Anticancer Activity of Extracellular Vesicles Derived From Adipose Mesenchymal Stem Cells of Macaca fascicularis on WiDr Colon Cancer Cells.

257Ticagrelor decreases concentrations of prothrombotic extracellular vesicles compared to clopidogrel

Citrate is the recommended anticoagulant for studies on plasma extracellular vesicles (EVs). Because citrate incompletely blocks platelet activation and the release of platelet-derived EVs, we compared EDTA and citrate in that regard. Blood from healthy individuals (n = 7) was collected and incubated with thrombin receptor-activating peptide-6 (TRAP-6) to activate platelets, subjected to pneumatic tube transportation (n = 6), a freeze-thaw cycle (n = 10), and stored before plasma preparation (n = 6). Concentrations of EVs from platelets (CD61+), activated platelets (P-selectin+), erythrocytes (CD235a+), and leukocytes (CD45+) were measured by flow cytometry. Concentrations of EVs from platelets and activated platelets increased 1.4-fold and 1.9-fold in EDTA blood upon platelet activation, and 4.2-fold and 9.6-fold in citrate blood. Platelet EV concentrations were unaffected by pneumatic tube transport in EDTA blood but increased in citrate blood, and EV concentrations of erythrocytes and leukocytes were comparable. The stability of EVs during a freeze-thaw cycle was comparable for both anticoagulants. Finally, the concentration of platelet EVs was stable during storage of EDTA blood for six hours, whereas this concentration increased 1.5-fold for citrate blood. Thus, EDTA improves the robustness of studies on plasma EVs.

Alzheimer’s disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies (DLB) are the three major neurodegenerative dementias. In this study, we provide evidence that an alteration in extracellular vesicles (EVs) release is common across the three most common neurodegenerative dementias, AD, DLB, and FTD. Specifically, we analyzed plasma EVs in three groups of patients affected by AD, DLB, and FTD, and we found a significant reduction in EVs concentration and larger EVs size in all patient groups. We then investigated whether the loss of neurotrophic factors is also a common pathogenic mechanism among FTD, DLB, and AD, and if levels of neurotrophic factors might affect EVs release. Plasma levels of progranulin and cystatin C (CysC) were partially altered; however, taking together all variables significantly associated with the diagnostic groups only EVs size and concentration were able to distinguish patients from controls. The diagnostic performance of these two EVs parameters together (ratio) was high, with a sensitivity of 83.3% and a specificity of 86.7%, able to distinguish patients from controls but not to differentiate the different forms of dementias. Among the candidate neurotrophic factors, only CysC levels were associated with EVs concentration. Our study suggests that an alteration in the intercellular communication mediated by EVs might be a common molecular pathway underlying neurodegenerative dementias. The identification of shared disease mechanisms is of pivotal importance to develop treatments to delay disease progression. To this aim, further studies investigating plasma EVs size and concentration as early biomarkers of dementia are required.

Background and aims: Saliva is essential for the proper dilution and distribution of taste molecules on the tongue. It harbors extracellular vesicles (EVs), which mediate cell–cell communication. Changes in the composition of salivary EVs may arise under obese conditions and may potentially be involved in taste sensation and dysregulated eating behavior. Therefore, this study addresses the relationship between the size and concentration of salivary EVs and metabolic shifts in obesity or factors of taste sensation. Materials and methods: A total of 119 participants in the Obese Taste Bud (OTB) Study were included, who performed a standardized taste test, underwent taste bud density assessment, and were phenotypically characterized for anthropometrics, blood- and saliva adipokine levels, and various metabolic factors. Utilizing size exclusion chromatography followed by ultrafiltration, EVs were extracted from 2 mL of actively secreted saliva. EVs were characterized using nanoparticle tracking analyses, Western blot, and scanning transmission electron microscopy. Finally, group comparisons and bivariate correlation analyses were conducted. Results: Among the total cohort, the median size of salivary EVs was 190.05 nm, and the overall concentration ranged from 1.4 × 107 to 1.76 × 109 per mL of saliva. The size range and concentration of EVs per mL are negatively correlated (p = 0.0002, r = −0.264). Comparing lean participants (mean rank of 45.98) with those presenting obesity (mean rank of 34.46), a significant difference in the salivary EV content was observed (p = 0.029). Body weight, BMI, arm and calf circumferences, as well as the percentage of body fat were all negatively related to the concentration of EVs in all study participants (all p < 0.05, r > −0.2). No associations were found between the EV parameters and taste perception but serum alkaline phosphatase levels were negatively correlated (p = 0.007, r = −0.284) and adiponectin serum levels were positively correlated to the EV concentration (p = 0.036, r = 0.208). Conclusion: The current study provides evidence for the relation between salivary EVs and anthropometric as well as metabolic parameters of obesity. This can provide the basis for further research on the cargo of salivary EVs and how they may influence taste sensation, and may elucidate their potential connection to altered eating habits in obesity.

