The Role of Extracellular Vesicles in the Treatment of Prostate Cancer.

Prostate cancer (PCa) has become a public health concern in elderly men due to an ever-increasing number of estimated cases. Unfortunately, the available treatments are unsatisfactory because of a lack of a durable response, especially in advanced disease states. Extracellular vesicles (EVs) are lipid-bilayer encircled nanoscale vesicles that carry numerous biomolecules (e.g., nucleic acids, proteins, and lipids), mediating the transfer of information. The past decade has witnessed a wide range of EV applications in both diagnostics and therapeutics. First, EV-based non-invasive liquid biopsies provide biomarkers in various clinical scenarios to guide treatment; EVs can facilitate the grading and staging of patients for appropriate treatment selection. Second, EVs play a pivotal role in pathophysiological processes via intercellular communication. Targeting key molecules involved in EV-mediated tumor progression (e.g., proliferation, angiogenesis, metastasis, immune escape, and drug resistance) is a potential approach for curbing PCa. Third, EVs are promising drug carriers. Naïve EVs from various sources and engineered EV-based drug delivery systems have paved the way for the development of new treatment modalities. This review discusses the recent advancements in the application of EV therapies and highlights EV-based functional materials as novel interventions for PCa.

Lab-on-a-Disc for Point-of-Care Infection Diagnostics.

Reliable, inexpensive, and rapid diagnostic tools are essential to control and prevent the spread of infectious diseases. Many commercial kits for coronavirus disease 2019 (COVID-19) diagnostics have played a crucial role in the fight against the COVID-19 pandemic. Most current standard in vitro diagnostic (IVD) protocols for infectious diseases are sensitive but time-consuming and require sophisticated laboratory equipment and specially trained personnel. Recent advances in biosensor technology suggest the potential to deliver point-of-care (POC) diagnostics that are affordable and provide accurate results in a short time. The ideal "sample-in-answer-out" type fully integrated POC infection diagnostic platforms are expected to be autonomous or easy-to-operate, equipment-free or infrastructure-independent, and high-throughput or easy to upscale. In this Account, we detail the recent progress made by our group and others in the development of centrifugal microfluidic devices or lab-on-a-disc (LOAD) systems. Unlike conventional pump-based fluid actuation, the centrifugal force generated by spinning the disc induces liquid pumping and no external fluidic interconnects are required. This allows a total fluidic network required for multiple steps of biological assays to be integrated on a disc, enabling fully automated POC diagnostics. Various applications have been demonstrated, including liquid biopsy for personalized cancer management, food applications, and environmental monitoring; here, we focus on IVD for infectious disease. First, we introduce various on-disc unit operation technologies, including reagent storage, sedimentation, filtration, valving, decanting, aliquoting, mixing, separation, serial dilution, washing, and calibration. Such centrifugal microfluidic technologies have already proved promising for micro-total-analysis systems for automated IVD ranging from molecular detection of pathogens to multiplexed enzyme-linked immunosorbent assays (ELISAs) that use raw samples such as whole blood or saliva. Some recent examples of LOAD systems for molecular diagnostics in which some or all steps of the assays are integrated on a disc, including pathogen enrichment, nucleic acid extraction, amplification, and detection, are discussed in detail. We then introduce fully automated ELISA systems with enhanced sensitivity. Furthermore, we demonstrate a toy-inspired fidget spinner that enables electricity-free and rapid analysis of pathogens from undiluted urine samples of patients with urinary tract infection symptoms and a phenotypic antimicrobial susceptibility test for an extreme POC diagnostics application. Considering the urgent need for cost-effective and reliable POC infection diagnostic tools, especially in the current pandemic crisis, the current limitations and future directions of fast and broad adaptation in real-world settings are also discussed. With proper attention to key challenges and leverage with recent advances in bio-sensing technologies, molecular biology, nanomaterials, analytical chemistry, miniaturization, system integration, and data management, LOAD systems hold the potential to deliver POC infection diagnostic tools with unprecedented performance regarding time, accuracy, and cost. We hope the new insight and promise of LOAD systems for POC infection diagnostics presented in this Account can spark new ideas and inspire further research and development to create better healthcare systems for current and future pandemics.

