Epstein-Barr viral product-containing exosomes promote fibrosis and nasopharyngeal carcinoma progression through activation of YAP1/FAPα signaling in fibroblasts.

BACKGROUND: The progression of nasopharyngeal carcinoma (NPC) is profoundly affected by Epstein-Barr virus (EBV) infection. However, the role of EBV in the intercommunication between NPC and surrounding stromal cells has yet to be explored. METHODS: NPC biopsies were obtained for immunohistochemical (IHC) analyses. Clinical correlations between the expression of active YAP1/FAPα and the fibrotic response and between YAP1/FAPα and the density of cytotoxic CD8a+ T lymphocytes were determined. Survival times based on IHC scores were compared between groups using Kaplan-Meier survival and log-rank tests. Independent prognostic factors for metastasis/recurrence-free survival and overall survival were identified using univariate and multivariate Cox regression models. Fibroblasts were isolated from human nasopharyngeal biopsies. Exosomes were purified from culture supernatants of EBV+-positive NPC cells. The effects of EBV product-containing exosomes on fibroblast activation, fibrotic response, tumor growth, immune response, and correlations between the expression of featured genes were investigated using gel contraction assays, ELISAs, EdU incorporation assays, real-time impedance assays, RNA sequencing, immunostaining, 3D cancer spheroid coculture systems, and an NPC xenograft model. RESULTS: NPC patients who developed metastasis had significantly higher levels of active YAP1 and FAPα in their tumor stroma, which was further correlated with tumor fibrosis and poorer metastasis-free survival. Exosomes released from EBV+-NPC cells contained abundant FAPα protein and EBV-encoded latent membrane protein 1. Viral product-containing exosomes markedly enhanced the fibrotic response and tumor growth in a mouse xenograft model. IHC analyses of human NPC and NPC xenografts revealed positive correlations between levels of active YAP1 and FAPα, YAP1 and the fibrotic response, and FAPα and the fibrotic response. Mechanistic studies showed that treatment of fibroblasts with viral product-containing exosomes promoted the characteristics of cancer-associated fibroblasts by stimulating YAP1 signaling and the production of the immunosuppressive cytokines IL8, CCL2, and IL6. Inhibition of YAP1 activation markedly reversed these exosome-mediated protumoral effects, resulting in reduced contractility, inactivation of YAP1 signaling, and decreased production of immunosuppressive cytokines in fibroblasts. Furthermore, fibroblasts stimulated with these viral product-containing exosomes promoted NPC resistance to T cell-mediated cytotoxicity within tumor spheroids. In NPC tissues, a significant negative correlation was found between YAP1/FAPα and the density of CD8a+ T lymphocytes with a granzyme B signature. CONCLUSION: EBV orchestrates interactions with the host and surrounding stroma by stimulating the functions of YAP1 and FAPα in fibroblasts through exosome cargos to create a more immunosuppressive, proinvasive microenvironment.

An integrated magneto-electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma.

Assays for cancer diagnosis via the analysis of biomarkers on circulating extracellular vesicles (EVs) typically have lengthy sample workups, limited throughput or insufficient sensitivity, or do not use clinically validated biomarkers. Here we report the development and performance of a 96-well assay that integrates the enrichment of EVs by antibody-coated magnetic beads and the electrochemical detection, in less than one hour of total assay time, of EV-bound proteins after enzymatic amplification. By using the assay with a combination of antibodies for clinically relevant tumour biomarkers (EGFR, EpCAM, CD24 and GPA33) of colorectal cancer (CRC), we classified plasma samples from 102 patients with CRC and 40 non-CRC controls with accuracies of more than 96%, prospectively assessed a cohort of 90 patients, for whom the burden of tumour EVs was predictive of five-year disease-free survival, and longitudinally analysed plasma from 11 patients, for whom the EV burden declined after surgery and increased on relapse. Rapid assays for the detection of combinations of tumour biomarkers in plasma EVs may aid cancer detection and patient monitoring.

Epstein-Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment.

