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Background: Since differentiating malignant ascites from benign ascites has always been a clinical difficult, recognition of novel biomarkers in malignant ascites of hepatocellular carcinoma (HCC) patients could be helpful for establishing a diagnosis for HCC patients with ascitic fluids., Methods: Thirty-five HCC patients with malignant ascites and chronic liver diseases patients with benign ascites were enrolled. Serum and ascites specimens were collected to determine TAN subpopulations and NETs concentration. Then, the correlation between ascitic NETs levels and clinical features were analyzed, and ROC curves were generated to evaluate the diagnostic value of NETs. For in vitro study, fresh neutrophils were employed to explore the underlying mechanism of TAN polarization and NETs formation using RNAseq analysis., Results: Significantly increased pro-tumor PD-L1 + TANs and higher lactate levels were measured in HCC ascites. RNAseq data showed that lactate regulated genes expression involving PD-L1 expression and NETs formation, suggesting that ascitic lactate might be responsible for tumor progression in TME. Then, NETs-related markers including calprotectin, dsDNA, CitH3, MPO and MPO-DNA were found dramatically elevated in malignant ascites. Next, correlation analysis revealed that ascitic NETs levels positively correlated with LDH, a classic ascitic biochemical indicator. Furthermore, we identified the diagnostic values of NETs in discriminating malignant ascites from benign ascites., Conclusions: Our findings highlighted that elevated ascitic NETs served as a biomarker in HCC patients with malignant ascites, which provided useful insights for both clinical and basic research for malignant ascites diagnosis and management., (© 2024. The Author(s).)

Background: Our hypothesis centred on the potential to mitigate ascites outbreaks in birds exposed to cold stress by inhibiting pulmonary artery contraction through dietary intervention., Objective: This study aimed to evaluate the effect of natural and synthetic medications on growth performance, ascites-related parameters and the expression of ascites-related genes in the lung tissue of broiler chickens under low ambient temperature., Methods: We randomly assigned 450 one-day-old male Ross 308 chicks to six dietary treatments across five replicate pens, each containing 15 chicks. The treatments included a basal diet (control), and the basal diet was supplemented with hydroalcoholic extracts of sumac (HES, 200 mg/kg), Syrian mesquite (HEM, 200 mg/kg), l-arginine (40% above requirement), captopril (15 mg/kg) and vitamin E (100 mg/kg)., Results: Diets containing HEM, l-arginine and vitamin E resulted in increased average daily gain on days 8-14 and 0-28, whereas HES showed a similar effect only during days 8-14 compared to the control diet (p < 0.05). Additionally, feed additives decreased packed cell volume, left and right ventricle volumes and systolic blood pressure (p < 0.05). Moreover, chickens fed the control and l-arginine diets exhibited higher levels of angiotensin converting enzyme (ACE) mRNA in lung tissue compared to those fed HES, HEM and captopril (p < 0.05). Meanwhile, supplementation with HEM and l-arginine increased the expression of inducible nitric oxide synthase (iNOS) mRNA in lung tissue compared to other treatments (p < 0.05). Regarding Cu/Zn-superoxide dismutase (Cu/Zn-SOD) expression, feed additives increased mRNA level in lung tissue, except for captopril (p < 0.05)., Conclusions: This study demonstrates that the plant extracts may reduce the incidence of ascites syndrome not only through their antioxidant properties but also by modulating the expression of ACE, iNOS and Cu/Zn-SOD genes., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Albumin infusions improve circulatory and renal function in patients with decompensated cirrhosis. However, there is no convincing evidence that hypoalbuminemia contributes to ascites formation in liver cirrhosis. The aim of our study is to determine the exact role of hypoalbuminemia in the formation of ascites caused by liver cirrhosis and its underlying mechanism. Clinical profiles of patients with liver cirrhosis retrospectively analyzed. The details of albumin involved in ascites formation were investigated in rat model and murine model. Statistical analysis demonstrated hypoalbuminemia was an independent risk factor for ascites formation in patients with liver cirrhosis (OR = 0.722, P < 0.001). In carbon tetrachloride (CCl 4 )-induced rat model of liver cirrhosis, a significant reduction in serum albumin was observed in rats with ascites (13.37 g/L) compared with rats without ascites (21.43 g/L, P < 0.001). In thioacetamide (TAA)-treated mice, ascites amount of heterozygous albumin (Alb +/- ) mice (112.0 mg) was larger than that of wild-type (Alb +/+ ) mice (58.46 mg, P < 0.001). In CCl 4 -induced chronic liver injury, ascites amounts of Alb +/- or Alb +/+ mice were 80.00 mg or 48.46 mg (P = 0.001). Further study demonstrated 24-h urinary sodium excretion in Alb +/- mice was lower than that of Alb +/+ mice in TAA/CCl 4 -induce murine models of liver cirrhosis. Additionally, serum sodium concentration of Alb +/- mice was lower than that of Alb +/+ mice. In cirrhotic mice, higher level of antidiuretic hormone was observed in Alb +/- mice compared with the control; and renal aquaporin (AQP2) expression in Alb +/- mice was significantly higher than that of WT mice. These revealed hypoalbuminemia contributed to the occurrence of ascites in liver cirrhosis through sodium and water retention., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ascites plays a key role in supporting the metastatic potential of ovarian cancer cells. Shear stress and carry-over of cancer cells by ascites flow support carcinogenesis and metastasis formation. In addition, soluble factors may participate in the procarcinogenic effects of ascites in ovarian cancer. This study aimed to determine the biological effects of cell-free ascites on carcinogenesis in ovarian cancer cells. Cell-free ascites from ovarian cancer patients (ASC) non-selectively induced cell proliferation in multiple models of ovarian cancer and untransformed primary human dermal fibroblasts. Furthermore, ASC induced a Warburg-type rearrangement of cellular metabolism in A2780 ovarian cancer cells characterized by increases in cellular oxygen consumption and glycolytic flux; increases in glycolytic flux were dominant. ASC induced mitochondrial uncoupling and fundamentally reduced fatty acid oxidation. Ascites-elicited effects were uniform among ascites specimens. ASC-elicited transcriptomic changes in A2780 ovarian cancer cells included induction of the TGFβ-ERK/MEK pathway, which plays a key role in inducing cell proliferation and oncometabolism. ASC-induced gene expression changes, as well as the overexpression of members of the TGFβ signaling system, were associated with poor survival in ovarian cancer patients. We provided evidence that the activation of the autocrine/paracrine of TGFβ signaling system may be present in bladder urothelial carcinoma and stomach adenocarcinoma. Database analysis suggests that the TGFβ system may feed forward bladder urothelial carcinoma and stomach adenocarcinoma. Soluble components of ASC support the progression of ovarian cancer. These results suggest that reducing ascites production may play an essential role in the treatment of ovarian cancer by inhibiting the progression and reducing the severity of the disease., (© 2024. The Author(s).)

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5β1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 CY Cergy Paris University. Published by Elsevier Inc. All rights reserved.)

Acute pancreatitis (AP) is an acute inflammatory reaction of the pancreatic tissue, which involves auto-digestion, edema, hemorrhage, and necrosis. AP can be categorized into mild, moderately severe, and severe AP, with severe pancreatitis also referred to as acute necrotizing pancreatitis (ANP). ANP is characterized by the accumulation of necrotic material in the peritoneal cavity. This can result in intestinal injury. However, the mechanism of ANP-associated intestinal injury remains unclear. We established an ANP-associated intestinal injury rat model (ANP-IR model) by injecting pancreatitis-associated ascites fluid (PAAF) and necrotic pancreatic tissue at various proportions into the triangular area formed by the left renal artery and ureter. The feasibility of the ANP-IR model was verified by comparing the similar changes in indicators of intestinal inflammation and barrier function between the two rat models. In addition, we detected changes in apoptosis levels and YAP protein expression in the ileal tissues of rats in each group and validated them in vitro in rat epithelial crypt cells (IEC-6) to further explore the potential injury mechanisms of ANP-associated intestinal injury. We also collected clinical data from patients with ANP to validate the effects of PAAF and pancreatic necrosis on intestinal injury. Our findings offer a theoretical basis for restricting the buildup of peritoneal necrosis in individuals with ANP, thus promoting the restoration of intestinal function and enhancing treatment efficacy. The use of the ANP-IR model in further studies can help us better understand the mechanism and treatment of ANP-associated intestinal injury. NEW & NOTEWORTHY We constructed a rat model of acute necrotizing pancreatitis-associated intestinal injury and verified its feasibility. In addition, we identified the mechanism by which necrotic pancreatic tissue and pancreatitis-associated ascites fluid (PAAF) cause intestinal injury through the HIPPO signaling pathway.