EV-Ident: Identifying Tumor-Specific Extracellular Vesicles by Size Fractionation and Single-Vesicle Analysis.

Tumor-derived extracellular vesicles (EVs) have emerged as a promising source of circulating biomarkers for liquid biopsies. However, understanding the heterogeneous physical and biochemical properties of EVs originating from multiple complex biogenesis pathways remains a major challenge. Here, we introduce EV-Ident for preparation of subpopulations of EVs in three different size fractions: large EVs (EV200 nm; 200-1 000 nm), medium EVs (EV100 nm; 100-200 nm), and small EVs (EV20 nm; 20-100 nm). Furthermore, this technology enables the in situ labeling of fluorescence markers for the protein profiling of individual EVs. As a proof-of-concept, we analyzed the presence of human epidermal growth factor receptor 2 (HER2) and prostate-specific membrane antigen (PSMA) in breast cancer and prostate cancer cell-derived EVs, respectively, using three different size fractions at the single-EV level. By reducing the complexity of EV heterogeneity in each size fraction, we found that HER2-positive breast cancer cells showed the greatest expression of HER2 in EV20 nm, whereas PSMA expression was the highest in EV200 nm derived from PSMA-expressing prostate cancer cells. This increase in HER2 expression in EV20 nm and PSMA expression in EV200 nm was further confirmed in plasma-derived nanoparticles (PNPs) obtained from breast and prostate cancer patients, respectively. Our study demonstrates that single-EV analysis using EV-Ident provides a practical way to understand EV heterogeneity and to successfully identify potent subpopulation of EVs for breast and prostate cancer, which has promising translational implications for cancer theranostics. Furthermore, these findings have the potential to address fundamental questions surrounding the biology and clinical applications of EVs.

Effects of poly(ethylene glycol) on the wetting behavior and director configuration of lyotropic chromonic liquid crystals confined in cylinders.

We investigate the effects of poly(ethylene glycol) (PEG) doping on nematic lyotropic chromonic liquid crystals (LCLCs) confined in a cylindrical cavity. First, PEG added to Sunset Yellow (SSY) renders confining glass surfaces nemato-phobic by adsorption. We also confirm that the grafting of PEG to bare glass surfaces changes them from nemato-philic to nemato-phobic. This change in the wetting behavior affects how nematic director configurations form and relax. Additionally, we observe that PEG-doped nematic SSY retains the double-twist director configuration as in the PEG-free case. However, the PEG-doped nematic SSY is accompanied by unprecedented domain-wall-like defects and heterogeneity in the director configuration. We propose multiple hypotheses on how PEG changes the director configuration, including the formation of meta-stable director configurations.

Emerging techniques in the isolation and characterization of extracellular vesicles and their roles in cancer diagnostics and prognostics.

Extracellular vesicles (EVs) are cell-derived nanovesicles, present in almost all types of body fluids, which play an important role in intercellular communication and are involved in the transport of biological signals for regulating diverse cellular functions. Due to the increasing clinical interest in the role of EVs in tumor promotion, various techniques for their isolation, detection, and characterization are being developed. In this review, we present an overview of the current EV isolation and characterization methods in addition to their applications and limitations. Furthermore, EVs as the potential emerging biomarkers in cancer management and their clinical implementation are briefly discussed.

All-in-one centrifugal microfluidic device for size-selective circulating tumor cell isolation with high purity.

Circulating tumor cells (CTCs) have gained increasing attention owing to their roles in cancer recurrence and progression. Due to the rarity of CTCs in the bloodstream, an enrichment process is essential for effective target cell characterization. However, in a typical pressure-driven microfluidic system, the enrichment process generally requires complicated equipment and long processing times. Furthermore, the commonly used immunoaffinity-based positive selection method is limited, as its recovery rate relies on EpCAM expression of target CTCs, which shows heterogeneity among cell types. Here, we propose a centrifugal-force-based size-selective CTC isolation platform that can isolate and enumerate CTCs from whole blood within 30 s with high purity. The device was validated using the MCF-7 breast cancer cell line spiked in phosphate-buffered saline and whole blood, and an average capture efficiency of 61% was achieved, which is typical for size-based filtration. The capture efficiency for whole blood samples varied from 44% to 84% under various flow conditions and dilution factors. Under the optimized operating conditions, a few hundred white blood cells per 1 mL of whole blood were captured, representing a 20-fold decrease compared to those obtained using a commercialized size-based CTC isolation device. In clinical validation, normalized CTC counts varied from 10 to 60 per 7.5 mL of blood from gastric and lung cancer patients, yielding a detection rate of 50% and 38%, respectively. Overall, our CTC isolation device enables rapid and label-free isolation of CTCs with high purity, which should greatly improve downstream molecular analyses of captured CTCs.