The most frequent location of metastatic EBV+ nasopharyngeal carcinoma (NPC) is the bone marrow, an adipocyte-dominant region. Several EBV-associated lymphoepithelioma-like carcinoma (LELC) types also grow in the anatomical vicinity of fat tissues. Here we show that in an adipose tissue-rich tumor setting, EBV targets adipocytes and remodels the tumor microenvironment. Positive immunoreactivity for EBV-encoded early antigen D was detected in adipose tissue near tumor beds of bone marrow metastatic NPC. EBV was capable of infecting primary human adipocytes in vitro, triggering expression of multiple EBV-encoded mRNA and proteins. In infected adipocytes, lipolysis was stimulated through enhanced expression of lipases and the AMPK metabolic pathway. The EBV-mediated imbalance in energy homeostasis was further confirmed by increased release of free fatty acids, glycerol, and expression of proinflammatory adipokines. Clinically, enhanced serum levels of free fatty acids in patients with NPC correlated with poorer recurrence-free survival. EBV-induced delipidation stimulated dedifferentiation of adipocytes into fibroblast-like cells expressing higher levels of S100A4, a marker protein of cancer-associated fibroblasts (CAF). IHC analyses of bone marrow metastatic NPC and salivary LELC revealed similar structural changes of dedifferentiated adipocytes located at the boundaries of EBV+ tumors. S100A4 expression in adipose tissues near tumor beds correlated with fibrotic response, implying that CAFs in the tumor microenvironment are partially derived from EBV-induced dedifferentiated adipocytes. Our data suggest that adipose tissue serves as an EBV reservoir, where EBV orchestrates the interactions between adipose tissues and tumor cells by rearranging metabolic pathways to benefit virus persistence and to promote a protumorigenic microenvironment. SIGNIFICANCE: This study suggests that Epstein-Barr virus hijacks adipocyte lipid metabolism to create a tumor-promoting microenvironment from which reactivation and relapse of infection could potentially occur.

Compact and Filter-Free Luminescence Biosensor for Mobile in Vitro Diagnoses.

We report a sensitive and versatile biosensing approach, LUCID (luminescence compact in vitro diagnostics), for quantitative molecular and cellular analyses. LUCID uses upconversion nanoparticles (UCNPs) as luminescent reporters in mutually exclusive photoexcitation and read-out sequences implemented on a smartphone. The strategy improves imaging signal-to-noise ratios, eliminating interference from excitation sources and minimizing autofluorescence, and thus enables filterless imaging. Here we developed a miniaturized detection system and optimized UCNPs for the system and biological applications. Nanoparticle luminescence lifetime was extended by controlling particle structure and composition. When tested with a range of biological targets, LUCID achieved high detection sensitivity (0.5 pM for protein and 0.1 pM for nucleic acids), differentiated bacterial samples, and allowed profiling of cells. In proof-of-concept clinical use, LUCID demonstrated effective screening of cancer cells in cervical brushing specimens, identifying patients at high risk for malignancy. These results suggest that LUCID could serve as a broadly applicable and inexpensive diagnostic platform.

Biocompatible Inhibitor Based on Chitosan and Amphiphilic Peptide against Mutant Huntingtin Toxicity.

Huntington's disease (HD) is classified as a protein-misfolding disease correlated with the mutant Huntingtin (mHtt) protein with abnormally expanded polyglutamine (polyQ) domains. Because no effective drugs have yet been reported, attempts to develop better therapy to delay the age of onset are in urgent demand. In this study, an amphiphilic peptide consisting of negatively charged hexaglutamic acid and a stretch of decaglutamine (E6 Q10 ) was chemically synthesized as an inhibitor against polyQ and mHtt toxicity. It is found that E6 Q10 selfassembles into spherical vesicles, as shown by means of TEM, cryoelectron microscopy, and dynamic light scattering. Assembled E6 Q10 prevented the polyQ-rich peptide (KKWQ20 AKK) from forming amyloid fibrils. To enable the cell-penetration ability of E6 Q10 , the E6 Q10 ⋅chitosan complex was generated. It is demonstrated that the complex penetrates cells, interferes with the mHtt oligomerization and aggregation process, and prevents mHtt cytotoxicity. By combining positively charged chitosan and amphiphilic peptides with a negatively charge moiety, a new strategy is provided to develop biocompatible and biodegradable inhibitors against mHtt toxicity.

Navigating the Landscape of Tumor Extracellular Vesicle Heterogeneity.

