Integrated microfluidic system for isolating exosome and analyzing protein marker PD-L1.

Exosomes are lipid-bilayered nanovesicles secreted by cells to mediate intercellular communication. Various kinds of biomolecules involved in exosomes offer non-invasive approaches for detecting or monitoring disease and developing targeted therapeutics. Here, we present an integrated microfluidic exosome isolation and detection system (EXID system) to analyze the abundance of the exosomal PD-L1 protein marker, which is a transmembrane protein expressed by tumors to suppress immune activation of T cells. By incorporating exosome isolation and biomarker labelling and quantification within a single microfluidic chip, our system reduced the total analysis time below 2 h. Using the EXID system, 7 categories of cell lines including cancer cell lines and control samples were profiled, where significant differences in the fluorescence intensity were observed with the limit of detection (LOD) down to 10.76 per microliter. Such noticeable variations in PD-L1 abundance among cancer cell lines highlighted the need of personalized treatments. Furthermore, 16 clinical samples from 7 post-treated cancer patients, 3 prior-treatment patients and 6 healthy controls, are tested, among which differences in sensitivity toward immune response were subsistent. Because the abundance of PD-L1 reflects the sensibility for immune response, our results provide useful guides to design immunotherapy strategies for different types of tumors.

Zeolite-Enhanced Portland Cement: Solution for Durable Wellbore-Sealing Materials.

Wellbore-plugging materials are threatened by challenging plugging and abandonment (P&A) conditions. Hence, the integrity and resilience of these materials and their ability to provide sufficient zonal isolation in the long-term are unknown. The present work focuses on investigating the potential to use zeolites as novel additives to the commonly used Class-H cement. Using four different zeolite-cement mixtures (0%, 5%, 15% and 30%, by weight of cement) where samples were cast as cylinders and cured at 90 °C and 95% relative humidity, the unconfined compressive strength (UCS) testing showed a 41% increase with the 5% ferrierite addition to the Class-H cement in comparison to neat Class-H cement. For triaxial compression tests at 90 °C, the highest strength achieved by the 5% ferrierite-added formulations was 68.8 MPa in comparison to 62.9 MPa for the neat Class-H cement. The 5% ferrierite formulation also showed the lowest permeability, 13.54 µD, which is in comparison to 49.53 µD for the neat Class-H cement. The overall results show that the 5% ferrierite addition is the most effective at improving the mechanical and petrophysical properties based on a water/cement ratio of 0.38 when tested after 28 days of curing in 95% relative humidity and 90 °C. Our results not only demonstrate that zeolite is a promising cement additive that could improve the long-term strength and petrophysical properties of cement formulations, but also provide a proposed optimal formulation that could be next utilized in a field trial.

Development and Assessment of Novel Predictive Nomograms Based on APRI for Hepatitis B Virus-associated Small Solitary Hepatocellular Carcinoma with Stereotactic Body Radiotherapy.

Background: The correlation between serum inflammatory marker before treatment and the survival of patients with hepatitis B virus (HBV)-associated small solitary hepatocellular carcinoma (HCC) after stereotactic body radiotherapy (SBRT) remains unclear. The objective of our study is to estimate survival in such patients using multivariable prediction models and investigate the prognostic value of aspartate aminotransferase-to-platelet index (APRI), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) for HBV-associated small solitary HCC patients treated with SBRT. Patients and methods: Patients with HBV-associated small solitary HCC who were newly treated with SBRT were retrospectively analysed in our hospital from 2009 to 2016. We counted the APRI, NLR, PLR, and LMR before treatment and calculated their cut-off values for predicting overall survival (OS) and progression-free survival (PFS) by receiver operating characteristic (ROC) analysis. The random forest model combined with least absolute shrinkage and selection operator (LASSO) regression model for OS and PFS were used to screen potentially prognostic factors from serum inflammatory markers, demographic data, and clinical characteristics. Predictive models for OS and PFS were developed by multivariable COX regression and nomograms were constructed. Discrimination was assessed using the C-index. Internal validation was assessed using the Bootstrap method. Survival analysis was carried out to assess the prognostic value of serum inflammatory markers, and OS and PFS curves were compared by Kaplan-Meier analysis and Log-Rank test, respectively. Results: A total of 72 patients with HBV-associated small solitary HCC were recruited for the study. The median follow-up time was 2015 days (range, 232-3823 days). Age, tumor size, NLR, PLR, and APRI were used to construct nomogram for OS, while gender, age, TNM stage, portal hypertension, AFP, APRI were for PFS. The two models displayed good discriminations with C-indexes of 0.738 (95% CI: 0.632-0.844) and 0.657 (95% CI: 0.538-0.777), and their C-indexes in the internal validation cohort reached 0.790 (95% CI: 0.684-0.896) and 0.739 (95% CI: 0.619-0.859). The multivariable cox analysis indicated that APRI<0.47 was favourable independent prognostic factors for OS and PFS. Compared to APRI≥0.47, APRI<0.47 predicts better OS (p=0.003) and PFS (p=0.003). Conclusions: Nomograms based on APRI are superior in predicting OS and PFS in HBV-associated small solitary HCC patients who have received SBRT. APRI before treatment is a feasible and convenient prognostic indicator for OS and PFS, which helpfully determines the beneficial population of SBRT for HBV-associated small solitary HCC.