Exosome Precipitation by Ionic Strength Modulation: ExoPRISM.

Extracellular vesicles (EVs) are emerging as crucial materials for precision theragnostic applications. However, current separation methods are time-consuming, costly, and not scalable and deliver limited yields or purity. Here, we present EV precipitation by ionic strength modulation (ExoPRISM), a simple, low-cost, user-friendly, and readily adaptable approach for separating EVs in high yields without compromising their biological functions. Adding an electrolyte solution to blood plasma in small increments generates the sequential precipitation of proteins and EVs, allowing for fractional separation of EVs using low-speed centrifugation. The coprecipitated electrolytes are easily washed away, and the entire EV separation and washing process takes less than an hour. This approach successfully separates EVs from a broad range of volumes and types of biological fluids, including culture medium, urine, plasma, and serum, showing promise as a robust tool for next-generation liquid biopsies and regenerative medicine.

Lab-on-a-disc for fully integrated multiplex immunoassays.

This paper presents a cost-effective, rapid, and fully automated lab-on-a-disc for simultaneous detection of multiple protein biomarkers in raw samples such as whole blood or whole saliva. For the diagnosis of cardiovascular disease, here, a novel centrifugal microfluidic layout was designed to conduct the simultaneous detection of high sensitivity C-reactive protein, cardiac troponin I, and N-terminal pro-B type natriuretic peptide based on a bead-based sandwich type enzyme-linked immunosorbent assay (ELISA). Three reaction chambers are initially interconnected for the common processes such as sample injection, incubation, and washing and then isolated on-demand for the independent processes such as substrate incubation and final detection. The assay performances such as the limit of detection and the dynamic range were comparable with those of the conventional ELISA despite the significant reduction of the minimum sample volume (200 µL), the amount of washing buffer (700 µL), and the total process time (20 min).

Flexible fabrication and applications of polymer nanochannels and nanoslits.

Fluidic devices that employ nanoscale structures (<100 nm in one or two dimensions, slits or channels, respectively) are generating great interest due to the unique properties afforded by this size domain compared to their micro-scale counterparts. Examples of interesting nanoscale phenomena include the ability to preconcentrate ionic species at extremely high levels due to ion selective migration, unique molecular separation modalities, confined environments to allow biopolymer stretching and elongation and solid-phase bioreactions that are not constrained by mass transport artifacts. Indeed, many examples in the literature have demonstrated these unique opportunities, although predominately using glass, fused silica or silicon as the substrate material. Polymer microfluidics has established itself as an alternative to glass, fused silica, or silicon-based fluidic devices. The primary advantages arising from the use of polymers are the diverse fabrication protocols that can be used to produce the desired structures, the extensive array of physiochemical properties associated with different polymeric materials, and the simple and robust modification strategies that can be employed to alter the substrate's surface chemistry. However, while the strengths of polymer microfluidics is currently being realized, the evolution of polymer-based nanofluidics has only recently been reported. In this critical review, the opportunities afforded by polymer-based nanofluidics will be discussed using both elastomeric and thermoplastic materials. In particular, various fabrication modalities will be discussed along with the nanometre size domains that they can achieve for both elastomer and thermoplastic materials. Different polymer substrates that can be used for nanofluidics will be presented along with comparisons to inorganic nanodevices and the consequences of material differences on the fabrication and operation of nanofluidic devices (257 references).

Exhausted mature dendritic cells exhibit a slower and less persistent random motility but retain chemotaxis against CCL19.

