Rapid Activation of Neuroinflammation in Stroke: Plasma and Extracellular Vesicles Obtained on a Mobile Stroke Unit.

BACKGROUND: Neuroinflammation is ubiquitous in acute stroke and worsens outcome. However, the precise timing of the inflammatory response is unknown, hindering the design of acute anti-inflammatory therapeutic interventions. We sought to identify the onset of the neuroinflammatory cascade using a mobile stroke unit. METHODS: The study is a proof-of-concept, cohort investigation of ultra-early blood- and extracellular vesicle-derived markers of neuroinflammation and outcome in acute stroke. Blood was obtained, prehospital, on an mobile stroke unit. Outcomes were biomarker concentrations, modified Rankin Scale score, and National Institutes of Health Stroke Scale score. RESULTS: Forty-one adults were analyzed, including 15 patients treated on the mobile stroke unit between August 2021 and April 2022, and 26 healthy controls to establish biomarker reference levels. Median patient age was 74 (range, 36-97) years, 60% were female, and 80% White. Ten (67%) were diagnosed as stroke, with 8 (53%) confirmed and 2 likely transient ischemic attack or stroke averted by thrombolysis; 5 were stroke mimics. For strokes, median initial National Institutes of Health Stroke Scale score was 11 (range, 4-19) and 6 (75%) received tPA (tissue-type plasminogen activator). Blood was obtained a median of 58 (range, 36-133) minutes after symptom onset. Within 36 minutes after stroke, plasma IL-6 (interleukin-6), neurofilament light chain, UCH-L1 (ubiquitin C-terminal hydrolase L1), and GFAP (glial fibrillary acidic protein) were elevated by as much as 10 times normal. In EVs, MMP-9 (matrix metalloproteinase-9), CXCL4 (chemokine (C-X-C motif) ligand 4), CRP (C-reactive protein), IL-6, OPN (osteopontin), and PECAM1 (platelet and endothelial cell adhesion molecule 1) were elevated. Inflammatory markers increased rapidly in the first 2 hours and continued rising for 24 hours. CONCLUSIONS: The neuroinflammatory cascade was found to be activated within 36 to 133 minutes after stroke and progresses rapidly. This is earlier than observed previously in humans and suggests injury from neuroinflammation occurs faster than had been surmised. These findings could inform development of acute immunomodulatory stroke therapies and lead to new diagnostic tools and improved outcomes.

Extracellular vesicle secretion is tissue-dependent ex vivo and skeletal muscle myofiber extracellular vesicles reach the circulation in vivo.

Extracellular vesicles (EVs) are biomarkers and modifiers of human disease. EVs secreted by insulin-responsive tissues like skeletal muscle (SkM) and white adipose tissue (WAT) contribute to metabolic health and disease but the relative abundance of EVs from these tissues has not been directly examined. Human Protein Atlas data and directly measuring EV secretion in mouse SkM and WAT using an ex vivo tissue explant model confirmed that SkM tissue secretes more EVs than WAT. Differences in EV secretion between SkM and WAT were not due to SkM contraction but may be explained by differences in tissue metabolic capacity. We next examined how many EVs secreted from SkM tissue ex vivo and in vivo are myofiber-derived. To do this, a SkM myofiber-specific dual fluorescent reporter mouse was created. Spectral flow cytometry revealed that SkM myofibers are a major source of SkM tissue-derived EVs ex vivo and EV immunocapture indicates that ∼5% of circulating tetraspanin-positive EVs are derived from SkM myofibers in vivo. Our findings demonstrate that 1) SkM secretes more EVs than WAT, 2) many SkM tissue EVs are derived from SkM myofibers, and 3) SkM myofiber-derived EVs reach the circulation in vivo. These findings advance our understanding of EV secretion between metabolically active tissues and provide direct evidence that SkM myofibers secrete EVs that can reach the circulation in vivo.

Diverse Populations of Extracellular Vesicles with Opposite Functions during Herpes Simplex Virus 1 Infection.