The last decade has seen a rapid expansion of interest in extracellular vesicles (EVs) released by cells and proposed to mediate intercellular communication in physiological and pathological conditions. Considering that the genetic content of EVs reflects that of their respective parent cell, many researchers have proposed EVs as a source of biomarkers in various diseases. So far, the question of heterogeneity in given EV samples is rarely addressed at the experimental level. Because of their relatively small size, EVs are difficult to reliably isolate and detect within a given sample. Consequently, standardized protocols that have been optimized for accurate characterization of EVs are lacking despite recent advancements in the field. Continuous improvements in pre-analytical parameters permit more efficient assessment of EVs, however, methods to more objectively distinguish EVs from background, and to interpret multiple single-EV parameters are lacking. Here, we review EV heterogeneity according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. In doing so, we also provide an overview of currently available and potentially applicable methods for single EV analysis. Finally, we examine the latest findings from experiments that have analyzed the issue at the single EV level and discuss potential implications.

Integrated Kidney Exosome Analysis for the Detection of Kidney Transplant Rejection.

Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.

Multiparametric plasma EV profiling facilitates diagnosis of pancreatic malignancy.

Pancreatic ductal adenocarcinoma (PDAC) is usually detected late in the disease process. Clinical workup through imaging and tissue biopsies is often complex and expensive due to a paucity of reliable biomarkers. We used an advanced multiplexed plasmonic assay to analyze circulating tumor-derived extracellular vesicles (tEVs) in more than 100 clinical populations. Using EV-based protein marker profiling, we identified a signature of five markers (PDACEV signature) for PDAC detection. In our prospective cohort, the accuracy for the PDACEV signature was 84% [95% confidence interval (CI), 69 to 93%] but only 63 to 72% for single-marker screening. One of the best markers, GPC1 alone, had a sensitivity of 82% (CI, 60 to 95%) and a specificity of 52% (CI, 30 to 74%), whereas the PDACEV signature showed a sensitivity of 86% (CI, 65 to 97%) and a specificity of 81% (CI, 58 to 95%). The PDACEV signature of tEVs offered higher sensitivity, specificity, and accuracy than the existing serum marker (CA 19-9) or single-tEV marker analyses. This approach should improve the diagnosis of pancreatic cancer.

Supramolecular Metallo-Bioadhesive for Minimally Invasive Use.

A novel metallo-bioadhesive to be used as tissue sealant in minimally invasive procedures is reported. Metal complexation can be used to render gelatin derivatives adhesive, which occurs in minutes, is efficient, and fully biodegradable within weeks.

Visibility enhancement of common bile duct for laparoscopic cholecystectomy by vivid fiber-optic indication: a porcine experiment trial.

Bile duct injury (BDI) is the most serious iatrogenic complication during laparoscopic cholecystectomy (LC) and occurs easily in inexperienced surgeons since the position of common bile duct (CBD) and its related ductal junctions are hard to precisely identify in the hepatic anatomy during surgery. BDI can be devastating, leading to chronic morbidity, high mortality, and prolonged hospitalization. In addition, it is the most frequent injury resulting in litigation and the most likely injury associated with a successful medical malpractice claim against surgeons. This study introduces a novel method for conveniently and rapidly indicating the anatomical location of CBD during LC by the direct fiber-optic illumination of 532-nm diode-pumped solid state laser through a microstructured plastic optical fiber to avoid the wrong identification of CBD and the injury from mistakenly cutting the CBD that can lead to permanent and even life threatening consequences. Six porcine were used for preliminary intra-CBD illumination experiments via laparotomy and direct duodenal incision to insert the invented CBD illumination laser catheter with nonharmful but satisfactory visual optical density.

Plasmon-induced optical switching of electrical conductivity in porous anodic aluminum oxide films encapsulated with silver nanoparticle arrays.

We report on plasmon induced optical switching of electrical conductivity in two-dimensional (2D) arrays of silver (Ag) nanoparticles encapsulated inside nanochannels of porous anodic aluminum oxide (AAO) films. The reversible switching of photoconductivity greatly enhanced by an array of closely spaced Ag nanoparticles which are isolated from each other and from the ambient by thin aluminum oxide barrier layers are attributed to the improved electron transport due to the localized surface plasmon resonance and coupling among Ag nanoparticles. The photoconductivity is proportional to the power, and strongly dependent on the wavelength of light illumination. With Ag nanoparticles being isolated from the ambient environments by a thin layer of aluminum oxide barrier layer of controlled thickness in nanometers to tens of nanometers, deterioration of silver nanoparticles caused by environments is minimized. The electrochemically fabricated nanostructured Ag/AAO is inexpensive and promising for applications to integrated plasmonic circuits and sensors.