Background: Peritoneal carcinomatosis was the main reason leading to gastric cancer (GC)-related death. We aimed to explore the roles of dysregulated microRNAs (miRNAs) and related immune regulation activities in GC-associated malignant ascites., Methods: GSE126399 were downloaded from GEO database. Differentially expressed miRNAs in GC ascites samples was firstly screened, and critical miRNAs were further investigated by LASSO (least absolute shrinkage and selection operator) logistic regression and random forest (RF) algorithm. Receiver operating characteristic of critical miRNAs was also constructed. Moreover, functional analysis, immune cell infiltration associated with differentially expressed mRNAs were further analyzed. After selecting key modules by weighted gene co-expression network analysis, mRNAs related with survival performance and transcription factor (TF)-miRNA-mRNA network were constructed., Results: Hsa-miR-181b-5p was confirmed as critical differentially expressed miRNAs in GC ascites. Then, the tumor samples were divided into high- and low- expression groups divided by mean expression levels of hsa-miR-181b-5p, and subjects with high hsa-miR-181b-5p levels had better survival outcomes. In total, 197 differentially expressed mRNAs associated with hsa-miR-181b-5p levels were obtained, and these mRNAs were mainly enriched in muscle activity and vascular smooth muscle contraction. Hsa-miR-181b-5 was positively related with activated CD4 T cells and negatively related with eosinophil. 17 mRNAs were selected as mRNAs significantly related with prognosis of GC, such as PDK4 and RAMP1. Finally, 75 TF-miRNA-mRNA relationships were obtained, including 15 TFs, hsa-miR-181b-5p, and five mRNAs., Conclusion: Our data suggest that the differentially expressed hsa-miR-181b-5p in ascites samples of GC patients may be a valuable prognostic marker and a potential target for therapeutic intervention, which should be validated in the near future., (© 2024. The Author(s).)

Platinum-resistant high-grade serous carcinoma (HGSC) is an incurable disease, so biomarkers that could help with timely treatment adjustments and personalized approach are extensively being sought. Tumor-derived extracellular vesicles (EVs) that can be isolated from ascites and blood of HGSC patients are such promising biomarkers. Epithelial cell adhesion molecule (EpCAM) expression is upregulated in most epithelium-derived tumors; however, studies on prognostic value of EpCAM overexpression in ovarian carcinoma have shown contradictory results. The aim of our study was to evaluate the potential of total and EpCAM-positive EVs as prognostic and predictive biomarkers for advanced HGSC. Flow cytometry was used to determine the concentration of total and EpCAM-positive EVs in paired pretreatment ascites and plasma samples of 37 patients with advanced HGSC who underwent different first-line therapy. We found that higher EpCAM-positive EVs concentration in ascites is associated with shorter progression-free survival (PFS) regardless of treatment strategy. We also found a strong correlation of EpCAM-positive EVs concentration between ascites and plasma. Our findings indicate that EpCAM-positive EVs in ascites of patients with advanced HGSC have the potential to serve as prognostic biomarkers for predicting early recurrence and thereby likelihood of more aggressive tumor biology and development of chemoresistance.

Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3 + T cells isolated from the peripheral blood ( n  = 20), malignant ascites ( n  = 16), and tumor tissue ( n  = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8 + T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1 high CD8 + T cell population was detected, which differed from PD-1 low CD8 + T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8 + T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors ( n  = 14) and malignant ascites ( n  = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24 + EpCAM + tumor cells showed a higher frequency of CD39 + or CD73 + cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8 + T cells., Competing Interests: The authors have no conflicts of interest to disclose., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

In this study, the impact of chitosan (CS) and maitake (GF) nanoparticles towards the renal toxicity induced by Ehrlich ascites carcinoma (EAC) in vivo model was conducted. Besides benchmark negative control group, EAC model was constructed by intraperitoneal injection (i.p.) of 2.5 × 10 6 cells. Alongside positive control, two groups of EAC-bearing mice received 100 mg/kg of CS and GF nanoparticles/body weight daily for 14 days. The kidney function was conducted by measuring urea, creatinine, ions, (anti)/oxidative parameters and DNA damage. Also, measuring immunoreactivity of P53, proliferating cell nuclear antigen (PCNA), and B-cell lymphoma 2 (Bcl-2) and apoptosis protein. The outcomes illustrated notable kidney toxicity, which indicated by elevations in urea, creatinine, oxidative stress, DNA damage and induction of apoptosis. These events were supported by the drastic alteration in kidney structure through histological examination. Administration of CS and GF nanoparticles was able to enhance the antioxidant power, which further reduced oxidative damage, DNA injury, and apoptosis. These results indicated the protective and therapeutic role of biogenic chitosan and maitake nanoparticles against nephrotoxicity., (© 2024 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society). Published by John Wiley & Sons Ltd.)

High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

The benefits of branched-chain amino acid (BCAA) administration after hepatic intervention in patients with liver diseases remain unclear. We conducted a systematic review and meta-analysis to evaluate the effects of BCAA on patients undergoing hepatectomy, trans-arterial embolisation and radiofrequency ablation. Relevant randomised controlled trials (RCT) were obtained from PubMed, EMBASE and Cochrane Library databases. A meta-analysis was performed to calculate the pooled effect size by using random-effects models. The primary outcomes were survival and tumour recurrence. The secondary outcomes were hospital stay, nutrition status, biochemistry profile, complication rate of liver treatment and adverse effect of BCAA supplementation. In total, eleven RCT involving 750 patients were included. Our meta-analysis showed no significant difference in the rates of tumour recurrence and overall survival between the BCAA and control groups. However, the pooled estimate showed that BCAA supplementation in patients undergoing hepatic intervention significantly increased serum albumin (mean difference (MD): 0·11 g/dl, 95 % CI: 0·02, 0·20; 5 RCT) at 6 months and cholinesterase level (MD: 50·00 U/L, 95 % CI: 21·08, 78·92; 1 RCT) at 12 months and reduced ascites incidence (risk ratio: 0·39, 95 % CI: 0·21, 0·71; 4 RCT) at 12 months compared with the control group. Additionally, BCAA administration significantly increased body weight at 6 months and 12 months and increased arm circumference at 12 months. In conclusion, BCAA supplementation significantly improved the liver function, reduced the incidence of ascites and increased body weight and arm circumference. Thus, BCAA supplementation may beneficial for selected patients undergoing liver intervention.

Background: Malignant ascites is one of the severe complications of hepatocellular carcinoma, which can be regarded as a unique tumor microenvironment of hepatocellular carcinoma. The identification of novel biomarkers in malignant ascites could be crucial to differentiate patients with hepatocellular carcinoma and cirrhotic ascites., Objective: The study aimed to distinguish the metabolomics of malignant ascites in patients with hepatocellular carcinoma from that of non-malignant ascites (cirrhotic ascites)., Methods: Liquid chromatography-mass spectrometry was performed to analyze the differentially distributed biomarkers in patients with malignant ascites and hepatocellular carcinoma (n = 39), as well as in patients with cirrhotic ascites, which were taken as controls (n = 36)., Results: A total of 20 differential metabolites associated with malignant ascites were identified, of which 8 metabolites were upregulated and 12 metabolites were downregulated (ratio < 0.5 or > 1.5, respectively). Moreover, pathway and enrichment analyses revealed nitrogen metabolism, urea cycle, phenylalanine, and tyrosine metabolism to be implicated in the formation of malignant ascites in patients with hepatocellular carcinoma., Conclusion: Our results suggest that the key factors associated with pathways, such as arachidonic acid, phenylalanine, and glutamic acid pathways, are potential ascitic fluidbased biomarkers for differentiating hepatocellular carcinoma with cirrhosis ascites; the results also provide a clinical pathophysiological interpretation of biomarkers and metabolic pathways relevant to disease status., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The vasopressin system has emerged as a therapeutic focus for lowering portal hypertension and reducing splanchnic vasodilation in patients with refractory ascites. Clinically available vasopressin agonists are limited by preferential selectivity for V1 receptors that also have steep concentration-response curves with potential risks of excess vasoconstriction and/or complete antidiuretic effects. OCE-205 is a novel, selective, partial V1a receptor agonist with mixed agonist/antagonist activity and no V2 receptor activation at therapeutic doses. We carried out two studies assessing the in vivo effects of OCE-205 in different rat models of cirrhosis and ascites. In a carbon tetrachloride rat cirrhosis model, OCE-205 administration produced a marked reduction in portal hypertension and hyperaldosteronism, along with robust diuretic and natriuretic effects. These effects were accompanied by marked decreases in ascites volume, with three of five animals experiencing total mobilization of ascites. There was no evidence of fluid overload or sodium or water retention, confirming OCE-205's lack of V2 receptor activity. In a second, corroborative study using a bile duct ligation rat model of ascites, OCE-205 produced significant decreases in ascites volume and body weight and a significant increase in urine volume versus vehicle. Urine sodium excretion increased significantly after the first administration of OCE-205 relative to vehicle; however, repeat administration over 5 days did not lead to hyponatremia. Thus, in separate in vivo models, the mixed agonist/antagonist OCE-205 demonstrated relevant and expected endpoint findings consistent with its known mechanism of action and in vitro pharmacology without apparent unwanted effects or nonspecific toxicities., Competing Interests: Declaration of Competing Interest WJ and GF-V report no conflicts of interest. GH is a founder of and consultant to Ocelot Bio, Inc. EC and GH were employees of Ferring Research Institute Inc., at the time of the study. PG has received grants from Gilead, Mallinckrodt, Grifols, and Ferring; and consulting fees from Grifols, Ferring, Gilead, Intercept, Martin Pharmaceuticals, Promethera, Sequana, Rallybio, and Behring. SB is the founding Chief Scientific and Medical Officer of Ocelot Bio, Inc., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: CD64 expression on neutrophils surface (CD64N) by flow cytometry has been validated as a rapid biomarker for bacterial infections in both peripheral blood and other biological fluids. Ascites is a common complication in cirrhotic patients that a variety of factors can cause, including bacterial infections. Manual counting of polymorphonuclear (PMN) cells in ascitic fluid and microbiologic culture are essential for its diagnosis. We aimed to validate the determination of CD64N by flow cytometry in ascitic fluid and assess its potential usefulness in the rapid identification of bacterial infections., Materials and Methods: A prospective unicentre study was conducted. Flow cytometry was used to analyse the expression of CD64N in 77 ascitic fluid samples from the initial paracentesis of 60 cirrhotic patients in different admission episodes from November 2021 to December 2022., Results: Seventeen samples were diagnosed with bacterial infection based on a positive microbiologic culture or by PMN count (>250 PMN/mm 3 in ascitic fluid). The median of CD64N MFI was significantly increased in the bacterial infection group (3690.5 MFI [1635.23-6521.18] vs. 1105.9 MFI [737.3-2048.2], p  < 0.001). The CD64 MFI ratio of granulocytes to lymphocytes was elevated in the bacterial infection group (13.06 [6.38-24.58] vs. 5.01 [3.38-7.36], p  < 0.001). A CD64N ratio higher than 9.9 identified those patients with bacterial infection with 70.6 and 86.7% sensitivity and specificity, with an area under the curve (AUC) of 79.4%., Conclusion: The CD64N determined by flow cytometry on ascitic fluid could help quickly identify bacterial infections in ascites patients, allowing early antibiotic treatment.

