Dual-Recognition-Mediated Autocatalytic Amplification Assay for the Subpopulations of PD-L1 Positive Extracellular Vesicle.

The PD-L1 protein on extracellular vesicles (EVs) is a promising biomarker for tumor immunotherapy. However, PD-L1+ EVs have various cell origins, so further analysis of the subpopulations is essential to help understand better their relationship with tumor immunotherapy. Different from the previous work which focus on the level of total PD-L1+ EVs expression, we, herein, report a dual-recognition mediated autocatalytic amplification (DRMAA) assay to detect the PD-L1 derived from tumors (EpCAM+), immune T cells (CD3+), and total (Lipids) EVs, respectively. The DRMAA assay employed proximity hybridization to construct a complete trigger sequence and then catalyzed the cross-hybridization of three hairpin probes, producing a three-way DNA junction (3-WJ) structure carrying the newly exposed trigger sequence. The 3-WJ complex subsequently initiated an autocatalytic amplification reaction and higher sensitivity than the traditional catalytic hairpin assembly assay was obtained. It was found that the EpCAM+ and PD-L1+ EVs were more effective than others in distinguishing lung cancer patients from healthy people. Surprisingly, the CD3+ and PD-L1+ EVs in lung cancer patients were also upregulated, indicating that immune cell-derived PD-L1+ EVs are also non-negligible marker in a tumor microenvironment. Our results suggested that the DRMAA assay would improve the study of subpopulations of PD-L1+ EVs to provide new insights for cancer immunotherapies.

Multivalent DNA Flowers for High-Performance Isolation, Detection, and Release of Tumor-Derived Extracellular Vesicles.

Tumor-derived extracellular vesicles (T-EVs) hold great promise for understanding cancer biology and improving cancer diagnostics and therapeutics. Herein, we developed multivalent DNA flowers (DFs) containing repeated and equidistant EpCAM aptamers for the efficient isolation of T-EVs. The multivalent aptamer chains in DFs had good flexibility to adapt to the surface morphology of T-EVs and achieved multivalent ligand-receptor interactions, thus showing enhanced isolation ability compared to monovalent aptamers. Compared with other materials for isolation of EVs, DFs were generated by rolling circle amplification (RCA) and self-assembled into microspheres in a one-pot reaction, and the recognition molecules (aptamers) were directly replicated and assembled during the RCA reaction instead of chemical modification and immobilization on the surface of solid materials. Moreover, as optically transparent biomaterials, the content of EpCAM+ EVs could be directly reflected via membrane-based hydrophobic assembly of signaling modules in DFs@EpCAM+ EVs complex, and we found that the amount of EpCAM+ EVs showed greater accuracy in cancer diagnosis than total EVs (88.3 vs 69.7%) and was also higher than the clinically commonly used marker carcinoembryonic antigen (CEA) (88.3 vs 76.7%). In addition, T-EVs could be released by lysis of DFs with the nuclease, gently and easily, keeping high intact and activity of EVs for downstream biological function studies. These results demonstrated that DFs are efficient and nondestructive tools for isolation, detection, and release of T-EVs.

Comprehensive Detection of PD-L1 Protein and mRNA in Tumor Cells and Extracellular Vesicles through a Real-Time qPCR Assay.

A growing number of studies have shown that tumor cells secrete extracellular vesicles (EVs) containing programmed death-ligand 1 (PD-L1) protein. These vesicles can travel to lymph nodes and remotely inactivate T cells, thereby evading immune system attack. Therefore, the simultaneous detection of PD-L1 protein expression in cells and EVs is of great significance in guiding immunotherapy. Herein, we developed a method based on qPCR for the simultaneous detection of PD-L1 protein and mRNA in EVs and their parental cells (PREC-qPCR assay). Lipid probes immobilized on magnetic beads were used to capture EVs directly from samples. For RNA assay, EVs were directly broken by heating and quantified with qPCR. As to protein assay, EVs were recognized and bound with specific probes (such as aptamers), which were used as templates in subsequent qPCR analysis. This method was used to analyze EVs of patient-derived tumor clusters (PTCs) and plasma samples from patients and healthy volunteers. The results revealed that the expression of exosomal PD-L1 in PTCs was correlated with tumor types and significantly higher in plasma-derived EVs from tumor patients than that of healthy individuals. When extended to cells and PD-L1 mRNAs, the results showed that the expression of PD-L1 protein was consistent with mRNA in cancer cell lines, while PTCs demonstrated significant heterogeneity. This comprehensive detection of PD-L1 at four levels (cell, EVs, protein, and mRNA) is believed to enhance our understanding of the relationship among PD-L1, tumors, and the immune system and to provide a promising tool for predicting the benefits of immunotherapy.

