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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

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID,celecoxib,to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. View Publication

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70,HSP90,GRP94,GRP58,HSP27,HSP40 and HSP60. OSU-03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1,receptors for Ebola/Marburg/Hepatitis A,Lassa fever,and Hepatitis B viruses,respectively. Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya,Mumps,Measles,Rubella,RSV,CMV,and Influenza viruses. OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU-03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU-03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus,Dna K and bacterial phosphodiesterases are novel antibiotic targets,and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. View Publication

The respiratory tract with its ease of access,vast surface area and dense network of antigen-presenting cells (APCs) represents an ideal target for immune-modulation. Bio-mimetic nanocarriers such as virosomes may provide immunomodulatory properties to treat diseases such as allergic asthma. In our study we employed a triple co-culture model of epithelial cells,macrophages and dendritic cells to simulate the human airway barrier. The epithelial cell line 16HBE was grown on inserts and supplemented with human blood monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) for exposure to influenza virosomes and liposomes. Additionally,primary human nasal epithelial cells (PHNEC) and EpCAM+ epithelial progenitor cell mono-cultures were utilized to simulate epithelium from large and smaller airways,respectively. To assess particle uptake and phenotype change,cell cultures were analyzed by flow cytometry and pro-inflammatory cytokine concentrations were measured by ELISA. All cell types internalized virosomes more efficiently than liposomes in both mono- and co-cultures. APCs like MDMs and MDDCs showed the highest uptake capacity. Virosome and liposome treatment caused a moderate degree of activation in MDDCs from mono-cultures and induced an increased cytokine production in co-cultures. In epithelial cells,virosome uptake was increased compared to liposomes in both mono- and co-cultures with EpCAM+ epithelial progenitor cells showing highest uptake capacity. In conclusion,all cell types successfully internalized both nanocarriers with virosomes being taken up by a higher proportion of cells and at a higher rate inducing limited activation of MDDCs. Thus virosomes may represent ideal carrier antigen systems to modulate mucosal immune responses in the respiratory tract without causing excessive inflammatory changes. View Publication

OBJECTIVE Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines,suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique,here we report the characterization of GBM cell lines including GBM variants. METHODS Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. RESULTS Thirteen oncosphere lines derived from GBM and GBM variants,including a GBM with PNET features and a GBM with oligodendroglioma component,were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas,including invasion,necrosis,abnormal vascularization,and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. CONCLUSIONS We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study. View Publication

Glioblastoma multiforme (GBM) is the most common primary malignant adult brain tumor and is associated with poor survival. Recently,stem-like cell populations have been identified in numerous malignancies including GBM. To identify genes whose expression is changed with differentiation,we compared transcript profiles from a GBM oncosphere line before and after differentiation. Bioinformatic analysis of the gene expression profiles identified podocalyxin-like protein (PODXL),a protein highly expressed in human embryonic stem cells,as a potential marker of undifferentiated GBM stem-like cells. The loss of PODXL expression upon differentiation of GBM stem-like cell lines was confirmed by quantitative real-time PCR and flow cytometry. Analytical flow cytometry of numerous GBM oncosphere lines demonstrated PODXL expression in all lines examined. Knockdown studies and flow cytometric cell sorting experiments demonstrated that PODXL is involved in GBM stem-like cell proliferation and oncosphere formation. Compared to PODXL-negative cells,PODXL-positive cells had increased expression of the progenitor/stem cell markers Musashi1,SOX2,and BMI1. Finally,PODXL expression directly correlated with increasing glioma grade and was a marker for poor outcome in patients with GBM. In summary,we have demonstrated that PODXL is expressed in GBM stem-like cells and is involved in cell proliferation and oncosphere formation. Moreover,high PODXL expression correlates with increasing glioma grade and decreased overall survival in patients with GBM. View Publication

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy,and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study,to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology,we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging,immunostaining,and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells,accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia,a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study,therefore,deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders. View Publication

Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton,adoption of stellate morphologies,and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial,but in the chronic injury phase,reactive astrocytes produce inhibitory factors (i.e.,chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS,we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However,the effects these compounds have on astrocytes are unknown. Specifically,we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis,we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44±0.03 (mean±standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative,but does not satisfy common metrics for a screenable" assay. As proof of principle we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis. View Publication

Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7,P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture,but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive,glucose-transporter-2-low (Ins(+)Glut2(LO)) cells,representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7,were retained into adulthood,and a subset differentiated into endocrine,ductal,and neural lineages,illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new,functional β-cells,and which may be potentially exploited for regenerative therapies in the future. View Publication

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine,disease modeling,and drug screening. However,their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research,only the best,competent clones should be used. The standard assays for pluripotency are based on genomic approaches,which take up to 1 week to perform and incur significant cost. Therefore,there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here,we describe a novel multiplexed,high-throughput,and sensitive peptide-based multiple reaction monitoring mass spectrometry assay,allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests. View Publication

Asthma is highly prevalent,but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study,we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore,deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17,miR-144,and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment,while it's expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally,we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies. View Publication

We inhale respiratory pathogens continuously,and the subsequent signaling events between host and microbe are complex,ultimately resulting in clearance of the microbe,stable colonization of the host,or active disease. Traditional in vitro methods are ill-equipped to study these critical events in the context of the lung microenvironment. Here we introduce a microscale organotypic model of the human bronchiole for studying pulmonary infection. By leveraging microscale techniques,the model is designed to approximate the structure of the human bronchiole,containing airway,vascular,and extracellular matrix compartments. To complement direct infection of the organotypic bronchiole,we present a clickable extension that facilitates volatile compound communication between microbial populations and the host model. Using Aspergillus fumigatus,a respiratory pathogen,we characterize the inflammatory response of the organotypic bronchiole to infection. Finally,we demonstrate multikingdom,volatile-mediated communication between the organotypic bronchiole and cultures of Aspergillus fumigatus and Pseudomonas aeruginosa. View Publication