Background: CXC-chemokine receptor 2 (CXCR2) expression was found to be down-regulated on circulating monocytes of cancer patients. Here, we analyze the percentage of CD14 + CXCR2 + monocyte subsets in hepatocellular carcinoma (HCC) patients, and investigate the mechanisms that regulate CXCR2 surface expression on monocytes and its biological function., Methods: Flow cytometry was used to analyze the proportion of the CD14 + CXCR2 + subset from the total circulating monocytes of HCC patients. Interleukin 8 (IL-8) levels were measured from serum and ascites, and their correlation with the CD14 + CXCR2 + monocyte subset proportion was calculated. THP-1 cells were cultured in vitro and treated with recombinant human IL-8 and CXCR2 surface expression was analyzed. CXCR2 was knocked down to examine how it affects the antitumor activity of monocytes. Finally, a monoacylglycerol lipase (MAGL) inhibitor was added to analyze its effect on CXCR2 expression., Results: A decrease in the proportion of the CD14 + CXCR2 + monocyte subset was observed in HCC patients compared with healthy controls. CXCR2 + monocyte subset proportion was associated with the AFP value, TNM stage, and liver function. Overexpression of IL-8 was observed in the serum and ascites of HCC patients, and negatively correlated with CXCR2 + monocyte proportion. IL-8 decreased CXCR2 expression in THP-1 cells, contributing to decreased antitumor activity toward HCC cells. MAGL expression in THP-1 cells was up-regulated after IL-8 treatment, and the MAGL inhibitor partially reversed the effects of IL-8 on CXCR2 expression., Conclusions: Overexpression of IL-8 drives CXCR2 down-regulation on circulating monocytes of HCC patients, which could be partially reversed by a MAGL inhibitor., (© 2023. The Author(s).)

Heavy metals are ubiquitous environmental pollutants that are extremely dangerous for public health, but the molecular mechanisms of their cytotoxic action are still not fully understood. In the present work, the possible contribution of the mitochondrial ATP-sensitive potassium channel (mK(ATP)), which is usually considered protective for the cell, to hepatotoxicity caused by heavy metals was investigated using polarography and swelling techniques as well as flow cytometry. Using isolated liver mitochondria from adult male Wistar rats and various potassium media containing or not containing penetrating anions (KNO 3 , KSCN, KAcet, KCl), we studied the effect of mK(ATP) modulators, namely its blockers (5-hydroxydecanoate, glibenclamide, ATP, ADP) and activators (diazoxide, malonate), on respiration and/or membrane permeability in the presence of hepatotoxins such as Cd 2+ , Hg 2+ , and Cu 2+ . It has been shown for the first time that, contrary to Hg 2+ and depending on media used, the mK(ATP) modulators affect Cd 2+ - and/or Cu 2+ -induced alterations in mitochondrial swelling and respiration rates, although differently, nevertheless, in the ways compatible with mK(ATP) participation in both these cases. On rat AS-30D ascites hepatoma cells, it was found that, unlike Cd 2+ , an increase in the production of reactive oxygen species was observed with the simultaneous use of Cu 2+ and diazoxide; in addition, there was no protective effect of diazoxide against cell death, which also occurred in the presence of Cu 2+ . In conclusion, the relationships (functional, structural and/or regulatory) between mK(ATP), components of the mitochondrial electron transport chain (CI, CII-CIII and/or ATP synthase, CV) and mitochondrial permeability transition pores were discussed, as well as the role of these molecular structures in the mechanisms of the cytotoxic action of heavy metals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background/aim: The management of refractory ascites is critical for the treatment of patients with decompensated cirrhosis. This study aimed to evaluate the feasibility and safety of cell-free and concentrated ascites reinfusion therapy (CART) in patients with cirrhosis and refractory ascites, with a focus on changes in coagulation and fibrinolytic factors in ascitic fluid following CART., Patients and Methods: This was a retrospective cohort study including 23 patients with refractory ascites undergoing CART. Serum endotoxin activity (EA) before and after CART and the levels of coagulation and fibrinolytic factors and proinflammatory cytokines in original and processed ascitic fluid were measured. The Ascites Symptom Inventory-7 (ASI-7) scale was used for subjective symptom assessment before and after CART., Results: Body weight and waist circumference significantly decreased after CART, whereas serum EA did not significantly change after CART. Similar to the previous reports, ascitic fluid concentrations of total protein, albumin, high-density lipoprotein cholesterol, γ-globulin, and immunoglobulin G levels were significantly increased after CART; mild elevations in body temperature and interleukin 6 and tumor necrosis factor-alpha levels in ascitic fluid were also observed. Importantly, the levels of antithrombin-III, factor VII, and X, which are useful for patients with decompensated cirrhosis, were markedly increased in the reinfused fluid during CART. Finally, the total ASI-7 score was significantly lower following CART, compared with the pre-CART score., Conclusion: CART is an effective and safe approach for the treatment of refractory ascites that allows the intravenous reinfusion of coagulation and fibrinolytic factors in the filtered and concentrated ascites., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective To explore the culture method of mass amplification for tumor-infiltrating lymphocytes (TILs) from malignant pleural/ascites in vitro, and identify the function and molecular phenotype of these amplified cells. Methods The pleural/ascites fluid was extracted under aseptic conditions, and lymphocytes were isolated by density gradient centrifugation. Then TILs were amplified by the program based on combined IFN-γ, OKT3 and IL-2, and the cell morphology and growth rate were recorded. The molecular phenotypes of the amplified lymphocytes were analyzed by Flow cytometry, and the killing ability against tumor cells was detected by CCK-8 assay. Results In this culture program, TILs remained in good condition until the 26th day, and the proliferation rate began to decrease on the 30th day. The proportions of CD4 - CD8 + and CD8 + CD56 + T cells gradually increased as cell culture time extended while the proportions of CD4 + CD25 + T cells decreased gradually. Unlike the proportions prior to amplification, the proportions of SLAMF7, CD45RO, PD-1 and granzyme B positive cells in T lymphocyte subpopulation were significantly increased, meanwhile, the expression of exhausted T-cell marker CD57 was also gradually increased. The cytotoxicity of amplified CD8 + T cells from TILs was significantly stronger than that from PBMC, and the cytotoxicity reached the peak at the effect-target ratio of 10:1 and was significantly different among tumor cell types. Conclusion A culture program for TILs amplification from cancerous thoracic/ascites is established. The method is simple and efficient. The effector cells are mainly CD8 + T lymphocytes with active phenotype.