Assessment of Drug Susceptibility for Patient-Derived Tumor Models through Lactate Biosensing and Machine Learning.

A patient-derived tumor model (PDM) is a practical tool to rapidly screen chemotherapeutics for individual patients. The evaluation method of cell viability directly determines the application of PDMs for drug susceptibility testing. As one of the metabolites of "glycosis", the lactate content was used to evaluate cell viability, but these assays were not specific for tumor cells. Based on the "Warburg effect", wherein tumor cells preferentially rely on "aerobic glycolysis" to produce lactate instead of pyruvate in "anaerobic glycolysis" of normal cells, we reported a gold lactate sensor (GLS) to estimate the cell viability of PDMs in drug susceptibility testing. It demonstrated high consistency between the GLS and commercial cell viability assay. Unlike either imaging or cell viability assay, the GLS characterizes the cell viability, enables dynamic monitoring, and distinguishes tumor cells from other cells. Moreover, machine learning (ML) was employed to perform a multi-index assessment for drug susceptibility of PDMs, which proved to be accurate and practical for clinical application. Therefore, the GLS provides an ideal drug susceptibility testing tool for individualized medicine.

Rapid enrichment and sensitive detection of extracellular vesicles through measuring the phospholipids and transmembrane protein in a microfluidic chip.

Extracellular vesicles (EVs) have attracted tremendous attention in recent years and quantification of EVs is a key issue in the evaluation of vesicle-based diagnostics and therapeutic development, but it's quite challenging to determine whether higher protein expression signals are due to larger vesicle amount or higher protein content within each vesicle. To solve this problem, herein, we proposed a strategy based on staining phospholipid bilayers of EVs with lipophilic dyes to evaluate their lipid amount, which was subsequently normalized as an internal standard for studying the expression of transmembrane protein (i.e., CD63) on EVs in different samples. In addition, a microfluidic platform based on electrophoresis technology was invented to effectively enrich and detect EVs. Small fluorescent labeling molecules (i.e., uncombined aptamers) were on-chip removed from EVs without pre-separation via ultracentrifugation or ultrafiltration which were indispensable in nanoparticle tracking analysis (NTA) and flow cytometry techniques and the performance of this assay is comparable to NTA. Finally, it was found obvious difference in the expression of CD63 on EVs before and after normalization based on lipid amount in plasma samples. This method is expected to provide more accurate information when comparing the expression levels of EVs biomarkers in different samples.

Snowflake-like DNA crystals templated Cu clusters as a fluorescent turn-on probe for sensing actin.

Herein, we synthesized snowflake-like DNA crystals (SDC) via hybridization chain reaction and used it for the first time in the synthesis of copper nanoclusters with enhanced fluorescence. Atomic force microscopy (AFM) and laser confocal microscopy characterization confirmed that SDC/CuNCs are self-assembled successfully on SDC. Aggregation induced emission allows SDC/CuNCs to exhibit better stability and stronger emission intensity. Thus, we developed the "turn-on" label-free fluorescence detection method of actin based on SDC/CuNCs which offer simplicity, low cost, good selectivity, and high sensitivity. The detection limit was determined to be 0.0124 µg mL-1, which was an order of magnitude lower than that of reported fluorescent methods (0.12 µg mL-1). Compared with previous method, the linear range is also much wider. We also performed standard recovery experiments in actual samples for evaluating the practicality of this strategy and proved that the capability of the proposed approach for the determination of actin is feasible and the interference from complex biological samples is negligible. These results indicate that SDC/CuNCs are expected to play a more important role in the field of biosensors.

DNA Nanoribbon-Templated Self-Assembly of Ultrasmall Fluorescent Copper Nanoclusters with Enhanced Luminescence.

Fluorescent copper nanoclusters (CuNCs) have been widely used in chemical sensors, biological imaging, and light-emitting devices. However, individual fluorescent CuNCs have limitations in their capabilities arising from poor photostability and weak emission intensities. As one kind of aggregation-induced emission luminogen (AIEgen), the formation of aggregates with high compactness and good order can efficiently improve the emission intensity, stability, and tunability of CuNCs. Here, DNA nanoribbons, containing multiple specific binding sites, serve as a template for in situ synthesis and assembly of ultrasmall CuNCs (0.6 nm). These CuNC self-assemblies exhibit enhanced luminescence and excellent fluorescence stability because of tight and ordered arrangement through DNA nanoribbons templating. Furthermore, the stable and bright CuNC assemblies are demonstrated in the high-sensitivity detection and intracellular fluorescence imaging of biothiols.

Functional modulation of cytochrome C upon specific binding to DNA nanoribbons.

We discovered that the function of cytochrome C can be modulated by DNA nanoribbons. Meanwhile, the interplay between the DNA nanoribbons and the native cytochrome C and the possible mechanisms are also discussed.