Extracellular vesicles (EVs) are released by all types of cells as a means of intercellular communication. Their significance lies in the fact that they can alter recipient cell functions, despite their limited capacity for cargo. We have previously demonstrated that herpes simplex virus 1 (HSV-1) infection influences the cargo and functions of EVs released by infected cells and that these EVs negatively impact a subsequent HSV-1 infection. In the present study, we have implemented cutting-edge technologies to further characterize EVs released during HSV-1 infection. We identified distinct EV populations that were separable through a gradient approach. One population was positive for the tetraspanin CD63 and was distinct from EVs carrying components of the endosomal sorting complexes required for transport (ESCRT). Nanoparticle tracking analysis (NTA) combined with protein analysis indicated that the production of CD63+ EVs was selectively induced upon HSV-1 infection. The ExoView platform supported these data and suggested that the amount of CD63 per vesicle is larger upon infection. This platform also identified EV populations positive for other tetraspanins, including CD81 and CD9, whose abundance decreased upon HSV-1 infection. The stimulator of interferon genes (STING) was found in CD63+ EVs released during HSV-1 infection, while viral components were found in ESCRT+ EVs. Functional characterization of these EVs demonstrated that they have opposite effects on the infection, but the dominant effect was negative. Overall, we have identified the dominant population of EVs, and other EV populations produced during HSV-1 infection, and we have provided information about potential roles.IMPORTANCE Extracellular vesicles mediate cell-to-cell communication and convey messages important for cell homeostasis. Pathways of EV biogenesis are often hijacked by pathogens to facilitate their dissemination and to establish a favorable microenvironment for the infection. We have previously shown that HSV-1 infection alters the cargo and functions of the released EVs, which negatively impact the infection. We have built upon our previous findings by developing procedures to separate EV populations from HSV-1-infected cells. We identified the major population of EVs released during infection, which carries the DNA sensor STING and has an antiviral effect. We also identified an EV population that carries selected viral proteins and has a proviral role. This is the first study to characterize EV populations during infection. These data indicate that the complex interactions between the virus and the host are extended to the extracellular environment and could impact HSV-1 dissemination and persistence in the host.

Revealing the presence of tear extracellular vesicles in Keratoconus.

Extracellular vesicles (EVs) are lipid-bound vesicles that originate from the endosomal system or budded off from the plasma membrane. EVs are involved in cell-cell communication via transporting DNA, RNA, and proteins from one cell to another. Tear EVs (tEVs) have been reported in dry eye, SjÓ§gren's Syndrome, and primary open-angle glaucoma. In this study, we sought to investigate the presence of tEVs in relation to keratoconus (KC). Tears were passively collected from the lateral meniscus from 10 healthy (5 males and 5 females) and 9 KC (4 males and 5 females) subjects. Tear samples were processed and analyzed using the ExoView™ R100. Statistical analysis was performed using a Mann-Whitney U non-parametric Student's t-test. All tEVs, in both Healthy and KC subjects, showed a CD9+ dominant tEV cohort independent of sex. A significant decrease in CD63+/CD9+ and CD63+/CD81+/CD9+ was found in the male KC tEVs (p < 0.05), but not in females compared to their healthy counterparts. Neither Healthy nor KC tEVs showed differences in the total number of tEVs, however significant differences were identified between the sexes (p < 0.05), with males having a higher number of tEVs. tEVs diameters ranged from 50 to 200 nm, in both Healthy and KC cohorts, with the majority in the 50-80 nm range suggesting exosome-dominant cohorts. To our knowledge, this is the first time, to date, that tEVs have been isolated and characterized in KCs. While further studies are warranted, the tEVs differences between KC and Healthy subjects suggest a potential role for tEVs in KC pathogenesis.

Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking.

We present a method that takes advantage of the fluorophore loading dependence of fluorescence nanoparticle tracking (fNTA) to determine the content of specific miRNA targets in extracellular vesicles (EVs) and their stoichiometry across the entire EV population. The method is based on an assay for detecting EV miRNA by hybridization to fluorescently labeled, miRNA-specific molecular beacons encapsulated in cationic lipoplex nanoparticles that fuse non-specifically with negatively charged EVs. To demonstrate the method, we carry out a stoichiometric analysis of miR-21 in EVs released from A549 lung cancer cells. We find approximately 2.3% of the A549 EVs have an average copy number of ~44 miR-21/A549 EV and contain at least a threshold number of 33 miR-21 copies/A549 EV required for fluorescence tracking. Potential applications of sizing, enumerating, and phenotyping EVs using this method include specifying dosages for therapeutic applications and identifying specific EV subpopulations in patient samples for diagnostic applications.

Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients.

