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.

Alix-normalized exosomal programmed death-ligand 1 analysis in urine enables precision monitoring of urothelial cancer.

Anti-programmed death-ligand 1 (PD-L1) Ab-based therapies have demonstrated potential for treating metastatic urothelial cancer with high PD-L1 expression. Urinary exosomes are promising biomarkers for liquid biopsy, but urine's high variability requires normalization for accurate analysis. This study proposes using the PD-L1/Alix ratio to normalize exosomal PD-L1 signal intensity with Alix, an internal exosomal protein less susceptible to heterogeneity concerns than surface protein markers. Extracellular vesicles were isolated using ExoDisc and characterized using various methods, including ExoView to analyze tetraspanins, PD-L1, and Alix on individual exosomes. On-disc ELISA was used to evaluate PD-L1 and Alix-normalized PD-L1 in 15 urothelial cancer patients during the initial treatment cycle with Tecentriq. Results showed that Alix signal range was relatively uniform, whereas tetraspanin marker intensity varied for individual exosome particles. On-disc ELISA was more reliable for detecting exosomal PD-L1 expression than standard plate ELISA-based measurement. Using exosomal Alix expression for normalization is a more reliable approach than conventional methods for monitoring patient status. Overall, the study provides a practical and reliable method for detecting exosomal PD-L1 in urine samples from patients with urothelial cancer.

Tonicity-induced cargo loading into extracellular vesicles.

The current challenge in using extracellular vesicles (EVs) as drug delivery vehicles is to precisely control their membrane permeability, specifically in the ability to switch between permeable and impermeable states without compromising their integrity and functionality. Here, we introduce a rapid, efficient, and gentle loading method for EVs based on tonicity control (TC) using a lab-on-a-disc platform. In this technique, a hypotonic solution was used for temporarily permeabilizing a membrane ("on" state), allowing the influx of molecules into EVs. The subsequent isotonic washing led to an impermeable membrane ("off" state). This loading cycle enables the loading of different cargos into EVs, such as doxorubicin hydrochloride (Dox), ssDNA, and miRNA. The TC approach was shown to be more effective than traditional methods such as sonication or extrusion, with loading yields that were 4.3-fold and 7.2-fold greater, respectively. Finally, the intracellular assessments of miRNA-497-loaded EVs and doxorubicin-loaded EVs confirmed the superior performance of TC-prepared formulations and demonstrated the impact of encapsulation heterogeneity on the therapeutic outcome, signifying potential opportunities for developing novel exosome-based therapeutic systems for clinical applications.

Platelet Membrane-Enclosed Bioorthogonal Catalysis for Combating Dental Caries.

Platelets have shown promise as a means to combat bacterial infections, fostering the development of innovative therapeutic approaches. However, several challenges persist, including cargo loading issues, limited efficacy against biofilms, and concerns regarding the impact of payloads on the platelet carriers. Here, human platelet membrane vesicles (h-PMVs) encapsulating supramolecular metal catalysts (SMCs) as "nanofactories" to convert prodrugs into antimicrobial compounds within close proximity to bacteria are introduced. Having established the feasibility and effectiveness of the SMCs within h-PMVs, referred to as the PLT-reactor, to activate pro-antibiotic drugs (pro-ciprofloxacin and pro-moxifloxacin) using model organisms (Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923), the investigation is subsequently extended to oral biofilms, with a particular emphasis on Streptococcus mutans 3065. This "bind and kill" strategy demonstrates the potent antimicrobial specificity of the PLT-reactor through localized antibiotic production. h-PMVs play a pivotal role by enabling precise targeting of pathogenic biofilms on natural teeth while minimizing potential hemolytic effects. The finding indicates that platelet membrane-cloaked surfaces exhibit robust, multifaceted, and pathogen-specific binding affinity with excellent biocompatibility, making them a promising alternative to antibody-based therapies for infectious diseases.

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.