We evaluated the association of disease outcome with T cell immune-related characteristics and T cell receptor (TCR) repertoire in malignant ascites from patients with high-grade epithelial ovarian cancer. Ascitic fluid samples were collected from 47 high-grade epithelial ovarian cancer patients and analyzed using flow cytometry and TCR sequencing to characterize the complementarity determining region 3 TCR β-chain. TCR functions were analyzed using the McPAS-TCR and VDJ databases. TCR clustering was implemented using Grouping of Lymphocyte Interactions by Paratope Hotspots software. Patients with poor prognosis had ascites characterized by an increased ratio of CD8+ T cells to regulatory T cells, which correlated with an increased productive frequency of the top 100 clones and decreased productive entropy. TCRs enriched in patients with an excellent or good prognosis were more likely to recognize cancer antigens and contained more TCR reads predicted to recognize epithelial ovarian cancer antigens. In addition, a TCR motif that is predicted to bind the TP53 neoantigen was identified, and this motif was enriched in patients with an excellent or good prognosis. Ascitic fluid in high-grade epithelial ovarian cancer patients with an excellent or good prognosis is enriched with TCRs that may recognize ovarian cancer-specific neoantigens, including mutated TP53 and TEAD1. These results suggest that an effective antigen-specific immune response in ascites is vital for a good outcome in high-grade epithelial ovarian cancer., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Yoshida-Court et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aim: To determine whether or not a decoction made from Qigu Zhushui has a suppressive impact on malignant ascites in mice., Background: Malignant ascites are one of the common complications of advanced malignant tumors. Patients with malignant ascites typically have a poor prognosis, with only 12 to 20 weeks of survival. Currently, the standard treatments for malignant ascites are systemic chemotherapy, which is ineffective in eradicating the disease and is associated with issues such as safety, short duration of sustained high-level drug concentration in localised regions, and drug resistance., Objective: To clarify the effect of Qigu Zhushui decoction on inhibiting malignant ascites in mice and provide the experimental basis for further research., Methods: The ascites model of liver cancer in mice was established by intraperitoneal injection of the H22-H8D8 cell line of liver cancer. ELISA detected the content of CEA, VEGF and TNF-α in ascites., Results: Qigu Zhushui decoction combined with cisplatin group and Qigu Zhushui decoction highdose group could significantly reduce the weight, abdominal circumference and ascites volume of mice, and their survival days and survival rate were also greatly improved; The levels of CEA and VEGF in the combination group decreased significantly, while the level of TNF-α increased; The level of TNF-a in the high dose group of Qigu Zhushui decoction was significantly increased, while the level of CEA and VEGF in the moderate dose group was decreased., Conclusion: Qigu Zhushui decoction can reduce the malignant ascites in mice, and the combination of Qigu Zhushui decoction and cisplatin has a significant anti-malignant ascites effect, which can significantly prolong the survival time and improve the survival rate., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Ascites syndrome occurs in growing broiler chickens in all parts of the world, which is one of the important causes of losses in many flocks, and its prevalence has been seen mostly in meat herds. The most important factor in the occurrence of ascites syndrome is the lack of oxygen in body tissues (hypoxia). Increasing the growth rate requires increasing the volume of blood flowing in the body to deliver nutrients to the organs and expel gases and metabolic products. Therefore, this experiment was conducted to compare the effect of peppermint extract and probiotics on the biochemical factors of the blood of chickens caused by ascites. The treatments were divided into 8 groups of 7 male chickens with 3 repetitions in each group at 21 days. The experimental treatments included control and treatments of peppermint, probiotic, peppermint and probiotic, induced ascites, induced ascites and peppermint, induced ascites and probiotics, induced ascites and peppermint and probiotics. At the end of 42 days, the blood factors of uric acid, triglyceride, glucose, cholesterol, ALT, ALP, and AST were measured by blood sampling. The experimental treatments significantly affected the investigated traits ( P <0.05). Considering that in the treatment of induced ascites+ peppermint compared to the treatment of induced ascites, weight gain was significant, and in the blood factors of cholesterol, triglyceride, uric acid, glucose, and functional liver enzymes including ALT, AST, ALP recorded a significant decrease. Therefore, the effectiveness of peppermint extract in improving induced ascites in chickens was determined. Peppermint extract had a positive effect on induced ascites and improved the performance indicators of broiler chickens, and this extract can be used as a preventive of ascites., Competing Interests: The authors declare that they have no conflict of interest.

Ovarian cancer (OC) is one of the most common and fatal types of gynecological cancer. In the early phase of OC detection, the current treatment and diagnostic methods are not efficient and sensitive enough. Therefore, it is crucial to explore the mechanisms of OC metastasis and discover valuable factors for early diagnosis of female cancers and novel therapeutic strategies for metastasis. Exosomes are known to be involved in the development, migration, and invasion of cancer cells, and their cargo could be useful for the non-invasive biopsy development. CD151- and Tspan8-positive exosomes are known to support the degradation of the extracellular matrix, and are involved in stroma remodeling, angiogenesis and cell motility, as well as the association of miR-24 and miR-101 with these processes. The objective of this study was to explore the relationship of these components of exosomal cargo, in patients with OC, to clarify the clinical significance of these markers in liquid biopsies. The levels of tetraspanins Tspan8+ and CD151+ exosomes were significantly higher in plasma exosomes of OC patients compared with healthy females (HFs). The relative levels of miR-24 and miR-101 in plasma exosomes of HFs were significantly higher than in plasma exosomes of OC patients, while the levels of these microRNAs in exosomes from plasma and ascites of ill females showed no difference. Our study revealed a strong direct correlation between the change in the ascites exosomes CD151+Tspan8+ subpopulation level and the expression levels of the ascites (R = 0.81, p < 0.05) and plasma exosomal miR-24 (R = 0.74, p < 0.05) in OC patients, which confirms the assumption that exosomal cargo act synergistically to increase cellular motility, affecting cellular processes and signaling. Bioinformatics analysis confirmed the involvement of CD151 and Tspan8 tetraspanins and genes controlled by miR-24-3p and miR-101 in signaling pathways, which are crucial for carcinogenesis, demonstrating that these tetraspanins and microRNAs are potential biomarkers for OC screening, and predictors of poor clinicopathological behavior in tumors.

Background: The poor prognosis of ovarian cancer patients is strongly related to peritoneal metastasis with the production of malignant ascites. However, it remains largely unclear how ascites in the peritoneal cavity influences tumor metabolism and recurrence. This study is an explorative approach aimed at for a deeper molecular and physical-chemical characterization of malignant ascites and to investigate their effect on in vitro ovarian cancer cell proliferation., Methods: This study included 10 malignant ascites specimens from patients undergoing ovarian cancer resection. Ascites samples were deeply phenotyped by 1 H-NMR based metabolomics, blood-gas analyzer based gas flow analysis and flow cytomertry based a 13-plex cytokine panel. Characteristics of tumor cells were investigated in a 3D spheroid model by SEM and metabolic activity, adhesion, anti-apoptosis, migratory ability evaluated by MTT assay, adhesion assay, flowcytometry and scratch assay. The effect of different pH values was assessed by adding 10% malignant ascites to the test samples., Results:  The overall extracellular (peritoneal) environment was alkaline, with pH of ascites at stage II-III = 7.51 ± 0.16, and stage IV = 7.78 ± 0.16. Ovarian cancer spheroids grew rapidly in a slightly alkaline environment. Decreasing pH of the cell culture medium suppressed tumor features, metabolic activity, adhesion, anti-apoptosis, and migratory ability. However, 10% ascites could prevent tumor cells from being affected by acidic pH. Metabolomics analysis identified stage IV patients had significantly higher concentrations of alanine, isoleucine, phenylalanine, and glutamine than stage II-III patients, while stage II-III patients had significantly higher concentrations of 3-hydroxybutyrate. pH was positively correlated with acetate, and acetate positively correlated with lipid compounds. IL-8 was positively correlated with lipid metabolites and acetate. Glutathione and carnitine were negatively correlated with cytokines IL-6 and chemokines (IL-8 & MCP-1)., Conclusion: Alkaline malignant ascites facilitated ovarian cancer progression. Additionally, deep ascites phenotyping by metabolomics and cytokine investigations allows for a refined stratification of ovarian cancer patients. These findings contribute to the understanding of ascites pathology in ovarian cancer., (© 2022. The Author(s).)

