Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition.

Acquired drug resistance prevents cancer therapies from achieving stable and complete responses. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the survival of residual cancer 'persister' cells. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance

Evaluation of pre-analytical factors affecting plasma DNA analysis.

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies

Controlling Nonspecific Adsorption of Proteins at Bio-Interfaces for Biosensor and Biomedical Applications

Partitioning of poly(ethyleneglycol) (PEG) molecules in 2-D and 3-D systems is presented as a self-assembly approach for controlling non-specific adsorption of proteins at interfaces. Lateral restructuring of multi-component Langmuir monolayers to accommodate adsorbing proteins was investigated as a model 2-D system. Ferritin adsorption to monolayers containing cationic, nonionic, and PEG bearing phospholipids induced protein sized binding pockets surrounded by PEG rich regions. The number, size, and distribution of protein imprint sites were controlled by the molar ratios, miscibility, and lateral mobility of the lipids. The influence of PEG chain length on the ternary monolayer restructuring and protein distribution was also investigated using DSPE-PEGx (x= 7, 16, 22). Monolayer miscibility analysis demonstrated that longer PEG chains diminished the condensed phase formation for a fixed ratio of lipids. Thus, incorporation of longer PEG chains, intended to diminish protein adsorption outside of the imprint sites of cationic / non-ionic lipids, leads to dramatic changes in monolayer phase behavior and protein distribution in this 2-D system. The assembly of PEG-amphiphiles at elastomer surfaces and subsequent protein adsorption was investigated as a model 3-D system. Polydimethylsiloxane (PDMS) substrates were modified with block copolymers comprised of PEG and PDMS segments by two methods: (1) the block copolymer was mixed with PDMS during polymerization; (2) the block copolymer diffused into solvent swollen PDMS monoliths. Hydrophilic surfaces resulted for both approaches that, for 600 D block copolymer, exhibited up to 85% reduction in fibrinogen adsorption as compared to native PDMS. Higher MW block copolymers (up to 3000 D) resulted in less hydrophilic surfaces and greater protein adsorption, presumably due to diffusion limitations of copolymer in the PDMS monolith. All modified PDMS surfaces were dynamic and restructured when cycled between air and water. PDMS transparency also decreased with increase in block copolymer concentration for both methods, limiting this modification protocol for applications requiring high polymer transparency. The 2-D system presents a bottom-up approach, where adsorbing protein constructs the binding site, while the 3-D system presents a top down approach, where protein-binding elements may be introduced into the PEG-bearing polymer for fabrication of surfaces with controlled protein adsorption

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) promotes glioblastoma cell chemotaxis via Lyn activation