Dendritic cells (DCs), which are immune sentinels in the peripheral tissues, play a number of roles, including patrolling for pathogens, internalising antigens, transporting antigens to the lymph nodes (LNs), interacting with T cells, and secreting cytokines. The well-coordinated migration of DCs under various immunological or inflammatory conditions is therefore essential to ensure an effective immune response. Upon maturation, DCs migrate faster and more persistently than immature DCs (iDCs), which is believed to facilitate CCR7-dependent chemotaxis. It has been reported that lipopolysaccharide-activated DCs produce IL-12 only transiently, and become resistant to further stimulation through exhaustion. However, little is known about the influence of DC exhaustion on cellular motility. Here, we studied the cellular migration of exhausted DCs in tissue-mimicked confined environments. We found that the speed of exhausted matured DCs (xmDCs) decreased significantly compared to active matured DCs (amDCs) and iDCs. In contrast, the speed fluctuation increased compared to that of amDCs and was similar to that of iDCs. In addition, the diffusivity of the xmDCs was significantly lower than that of the amDCs, which implies that DC exhaustion reduces the space exploration ability. Interestingly, CCR7-dependent chemotaxis against CCL19 in xmDCs was not considerably different from that observed in amDCs. Taken together, we report a unique intrinsic cell migration behaviour of xmDCs, which exhibit a slower, less persistent, and less diffusive random motility, which results in the DCs remaining at the site of infection, although a well-preserved CCR7-dependent chemotactic motility is maintained.

Prediction of tumor metastasis via extracellular vesicles-treated platelet adhesion on a blood vessel chip.

In preclinical and clinical studies, it has been demonstrated that tumor-educated platelets play a critical role in tumorigenesis, cancer development, and metastasis. Unlike the role of cancer-derived chemokines in platelet activation, the role of cancer-derived extracellular vesicles (EVs) has remained elusive. Here, we found that interleukin-8 (IL-8) in cancer-derived EVs contributed to platelet activation by increasing P-selectin expression and ligand affinity, resulting in increased platelet adhesion on the human vessel-mimicking microfluidic system. Furthermore, platelet adhesion levels on vessels treated with human plasma-derived EVs demonstrated good discrimination between breast cancer patients with metastasis and those without, with the area under the curve (AUC) value of 0.88. While EpCAM expression on EVs could detect the existence of a tumor (AUC = 0.89), it performed poorly in predicting metastasis (AUC = 0.42). We believe that these findings shed light on the role of the interaction between cancer-derived EVs and platelets in pre-metastatic niche formation and tumor metastasis, potentially leading to the development of platelet-tumor interaction-based novel diagnostic and therapeutic strategies.

Fully automated platelet isolation on a centrifugal microfluidic device for molecular diagnostics.

Platelets play crucial roles in hemostasis and immunity. Over the last decades, clinical evidence has revealed the significance of platelets as complementary biomarkers for the detection and treatment of various diseases, including cancer. Due to a lack of well standardized convenient isolation methods for platelets, pre-analytical factors such as complex handling procedures negatively impact the quality of the platelet samples, including overactivation, low purity, and poor reproducibility. This may lead to biased interpretation of various downstream analyses, such as proteomic and genomic analyses. Herein, we describe a fully automated lab-on-a-disc-based method of platelet isolation from a small volume of blood (<1 mL). This method provides higher yields (>4 folds) and purity (>99%) and lower platelet activation than the conventional method. Moreover, it was also superior in the detection of platelet-related RNAs CD41, PF4, and P2Y12 due to lower contamination with white blood cells.

Three-Dimensional Human Liver-Chip Emulating Premetastatic Niche Formation by Breast Cancer-Derived Extracellular Vesicles.

The liver is one of the most common sites of breast cancer metastasis and is associated with high lethality. Although the interaction between tumor cells and their microenvironment at metastatic sites has been recognized as a key regulator of tumor progression, the underlying mechanism is not fully elucidated. Here, we describe a three-dimensional (3D) microfluidic human liver-on-a-chip (liver-chip) that emulates the formation of a premetastatic niche to investigate the roles of breast cancer-derived extracellular vesicles (EVs) in liver metastasis. We demonstrate that breast cancer-derived EVs activate liver sinusoidal endothelial cells (LSECs) in the liver-chip, inducing endothelial to mesenchymal transition and destruction of vessel barriers. In addition, we show that transforming growth factor ß1 (TGFß1) in breast cancer-derived EVs upregulates fibronectin, an adhesive extracellular matrix protein, on LSECs, which facilitates the adhesion of breast cancer cells to the liver microenvironment. Furthermore, we observed that EVs isolated from triple-negative breast cancer (TNBC) patients with liver metastasis contain higher TGFß1 levels and induce adhesion of more breast cancer cells to the 3D human liver-chip than do EVs isolated from healthy donors or nonmetastatic TNBC patients. These findings provide a better understanding of the mechanisms through which breast cancer-derived EVs guide secondary metastasis to the liver. Furthermore, the 3D human liver-chip described in this study provides a platform to investigate the mechanisms underlying secondary metastasis to the liver and possible therapeutic strategies.