The ascites ecosystem in ovarian cancer is inhabited by complex cell types and is bathed in an environment rich in cytokines, chemokines, and growth factors that directly and indirectly impact metabolism of cancer cells and tumor associated cells. This milieu of malignant ascites, provides a 'rich' environment for the disease to thrive, contributing to every aspect of advanced ovarian cancer, a devastating gynecological cancer with a significant gap in targeted therapeutics. In this perspective we focus our discussions on the 'acellular' constituents of this liquid malignant tumor microenvironment, and how they influence metabolic pathways. Growth factors, chemokines and cytokines are known modulators of metabolism and have been shown to impact nutrient uptake and metabolic flexibility of tumors, yet few studies have explored how their enrichment in malignant ascites of ovarian cancer patients contributes to the metabolic requirements of ascites-resident cells. We focus here on TGF-βs, VEGF and ILs, which are frequently elevated in ovarian cancer ascites and have all been described to have direct or indirect effects on metabolism, often through gene regulation of metabolic enzymes. We summarize what is known, describe gaps in knowledge, and provide examples from other tumor types to infer potential unexplored roles and mechanisms for ovarian cancer. The distribution and variation in acellular ascites components between patients poses both a challenge and opportunity to further understand how the ascites may contribute to disease heterogeneity. The review also highlights opportunities for studies on ascites-derived factors in regulating the ascites metabolic environment that could act as a unique signature in aiding clinical decisions in the future., Competing Interests: Declaration of competing interest Authors have no conflicts to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This study investigated the anti-ascites effect of the total saponins of Phytolaccae Radix(PRTS) and the mechanism.H22 cell suspension was used(ip) to induce ascites in ICR male mice, and the model mice were randomized into model group, positive drug group(furosemide, 6 mg·kg~(-1)), total extract of Phytolaccae Radix(PRTE) group, and PRTS(1.29 g·kg~(-1)).Another 10 male mice were selected as the blank group.Mice in the blank group and model group were given(ig) normal saline containing 0.5% CMC-Na, and those in the positive drug group, PRTE group, and PRTS group received(ig) corresponding doses of drugs, once a day, for 8 consecutive days.The ascites volume, urine volume, and fecal water content in mice with ascites, serum levels of antidiure-tic hormone(ADH), renin in renin-angiotensin-aldosterone system(RAAS), angiotensin Ⅱ(AngⅡ), and aldosterone(ALD), expression of aquaporin(AQP)1-AQP4 in kidney, expression of AQP1, AQP3 in colon, and expression of phosphatidylinositol 3-kinase/protein kinase B(PI3 K/Akt) pathway-related proteins were detected to explore the anti-ascites mechanism of PRTS.The results showed that the PRTS can increase the urine volume and fecal water content and decrease the ascites volume of ascites mice.Moreover, PRTS significantly reduced the expression of AQP1-AQP4 in kidney and AQP1, AQP3 in colon, serum levels of renin, AngⅡ, ALD, and ADH, and the expression of p-PI3 K and p-Akt in the kidney of ascites mice.PRTS exerts anti-ascites effect by promoting urination and defecation.The mechanism is that it inhibits the activities of RAAS and ADH and suppresses the phosphorylation of PI3 K/Akt signaling pathway, thereby restricting the expression of AQPs in the kidney and colon.

Ascites is a common complication of decompensated liver cirrhosis, and yet relatively little is known about its biochemical composition. We conducted two metabolomic investigations, comparing the profile of ascites from 33 cirrhotic patients and postoperative peritoneal drainage fluid from 33 surgical patients (Experiment 1). The profile of paired ascites and plasma was also compared in 17 cirrhotic patients (Experiment 2). Gas chromatography−mass spectrometry-based metabolomics identified 29 metabolites that significantly characterized ascites fluid, whether postoperative drainage fluid or plasma were used as controls. Ten elevated amino acids (glutamine, proline, histidine, tyrosine, glycine, valine, threonine, methionine, lysine, phenylalanine) and seven diminished lipids (laurate, myristate, palmitate, oleate, vaccenate, stearate, cholesterol) largely comprised the cirrhotic ascites metabolomic phenotype that differed significantly (adjusted p < 0.002 to 0.03) from peritoneal drainage fluid or plasma. The pattern of upregulated amino acids in cirrhotic ascites did not indicate albumin proteolysis by peritoneal bacteria. Bidirectional clustering showed that the more severe the cirrhosis, the lower the lipid concentration in ascitic fluid. The metabolomic compartment of ascites in patients with decompensated cirrhosis is characterized by increased amino acids and decreased lipids. These novel findings have potential relevance for diagnostic purposes.

Malignant ascites is a common clinical problem in ovarian cancer. NK cells are present in the ascites, but their antitumor activity is inhibited. The underlying mechanisms of the inhibition have yet to be fully elucidated. Using an Fcγ receptor-mediated NK cell activation assay, we show that ascites from ovarian cancer patients potently inhibits NK cell activation. Part of the inhibitory activity is mediated by CA125, a mucin 16 fragment shed from ovarian cancer tumors. Moreover, transcriptional analyses by RNA sequencing reveal upregulation of genes involved in multiple metabolic pathways but downregulation of genes involved in cytotoxicity and signaling pathways in NK cells purified from ovarian cancer patient ascites. Transcription of genes involved in cytotoxicity pathways are also downregulated in NK cells from healthy donors after in vitro treatment with ascites or with a CA125-enriched protein fraction. These results show that ascites and CA125 inhibit antitumor activity of NK cells at transcriptional levels by suppressing expression of genes involved in NK cell activation and cytotoxicity. Our findings shed light on the molecular mechanisms by which ascites inhibits the activity of NK cells and suggest possible approaches to reactivate NK cells for ovarian cancer immunotherapy., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Cancer-associated fibroblasts (CAFs) play an important role in cancer progression. However, the origin of CAFs remains unclear. This study shows that macrophages in malignant ascites and pleural effusions (cavity fluid-associated macrophages: CAMs) transdifferentiate into fibroblast-like cells. CAMs obtained from gastrointestinal cancer patients were sorted by flow cytometry and cultured in vitro. CD45 + CD14 + CAMs transdifferentiated into CD45 - CD90 + fibroblast-like cells that exhibited spindle shapes. Then, cDNA microarray analysis showed that the CD45 - CD90 + fibroblast-like cells (macrophage-derived CAFs: MDCAFs) had a fibroblast-specific gene expression signature and produced growth factors for epithelial cell proliferation. Human colon cancer cells transplanted into immunodeficient mice with MDCAFs formed larger tumors than cancer cells alone. Gene ontology analyses showed the involvement of TGFβ signaling and cell-matrix adhesion in MDCAFs, and transdifferentiation of CAMs into MDCAFs was canceled by inhibiting TGFβ and cell adhesion. Furthermore, the acquired genetic alterations in hematopoietic stem cells (HSCs) were shared in CAMs and MDCAFs. Taken together, CAMs could be a source of CAFs and might originate from HSCs. We propose the transdifferentiation process of CAMs into MDCAFs as a new therapeutic target for fibrosis associated with gastrointestinal cancer., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Understanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies. Tumor cells often undergo spontaneous apoptotic cell death in tumor microenvironment, these apoptotic cells are histologically co-localized with immunosuppressive macrophages. However, the mechanism by which tumor cell apoptosis modulates macrophage polarization is not fully understood. In this study, we aimed to explore the tumor promoting effects of apoptotic tumor cells and the signal pathways involved., Methods: Apoptotic cells and macrophages in tumors were detected by immunohistochemical staining. Morphological analysis was performed with Giemsa staining. Lipids generated from apoptotic cells were detected by liquid chromatography-mass spectrometry. Phosphatidylserine-containing liposomes were prepared to mimic apoptotic cells. The expression of protein was determined by real-time PCR, immunohistochemistry enzyme-linked immunosorbent assay and Western blotting. Mouse malignant ascites and subcutaneous tumor models were designed for in vivo analysis. Transgenic mice with specific genes knocked out and inhibitors specific to certain proteins were used for the mechanistic studies., Results: The location and the number of apoptotic cells were correlated with that of macrophages in several types of carcinomas. Phosphatidylserine, a lipid molecule generated in apoptotic cells, induced polarization and accumulation of M2-like macrophages in vivo and in vitro. Moreover, sustained administration of phosphoserine promoted tumor growth in the malignant ascites and subcutaneous tumor models. Further analyses suggested that phosphoserine induced a M2-like phenotype in macrophages, which was related to the activation of phosphoserine receptors including T-cell immunoglobin mucin 4 (TIM4) and the FAK-SRC-STAT3 signaling pathway as well as elevated the expression of the histone demethylase Jumonji domain-containing protein 3 (JMJD3). Administration of specific inhibitors of these pathways could reduce tumor progression., Conclusions: This study suggest that apoptotic cell-generated phosphoserine might be a notable signal for immunosuppressive macrophages in tumors, and the related pathways might be potential therapeutic targets for cancer therapy., (© 2022 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.)

Utilizing metabolomics technology, this study explored the change of fecal endogenous metabolites in Walker-256 rats with malignant ascites after the administration with Kansui Radix(KR) stir-fried with vinegar(VKR), sought the potential biomarkers in feces which were related to the treatment of malignant ascites by VKR and revealed the biological mechanism of water-expelling effect of VKR. Ultra-fast liquid chromatography-quadrupole-time-of-flight mass spectrometry(UFLC-Q-TOF-MS) was employed to detect the feces of rats in all groups. Principle component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) were conducted to achieve pattern recognition. Combining t-test and variable importance in the projection(VIP) enabled the screening of potential biomarkers for the malignant ascites. Metabolic pathway analysis was accomplished with MetaboAnalyst. Correlation analysis was finally conducted integrating the sequencing data of gut microbiota to elucidate the mechanism underlying the water-expelling effect of VKR. The results showed that both KR and VKR could restore the abnormal metabolism of model rats to some extent, with VKR being inferior to KR in the regulation. Eleven potential biomarkers were identified to be correlated with the malignant ascites and five metabolic pathways were then enriched. Four kinds of gut microbiota were significantly related to the potential biomarkers. The water-expelling effect of VKR may be associated with the regulation of phenylalanine metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, tryptophan metabolism, glycerophospholipid metabolism, and glycosylphosphatidylinositol(GPI)-anchor biosynthesis. This study can provide a scientific basis for comprehensive understandings of the interaction between gut microbiota and host which has relation to the water-expelling effect of VKR and guide the reasonable clinical application of VKR.

