We present a simple cell docking method induced by receding meniscus to capture non-adherent yeast cells onto microwells inside a microfluidic channel. Microwells were fabricated either by capillary moulding of UV curable polyurethane acrylate (PUA) onto glass substrate or direct replica moulding of poly(dimethyl siloxane) (PDMS). A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into the microfluidic channel by surface tension driven capillary flow and a receding meniscus was subsequently generated by evaporation. As the meniscus progressed, one to multiple yeast cells were spontaneously captured onto microwells by lateral capillary force created at the bottom of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (a-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that a-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for an additional 60 min without changes.

Tumor cells shed an abundance of extracellular vesicles (EVs) to body fluids containing bioactive molecules including DNA, RNA, and protein. Investigations in the field of tumor-derived EVs open a new horizon in understanding cancer biology and its potential as cancer biomarkers as well as platforms for personalized medicine. This study demonstrates that successfully isolated EVs from plasma and bronchoalveolar lavage fluid (BALF) of non-small cell lung cancer (NSCLC) patients contain DNA that can be used for EGFR genotyping through liquid biopsy. In both plasma and BALF samples, liquid biopsy results using EV DNA show higher accordance with conventional tissue biopsy compared to the liquid biopsy of cfDNA. Especially, liquid biopsy with BALF EV DNA is tissue-specific and extremely sensitive compared to using cfDNA. Furthermore, use of BALF EV DNA also demonstrates higher efficiency in comparison to tissue rebiopsy for detecting p.T790 M mutation in the patients who developed resistance to EGFR-TKIs. These finding demonstrate possibility of liquid biopsy using EV DNA potentially replacing the current diagnostic methods for more accurate, cheaper, and faster results.Electronic supplementary materialThe online version of this article (10.1186/s12943-018-0772-6) contains supplementary material, which is available to authorized users.

BackgroundEGFR genotyping in pulmonary adenocarcinoma patients who develop pleural effusions is mostly performed using cytology or cell block slides with low sensitivity. Liquid biopsy using the supernatant of pleural effusions may be more effective because they contain many components released by cancer cells. Extracellular vesicles (EVs) are known to carry oncogenic double-stranded DNA that is considered a notable biomarker. Here, we investigate the efficiency of liquid biopsy using cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (EV-derived DNA) from the supernatant of pleural effusions for EGFR genotyping in patients with pulmonary adenocarcinoma.MethodsFifty pleural effusion samples from patients with pulmonary adenocarcinoma were evaluated. The supernatant, after removing the cell pellet by centrifugation, was used for liquid biopsy, and EVs were isolated from the pleural effusion by ultracentrifugation. EV-derived DNA and cfDNA were extracted separately, and EGFR genotyping was performed by the PNA clamping method.ResultsAmong 32 patients who were EGFR-tyrosine kinase inhibitor (TKI) naïve with a known tissue EGFR genotype, liquid biopsy using EV-derived DNA from the pleural effusion supernatant showed 100% matching results with tissue EGFR genotyping in 19 EGFR mutant cases and detected three additional EGFR mutations in patients with wild-type (WT) tissue. Liquid biopsy using cfDNA from pleural effusion supernatants missed two cases of tissue-based EGFR mutations and found two additional EGFR mutation cases. In 18 patients who acquired resistance to EGFR-TKI, EGFR genotyping using EV-derived DNA from the pleural effusion supernatant detected the T790 M mutation in 13 of 18 (72.2%) patients, and this mutation was detected in 11 (61.1%) patients using cfDNA. By contrast, only three patients were found to present the T790 M mutation when using cell block or cytology slides.ConclusionsLiquid biopsy using the supernatant of pleural effusions showed significantly improved results for EGFR genotyping compared to those using conventional cell block or cytology samples. Liquid biopsy using EV-derived DNA is promising for EGFR genotyping, including T790 M detection in pulmonary adenocarcinoma patients who develop pleural effusions.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-5138-3) contains supplementary material, which is available to authorized users.

The relationship between atopic dermatitis (AD) and psychological distress has been well established for children and adolescents. However, it is unclear whether this relationship exists in young adults. This study aimed to assess the relationship between AD and psychological distress in young male adults in South Korea.A cross-sectional study was conducted using regional conscription data from 2008 to 2012. A dermatologist diagnosed AD based on historical and clinical features, and determined severity using the eczema area and severity index. A psychiatrist used medical records, an interview, and a psychological test to examine psychological distress (depression, anxiety, and somatization). The relationship between psychological distress and AD was assessed by multivariate logistic regression analyses.Among the 120,508 conscripts, 1517 (1.2%) presented with AD. The odds of having each type of psychological distress were significantly greater for individuals with AD compared with those without AD. The adjusted odds ratios for depression, anxiety, and somatization were 1.79 (95% CI 1.40–2.29), 1.38 (95% CI 1.08–1.76), and 1.75 (95% CI 1.40–2.20), respectively. Moderate-to-severe AD was significantly related to depression and somatization to a greater extent compared with mild AD.Depression, anxiety, and somatization are strongly and independently associated with AD in young adult males. Early treatment of skin inflammation might modify the risk of psychiatric problems. Prospective cohort studies are needed to verify causal relationships.

