JNK pathway inhibition selectively primes pancreatic cancer stem cells to TRAIL-induced apoptosis without affecting the physiology of normal tissue resident stem cells

Lipids are increasingly recognized as bioactive mediators of extracellular vesicle (EV) functions. However, while EV proteins and nucleic acids are well described, EV lipids are insufficiently understood due to lack of adequate quantitative methods. We adapted an established targeted and quantitative mass spectrometry (LC-MS/MS) method originally developed for analysis of 94 eicosanoids and seven polyunsaturated fatty acids (PUFA) in human plasma. Additionally, the influence of freeze–thaw (FT) cycles, injection volume, and extraction solvent were investigated. The modified protocol was applied to lipidomic analysis of differently polarized macrophage-derived EVs. We successfully quantified three PUFAs and eight eicosanoids within EVs. Lipid extraction showed reproducible PUFA and eicosanoid patterns. We found a particularly high impact of FT cycles on EV lipid profiles, with significant reductions of up to 70%. Thus, repeated FT will markedly influence analytical results and may alter EV functions, emphasizing the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs. EV lipid profiles differed largely depending on the polarization of the originating macrophages. Particularly, we observed major changes in the arachidonic acid pathway. We emphasize the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs.

Background: The “Liquid Biopsy” has become a powerful tool for cancer research during the last decade. Circulating cell-free DNA (cfDNA) that originates from tumors has emerged as one of the most promising analytes. In contrast to plasma-derived cfDNA, only a few studies have investigated urinary cfDNA. One reason might be rapid degradation and hence inadequate concentrations for downstream analysis. In this study, we examined the stability of cfDNA in urine using different methods of preservation under various storage conditions. Methodology: To mimic patient samples, a pool of healthy male and female urine donors was spiked with a synthetic cfDNA reference standard (fragment size 170 bp) containing the T790M mutation in the EGFR gene. Spiked samples were preserved with three different buffers and with no buffer over four different storage periods (0 h; 4 h; 12 h; 24 h) at room temperature vs. 4 °C. The preservatives used were Urinary Analyte Stabilizer (UAS, Novosanis, Wijnegem, Belgium), Urine Conditioning Buffer (UCB, Zymo, Freiburg, Germany) and a self-prepared buffer called “AlloU”. CfDNA was extracted using the QIAamp MinElute ccfDNA Mini Kit (Qiagen, Hilden, Germany). CfDNA concentration was measured using the Qubit™ 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Droplet digital PCR (ddPCR) was used for detection and quantification of the T790M mutation. Results: Almost no spiked cfDNA was recoverable from samples with no preservation buffer and the T790M variant was not detectable in these samples. These findings indicate that cfDNA was degraded below the detection limit by urinary nucleases. Stabilizing buffers showed varying efficiency in preventing this degradation. The most effective stabilizing buffer under all storage conditions was the UAS, enabling adequate recovery of the T790M variant using ddPCR. Conclusion: From a technical point of view, stabilizing buffers and adequate storage conditions are a prerequisite for translation of urinary cfDNA diagnostics into clinical routine.

Background: Urinary ethyl glucuronide (EtG) has emerged as the biomarker of choice for alcohol abstinence monitoring in forensic toxicology and is now used in the listing decision process for liver transplantations (LTs) in the German transplant program. However, EtG analysis in this patient group is challenging due to severely impaired liver function, renal failure, co-morbidities and multidrug regimens. The aim of our study was to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based EtG analysis for a precise abstinence monitoring in transplant candidates. Methods: EtG and ethyl sulfate (EtS) were analyzed by a commercial LC-MS/MS assay in 1787 spot urine samples of 807 patients (>85% from the Department of Hepatology) using a combination of quantifier and two qualifier mass transitions for each analyte. Influences of bacterial contamination, kidney and liver function were investigated. Results: Two hundred and sixty-four urine samples had elevated (≥0.5 mg/L) EtG concentrations when only analyzing one quantifier mass transition. Eleven results (4.2%) were found to be false positive after combining three mass transitions for EtG quantification and verification with parallel analysis of EtS. Decreased kidney function was associated with a significantly higher rate of positive EtG samples. One of the false positive results was caused by bacterial metabolism. Conclusions: Multimorbid pre-transplant patients have a high risk of individual analytical disturbances of EtG results obtained by LC-MS/MS. Therefore, EtG and EtS should always be measured by a combination of one quantifier and two qualifiers each and evaluated together.