Fully Automated, Label-Free Isolation of Extracellular Vesicles from Whole Blood for Cancer Diagnosis and Monitoring.

Extracellular vesicles (EVs) that circulate in body fluids possess significant potential for disease diagnosis. Their use in clinical settings, however, has been limited owing to lack of simple and robust isolation methods. To rectify this problem, a centrifugal device for automatic, fast, and efficient isolation of EVs from whole-blood, called Exodisc-B is presented in this paper. Methods: The device comprises a built-in chamber to facilitate plasma separation and two nanoporous filters-one for removing larger particles and the other for enriching EVs. The performance of the device in comparison to ultracentrifugation (UC) was evaluated by analyzing the yield, purity, protein and RNA content of the isolated EVs. Additionally, the EV protein marker expressions were measured by ELISA and statistically analyzed to differentiate prostate cancer patients from healthy donors. Results: Compared with the UC technique, the proposed device is capable of isolating at least an order of magnitude higher number of EVs with about 30-fold higher mRNA count within 40 min. Sandwich ELISA of EV-specific membrane proteins-CD9-CD81-confirmed that Exodisc-B can isolate EVs from a volume of whole blood as low as 30 µL with a capture efficiency exceeding 75%. The device also facilitates temporal monitoring of tumor progression within live mouse xenograft models over a period of 13 weeks while using minimal volumes of weekly collected blood samples. Further, in ELISA analyses of multiple cancer-related proteins, such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor 1 (EGFR1), and heat shock protein 90 (HSP90), extracted from EVs isolated from human plasma, 43 patients were differentiated from 30 healthy donors. Conclusion: The results demonstrated the ability of Exodisc-B to provide a rapid, sensitive, and point-of-care-type method for extracting intact EVs from small volumes of clinical blood samples for disease diagnosis and monitoring.

Liquid Biopsy in Lung Cancer: Clinical Applications of Circulating Biomarkers (CTCs and ctDNA).

Lung cancer is by far the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Recent advances in the understanding of the biology of tumors and in highly sensitive detection technologies for molecular analysis offer targeted therapies, such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. However, our understanding of an individual patient's lung cancer is often limited by tumor accessibility because of the high risk and invasive nature of current tissue biopsy procedures. "Liquid biopsy", the analysis of circulating biomarkers from peripheral blood, such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), offers a new source of cancer-derived materials that may reflect the status of the disease better and thereby contribute to more personalized treatment. In this review, we examined the clinical significance and uniqueness of CTCs and ctDNA from NSCLC patients, isolation and detection methods developed to analyze each type of circulating biomarker, and examples of clinical studies of potential applications for early diagnosis, prognosis, treatment monitoring, and prediction of resistance to therapy. We also discuss challenges that remain to be addressed before such tools are implemented for routine use in clinical settings.

Urine-based liquid biopsy: non-invasive and sensitive AR-V7 detection in urinary EVs from patients with prostate cancer.