Background: Cachexia-associated skeletal muscle wasting or 'sarcopenia' is highly prevalent in ovarian cancer and contributes to poor outcome. Drivers of cachexia-associated sarcopenia in ovarian cancer remain elusive, underscoring the need for novel and better models to identify tumour factors inducing sarcopenia. We aimed to assess whether factors present in ascites of sarcopenic vs. non-sarcopenic ovarian cancer patients differentially affect protein metabolism in skeletal muscle cells and to determine if these effects are correlated to cachexia-related patient characteristics., Methods: Fifteen patients with an ovarian mass and ascites underwent extensive physical screening focusing on cachexia-related parameters. Based on computed tomography-based body composition imaging, six cancer patients were classified as sarcopenic and six were not; three patients with a benign condition served as an additional non-sarcopenic control group. Ascites was collected, and concentrations of cachexia-associated factors were assessed by enzyme-linked immunosorbent assay. Subsequently, ascites was used for in vitro exposure of C2C12 myotubes followed by measurements of protein synthesis and breakdown by radioactive isotope tracing, qPCR-based analysis of atrophy-related gene expression, and NF-κB activity reporter assays., Results: C2C12 protein synthesis was lower after exposure to ascites from sarcopenic patients (sarcopenia 3.1 ± 0.1 nmol/h/mg protein vs. non-sarcopenia 5.5 ± 0.2 nmol/h/mg protein, P < 0.01), and protein breakdown rates tended to be higher (sarcopenia 31.2 ± 5.2% vs. non-sarcopenia 20.9 ± 1.9%, P = 0.08). Ascites did not affect MuRF1, Atrogin-1, or REDD1 expression of C2C12 myotubes, but NF-κB activity was specifically increased in cells exposed to ascites from sarcopenic patients (sarcopenia 2.2 ± 0.4-fold compared with control vs. non-sarcopenia 1.2 ± 0.2-fold compared with control, P = 0.01). Protein synthesis and breakdown correlated with NF-κB activity (r s  = -0.60, P = 0.03 and r s  = 0.67, P = 0.01, respectively). The skeletal muscle index of the ascites donors was also correlated to both in vitro protein synthesis (r s  = 0.70, P = 0.005) and protein breakdown rates (r s  = -0.57, P = 0.04)., Conclusions: Ascites of sarcopenic ovarian cancer patients induces pronounced skeletal muscle protein metabolism changes in C2C12 cells that correlate with clinical muscle measures of the patient and that are characteristic of cachexia. The use of ascites offers a new experimental tool to study the impact of both tumour-derived and systemic factors in various cachexia model systems, enabling identification of novel drivers of tissue wasting in ovarian cancer., (© 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Adipocyte-rich omentum offers "good soil" for disseminating ovarian cancer (OvCa), contributing to therapeutic difficulty. However, little is understood about the association between adipocytes and tumor growth at peritoneal dissemination site. Herein, we report the induction of adipocyte dedifferentiation by OvCa cells and pro-tumorigenic effects of resulted adipocyte-derived fibroblasts. We confirmed that malignant ascites promoted the dedifferentiation of the primary human adipocytes obtained from surgical omental specimen into omental adipocyte-derived fibroblast (O-ADF) that possess both mesenchymal stem cell and myofibroblast-like features. This promotion of dedifferentiation by malignant ascites was blocked by addition of Wnt signaling inhibitor. The effects of dedifferentiated adipocytes in proliferation and migration of OvCa cells were analyzed with in vitro coculturing experimental models and in vivo mice model, and we demonstrated that OvCa cell lines showed enhanced proliferative characteristics, as well as increased migratory abilities upon coculturing with O-ADF. Additionally, exogenous transforming growth factor-β1 augmented desmoplastic morphological change of O-ADF, leading to higher proliferative ability. Our results suggest that OvCa cells promote dedifferentiation of peritoneal adipocytes by activating Wnt/β-catenin signaling, and generated O-ADFs exhibit pro-tumoral hallmarks., (© 2021 UICC.)

The exact role of malignant ascites in the development of intraperitoneal metastases remains unclear, and the mechanisms by which extracellular vesicles (EVs) promote tumor progression in the pre-metastatic niche have not been fully discovered. In this study, we characterized ascites from high-grade epithelial ovarian cancer patients. Small-EVs (30-150 nm) were isolated from two sources-the bulk ascites and the ascitic fluid-derived tumor cell cultures-and assessed with a combination of imaging, proteomic profiling, and protein expression analyses. In addition, Gene Ontology and pathway analysis were performed using different databases and bioinformatic tools. The results proved that the small-EVs derived from the two sources exhibited significantly different stiffness and size distributions. The bulk ascitic fluid-derived small-EVs were predominantly involved in the complement and coagulation cascade. Small-EVs derived from ascites cell cultures contained a robust proteomic profile of extracellular matrix remodeling regulators, and we observed an increase in transforming growth factor-β-I (TGFβI), plasminogen activator inhibitor 1 (PAI-1), and fibronectin expression after neoadjuvant chemotherapy. When measured in the two sources, we demonstrated that fibronectin exhibited opposite expression patterns in small-EVs in response to chemotherapy. These findings highlight the importance of an ascites cell isolation workflow in investigating the treatment-induced cancer adaption processes., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In parallel to medical treatment of ovarian cancer, methods for the early detection of cancer tumors are being sought. In this contribution, the use of non-invasive static (SLS) and dynamic light scattering (DLS) for the characterization of extracellular nanoparticles (ENPs) in body fluids of advanced serous ovarian cancer (OC) and benign gynecological pathology (BP) patients is demonstrated and critically evaluated. Samples of plasma and ascites (OC patients) or plasma, peritoneal fluid, and peritoneal washing (BP patients) were analyzed. The hydrodynamic radius ( R h ) and the radius of gyration ( R g ) of ENPs were calculated from the angular dependency of LS intensity for two ENP subpopulations. R h and R g of the predominant ENP population of OC patients were in the range 20-30 nm (diameter 40-60 nm). In thawed samples, larger particles ( R h mostly above 100 nm) were detected as well. The shape parameter ρ of both particle populations was around 1, which is typical for spherical particles with mass concentrated on the rim, as in vesicles. The R h and R g of ENPs in BP patients were larger than in OC patients, with ρ ≈ 1.1-2, implying a more elongated/distorted shape. These results show that SLS and DLS are promising methods for the analysis of morphological features of ENPs and have the potential to discriminate between OC and BP patients. However, further development of the methodology is required.

Altered lipid metabolism has emerged as an important feature of ovarian cancer (OC), yet the translational potential of lipid metabolites to aid in diagnosis and triage remains unproven. We conducted a multi-level interrogation of lipid metabolic phenotypes in patients with adnexal masses, integrating quantitative lipidomics profiling of plasma and ascites with publicly-available tumor transcriptome data. Using Sciex Lipidyzer, we assessed concentrations of > 500 plasma lipids in two patient cohorts-(i) a pilot set of 100 women with OC (50) or benign tumor (50), and (ii) an independent set of 118 women with malignant (60) or benign (58) adnexal mass. 249 lipid species and several lipid classes were significantly reduced in cases versus controls in both cohorts (FDR < 0.05). 23 metabolites-triacylglycerols, phosphatidylcholines, cholesterol esters-were validated at Bonferroni significance (P < 9.16 × 10 -5 ). Certain lipids exhibited greater alterations in early- (diacylglycerols) or late-stage (lysophospholipids) cases, and multiple lipids in plasma and ascites were positively correlated. Lipoprotein receptor gene expression differed markedly in OC versus benign tumors. Importantly, several plasma lipid species, such as DAG(16:1/18:1), improved the accuracy of CA125 in differentiating early-stage OC cases from benign controls, and conferred a 15-20% increase in specificity at 90% sensitivity in multivariate models adjusted for age and BMI. This study provides novel insight into systemic and local lipid metabolic differences between OC and benign disease, further implicating altered lipid uptake in OC biology, and advancing plasma lipid metabolites as a complementary class of circulating biomarkers for OC diagnosis and triage., (© 2021. The Author(s).)

The isolation of a patient's metastatic cancer cells is the first, enabling step toward treatment of that patient using modern personalized medicine techniques. Whereas traditional standard-of-care approaches select treatments for cancer patients based on the histological classification of cancerous tissue at the time of diagnosis, personalized medicine techniques leverage molecular and functional analysis of a patient's own cancer cells to select treatments with the highest likelihood of being effective. Unfortunately, the pure populations of cancer cells required for these analyses can be difficult to acquire, given that metastatic cancer cells typically reside in fluid containing many different cell populations. Detection and analyses of cancer cells therefore require separation from these contaminating cells. Conventional cell sorting approaches such as Fluorescence Activated Cell Sorting or Magnetic Activated Cell Sorting rely on the presence of distinct surface markers on cells of interest which may not be known nor exist for cancer applications. In this work, we present a microfluidic platform capable of label-free enrichment of tumor cells from the ascites fluid of ovarian cancer patients. This approach sorts cells based on differences in biomechanical properties, and therefore does not require any labeling or other pre-sort interference with the cells. The method is also useful in the cases when specific surface markers do not exist for cells of interest. In model ovarian cancer cell lines, the method was used to separate invasive subtypes from less invasive subtypes with an enrichment of ~ sixfold. In ascites specimens from ovarian cancer patients, we found the enrichment protocol resulted in an improved purity of P53 mutant cells indicative of the presence of ovarian cancer cells. We believe that this technology could enable the application of personalized medicine based on analysis of liquid biopsy patient specimens, such as ascites from ovarian cancer patients, for quick evaluation of metastatic disease progression and determination of patient-specific treatment., (© 2021. The Author(s).)