INTRODUCTION: Circulating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or it can be initially not present. We designed the present prospective trial to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM-dependent enrichment and/or isolation technology. METHODS: A total of 32 patients with metastatic breast cancer were enrolled, and blood samples were processed using a previously described negative depletion immunomagnetic methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep sequential labeling was performed to label and fix cell-surface markers followed by permeabilization for cytokeratins (CK) 8, 18 and 19. Multiparametric flow cytometry (FCM) analysis was conducted using a BD LSR II flow cytometer or a BD FACSAria II or FACSAria III cell sorter. Immunocytochemical staining on postenrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM). RESULTS: CD45-negative/CK-positive (CD45- CK+) populations with EpCAM + and EpCAM - expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM + and EpCAM - populations that were CK + and coexpressing the pan-hematopoietic marker CD45 were also noted. There were more CK + EpCAM - events/ml than CK + EpCAM + events/ml in both the CD45- and CD45+ fractions (both statistically significant at P ≤ 0.0005). The number of CK + CD45- and CK + CD45+ events per milliliter in blood samples (regardless of EpCAM status) was higher in patient samples than in normal control samples (P ≤ 0.0005 and P ≤ 0.026, respectively). Further, a significant fraction of the CK + CD45+ events also expressed CD68, a marker associated with tumor-associated macrophages. Higher levels of CD45-CK + EpCAM - were associated with worse overall survival (P = 0.0292). CONCLUSIONS: Metastatic breast cancer patients have atypical cells that are CK + EpCAM - circulating in their blood. Because a substantial number of these patients do not have EpCAM + CTCs, additional studies are needed to evaluate the role of EpCAM - circulating cells as a prognostic and predictive marker.

Isolation and analysis of rare cells in the blood of cancer patients using a negative depletion methodology.

A variety of enrichment/isolation technologies exist for the characterization of rare cells in the blood of cancer patients. In this article, a negative depletion process is presented and discussed which consists of red blood cell (RBC) lysis and the subsequent removal of CD45 expressing cells through immunomagnetic depletion. Using this optimized assembly on 120 whole blood specimens, from 71 metastatic breast cancer patients, after RBC lysis, the average nucleated cell log depletion was 2.56 with a 77% recovery of the nucleated cells. The necessity of exploring different anti-CD45 antibody clones to label CD45 expressing cells in this enrichment scheme is also presented and discussed. An optimized, four-color immunofluorescence staining is conducted on the cells retained after the CD45-based immunomagnetic depletion process. Different types of rare non-hematopoietic cells are found in these enriched peripheral blood samples and a wide range of external and internal markers have been characterized, which demonstrates the range and heterogeneity of the rare cells.

Lineage specific extracellular vesicle-associated protein biomarkers for the early detection of high grade serous ovarian cancer.

High grade serous ovarian carcinoma (HGSOC) accounts for ~ 70% of ovarian cancer cases. Non-invasive, highly specific blood-based tests for pre-symptomatic screening in women are crucial to reducing the mortality associated with this disease. Since most HGSOCs typically arise from the fallopian tubes (FT), our biomarker search focused on proteins found on the surface of extracellular vesicles (EVs) released by both FT and HGSOC tissue explants and representative cell lines. Using mass spectrometry, 985 EV proteins (exo-proteins) were identified that comprised the FT/HGSOC EV core proteome. Transmembrane exo-proteins were prioritized because these could serve as antigens for capture and/or detection. With a nano-engineered microfluidic platform, six newly discovered exo-proteins (ACSL4, IGSF8, ITGA2, ITGA5, ITGB3, MYOF) plus a known HGSOC associated protein, FOLR1 exhibited classification performance ranging from 85-98% in a case-control study using plasma samples representative of early (including stage IA/B) and late stage (stage III) HGSOCs. Furthermore, by linear combination of IGSF8 and ITGA5 based on logistic regression analysis, we achieved a sensitivity of 80% (99.8% specificity). These lineage-associated exo-biomarkers have potential to detect cancer while localized to the FT when patient outcomes are more favorable.

Lineage specific extracellular vesicle-associated protein biomarkers for the early detection of high grade serous ovarian cancer.