Serologic and Molecular Prevalence of Severe Fever with Thrombocytopenia Syndrome Virus Among Poultry in the Republic of Korea.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by Dabie bandavirus, which belongs to the genus Bandavirus, family Phenuiviridae, and order Bunyavirales. It has been found in tick species, various animals, and humans. The aim of this study was to detect RNA of antigens and antibodies against SFTS virus (SFTSV) among poultry such as chickens, ducks, and wild geese from five provinces in the Republic of Korea (ROK). Materials and Methods: A one-step reverse transcriptase (RT)-PCR and nested PCR were performed after viral RNA extraction. The phylogenetic tree was constructed after sequencing data were analyzed and aligned. An indirect enzyme-linked immunosorbent assay (ELISA) and a neutralization test (NT) were performed to test for IgG antibodies of SFTSV. Results: Of a total of 606 poultry serum samples collected, 568 and 539 serum samples were used to perform ELISA and NT, respectively. Of a total of 606 serum samples tested by RT-PCR targeting the S segment, 15 (2.5%) were positive for SFTSV. From the 15 positive serum samples for the SFTSV antigen, three from chickens, three from ducks, and one from wild geese were classified as genotype B-2; one from chickens was classified as genotype B-3; and three from chickens and four from wild geese were classified as genotype D. Of the 568 serum samples tested by ELISA, 83 (28.0%) from chickens, 81 (32.9%) from ducks, and 8 (30.8%) from wild geese were seropositive. Of the 539 serum samples for which an NT was performed, 113 (38.6%) from chickens and 75 (30.5%) from ducks were positive for SFTSV antibodies. Conclusions: The results of this study provide useful information regarding detection of SFTSV RNA and antibodies among poultry and the possibility of SFTSV transmission in various types of poultry, including chickens, ducks, and wild geese, in the ROK.

Molecular and Serological Survey of Severe Fever with Thrombocytopenia Syndrome Virus in Horses from the Republic of Korea.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic tick-borne disease in East Asia caused by the SFTS virus (SFTSV). It is to investigate the presence of SFTSV RNA and antibodies in horses from a slaughterhouse and equestrian centers in the Republic of Korea (ROK). A prevalence study of SFTSV-specific RNA and antibodies was designed from 889 horses in the ROK. Materials and Methods: Serum samples were collected from horses at a slaughterhouse and equestrian centers from 2018 to 2020. To detect the presence of SFTSV, RNA was extracted from the serum samples, and a nested reverse transcription-polymerase chain reaction (RT-PCR) was conducted. Sequencing data were analyzed, and a phylogenetic tree was constructed using the maximum-likelihood method with Molecular Evolutionary Genetics Analysis Version 7.0 software. The horse sera were also tested for SFTSV-specific immunoglobulin G antibodies using enzyme-linked immunosorbent assay (ELISA). Results: Twelve of 889 (1.3%) horse sera were positive for SFTSV RNA, and 452 of 887 (51.0%) horse sera were seropositive by ELISA. Among the RT-PCR-positive samples, 12 of the SFTSV S-segment sequences were classified as sub-genotypes B-2 (n = 6) and B-3 (n = 6). ELISA analysis was evaluated by comparison with neutralization test. We investigated SFTSV infection in horses over a 3-year period, but sampling was not performed evenly by season; continuous surveillance of SFTSV in horses is needed. Conclusions: We report the detection of SFTSV RNA and provide serological data on SFTSV prevalence in horses in the ROK. The detection of SFTSV-specific RNA and antibodies in horses, which are in close proximity to humans, suggests that SFTS is an emerging and important health issue, indicating that more attention to its relevance for equestrian workers is needed.

Nuclear morphology predicts cell survival to cisplatin chemotherapy.