The controlled assembly of nanomaterials into desired architectures presents many opportunities; however, current preparations lack spatial precision and versatility in developing complex nano-architectures. Inspired by the amphiphilic nature of surfactants, we develop a facile approach to guide nanomaterial integration - spatial organization and distribution - in metal-organic frameworks (MOFs). Named surfactant tunable spatial architecture (STAR), the technology leverages the varied interactions of surfactants with nanoparticles and MOF constituents, respectively, to direct nanoparticle arrangement while molding the growing framework. By surfactant matching, the approach achieves not only tunable and precise integration of diverse nanomaterials in different MOF structures, but also fast and aqueous synthesis, in solution and on solid substrates. Employing the approach, we develop a dual-probe STAR that comprises peripheral working probes and central reference probes to achieve differential responsiveness to biomarkers. When applied for the direct profiling of clinical ascites, STAR reveals glycosylation signatures of extracellular vesicles and differentiates cancer patient prognosis.

Background/aim: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant tumor. Research using an innovative research approach is needed to identify effective biomarkers or therapeutic targets for PDAC. We aimed to identify proteins related to the peritoneal dissemination of PDAC., Materials and Methods: We performed proteomic analysis using ascites samples from patients with advanced PDAC and peritoneal dissemination and patients with liver cirrhosis (LC). Proteins specific to PDAC were identified in comparison to the findings for ascites from patients with LC as a control group., Results: In total, 336 proteins were identified in ascites from patients with PDAC. We identified 18 specific proteins in ascites from patients with advanced PDAC. Among these proteins, CD13, lymphatic vessel endothelial hyaluronan receptor 1, ficolin-3, and V-set and immunoglobulin domain containing 4 were the most frequently detected. In addition, these 18 proteins could be classified into four categories: extracellular matrix, immunity, metabolism, and others., Conclusion: The identified proteins could be informative for developing treatment strategies for patients with PDAC and peritoneal dissemination., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Ovarian cancer is a highly fatal malignancy characterized by early chemotherapy responsiveness but the eventual development of resistance. Immune targeting therapies are changing treatment paradigms for numerous cancer types but have had minimal success in ovarian cancer. Through retrospective patient sample analysis, we have determined that high human epididymis protein 4 (HE4) production correlates with multiple markers of immune suppression in ovarian cancer, including lower CD8 + T cell infiltration, higher PD-L1 expression, and an increase in the peripheral monocyte to lymphocyte ratio. To further understand the impact that HE4 has on the immune microenvironment in ovarian cancer, we injected rats with syngeneic HE4 high- and low-expressing cancer cells and analyzed the differences in their tumor and ascites immune milieu. We found that high tumoral HE4 expression promotes an ascites cytokine profile that is rich in myeloid-recruiting and differentiation factors, with an influx of M2 macrophages and increased arginase 1 production. Additionally, CTL activation is significantly reduced in the ascites fluid, and there is a trend toward lower CTL infiltration of the tumor, whereas NK cell recruitment to the ascites and tumor is also reduced. PD-L1 expression by tumor cells and macrophages is increased by HE4 through a novel posttranscriptional mechanism. Our data have identified HE4 as a mediator of tumor-immune suppression in ovarian cancer, highlighting this molecule as a potential therapeutic target for the treatment of this devastating disease., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Fluid accumulation in the abdominal cavity is commonly found in advanced-stage ovarian cancer patients, which creates a specialized tumor microenvironment for cancer progression. Using single-cell RNA sequencing (scRNA-seq) of ascites cells from five patients with ovarian cancer, we identified seven cell types, including heterogeneous macrophages and ovarian cancer cells. We resolved a distinct polarization state of macrophages by MacSpectrum analysis and observed subtype-specific enrichment of pathways associated with their functions. The communication between immune and cancer cells was predicted through a putative ligand-receptor pair analysis using NicheNet. We found that CCL5, a chemotactic ligand, is enriched in immune cells (T cells and NK cells) and mediates ovarian cancer cell survival in the ascites, possibly through SDC4. Moreover, SDC4 expression correlated with poor overall survival in ovarian cancer patients. Our study highlights the potential role of T cells and NK cells in long-term survival patients with ovarian cancer, indicating SDC4 as a potential prognostic marker in ovarian cancer patients., (© 2021 Wiley Periodicals LLC.)

Pyrazole or 1,2-Diazole is a five-membered heteroaromatic ring with two nitrogen atoms which is widely used in pharmacological research and organic synthesis. Several natural and synthetic pyrazole derivatives possess anti-cancer potential and some of them have underwent clinical trials. In this aspect, an investigation into the efficiency of the pyrazole nucleus to inhibit the growth and progression of various cancer cell lines/ experimental tumours would help in giving a better clarity to the anti-cancer behaviour of pyrazole containing drugs. This paper investigates the efficiency of pyrazole against Dalton's Lymphoma Ascites (DLA) cell line. Pyrazole inhibited the growth of DLA cells in vitro by committing them towards apoptosis. In vitro results were consistent in DLA induced murine solid tumour in vivo systems. Drug-treatment improved survival, reduced tumour loads, stabilized body weights and improved the haematological and serum biochemical parameters of DLA solid tumour bearing mice, thereby improving their overall survivability. Drug administration contained the aggravation of solid tumour by targeted downregulation of Cyclin-D1 and Ki-67. In addition, the mRNA expression levels of anti-apoptotic genes, BCL-2 and BCL-XL were downregulated in solid tumours, corroborating the in vitro results that pyrazole encourage apoptotic cell death in DLA cells. The new findings establish pyrazole as a potential anti-cancer drug candidate. The results must encourage future investigations into the efficacy of the drug against various cancer types., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Although malignant ascites (MAs) are known to contribute to various aspects of ovarian cancer progression, knowledge regarding their role in the adhesion of cancer cells to normal peritoneal cells is incomplete. Here, we compared the effect of MAs and benign ascites (BAs) on the adhesion of A2780 and OVCAR-3 cancer cells to omentum-derived peritoneal mesothelial cells (PMCs) and peritoneal fibroblasts (PFBs). The results showed that MAs stimulated the adhesion of A2780 and OVCAR-3 cells to PMCs and PFBs more efficiently than did BAs, and the strongest binding occurred when both cancer and normal cells were exposed to the fluid. Intervention studies showed that MAs-driven adhesion of A2780 cells to PMCs/PFBs depends on the presence of TGF-β1 and HGF, whereas binding of OVCAR-3 cells was mediated by TGF-β1, GRO-1, and IGF-1. Moreover, MAs upregulated α5β1 integrin expression on PFBs but not on PMCs or cancer cells, vimentin expression in all cells tested, and ICAM-1 only in cancer cells. When integrin-linked kinase was neutralized in PMCs or PFBs, cancer cell adhesion to PMCs and PFBs decreased. Collectively, our report shows that MAs may contribute to the early stages of ovarian cancer metastasis by modulating the proadhesive interplay between normal and cancer cells.

Ethnopharmacological Relevance: Malignant ascites (MA) effusion is mainly caused by hepatocellular, ovarian, and breast cancer etc. It has been reported that Euphorbia kansui (EK), the root of Euphorbia kansui S.L.Liou ex S.B.Ho, possessing a therapeutic effect on MA. However, the clinical applications of EK are seriously restricted for its severe toxicity. Although studies demonstrated that vinegar-processing can reduce the toxicity and retain the water expelling effect of EK, its specific mechanism remains unknown., Aim of the Study: This study aims to explore the underlying mechanisms of toxicity reduction without compromising the pharmacological effects of EK stir-fried with vinegar (VEK)., Materials and Methods: 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3-O-EZ), a major diterpenoid of EK, could convert into ingenol after processing EK with vinegar. The H22 mouse hepatoma ascites model was replicated, and were given 3-O-EZ and ingenol seven days (110.14, 50.07 and 27.54 mg/kg). The histopathological observation, serum liver enzymes, serum Renin-Angiotensin-Aldosterone System (RAAS) levels, ascites volumes, pro-inflammatory cytokines levels and H22 cells apoptosis in ascites were examined. Then the intestine (Aquaporin 8, AQP8) and kidney (Aquaporin 2, AQP2; Vasopressin type 2 receptor, V2R) protein expression were detected, as well as the metabolomics of serum were analyzed. Finally, the content of 3-O-EZ and ingenol in EK and VEK were investigated., Results: 3-O-EZ and ingenol can relieve hepatic and gastrointestinal injuries, reduce ascites volumes, enhance the H22 cells apoptosis, ameliorate abnormal pro-inflammatory cytokines and RAAS levels, and down-regulate the expression of AQP8, AQP2, V2R. The involved metabolic pathways mainly included glycerophospholipid metabolism and arachidonic acid metabolism. And the decreasing rate of 3-O-EZ in VEK was 19.14%, the increasing rate of ingenol in VEK was 92.31%., Conclusion: 3-O-EZ and ingenol possess significant effect in treating MA effusion, while ingenol has lower toxicity compared with 3-O-EZ. And provide evidence for the mechanism of attenuation in toxicity without compromising the pharmacological effects of VEK., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The poor prognosis of ovarian cancer is mainly due to metastasis, and the specific mechanism underlying ovarian cancer metastasis is not clear. Ascites-derived exosomes (ADEs) play an important role in the progression of ovarian cancer, but the mechanism is unknown. Here, we found that ADEs promoted ovarian cancer metastasis not only in vitro but also in vivo. This promotive function was based on epithelial-mesenchymal transition (EMT) of ovarian cancer cells. Bioinformatics analysis of RNA sequencing microarray data indicated that miR-6780b-5p may be the key microRNA (miRNA) in ADEs that facilitates cancer metastasis. Moreover, the expression of exosomal miR-6780b-5p correlated with tumor metastasis in ovarian cancer patients. miR-6780b-5p overexpression promoted and miR-6780b-5p downregulation suppressed EMT of ovarian cancer cells. These results suggest that ADEs transfer miR-6780b-5p to ovarian cancer cells, promoting EMT and finally facilitating ovarian cancer metastasis.