To characterize the size and procoagulant activity of extracellular vesicles (EV) that accumulate in canine packed red blood cells (pRBCs) over time and the effect of leukocyte reduction on these characteristics. Prospective cohort study. Private small animal specialty referral hospital and university research laboratories. Ten healthy blood donor dogs. Five pRBCs units were obtained according to standard protocols, and 5 were leukocyte-reduced prior to processing. Platelet-free supernatant from the pRBC units was collected on days 0, 10, 20, 32, and 42. Nanoparticle tracking analysis was performed to determine the size and concentration of EVs. Thrombin generation associated with phosphatidylserine-positive EVs was determined using a capture assay. Factor Xa generation associated with phosphatidylserine-positive EVs and tissue factor-positive EVs was measured in a subset of EVs isolated by centrifugation of the supernatant at 20,000 × g. R package nparLD and the Mann-Whitney U-test were used to determine the effect of duration of storage and the effect of leukocyte reduction, respectively. Small (mean<125nm) procoagulant EVs accumulated over time, with significant increases occurring on or after day 20 in both non-leukocyte reduced and leukocyte-reduced units. The procoagulant activity of the EVs was due to phosphatidylserine, not tissue factor. Increases in EV concentration and procoagulant activity occurred earlier in non-leukocyte reduced units. Extracellular vesicle accumulation and procoagulant activity were not decreased at any individual time point by leukocyte reduction. Further studies characterizing and determining the clinical relevance of small procoagulant EVs in pRBCs are warranted.

Extracellular vesicles (EVs) are mediators of physiological changes that occur during physical exertion. This study examined the effects of physical exertion with and without sleep and caloric restriction on EV size, concentration, and surface proteins in men and women. Twenty participants (10 men) completed a 5-day simulated military operational stress protocol with daily physical exertion. Blood was drawn before and immediately after exertion at baseline (D1) and following 48-h of sleep and caloric restriction (D3). EV size and concentration were assessed using nanoparticle tracking analysis. EVs were identified with markers associated with exosomes (CD63), microvesicles (VAMP3), apoptotic bodies (THSD1), and skeletal muscle-derived EVs (SGCA) and quantified using imaging flow cytometry. Interactive and main effects of sex, day, and time on EVs were assessed using three-way ANOVAs. EV concentration declined pre to postexertion in women on D1 and D3 but was stable in men. EV size increased from pre to postexertion and from D1 to D3 in men and women. Physical exertion following sleep and caloric restriction increased CD63+ EV concentration, proportion of total EVs, and CD63 surface protein expression regardless of sex. The proportion of SGCA+ EVs increased in men and women following exertion and from D1 to D3 but was higher in women than in men. No differences were observed in VAMP3+ and THSD1+ EVs. This study identified sexually dimorphic EV profiles in response to various stressors. Further investigations are necessary to determine if dimorphic EV responses affect health and performance outcomes during stress.NEW & NOTEWORTHY Sex is understudied in EV research, and most studies limit EV analysis to single stress conditions such as exercise. Multistress conditions consisting of physical exertion and sleep and caloric restriction are common in real-world settings. We demonstrate that physical exertion results in sex-specific EV signatures and that EV profiles vary according to single versus multistress conditions. Our data highlight important biological and ecological characteristics that should be considered in EV research.

