技术资料

The quantification of cellular mechanical properties is of tremendous interest in biology and medicine. Recent microfluidic technologies that infer cellular mechanical properties based on analysis of cellular deformations during microchannel traversal have dramatically improved throughput over traditional single-cell rheological tools,yet the extraction of material parameters from these measurements remains quite complex due to challenges such as confinement by channel walls and the domination of complex inertial forces. Here,we describe a simple microfluidic platform that uses hydrodynamic forces at low Reynolds number and low confinement to elongate single cells near the stagnation point of a planar extensional flow. In tandem,we present,to our knowledge,a novel analytical framework that enables determination of cellular viscoelastic properties (stiffness and fluidity) from these measurements. We validated our system and analysis by measuring the stiffness of cross-linked dextran microparticles,which yielded reasonable agreement with previously reported values and our micropipette aspiration measurements. We then measured viscoelastic properties of 3T3 fibroblasts and glioblastoma tumor initiating cells. Our system captures the expected changes in elastic modulus induced in 3T3 fibroblasts and tumor initiating cells in response to agents that soften (cytochalasin D) or stiffen (paraformaldehyde) the cytoskeleton. The simplicity of the device coupled with our analytical model allows straightforward measurement of the viscoelastic properties of cells and soft,spherical objects. View Publication

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM),and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors,and both EGFR and Rictor are associated with increased proliferation,invasion,metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines,including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing,tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan,temozolomide and vincristine. In LN229,co-silencing of EGFR and Rictor resulted in reduced cell migration,and increased sensitivity to vincristine and temozolomide. In U118MG,silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo,while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth,silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM. 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 Glioblastoma (GBM),being a highly vascularised and locally invasive tumour,is an attractive target for anti-angiogenic and anti-invasive therapies. The GBM/endothelial cell response to gossypol/temozolomide (TMZ) treatment was investigated with a particular aim to assess treatment effects on cancer hallmarks. METHODS Cell viability,endothelial tube formation and GBM tumour cell invasion were variously assessed following combined treatment in vitro. The U87MG-luc2 subcutaneous xenograft model was used to investigate therapeutic response in vivo. Viable tumour response to treatment was interrogated using immunohistochemistry. Combined treatment protocols were also tested in primary GBM patient-derived cultures. RESULTS An endothelial/GBM cell viability inhibitory effect,as well as an anti-angiogenic and anti-invasive response,to combined treatment have been demonstrated in vitro. A significantly greater anti-proliferative (P=0.020,P=0.030),anti-angiogenic (P=0.040,P<0.0001) and pro-apoptotic (P=0.0083,P=0.0149) response was observed when combined treatment was compared with single gossypol/TMZ treatment response,respectively. GBM cell line and patient-specific response to gossypol/TMZ treatment was observed. CONCLUSIONS Our results indicate that response to a combined gossypol/TMZ treatment is related to inhibition of tumour-associated angiogenesis,invasion and proliferation and warrants further investigation as a novel targeted GBM treatment strategy. View Publication

Giant cell tumor of bone (GCTB) is a very rare tumor entity,which is little examined owing to the lack of established cell lines and mouse models and the restriction of available primary cell lines. The stromal cells of GCTB have been made responsible for the aggressive growth and metastasis,emphasizing the presence of a cancer stem cell population. To identify and target such tumor-initiating cells,stromal cells were isolated from eight freshly resected GCTB tissues. Tumorigenic properties were examined by colony and spheroid formation,differentiation,migration,MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,immunohistochemistry,antibody protein array,Alu in situ hybridization,FACS analysis and xenotransplantation into fertilized chicken eggs and mice. A sub-population of the neoplastic stromal cells formed spheroids and colonies,differentiated to osteoblasts,migrated to wounded regions and expressed the metastasis marker CXC-chemokine receptor type 4,indicating self-renewal,invasion and differentiation potential. Compared with adherent-growing cells,markers for pluripotency,stemness and cancer progression,including the CSC surface marker c-Met,were enhanced in spheroidal cells. This c-Met-enriched sub-population formed xenograft tumors in fertilized chicken eggs and mice. Cabozantinib,an inhibitor of c-Met in phase II trials,eliminated CSC features with a higher therapeutic effect than standard chemotherapy. This study identifies a c-Met(+) tumorigenic sub-population within stromal GCTB cells and suggests the c-Met inhibitor cabozantinib as a new therapeutic option for targeted elimination of unresectable or recurrent GCTB. View Publication

BACKGROUND Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However,resistance inevitably emerges against these therapies,particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets. PROCEDURE High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels,p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer,neurosphere,and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics,100 mg/kg was I.P. injected into mice and tissues were collected at various time points. RESULTS Daoy,ONS76,UW228,and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth,induced apoptosis,and sensitized cells to SHH agents. Notably,RSK expression is correlated with SHH patients. In mice,BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB). CONCLUSIONS RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly,BI-D1870 crosses the BBB,acting as a scaffold for development of more long-lived RSK inhibitors. View Publication

Medulloblastoma (MB) is a highly malignant pediatric brain tumor. Despite aggressive therapy,many patients succumb to the disease,and survivors experience severe side effects from treatment. MYC-driven MB has a particularly poor prognosis and would greatly benefit from more effective therapies. We used an animal model of MYC-driven MB to screen for drugs that decrease viability of tumor cells. Among the most effective compounds were histone deacetylase inhibitors (HDACIs). HDACIs potently inhibit survival of MYC-driven MB cells in vitro,in part by inducing expression of the FOXO1 tumor suppressor gene. HDACIs also synergize with phosphatidylinositol 3-kinase inhibitors to inhibit tumor growth in vivo. These studies identify an effective combination therapy for the most aggressive form of MB. View Publication

Gliomas represent approximately 30% of all central nervous system tumors and 80% of malignant brain tumors. To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1),we studied paired tumor samples from 41 patients,comparing higher-grade,progressed samples to their lower-grade counterparts. Integrated genomic analyses,including whole-exome sequencing and copy number,gene expression and DNA methylation profiling,demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions,as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach. View Publication