This study evaluated the anti-ascites effect of total diterpenoids extracted from Euphorbiae Ebracteolatae Radix (TDEE) on malignant ascitic mice and elucidated its underlying mechanism. TDEE was extracted by dichloromethane and subjected to column chromatography. The purity of six diterpenoids isolated from TDEE was determined to be 77.18% by HPLC. TDEE (3 and 0.6 g raw herbs/kg, p.o.) reduced ascites and increased urine output. Meanwhile, analysis of tumor cell viability, cycle and apoptosis indicated that TDEE had no antitumor activity. In addition, the expression levels of aquaporins (AQPs) and the membrane translocation levels of protein kinase C (PKC) α and PKCβ in kidney and cells were measured. TDEE reduced the levels of AQP1-4, and inhibited PKCβ expression in membrane fraction. Four main diterpenoids, except compound 2 , reduced AQP1 level in human kidney-2 cells. Compounds 4 and 5 inhibited AQP2-4 expression in murine inner medullary collecting duct cells. The diterpenoid-induced inhibition of AQP1-4 expression was blocked by phorbol-12-myristate-13-acetate (PMA; agonist of PKC). The diterpenoids from TDEE are the main anti-ascites components. The anti-ascites effect of diterpenoids may be associated with alterations in AQPs in the kidneys to promote diuresis. The inhibition of AQP1-4 expression by TDEE is related to the inhibition of PKCβ activation.

Peritoneal dissemination and malignant ascites in pancreatic ductal adenocarcinoma (PDAC) patients represent a major clinical issue. Lysophosphatidic acid (LPA) is a lipid mediator that modulates the progression of various cancers. Based on the increasing evidence showing that LPA is abundant in malignant ascites, we focused on autotaxin (ATX), which is a secreted enzyme that is important for the production of LPA. This study aimed to elucidate the importance of the ATX-LPA axis in malignant ascites in PDAC and to determine whether ATX works as a molecular target for treating peritoneal dissemination. In a PDAC peritoneal dissemination mouse model, the amount of ATX was significantly higher in ascites than in serum. An in vitro study using two PDAC cell lines, AsPC-1 and PANC-1, showed that ATX-LPA signaling promoted cancer cell migration via the activation of the downstream signaling, and this increased cell migration was suppressed by an ATX inhibitor, PF-8380. An in vivo study showed that PF-8380 suppressed peritoneal dissemination and decreased malignant ascites, and these results were validated by the biological analysis as well as the in vitro study. Moreover, there was a positive correlation between the amount of ATX in ascites and the degree of disseminated cancer progression. These findings demonstrated that ATX in ascites works as a promotor of peritoneal dissemination, and the targeting of ATX must represent a useful and novel therapy for peritoneal dissemination of PDAC., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The development of ascites correlates with advanced stage disease and poor prognosis in ovarian cancer. Vascular permeability is the key pathophysiological change involved in ascites development. Previously, we provided evidence that perivascular M2-like macrophages protect the vascular barrier through direct contact with endothelial cells (ECs). Here, we investigated the molecular mechanism and its clinical significance in the ovarian cancer setting. We found that upon direct coculture with the endothelium, M2 macrophages tuned down their VLA4 and reduced the levels of VCAM1 in ECs. On the other hand, ectopically overexpressing VLA4 in macrophages or VCAM1 in ECs induced hyperpermeability. Mechanistically, downregulation of VLA4 or VCAM1 led to reduced levels of RAC1 and ROS, which resulted in decreased phosphorylation of PYK2 (p-PYK2) and VE-cadherin (p-VE-cad), hence enhancing cell adhesion. Furthermore, targeting the VLA4/VCAM1 axis augmented vascular integrity and abrogated ascites formation in vivo. Finally, VLA4 expression on the macrophages isolated from ascites dictated permeability ex vivo. Importantly, VLA4 antibody acted synergistically with bevacizumab to further enhance the vascular barrier. Taking these data together, we reveal here that M2 macrophages regulate the vascular barrier though the VCAM1/RAC1/ROS/p-PYK2/p-VE-cad cascade, which provides specific therapeutic targets for the treatment of malignant ascites.

The development of malignant effusions such as ascites reflects a massive progression of a malignant disease. In patients with ovarian carcinoma, a high amount of ascites (>500 mL) is an independent negative prognostic marker. The composition and constituents of ascites reflect the inflammatory environment of the underlying tumor. Increased cellular resistance of ascites-derived tumor cells and the development of venous thromboembolic events (VTE) are major risks for these patients, especially in patients with advanced ovarian carcinoma. In this study, we discuss the release of tissue factor-bearing extracellular vesicles (TF + EVs) from tumor cells into the environment (ascites fluid) and their systemic spreading as a possible causal explanation of the pathologic coagulation status in these patients. We obtained ascites from patients with advanced ovarian carcinoma, collected during surgery or therapeutic paracentesis (n = 20). Larger ectosome-like EVs were isolated using sequential centrifugation, quantified by high-resolution flow cytometry and analyzed using nanoparticle tracking analysis. Furthermore, the pro-coagulant properties (TF activity) of EVs were determined. Compared to published TF activities of EVs from healthy persons, TF activities of EVs derived from ascites of patients with ovarian cancer were very high, with a median of 80 pg/mL. The rate of VTE, as reported in the patient files, was high as well (35%, 7 out of 20). Furthermore, all but one patient with VTE had EV concentrations above the median within their ascetic fluid ( p < 0.02). Since VTE continues to be a frequent cause of death in cancer patients, prophylactic antithrombotic treatment might be worth considering in these patients. However, given the risk of bleeding, more clinical data are warranted. Although the study is too small to enable reaching a conclusion on direct clinical implementation, it can well serve as a proof of principle and a rationale to initiate a prospective clinical study with different patient subgroups. We also show ex vivo that these larger ectosome-like EVs induce intracellular ERK phosphorylation and tumor cell migration, which is not directly related to their pro-coagulative potency, but might help to understand why cancer patients with thromboembolic events have a poorer prognosis.

The presence of ascites in the peritoneal cavity leads to morphological and functional changes of the peritoneal mesothelial cell layer. Cells loose cell-cell interactions, rearrange their cytoskeleton, activate the production of fibronectin, and change their cell surface morphology in a proinflammatory environment. Moreover, ovarian cancer cell adhesion has been shown to be facilitated by these changes due to increased integrin- and CD44-mediated binding sites. In this study, the biological responsiveness of the human pleural mesothelial cell line MeT-5A to patient-derived and artificial ascites was studied in vitro and adhesion of ovarian cancer cells, i.e. SKOV-3 cells, investigated. Changes were mainly observed in cells exposed to artificial ascites containing higher cytokine concentrations than patient-derived ascites. Interestingly, reduced cell-cell interactions were already observed in untreated MeT-5A cells and effects on tight junction protein expression and permeability upon exposure to ascites were minor. Ascites induced upregulation of CDC42 effector protein 2 expression, which affects stress fiber formation, however significant F-actin reorganization was not observed. Moreover, fibronectin production remained unchanged. Analysis of mesothelial cell surface characteristics showed upregulated expression of intercellular adhesion molecule 1, slightly increased hyaluronic acid secretion and decreased microvillus expression upon exposure to ascites. Nevertheless, the observed changes were not sufficient to facilitate adhesion of SKOV-3 cells on MeT-5A cell layer. This study revealed that MeT-5A cells show a reduced biological responsiveness to the presence of ascites, in contrast to published studies on primary human peritoneal mesothelial cells., Competing Interests: The authors have declared that no competing interests exist.