher than ALDH cells. ALDH(+) cells generate neurospheres with high proliferative potential,express neural stem cell markers and differentiate into multiple nervous system lineages. ALDH(+) cells tend to show high expression of induced pluripotent stem cell-related genes. Notably,targeted knockdown of ALDH1 by shRNA interference in BTICs potently disturbed their self-renewing ability. After 3months,ALDH(+) cells gave rise to tumours in 93% of mice whereas ALDH cells did not. The characteristic pathology of mice brain tumours from ALDH(+) cells was similar to that of human brain tumours,and these cells are highly proliferative in vivo. Our data suggest that primary brain tumours contain distinct subpopulations of cells that have high expression levels of ALDH and BTIC characteristics. ALDH might be a potential therapeutic target applicable to primary brain tumours. View Publication

The Hedgehog (Hh) pathway regulates the growth of a subset of adult gliomas and better definition of Hh-responsive subtypes could enhance the clinical utility of monitoring and targeting this pathway in patients. Somatic mutations of the isocitrate dehydrogenase (IDH) genes occur frequently in WHO grades II and III gliomas and WHO grade IV secondary glioblastomas. Hh pathway activation in WHO grades II and III gliomas suggests that it might also be operational in glioblastomas that developed from lower-grade lesions. To evaluate this possibility and to better define the molecular and histopathological glioma subtypes that are Hh-responsive,IDH genes were sequenced in adult glioma specimens assayed for an operant Hh pathway. The proportions of grades II-IV specimens with IDH mutations correlated with the proportions that expressed elevated levels of the Hh gene target PTCH1. Indices of an operational Hh pathway were measured in all primary cultures and xenografts derived from IDH-mutant glioma specimens,including IDH-mutant glioblastomas. In contrast,the Hh pathway was not operational in glioblastomas that lacked IDH mutation or history of antecedent lower-grade disease. IDH mutation is not required for an operant pathway however,as significant Hh pathway modulation was also measured in grade III gliomas with wild-type IDH sequences. These results indicate that the Hh pathway is operational in grades II and III gliomas and glioblastomas with molecular or histopathological evidence for evolvement from lower-grade gliomas. Lastly,these findings suggest that gliomas sharing this molecularly defined route of progression arise in Hh-responsive cell types. View Publication

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis,tumor maintenance and therapeutic resistance. Thus,to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse,at least in part,the gene expression signature of GBM and GSCs,this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here,we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened,thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine,we found that thioridazine induces autophagy in GBM cell lines,and upregulates AMPK activity. Moreover,LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition,thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent,but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties. View Publication