Androgen-receptor splice variant 7 (AR-V7) is associated with castration-resistant prostate cancer (CRPC) and resistance to anti-androgen therapy. Despite its clinical importance, the lack of efficient methods for AR-V7 analysis remains a challenge for broader use of this biomarker in routine clinical practice. Herein, we suggest a practical and non-invasive liquid biopsy method for analysis of AR-V7 in the RNA of urine-derived extracellular vesicles (EVs) without the need for blood withdrawal. Urine-derived EVs were isolated by a lab-on-a-disc integrated with six independent nanofiltration units (Exo-Hexa) allowing simultaneous processing of six individual samples. Rapid enrichment of EVs (<30 min) from each 4 mL urine sample was followed by mRNA extraction, and AR-V7 and androgen receptor full-length (AR-FL) mRNA levels in the urinary EVs were quantified by droplet digital polymerase chain reaction (ddPCR) as absolute concentrations (copies per mL). Higher AR-V7 and lower AR-FL expressions were detected in urine-derived EVs from 14 patients with CRPC than in those from 22 patients with hormone-sensitive prostate cancer. Additionally, we found that AR-V7 transcript levels and the AR-V7/AR-FL ratio in urinary EVs were higher in patients with advanced prostate cancer. This study is the first to report that RNA of urine-derived EVs is a reliable source for AR-V7 expression analysis. The proposed method for quantifying AR-V7 in urinary EVs prepared by a lab-on-a-disc is therefore a simple and promising approach to liquid biopsy with great potential for therapeutic impact on prostate cancer.

Fully automated, on-site isolation of cfDNA from whole blood for cancer therapy monitoring.

The potential utility of circulating tumour DNA (ctDNA) in patient blood for cancer diagnostics and real-time monitoring of disease progression is highly recognized. However, the lack of automated and efficient methods for cell-free DNA (cfDNA) isolation from peripheral blood has remained a challenge for broader acceptance of liquid biopsy in general clinical settings. Here, we demonstrate a lab-on-a-disc system equipped with newly developed, electromagnetically actuated, and reversible diaphragm valves that allows fully automated and rapid (<30 min) isolation of cfDNA from whole blood (>3 ml) to achieve high detection sensitivity by minimizing the degradation of fragile ctDNA as well as contamination of wild-type DNA from abundant blood cells. As a proof of concept study, we used the lab-on-a-disc to isolate cfDNA from patients with non-small cell lung cancer and successfully detected epidermal growth factor receptor gene mutations (L858R, T790M) during targeted drug therapy. The proposed lab-on-a-disc enables a fully automated, rapid, and point-of-care cfDNA enrichment starting from whole blood to facilitate the wide use of liquid biopsy in routine clinical practice.

Sensitivity enhancement of DNA microarray on nano-scale controlled surface by using a streptavidin-fluorophore conjugate.

High throughput analysis of DNA in low concentration and small volume is an important issue and a continuing challenge in the field of DNA microarray and sensor. Recently, we have demonstrated that the DNA microarray on nano-scale controlled surface provides ample space for hybridization resulting in the best discrimination efficiency for SNP analysis. Here, we report the utility of the nano-scale controlled surface in conjunction with a multiply tagged protein. Application of streptavidin-fluorophore conjugates in combination with the highly controlled surface that suppresses non-specific binding of DNA allows highly sensitive detection of DNA while maintaining superior SNP discrimination efficiency comparable to our earlier results. The sensitivity of DNA microarray on the mesospaced surface is two orders of magnitude higher than that of the generic surface when a streptavidin-fluorophore conjugate was employed, and the detection limit on the former surface was found to be 50 fM of 15-mer target DNA. Various streptavidin-fluorophore conjugates including streptavidin-Cy3, streptavidin-Cy5, streptavidin-Alexa Flour 555 and streptavidin-phycoerythrin were examined.

DNA microarrays on nanoscale-controlled surface.

We have developed new surface to ensure a proper spacing between immobilized biomolecules. While DNA microarray on this surface provided each probe DNA with ample space for hybridization with incoming target DNAs, the microarray showed enhanced discrimination efficiency for various types of single nucleotide polymorphism. The high discrimination efficiency holds for all tested cases (100:<1 for internal mismatched cases; 100:<28 for terminal mismatched ones). In addition, by investigating influence of hybridization temperature and washing condition on the fluorescence intensity and the discrimination efficiency with and without controlled mesospacing, it was observed that the nanoscale-controlled surface showed good discrimination efficiency in a wide range of temperature (37-50 degrees C), and hybridization behavior on the surface was in agreement with the solution one. Intriguingly, it was found that washing process after the hybridization was critical for the high discrimination efficiency. For the particular case, washing process was so efficient that only 30 s washing was sufficient to reach the optimal discrimination ratio.