The emergence of chemotherapy resistance drives cancer lethality in cancer patients, with treatment initially reducing overall tumor burden followed by resistant recurrent disease. While molecular mechanisms underlying resistance phenotypes have been explored, less is known about the cell biological characteristics of cancer cells that survive to eventually seed the recurrence. To identify the unique phenotypic characteristics associated with survival upon chemotherapy exposure, we characterized nuclear morphology and function as prostate cancer cells recovered following cisplatin treatment. Cells that survived in the days and weeks after treatment and resisted therapy-induced cell death showed increasing cell size and nuclear size, enabled by continuous endocycling resulting in repeated whole genome doubling. We further found that cells that survive after therapy release were predominantly mononucleated and likely employ more efficient DNA damage repair. Finally, we show that surviving cancer cells exhibit a distinct nucleolar phenotype and increased rRNA levels. These data support a paradigm where soon after therapy release, the treated population mostly contains cells with a high level of widespread and catastrophic DNA damage that leads to apoptosis, while the minority of cells that have successful DDR are more likely to access a pro-survival state. These findings are consistent with accession of the polyaneuploid cancer cell (PACC) state, a recently described mechanism of therapy resistance and tumor recurrence. Our findings demonstrate the fate of cancer cells following cisplatin treatment and define key cell phenotypic characteristics of the PACC state. This work is essential for understanding and, ultimately, targeting cancer resistance and recurrence.

A machine learning approach to discover migration modes and transition dynamics of heterogeneous dendritic cells.

Dendritic cell (DC) migration is crucial for mounting immune responses. Immature DCs (imDCs) reportedly sense infections, while mature DCs (mDCs) move quickly to lymph nodes to deliver antigens to T cells. However, their highly heterogeneous and complex innate motility remains elusive. Here, we used an unsupervised machine learning (ML) approach to analyze long-term, two-dimensional migration trajectories of Granulocyte-macrophage colony-stimulating factor (GMCSF)-derived bone marrow-derived DCs (BMDCs). We discovered three migratory modes independent of the cell state: slow-diffusive (SD), slow-persistent (SP), and fast-persistent (FP). Remarkably, imDCs more frequently changed their modes, predominantly following a unicyclic SD→FP→SP→SD transition, whereas mDCs showed no transition directionality. We report that DC migration exhibits a history-dependent mode transition and maturation-dependent motility changes are emergent properties of the dynamic switching of the three migratory modes. Our ML-based investigation provides new insights into studying complex cellular migratory behavior.

Tuning the Extracellular Vesicles Membrane through Fusion for Biomedical Applications.

Membrane fusion is one of the key phenomena in the living cell for maintaining the basic function of life. Extracellular vesicles (EVs) have the ability to transfer information between cells through plasma membrane fusion, making them a promising tool in diagnostics and therapeutics. This study explores the potential applications of natural membrane vesicles, EVs, and their fusion with liposomes, EVs, and cells and introduces methodologies for enhancing the fusion process. EVs have a high loading capacity, bio-compatibility, and stability, making them ideal for producing effective drugs and diagnostics. The unique properties of fused EVs and the crucial design and development procedures that are necessary to realize their potential as drug carriers and diagnostic tools are also examined. The promise of EVs in various stages of disease management highlights their potential role in future healthcare.

Integrated technologies for continuous monitoring of organs-on-chips: Current challenges and potential solutions.

Organs-on-chips (OoCs) are biomimetic in vitro systems based on microfluidic cell cultures that recapitulate the in vivo physicochemical microenvironments and the physiologies and key functional units of specific human organs. These systems are versatile and can be customized to investigate organ-specific physiology, pathology, or pharmacology. They are more physiologically relevant than traditional two-dimensional cultures, can potentially replace the animal models or reduce the use of these models, and represent a unique opportunity for the development of personalized medicine when combined with human induced pluripotent stem cells. Continuous monitoring of important quality parameters of OoCs via a label-free, non-destructive, reliable, high-throughput, and multiplex method is critical for assessing the conditions of these systems and generating relevant analytical data; moreover, elaboration of quality predictive models is required for clinical trials of OoCs. Presently, these analytical data are obtained by manual or automatic sampling and analyzed using single-point, off-chip traditional methods. In this review, we describe recent efforts to integrate biosensing technologies into OoCs for monitoring the physiologies, functions, and physicochemical microenvironments of OoCs. Furthermore, we present potential alternative solutions to current challenges and future directions for the application of artificial intelligence in the development of OoCs and cyber-physical systems. These "smart" OoCs can learn and make autonomous decisions for process optimization, self-regulation, and data analysis.

Bridging the Gap between Nonliving Matter and Cellular Life.