PurposeThis article investigates the relationship between cancer cells and stromal cells in carcinoma cell living microenvironment and elucidates the mechanism that interleukin-1 receptor antagonist (IL-1RA) blocks metastatic potential in colon cancer.MethodsWestern blot and RT-PCR assay were used to determine the expression of hepatocyte growth factor (HGF) and IL-1α in colon carcinoma cells and stromal cells. Effect of IL-1RA and HGF on metastatic potential of colon cancer cells were examined by proliferation, invasion, and angiogenesis assays. The interactional role of IL-1RA and HGF were detected by ELISA assay, invasion, and angiogenesis assay making up a co-culture system consisting of stromal and colon cancer cells in cells living microenvironment.ResultsIL-1α was expressed in human umbilical vein endothelial cells (HUVECs) and HT-29 and WiDr (colon cancer cell lines with higher liver metastatic potential). HGF was expressed only in fibroblast. HGF secretion from fibroblasts was significantly inhibited by IL-1RA (P<0.01). Furthermore, IL-1RA could significantly inhibit migration, proliferation, and angiogenesis of HUVECs (P<0.01). In the double co-culture system, there is a high liver metastatic potential of colon cancer cell line (HT-29) because it can secrete autocrine IL-1α, significantly enhanced angiogenesis compared with low liver metastatic cell line (CaCo-2) (P<0.01), which does not secrete IL-1α. On the contrary, blockage of autocrine IL-1α by IL-1RA might significantly decrease metastatic potential of colon carcinoma cells through downregulation of IL-1α/PI3K/NF-κB pathway.ConclusionIL-1 receptor antagonist (IL-1RA) is an important inhibitor in metastatic process of colon carcinoma cell. Based on the above results, we suggest that IL-1RA may be a promising new therapeutic approach in inhibiting colon cancer with IL-1-producing patients.

BackgroundUnderstanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations.Materials and MethodsA standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets.ResultsPCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs.ConclusionThe results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.

BACKGROUNDThe incidence of colon cancer (CC) is currently high, and is mainly treated with chemotherapy. Oxaliplatin (L-OHP) is a commonly used drug in chemotherapy; however, long-term use can induce drug resistance and seriously affect the prognosis of patients. Therefore, this study investigated the mechanism of Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) on L-OHP resistance by determining the expression of OIP5-AS1 and microRNA-137 (miR-137) in CC cells and the effects on L-OHP resistance, with the goal of identifying new targets for the treatment of CC.AIMTo study the effects of long non-coding RNA OIP5-AS1 on L-OHP resistance in CC cell lines and its regulation of miR-137.METHODSA total of 114 CC patients admitted to China-Japan Union Hospital of Jilin University were enrolled, and the expression of miR-137 and OIP5-AS1 in tumor tissues and corresponding normal tumor-adjacent tissues was determined. The influence of OIP5-AS1 and miR-137 on the biological behavior of CC cells was evaluated. Resistance to L-OHP was induced in CC cells, and their activity was determined and evaluated using cell counting kit-8. Flow cytometry was used to analyze the apoptosis rate, Western blot to determine the levels of apoptosis-related proteins, and dual luciferase reporter assay combined with RNA-binding protein immunoprecipitation to analyze the relationship between OIP5-AS1 and miR-137.RESULTSOIP5-AS1 was up-regulated in CC tissues and cells, while miR-137 was down-regulated in CC tissues and cells. OIP5-AS1 was inversely correlated with miR-137 (P < 0.001). Silencing OIP5-AS1 expression significantly hindered the proliferation, invasion and migration abilities of CC cells and markedly increased the apoptosis rate. Up-regulation of miR-137 expression also suppressed these abilities in CC cells and increased the apoptosis rate. Moreover, silencing OIP5-AS1 and up-regulating miR-137 expression significantly intensified growth inhibition of drug-resistant CC cells and improved the sensitivity of CC cells to L-OHP. OIP5-AS1 targetedly inhibited miR-137 expression, and silencing OIP5-AS1 reversed the resistance of CC cells to L-OHP by promoting the expression of miR-137.CONCLUSIONHighly expressed in CC, OIP5-AS1 can affect the biological behavior of CC cells, and can also regulate the resistance of CC cells to L-OHP by mediating miR-137 expression.