Daya tarik wisata religi Indonesia merupakan anugerah yang dimiliki bangsa Indonesia, demikian keragaman budaya dengan potensi yang demikian sempurna. Pengembangan pariwisata religi dapat memberikan manfaat sebesar-besarnya bagi pembangunan, maka dalam pelaksanaannya dibutuhkan strategi yang terencana dan sistematis agar kepuasan serta loyalitas pengunjung dapat terbentuk. Penelitian ini merupakan penelitian lapangan (field research) dengan pendekatan kuantitatif. Data yang digunakan adalah data primer dan sekunder dengan metode survei melalui penyebaran kuesioner. Terdapat tiga variabel independen dalam penelitian ini yaitu (X1) kualitas layanan syariah, (X2) nilai-nilai Islam, dan (X3) citra destinasi. Variabel dependen (Y1) dalam penelitian ini adalah kepuasan pengunjung. Sedangkan variabel mediasi (Y2) adalah loyalitas. Populasi dalam penelitian ini adalah seluruh pengunjung atau peziarah wisata Masjid Agung Demak dalam kurun waktu satu tahun terakhir. Teknik pengambilan sampel dilakukan dengan purposive sampling. Pengumpulan data dalam penelitian ini dilakukan dengan membagikan kuesioner sejumlah 167 kepada para responden. Penelitian ini menggunakan alat analisis SEM-PLS dengan bantuan program WarpPLS 3.0. Hasil penelitian ini menunjukkan bahwa: (1) kualitas layanan syariah tidak berpengaruh terhadap loyalitas dengan koefisien ß=0,03 dan p=0,39 (2) nilai-nilai Islam berpengaruh positif dan signifikan secara langsung terhadap loyalitas dengan koefisien ß=0,34 dan p<0,01 (3) citra destinasi berpengaruh positif dan signifikan secara langsung terhadap loyalitas dengan koefisien ß=0,38 dan p<0,01 (4) Kualitas layanan syariah tidak berpengaruh terhadap kepuasan pengunjung dengan koefisien ß=0,16 dan p=0,1 (5) nilai-nilai Islam tidak berpengaruh terhadap kepuasan pengunjung dengan koefisien ß=0,12 dan p=0,07 (6) citra destinasi berpengaruh positif dan signifikan secara langsung terhadap kepuasan pengunjung dengan koefisien ß=0,64 dan p<0,01 (7) kepuasan pengunjung berpengaruh positif dan signifikan terhadap loyalitas dengan koefisien ß=0,24 dan p<0,01 (8) kepuasan pengunjung tidak memediasi hubungan antara kualitas layanan syariah dan loyalitas ditunjukkan pada model direct dan indirect tidak signifikan p=0,39 dan p=0,47 (9) kepuasan pengunjung tidak memediasi hubungan antara nilai-nilai Islam dan loyalitas ditunjukkan pada model direct dan indirect koefisien naik dari ß=0,34 menjadi ß=0,35 (10) kepuasan pengunjung memediasi sebagian hubungan antara citra destinasi dan loyalitas dengan koefisien turun dari ß=0,38 menjadi ß=0,26 serta tetap signifikan p<0,01

Phase 0 Trial of AZD1775 in First-Recurrence Glioblastoma Patients

AZD1775 is a first-in-class Wee1 inhibitor with dual function as a DNA damage sensitizer and cytotoxic agent. A phase I study of AZD1775 for solid tumors suggested activity against brain tumors, but a preclinical study indicated minimal blood-brain barrier penetration in mice. To resolve this controversy, we examined the pharmacokinetics and pharmacodynamics of AZD1775 in patients with first-recurrence, glioblastoma. Twenty adult patients received a single dose of AZD1775 prior to tumor resection and enrolled in either a dose-escalation arm or a time-escalation arm. Sparse pharmacokinetic blood samples were collected, and contrast-enhancing tumor samples were collected intraoperatively. AZD1775 total and unbound concentrations were determined by a validated LC/MS-MS method. Population pharmacokinetic analysis was performed to characterize AZD1775 plasma pharmacokinetic profiles. Pharmacodynamic endpoints were compared to matched archival tissue. The AZD1775 plasma concentration-time profile following a single oral dose in patients with glioblastoma was well-described by a one-compartment model. Glomerular filtration rate was identified as a significant covariate on AZD1775 apparent clearance. AZD1775 showed good brain tumor penetration, with a median unbound tumor-to-plasma concentration ratio of 3.2, and achieved potential pharmacologically active tumor concentrations. Wee1 pathway suppression was inferred by abrogation of G arrest, intensified double-strand DNA breakage, and programmed cell death. No drug-related adverse events were associated with this study. In contrast to recent preclinical data, our phase 0 study of AZD 1775 in recurrent glioblastoma indicates good human brain tumor penetration, provides the first evidence of clinical biological activity in human glioblastoma, and confirms the utility of phase 0 trials as part of an accelerated paradigm for drug development in patients with glioma.

