Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer.

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.

Ehrlich ascites carcinoma provokes renal toxicity and DNA injury in mice: Therapeutic impact of chitosan and maitake nanoparticles.

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 × 106 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.

Effects of branched-chain amino acids supplementation on patients undergoing hepatic intervention: a meta-analysis of randomised controlled trials.

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.

IL-8-induced CXCR2 down-regulation in circulating monocytes in hepatocellular carcinoma is partially dependent on MAGL.

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.

Partial vasopressin 1a receptor agonism reduces portal hypertension and hyperaldosteronism and induces a powerful diuretic and natriuretic effect in rats with cirrhosis and ascites.

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.

CD64 expression on neutrophils as a potential biomarker for bacterial infection in ascitic fluid of cirrhotic patients.

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/mm3 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.

Changes in Coagulation and Fibrinolytic Factors in Patients With Cirrhotic Refractory Ascites Undergoing Cell-free and Concentrated Ascites Reinfusion Therapy: A Retrospective Observational Study in Japan.

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.

[Establishment of culture system for selective amplification of CD8+ T cells in vitro and analysis of its functional and molecular phenotypes from tumor-infiltrating lymphocytes in malignant pleural/ascites].

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.

An Investigation into the Potential Therapeutic Effects of the Qigu Zhushui Decoction on Mouse Models of Malignant Ascites.

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.

Modulators of mitochondrial ATP-sensitive potassium channel affect cytotoxicity of heavy metals: Action on isolated rat liver mitochondria and AS-30D ascites hepatoma cells.

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 (KNO3, 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 Cd2+, Hg2+, and Cu2+. It has been shown for the first time that, contrary to Hg2+ and depending on media used, the mK(ATP) modulators affect Cd2+- and/or Cu2+-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 Cd2+, an increase in the production of reactive oxygen species was observed with the simultaneous use of Cu2+ and diazoxide; in addition, there was no protective effect of diazoxide against cell death, which also occurred in the presence of Cu2+. 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.

Immune environment and antigen specificity of the T cell receptor repertoire of malignant ascites in ovarian cancer.

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 ß-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.

Establishment of culture system for selective amplification of CD8+ T cells in vitro and analysis of its functional and molecular phenotypes from tumor-infiltrating lymphocytes in malignant pleural/ascites / 细胞与分子免疫学杂志

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.

Acidic ascites inhibits ovarian cancer cell proliferation and correlates with the metabolomic, lipidomic and inflammatory phenotype of human patients.

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 1H-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.

[Anti-ascites effect of total saponins of Phytolaccae Radix on mice with ascites and mechanism].

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.

A Metabolomic Analysis of Cirrhotic Ascites.

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.

Ovarian Cancer Ascites Inhibits Transcriptional Activation of NK Cells Partly through CA125.

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.

[Effect of Kansui Radix stir-fried with vinegar on fecal metabolomics in Walker-256 rats with ascites].

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.

Emerging perspectives on growth factor metabolic relationships in the ovarian cancer ascites environment.

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-ßs, 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.

Macrophages are primed to transdifferentiate into fibroblasts in malignant ascites and pleural effusions.

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 TGFß signaling and cell-matrix adhesion in MDCAFs, and transdifferentiation of CAMs into MDCAFs was canceled by inhibiting TGFß 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.

Phosphatidylserine released from apoptotic cells in tumor induces M2-like macrophage polarization through the PSR-STAT3-JMJD3 axis.

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.