Extracting single-cell information during cellular responses to external signals in a high-throughput manner is an essential step for quantitative single-cell analyses. Here, we have developed a simple yet robust microfluidic platform for measuring time-course single-cell response on a large scale. Our method combines a simple microwell-based cell docking process inside a patterned microfluidic channel, with programmable time-course live-cell imaging and software-aided fluorescent image processing. The budding yeast, Saccharomyces cerevisiae (S. cerevisiae), cells were individually captured in microwells by multiple sweeping processes, in which a cell-containing solution plug was actively migrating back and forth several times by a finger-pressure induced receding meniscus. To optimize cell docking efficiency while minimizing unnecessary flooding in subsequent steps, circular microwells of various channel dimensions (4-24 µm diameter, 8 µm depth) along with different densities of cell solution (1.5-6.0 × 10(9) cells per mL) were tested. It was found that the microwells of 8 µm diameter and 8 µm depth allowed for an optimal docking efficiency (>90%) without notable flooding issues. For quantitative single-cell analysis, time-course (time interval 15 minute, for 2 hours) fluorescent images of the cells stimulated by mating pheromone were captured using computerized fluorescence microscope and the captured images were processed using a commercially available image processing software. Here, real-time cellular responses of the mating MAPK pathway were monitored at various concentrations (1 nM-100 µM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics.

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Background: Extracellular vesicles (EV) have been proven to contain double-stranded DNA reflecting the mutational status of the parental tumor cells in non-small cell lung cancer (NSCLC), which can be translated into clinically useful EV-based liquid biopsy for Epidermal growth factor receptor (EGFR) genotyping using bronchoalveolar lavage fluid (BALF) obtained from tumor site.Methods: Patients subjected for an initial lung cancer work-up underwent bronchoscopy and BALF was obtained from tumor site. After isolating EVs from BALF by ultracentrifugation, EV-derived DNA (EV DNA) was extracted for subsequent EGFR genotyping performed through peptide nucleic acid (PNA)mediated Real-Time PCR. The sensitivity, specificity, and concordance rate of BALF EV-based EGFR genotyping were calculated in comparison to tissue genotyping. Results:The average sensitivity and specificity of BALF EV-based EGFR genotyping were 76% and 87%, respectively, while the sensitivity significantly increased as the stage progressed. Especially, in stage IV, BALF EV-based EGFR typing identified all tissue-proven EGFR mutant cases (n=31) and detected 6 additional mutant cases. The concordance rate was 79% in stage I, 100% in stage II, 74% in stage III, and 92% in stage IV. As TNM stage advanced, especially in the presence of metastasis, concordance rate significantly increased (P<0.05). Conclusions:The use of BALF for the collection of EV DNA in lung cancer patients resulted in a highly accurate diagnosis. The establishment of a fast and reliable method to identify target genes using EV DNA illustrated that it can overcome the problems of low sensitivity and instability in using cell-free DNA (cfDNA).

Molecular analysis is traditionally performed on tumor tissue. Although the number of mandatory tests for treatment decisions increases in patients with advanced non-small cell lung cancer (NSCLC), it is difficult to secure adequate tumor tissue for this purpose [1]. Small biopsy specimens, cell blocks, or aspirates are often the only available samples in patients with advanced NSCLC [2,3]. It is difficult to repeat tissue biopsies because they are invasive. Liquid biopsy could be an alternative or a complementary minimally invasive method for detecting molecular changes in NSCLC [1,2,4,5]. The clinical use of liquid biopsy to select patients with advanced NSCLC who are candidates for third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy has been demonstrated in many clinical trials [6-10]. The United States Food and Drug Administration (FDA) approved Cobas EGFR Mutation Test v2 (Roche, Indianapolis, IN, USA) in 2018 as a companion diagnostic for third-generation EGFR TKI based on these results [11]. The Korea National Health Insurance Service (NHIS) has covered circulating cell-free tumor DNA (ctDNA) tests for EGFR mutations in advanced NSCLC since 2018. In this review, we present the current status and future perspectives of liquid biopsy in patients with NSCLC. BIOLOGY OF CIRCULATING TUMOR DNA Liquid biopsy refers to the collection and analysis of analytes from various body fluids such as blood, urine, sputum, and pleural fluid [12-14]. Different analytes can be present in a liquid biopsy including circulating tumor cells (CTCs), circulating cell-free DNAs (cfDNAs), circulating tumor RNAs (ctRNAs), circulating exosomes, tumor-educated platelets, proteins, and metabolites [15,16]. CTCs are intact, viable tumor cells circulating in the blood [12]. Cancer releases single or clusters of CTCs into the bloodstream during the course of hematogenous spread. cfDNA refers to all circulating DNA in body fluids. cfDNA can be derived from neoplastic as well as non-neoplastic cells [15,16]. cfDNA can be detected in other body fluids, including urine, saliva, or cerebrospinal fluid. ctDNA refers to a subgroup