High grade serous ovarian carcinoma (HGSOC) accounts for ~ 70% of ovarian cancer cases. Non-invasive, highly specific blood-based tests for pre-symptomatic screening in women are crucial to reducing the mortality associated with this disease. Since most HGSOCs typically arise from the fallopian tubes (FT), our biomarker search focused on proteins found on the surface of extracellular vesicles (EVs) released by both FT and HGSOC tissue explants and representative cell lines. Using mass spectrometry, 985 EV proteins (exo-proteins) were identified that comprised the FT/HGSOC EV core proteome. Transmembrane exo-proteins were prioritized because these could serve as antigens for capture and/or detection. With a nano-engineered microfluidic platform, six newly discovered exo-proteins (ACSL4, IGSF8, ITGA2, ITGA5, ITGB3, MYOF) plus a known HGSOC associated protein, FOLR1 exhibited classification performance ranging from 85 to 98% in a case-control study using plasma samples representative of early (including stage IA/B) and late stage (stage III) HGSOCs. Furthermore, by a linear combination of IGSF8 and ITGA5 based on logistic regression analysis, we achieved a sensitivity of 80% with 99.8% specificity and a positive predictive value of 13.8%. Importantly, these exo-proteins also can accurately discriminate between ovarian and 12 types of cancers commonly diagnosed in women. Our studies demonstrate that these lineage-associated exo-biomarkers can detect ovarian cancer with high specificity and sensitivity early and potentially while localized to the FT when patient outcomes are more favorable.

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics.

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

WJMSC-derived small extracellular vesicle enhance T cell suppression through PD-L1.

Both mesenchymal stem cells (MSCs) and their corresponding small extracellular vesicles (sEVs, commonly referred to as exosomes) share similar immunomodulatory properties that are potentially beneficial for the treatment of acute graft versus host disease (aGvHD). We report that clinical grade Wharton's Jelly-derived MSCs (WJMSCs) secrete sEVs enriched in programmed death-ligand 1 (PD-L1), an essential ligand for an inhibitory immune checkpoint. A rapid increase in circulating sEV-associated PD-L1 was observed in patients with aGvHD and was directly associated with the infusion time of clinical grade WJMSCs. In addition, in vitro inhibitory antibody mediated blocking of sEV-associated PD-L1 restored T cell activation (TCA), suggesting a functional inhibitory role of sEVs-PD-L1. PD-L1-deficient sEVs isolated from WJMSCs following CRISPR-Cas9 gene editing fail to inhibit TCA. Furthermore, we found that PD-L1 is essential for WJMSC-derived sEVs to modulate T cell receptors (TCRs). Our study reveals an important mechanism by which therapeutic WJMSCs modulate TCR-mediated TCA through sEVs or sEV-carried immune checkpoints. In addition, our clinical data suggest that sEV-associated PD-L1 may be not only useful in predicting the outcomes from WJMSC clinical administration, but also in developing cell-independent therapy for aGvHD patients.

Multiparameter Evaluation of the Heterogeneity of Circulating Tumor Cells Using Integrated RNA In Situ Hybridization and Immunocytochemical Analysis.

Circulating tumor cells (CTCs) are routinely identified as cytokeratin (CK)-positive, epithelial cell adhesion molecule (EpCAM)-positive, and CD45-negative and are enriched based on EpCAM. However, there are a number of methodological challenges regarding both isolation and characterization of these rare CTCs including downregulation or absence of EpCAM in a variety of solid tumors leading to the omission of subpopulations of CTCs, difficulties in analyzing RNA and protein targets in CTCs due to the rarity of these cells, and low levels of targets and technological limitations of visualizing the targets of interest on each individual cell. Building on our previous CTC research on CD45-based negative magnetic separation and four-color fluorescent immunocytochemical (ICC) staining, RNA in situ hybridization (ISH) was applied to fluorescently target mRNA sequences corresponding to tumor-related genes at the single CTC level. Multiple categories of markers are targeted including CK, human epidermal growth factor receptor family markers, Hedgehog pathway markers, human papillomavirus markers, and protein arginine methyltransferase 5. In addition, an integrated method of RNA ISH and fluorescent ICC staining was developed to visualize CTCs on both mRNA and protein levels. The robustness of the integrated co-ICC and RNA ISH staining was demonstrated by a series of tests on representative tumor markers of different categories. The integrated staining can incorporate the advantages of both RNA ISH and fluorescent ICC staining and provide more intense signals and more specific bindings. With this integrated staining methodology, distinct staining patterns were applied in this report to facilitate the searching and characterization of rare subgroups of CTCs. These results support the existence of diverse groups of CTCs at both protein and mRNA transcript levels and provide an analytical tool for the research on CTCs of rare subgroups.