A cell, the fundamental unit of life, contains the requisite blueprint information necessary to survive and to build tissues, organs, and systems, eventually forming a fully functional living creature. A slight structural alteration can result in data misprinting, throwing the entire life process off balance. Advances in synthetic biology and cell engineering enable the predictable redesign of biological systems to perform novel functions. Individual functions and fundamental processes at the core of the biology of cells can be investigated by employing a synthetically constrained micro or nanoreactor. However, constructing a life-like structure from nonliving building blocks remains a considerable challenge. Chemical compartments, cascade signaling, energy generation, growth, replication, and adaptation within micro or nanoreactors must be comparable with their biological counterparts. Although these reactors currently lack the power and behavioral sophistication of their biological equivalents, their interface with biological systems enables the development of hybrid solutions for real-world applications, such as therapeutic agents, biosensors, innovative materials, and biochemical microreactors. This review discusses the latest advances in cell membrane-engineered micro or nanoreactors, as well as the limitations associated with high-throughput preparation methods and biological applications for the real-time modulation of complex pathological states.

Alpha-2-macroglobulin as a novel diagnostic biomarker for human bladder cancer in urinary extracellular vesicles.

Extracellular vesicles (EVs) derived from urine are promising tools for the diagnosis of urogenital cancers. Urinary EVs (uEVs) are considered potential biomarkers for bladder cancer (BC) because urine is in direct contact with the BC tumor microenvironment and thus reflects the current state of the disease. However, challenges associated with the effective isolation and analysis of uEVs complicate the clinical detection of uEV-associated protein biomarkers. Herein, we identified uEV-derived alpha-2-macroglobulin (a2M) as a novel diagnostic biomarker for BC through comparative analysis of uEVs obtained from patients with BC pre- and post-operation using an antibody array. Furthermore, enzyme-linked immunosorbent assay of uEVs isolated from patients with BC (n=60) and non-cancer control subjects (n=23) validated the significant upregulation of a2M expression in patient uEVs (p<0.0001). There was no significant difference in whole urine a2M levels between patients with BC and controls (p=0.317). We observed that compared to classical differential centrifugation, ExoDisc, a centrifugal microfluidic tangential flow filtration device, was a significantly more effective separation method for uEV protein analysis. We expect that our approach for EV analysis will provide an efficient route for the identification of clinically meaningful uEV-based biomarkers for cancer diagnosis.

Clinical features and epidemiology of severe fever with thrombocytopenia syndrome in dogs in the Republic of Korea: an observational study (2019-2020).

Severe fever with thrombocytopenia syndrome (SFTS) is a zoonotic disease with a high mortality rate for humans and cats. The clinical course and prognosis of SFTS in dogs remains unclear. In the present study, we investigated the clinical and epidemiological characteristics of SFTS virus (SFTSV) infection in dogs. All evaluated dogs exhibited an acute course and symptoms including fever (57.1%), anorexia (57.1%), depression (42.9%), and vomiting (35.7%). Thrombocytopenia was present in 45.5% of dogs, while jaundice was not observed. C-reactive protein, alanine transaminase, and alkaline phosphatase were elevated in some cases. Viral clearance occurred within 6 to 26 days. Phylogenetic analysis revealed that the SFTSV sequences were consistent with viruses circulating in the Republic of Korea. As dogs often live in close contact with humans, awareness of the clinical and epidemiological features of SFTS in dogs is crucial. Further large-scale studies are necessary to investigate SFTSV infection in dogs.

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.

SEEDING to Enable Sensitive Electrochemical Detection of Biomarkers in Undiluted Biological Samples.

Electrochemical biosensors have shown great potential for simple, fast, and cost-effective point-of-care diagnostic tools. However, direct analysis of complex biological fluids such as plasma has been limited by the loss of sensitivity caused by biofouling. By increasing the surface area, the nanostructured electrode can improve detection sensitivity. However, like a double-edged sword, a large surface area increases the nonspecific adsorption of contaminating proteins. The use of nanoporous structures may prevent fouling proteins. However, there is no straightforward approach for creating nanostructured and nanoporous surfaces compatible with microfabricated thin-film electrodes. Herein, the preferential etching of chloride and surfactant-assisted anisotropic gold reduction to create homogeneous, nanostructured, and nanoporous gold electrodes is demonstrated, yielding a 190 ± 20 times larger surface area within a minute without using templates. This process, "surfactant-based electrochemical etch-deposit interplay for nanostructure/nanopore growth" (SEEDING), on electrodes enhances the sensitivity and antibiofouling capabilities of amperometric biosensors, enabling direct analysis of tumor-derived extracellular vesicles (tEVs) in complex biofluids with a limit of detection of 300 tEVs µL-1  from undiluted plasma and good discrimination between patients with prostate cancer from healthy ones with an area under the curve of 0.91 in urine and 0.90 in plasma samples.