Extracellular vesicles (EVs) have recently come into the spotlight as potential cancer biomarkers. Isolation of pure EVs is complex, so wider use requires reliable and time-efficient isolation methods. In the present study, galectin-based magnetic glycan recognition particles, EXÖBead® were investigated for their practicality as a novel EV isolation technique, exemplified here for squamous cell carcinoma of the head and neck. Analysis of the isolation method showed a high concentration of pure EVs with detection of specific EV markers such as CD9, CD63, CD81 and TSG101. No apolipoprotein A1 was shown in the isolates, indicating low contamination of this isolation technique compared with size exclusion chromatography. In addition, common leukocyte antigen (CD45), three HNSCC [epithelial cell adhesion molecule (EpCAM), pan-cytokeratin and programmed death-ligand 1 (PD-L1)] and PanEV markers (premixed CD9, CD63 and CD81 anti-bodies) were measured by bead-based flow cytometry (BFC). BFC revealed that CD45Neg PanEV+, EpCAM+ PanEV+ and PD-L1+ PanEV+ were significantly higher in tumor patients compared with healthy control plasma. CD45Neg PanEV+ and CD45+ PanEV+ carrying two or three HNSCC biomarkers were also significantly higher in tumor patients compared with healthy controls (BFC). Comparison of the functional immunosuppression effect of eluted tumor patient plasma EVs from EXÖBead® and commercial polyethylene glycol isolation showed a significant tumor-dependent increase in concentration of EVs. A peripheral blood mononuclear cell activation assay also showed that the T-cell functionality of tumor patient plasma EVs isolated with EXÖBead® was preserved in vitro. In conclusion, isolation using galectin-based magnetic glycan recognition particles is a novel method for isolating plasma EVs with low lipoprotein contamination. Bead-based flow cytometry provided an easy way to understand EV subpopulations. EXÖBead® therefore showed great potential as a new isolation tool with high throughput capacity that could potentially be used in a clinical setting.

Abstract. Kristiani EBE, Kasmiyati S, Herawati MM. 2021. The cytotoxic and apoptotic effects of wild and polyploidy genotype of Artemisia cina extracts on the WiDr colon and HTB-183 lung cancer cell lines. Biodiversitas 22: 2844-2852. Artemisia belongs to the Asteraceae family, usually used for traditional treatments of various diseases in China. Artemisia cina Berg ex Poljakov, which lots found in Indonesia, has not been studied much like a cancer drug. The research aims were (i) to compare the bioactive content of hexane and ethyl acetate extract of wild (TWN and KJT) and polyploidy type (J and M) of A. cina and its cytotoxicity on WiDr and HTB-183 cancer cells, (ii) to evaluate the cytotoxicity mechanism of the most substantial extract of it. All of the extracts were prepared by maceration methods using hexane and ethyl acetate separately. The research method used was quantitative with experimental design. The determination of quercetin, kaempferol, and artemisinin used HPLC. The cytotoxicity of the extract determined using the MTT method. The assay of the specific protein related to apoptosis using ICC assay. The content of three bioactive compounds on ethyl acetate extracts higher than hexane extract, in which the extract of J and KJT genotypes had higher levels than M and TWN genotypes. The IC50 value (µg/mL) of extracts against cancer cell lines tested was about 400-700 hexane extracts and 200-500 ethyl acetate extracts. The BCl-2 expressions of both cancer cell lines decreased by treating TWN-EA and M-EA, while the P53 expressions increased. The TWN-EA induced apoptosis of HTB-183 cell lines through caspase-8 and caspase-9 pathways, but M-EA induced caspase-9 pathway only. The TWN and M genotypes were potential to use as anticancer agents on colon and lung cancer.

