The Challenge to Stabilize, Extract and Analyze Urinary Cell-Free DNA (ucfDNA) during Clinical Routine.

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.

Targeted Lipidomics for Characterization of PUFAs and Eicosanoids in Extracellular Vesicles.

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.

Challenges of LC-MS/MS ethyl glucuronide analysis in abstinence monitoring of liver transplant candidates.

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.

Sequential Filtration: A Gentle Method for the Isolation of Functional Extracellular Vesicles.

A prevalent challenge in isolating extracellular vesicles (EVs) from biological fluids is the reliable depletion of abundant contaminants-including free proteins and biomolecules, as well as nontarget vesicle subpopulations and other nanoparticulates-from the sample matrix while maximizing recovery. Sequential Filtration is a recently published approach for the size-based isolation of exosomes that is ideally suited for large-volume biofluid samples such as ascites , urine , lavage fluid, or cell-conditioned media. We describe a straightforward, three-step protocol comprising back-to-back steps of dead-end (normal) filtration, tangential-flow filtration, and track-etched membrane filtration that can be applied to yield a homogeneous population of exosome-sized extracellular vesicles. The approach is scalable and employs relatively gentle manipulation forces to fractionate and concentrate extracellular vesicles with good purity and functional integrity.

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

OBJECTIVE: Successful treatment of solid cancers mandates targeting cancer stem cells (CSC) without impact on the physiology of normal tissue resident stem cells. C-Jun N-terminal kinase (JNK) signaling has been shown to be of importance in cancer. We test whether JNK inhibition would sensitize pancreatic CSCs to induction of apoptosis via low-dose TNFα-related apoptosis-inducing ligand (TRAIL). DESIGN: Effects of JNK inhibition (JNKi) were evaluated in vitro in functional assays, through mRNA and protein expression analysis, and in in vivo mouse studies. CSCs were enriched in anoikis-resistant spheroid culture and analyzed accordingly. RESULTS: We confirmed that the JNK pathway is an important regulatory pathway in pancreatic cancer stem cells and further found that JNK inhibition downregulates the decoy receptor DcR1 through IL-8 signaling while upregulating pro-apoptotic death receptors DR4/5, thereby sensitizing cells - even with acquired TRAIL-resistance - to apoptosis induction. Treatment of orthotopic pancreatic cancer xenografts with either gemcitabine, JNKi or TRAIL alone for 4 weeks showed only modest effects compared to control, while the combination of JNKi and TRAIL resulted in significantly lower tumor burden (69%; p < 0.04), reduced numbers of circulating tumor cells, and less distant metastatic events, without affecting the general health of the animals. CONCLUSIONS: The combination of JNKi and TRAIL significantly impacts on CSCs, but leaves regular tissue-resident stem cells unaffected - even under hypoxic stress conditions. This concept of selective treatment of pancreatic CSCs warrants further evaluation.

Benchtop isolation and characterization of functional exosomes by sequential filtration.

Early and minimally invasive detection of malignant events or other pathologies is of utmost importance in the pursuit of improved patient care and outcomes. Recent evidence indicates that exosomes and extracellular vesicles in serum and body fluids can contain nucleic acid, protein, and other biomarkers. Accordingly, there is great interest in applying these clinically as prognostic, predictive, pharmacodynamic, and early detection indicators. Nevertheless, existing exosome isolation methods can be time-consuming, require specialized equipment, and/or present other inefficiencies regarding purity, reproducibility and assay cost. We have developed a straightforward, three-step protocol for exosome isolation of cell culture supernatants or large volumes of biofluid based on sequential steps of dead-end pre-filtration, tangential flow filtration (TFF), and low-pressure track-etched membrane filtration that we introduce here. Our approach yields exosome preparations of high purity and defined size distribution and facilitates depletion of free protein and other low-molecular-weight species, extracellular vesicles larger than 100nm, and cell debris. Samples of exosomes prepared using the approach were verified morphologically by nanoparticle tracking analysis and electron microscopy, and mass spectrometry analyses confirmed the presence of previously reported exosome-associated proteins. In addition to being easy-to-implement, sequential filtration yields exosomes of high purity and, importantly, functional integrity as a result of the relatively low-magnitude manipulation forces employed during isolation. This answers an unmet need for preparation of minimally manipulated exosomes for investigations into exosome function and basic biology. Further, the strategy is amenable to translation for clinical exosome isolations because of its speed, automatability, scalability, and specificity for isolating exosomes from complex biological samples.