Recent advances in spheroid-based microfluidic models to mimic the tumour microenvironment.

Three-dimensional (3D) multicellular spheroid models can recapitulate the human tumour microenvironment with more accuracy than conventional cell culture models, as they include complex architectural structures and dynamic cellular interactions. Among the diverse platforms for spheroid formation, microfluidic platforms have been extensively applied to study spheroids because they can mimic the in vivo microenvironment. This review provides an overview of the advantages of 3D spheroid cultures with a summary of the recent applications for tumour microenvironment-focused cellular interactions, as well as the studies on spheroids and external stimuli. These 3D tumour spheroid-based microfluidic devices will provide a platform for a better understanding of cellular and external interactions, as well as the discovery of cancer therapeutics.

Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a zoonotic, tick-borne RNA virus of the genus Bandavirus (Family Phenuiviridae), mainly reported in China, Japan, and the Republic of Korea (Korea). For the purpose of this study, a total of 3,898 adult and nymphal ticks of species Haemaphysalis longicornis (94.2%), Haemaphysalis flava (5.0%), Ixodes nipponensis (0.8%), and 1 specimen of Ixodes ovatus, were collected from the Deogyusan National Park, Korea, between April 2016 and June 2018. A single-step reverse transcriptase-nested PCR was performed, targeting the S segment of the SFTSV RNA. Total infection rate (IR) of SFTSV in individual ticks was found to be 6.0%. Based on developmental stages, IR was 5.3% in adults and 6.0% in nymphs. The S segment sequences obtained from PCR were divided into 17 haplotypes. All haplotypes were phylogenetically clustered into clades B-2 and B-3, with 92.7% sequences in B-2 and 7.3% in B-3. These observations indicate that the Korean SFTSV strains were closer to the Japanese than the Chinese strains. Further epidemiological studies are necessary to better understand the characteristics of the Korean SFTSV and its transmission cycle in the ecosystem.

Serological evidence of Anaplasma spp., Borrelia burgdorferi and Ehrlichia canis in dogs from the Republic of Korea by rapid diagnostic test kits.

BACKGROUND: Emergent and re-emergent canine tick-borne infections are attracting increasing attention worldwide. The rise in pet ownership and the close relationship between dogs and their owners are the most concerning factors because dogs may act as competent reservoirs for human tick-transmitted infectious agents. OBJECTIVES: This study contributes to the epidemiological surveillance of canine tick-transmitted infections with zoonotic risk in the Republic of Korea (ROK) by investigating the seroprevalence of the pathogens, Anaplasma spp., Borrelia burgdorferi, and Ehrlichia canis. METHODS: Four hundred and thirty whole blood samples from domestic dogs were collected in seven metropolitan cities and nine provinces in the ROK and tested using SensPERT Ab test kits (VetAll Laboratories®) to detect seroreactive animals. RESULTS: The seroprevalence rates identified were 9.8% (42/430) for Anaplasma spp., 2.8% (12/430) for B. burgdorferi, and 1.4% (6/430) for E. canis. The risk factors evaluated in this study that could be associated with the development of a humoral immune response, such as sex, age, and history of tick exposure, were similar. There was only one exception for dogs seroreactive to Anaplasma spp., where the risk factor "tick exposure" was statistically significant (p = 0.047). CONCLUSIONS: This serological survey exhibited the widespread presence of Anaplasma spp., B. burgdorferi, and E. canis throughout the ROK. Hence, dogs may play a key role as the sentinel animals of multiple zoonotic infectious agents in the country.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis.

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.