Reciprocal Activation of Transcription Factors Underlies the Dichotomy between Proliferation and Invasion of Glioma Cells

<div><p>Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the “Go or Grow” hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the “Go or Grow” hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens <i>in situ</i> (n = 44) revealed higher proliferation (Ki67 labeling index) in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-κB activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-κB- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-κB activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-κB staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation <i>in vitro</i>, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-κB by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration <i>in vitro</i> and invasion <i>ex vivo</i>. Notably, inhibition of NF-κB was found to have no effect on the proliferation rate of glioma cells. These findings suggest that the reciprocal and coordinated suppression/activation of transcription factors, such as c-Myc and NF-κB may underlie the shift of glioma cells from a “growing-to-going” phenotype.</p></div

Transcription Factor Profiling of Migrating Glioma Cells vs.

<p><b>Migration-Restricted Glioma Cells.</b> Tumor cells were seeded in either migration-activated “sparse” condition or in migration-restricted “dense” condition on SF767 glioma-derived ECM. Nuclear lysates were collected and hybridized with biotinylated-DNA probes specific to 19 transcription factors as per Marligen’s transcription kit and assayed using the Luminex 200. Two independent biological replicates were performed with each sample in triplicate. Ratios of the averaged mean fluorescent intensities for each transcription factor for sparse over dense were calculated for each biological set and average values of two biological replicates are plotted in the heat map above (For ratios of the average MFI for each biological replicate is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072134#pone.0072134.s002" target="_blank">Figure S2</a>). The heat map was constructed using a conditionally formatted color range. Green boxes represent the transcription factors activated when cells were in a migration-activated condition (sparse/dense ratios ≥1.5). Red boxes represent transcription factors activated when cells were in a migration-restricted condition (sparse/dense ratios ≤0.6). Yellow boxes indicate no change in transcription activity (sparse/dense ratios between 0.65 and 1.5).</p

Glioma tumor specimens show differential activation of c-Myc and NF-κB in core and invasive rim.

<p>Immunohistochemistry of matched glioma core and rim sample from a glioma invasion specific tissue microarray (n = 45). Phosphorylated c-Myc nuclear protein expression is greater at the glioma tumor core than the rim regions of tumor. Representative 10X images of core (A) and rim (C) of a GBM sample. 20X images of core (B) and rim (D) of the same GBM sample. Phosphorylated NFκB nuclear protein expression is greater at the glioma tumor rim than the core regions of tumor. Representative 10X images of core (E) and rim (G) of a GBM sample. 20X images of core (F) and rim (H) of the same GBM sample. Black arrows represent positively stained nuclear regions of the glioma tumor cells.</p

Migrating glioma cells promote activation of the transcription factor NF-κB whereas migration-restricted glioma cells display high c-Myc activation.

<p>T98G and SNB19 glioma cells were infected with lentivirus expressing the binding element for either the transcription factor NF-kB and a green fluorescent protein (GFP) reporter or the transcription factor c-Myc and a red fluorescent protein (tdTomato) reporter. Cells were plated in a migration stimulating environment and imaged after 48 hrs. (A) & (B) Quantitative analysis of GFP positive T98G and SNB19 glioma cells respectively, at the core and the rim in radial monolayer assay (n = 5). (C) & (D) Quantitative analysis of tdTomato positive T98G and SNB19 glioma cells respectively, at the core and the rim in radial monolayer assay (n = 5). (E) & (F) Fluorescent micrographs (20X) of mCMV control GFP vector infected T98G and SNB19 glioma cells respectively. (G) & (H) Fluorescent micrographs (20X) of control tdTomato vector infected T98G and SNB19 glioma cells respectively. (I) & (J) Fluorescent micrographs (20X) of NF-κB reporter vector infected T98G and SNB19 glioma cells respectively. (K) & (L) Fluorescent micrographs (20X) of tdTomato reporter vector infected T98G and SNB19 glioma cells respectively. Green cells are GFP positive and blue cells are not expressing the GFP protein but are stained with Hoescht stain. Red cells are tdTomato positive and blue cells are not expressing the tdTomato protein but are stained with Hoescht stain. Fluorescent micrographs of core regions are depicted by “C” and corresponding rim regions are depicted by “R”. Error bar represent the standard deviation of n = 5 observations. Asterisk (*) represents p value <0.05.</p