Absolute quantification and analysis of extracellular vesicle lncRNAs from the peripheral blood of patients with lung cancer based on multi-colour fluorescence chip-based digital PCR.

Emerging evidence indicates that extracellular vesicle (EV) long non-coding ribonucleic acids (lncRNAs) in lung cancer may be clinically useful biomarkers for early diagnosis using liquid biopsy. However, the extremely low quantities of EV-lncRNAs in peripheral blood are a major challenge for multi-target detection. In this study, we developed a new multi-colour fluorescence digital PCR EV-lncRNA (miDER) analysis chip, and then demonstrated its ability to quickly and accurately analyse the levels of two target genes and one reference gene from peripheral blood. Under the miDER assay, the limit of detection of the target gene from peripheral blood was 10 copies/µL. Based on multiplex assay, the expression levels of two lung cancer-related genes (SLC9A3-AS1 and PCAT6) in patients with lung cancer (n = 32) and healthy controls (n = 30) showed a significant difference between the two groups (P < 0.001; two-tailed t-test). A receiver operating characteristic (ROC) curve analysis was used to evaluate the discrimination ability of these lncRNAs. The combination of two lncRNAs in the miDER assay yielded a higher area under curve (AUC) value of 0.811 (95% CI = 0.705-0.918). Moreover, to determine the absolute quantitation capacity of the miDER assay, we compared the results to those obtained by quantitative real-time polymerase chain reaction (qPCR), demonstrating that the miDER assay is more sensitive than qPCR. The multiplex assay based on the miDER could provide a new solution for the multi-index combined detection of trace EV-lncRNAs in body fluids and demonstrate the use of EV-lncRNAs as biomarkers for lung tumour biopsy.

A microfluidic platform for high-purity separating circulating tumor cells at the single-cell level.

Circulating tumor cells (CTCs) are rare cancer cells that are shed from the tumors into the peripheral blood and are instrumental in distant metastasis. Early detection of CTCs can therefore improve prognoses and help design patient-specific treatment regimen. However, the current CTC isolation techniques have poor efficacy and selectivity, owing to the rarity and heterogeneity of the CTCs. We designed a microchip for integrated single-cell isolation of CTCs - based on cell size and immuno-phenotype - and analysis. Each isolation unit consisted of a trap channel, a bypass channel, and a release channel. The larger cells were preferentially captured at the trap channels and flushed out selectively via release microvalves according to their immuno-phenotype. The average recovery rate and purity of lung cancer cells isolated from a spiked WBC population were respectively 92.5% and 94% using the microchip, which were significantly higher compared to that obtained using anti-CD45 magnetic beads. In addition, the isolated cancer cells were analyzed on chip for the surface markers of epithelial mesenchymal transition. Taken together, the integrated microchip is a promising tool for the isolation and analysis of CTCs in the clinical setting.

Rapid Isolation and Multiplexed Detection of Exosome Tumor Markers Via Queued Beads Combined with Quantum Dots in a Microarray.

Tumor-derived exosomes are actively involved in cancer progression and metastasis and have emerged as a promising marker for cancer diagnosis in liquid biopsy. Because of their nanoscale size, complex biogenesis, and methodological limitations related to exosome isolation and detection, advancements in their analysis remain slow. Microfluidic technology offers a better analytic approach compared with conventional methods. Here, we developed a bead-based microarray for exosome isolation and multiplexed tumor marker detection. Using this method, exosomes are isolated by binding to antibodies on the bead surface, and tumor markers on the exosomes are detected through quantum dot (QD) probes. The beads are then uniformly trapped and queued among micropillars in the chip. This design benefits fluorescence observation by dispersing the signals into every single bead, thereby avoiding optical interference and enabling more accurate test results. We analyzed exosomes in the cell culture supernatant of lung cancer and endothelial cell lines, and different lung cancer markers labeled with three QD probes were used to conduct multiplexed detection of exosome surface protein markers. Lung cancer-derived samples showed much higher (~ sixfold-tenfold) fluorescence intensity than endothelial cell samples, and different types of lung cancer samples showed distinctive marker expression levels. Additionally, using the chip to detect clinical plasma samples from cancer patients showed good diagnostic power and revealed a well consistency with conventional tests for serological markers. These results provide insight into a promising method for exosome tumor marker detection and early-stage cancer diagnosis.