Tissue Factor Positive Extracellular Vesicles and Risk of Thromboembolism in Diffuse Large B-Cell Lymphoma: A Prospective Exploratory Study

The benefits of regular physical exercise on cancer prevention, as well as reducing fatigue, treatment side effects and recurrence, and improving quality of life and overall survival of cancer patients, are increasingly recognised. Initial studies showed that the concentration of extracellular vesicles (EVs) increases during physical activity and that EVs carry biologically active cargo. These EVs are released by blood cells, skeletal muscle and other organs involved in exercise, thus suggesting that EVs may mediate tissue crosstalk during exercise. This possibility triggered a great interest in the study of the roles of EVs in systemic adaptation to exercise and in their potential applications in the prevention and treatment of various diseases, including cancer. This review presents studies exploring the concentration and molecular cargo of EVs released during exercise. Furthermore, we discuss putative stimuli that may trigger EV release from various cell types, the biological functions and the impact of exercise-induced EVs on cancer development and progression. Understanding the interplay between exercise, EVs, and cancer biology may offer insights into novel therapeutic strategies and preventive measures for cancer.

INTRODUCTION Despite recent advances, the therapies available for glioblastoma (GBM) still fall short of delivering a cure. Meaningful progress now rests on precision-medicine approaches that depend on robust biomarkers to diagnose, characterize, and monitor the tumor while supporting patient stratification. Unfortunately, an effective liquid biopsy able to fulfill these roles for GBM has yet to be realized. This study tests plasma extracellular vesicles (EVs) as non-invasive GBM biomarkers. METHODS EVs were isolated from plasma by SEC and characterized. EVs concentration and size were quantified with TRPS. Baseline EVs levels from 62 pre-operative GBM patients were compared with two control groups: 79 healthy volunteers and 50 patients harboring other brain malignancies that radiologically mimic GBM. Serial plasma samples for longitudinal EV analysis were collected from 44 of the GBM patients. Surface-marker profiling and proteomic characterization of EVs were performed by mass spectrometry in a subset of plasma specimens (27 GBM patients and 38 healthy controls) RESULTS Plasma EV concentration and mean size significantly differ between GBM patients and controls. Following surgery, both parameters become comparable to healthy controls but increase again upon tumor recurrence. EVs size and concentration effectively distinguish GBM from control groups (C1 and C2), achieving near-perfect AUC values. EV size is a predictive biomarker for response to radio- and chemotherapy. Longitudinal monitoring of EV concentration reflects GBM progression, even preceding radiological evidence on MRI. Proteomic analysis identified over 2,000 proteins across all samples, with 117 upregulated in GBM patients and enriched in complement cascade pathways. CONCLUSIONS Monitoring circulating EVs offers a highly sensitive and specific, non-invasive tool for the differential diagnosis of GBM, patient stratification, and longitudinal follow-up. Integrating EV concentration, size, and proteomic signatures yields a robust biomarker panel that can track disease dynamics over time, underscoring the promise of liquid-biopsy strategies in routine GBM management.

Colon cancer is one of the most common death-caused cancer. The high mortality rate indicates that chemotherapy has not overcome cancer disease. Strategies and development of colon cancer treatment should be pursued. Compound 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone is the 2ʹ,5ʹ-dihydroxychalcone derivative, of which the B ring was substituted with 2-pyridine ring. Chalcone and its derivatives have been reported to have several biological activities, such as cytotoxic, anti-inflammatory, antiHIV, and as a tyrosine kinase inhibitor. The objectives of this research was to determine the cytotoxic activity on WiDr colon cancer cells of 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone. Cytotoxic activity was measured using MTT assay. Compound 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone inhibited WiDr cell growth with the IC50 of 16 µM. Morphology of WiDr cell showed that compound 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone inhibited cell growth in dose dependent.Keywords: compound 1-(2,5-dihydroxyphenyl)-3-pyridine-2-yl-propenone, WiDr colon cancer cell line, cytotoxic activity