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1.  Osteoprotegerin (OPG) protects ovarian cancer cells from TRAIL-induced apoptosis but does not contribute to malignant ascites-mediated attenuation of TRAIL-induced apoptosis 
Resistance to apoptosis is a major problem in ovarian cancer and correlates with poor prognosis. Osteoprotegerin (OPG) is a secreted factor in malignant ascites and acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL promotes apoptosis in ovarian cancer cells. Ovarian cancer ascites attenuate TRAIL-induced apoptosis raising the possibility that OPG contained in ascites may abrogate the anti-tumor activity of TRAIL.
Determination of OPG levels in ascites was measured by ELISA. Effect of OPG on TRAIL-induced cell death was determined by XTT and colony forming assays in ovarian cancer cell lines and primary tumor cells. Apoptosis was assessed by ELISA.
We found that recombinant OPG and malignant ascites attenuates TRAIL-induced cell death and apoptosis in a dose-dependent manner in ovarian cancer cell lines and primary ovarian tumor cells. OPG is present at high levels in the ascites of patients with ovarian cancer. We found a positive correlation between the levels of OPG in ascites and the ability of the ascites to attenuate TRAIL-induced cell death. The anti-apoptotic effect of ascites was not reversed by co-incubation with an OPG blocking antibody.
OPG and malignant ascites protect ovarian cancer cells from TRAIL-induced apoptosis. Although malignant ascites contain high levels of OPG, OPG is not a critical component that contributes to ascites-mediated attenuation of TRAIL-induced apoptosis.
PMCID: PMC3507713  PMID: 23153223
Osteoprotegerin; TRAIL; Ovarian carcinoma; Resistance; Ascites; Apoptosis
2.  Procalcitonin, and cytokines document a dynamic inflammatory state in non-infected cirrhotic patients with ascites 
AIM: To quantitate the simultaneous serum and ascitic fluid levels of procalcitonin and inflammatory markers in cirrhotics with and without ascites.
METHODS: A total of 88 consecutive severe cirrhotic patients seen in a large city hospital liver clinic were studied and divided into two groups, those with and without ascites. Group 1 consisted of 41 cirrhotic patients with massive ascites, as demonstrated by necessity for therapeutic large-volume paracentesis. Group 2 consisted of 47 cirrhotic patients without any clinically documented ascites to include either a recent abdominal computed tomography scan or ultrasound study. Serum and ascitic fluid levels of an array of inflammatory markers, including procalcitonin, were measured and compared to each other and a normal plasma panel (NPP).
RESULTS: The values for inflammatory markers assayed in the serum of Groups 1 and 2, and ascitic fluid of the Group 1. The plasma levels of the inflammatory cytokines interleukin (IL)-2, IL-4, IL-6, IL-8, interferon gamma (IFNγ) and epidermal growth factor (EGF) were all significantly greater in the serum of Group 1 as compared to that of the serum obtained from the Group 2 subjects (all P < 0.05). There were significantly greater serum levels of IL-6, IL-8, IL-10, monocyte chemoattractant protein-1, tumor necrosis factor-α, vascular endothelial growth factor and EGF when comparing Group 2 to the NPP. There was no significant difference for IL-1A, IL-1B, IL-2, IL-4 and IFNγ levels between these two groups. Serum procalcitonin levels were increased in cirrhotics with ascites compared to cirrhotics without ascites, but serum levels were similar to ascites levels within the ascites group. Furthermore, many of these cytokines, but not procalcitonin, demonstrate an ascites-to-serum gradient. Serum procalcitonin does not demonstrate any significant difference segregated by liver etiology in the ascites group; but ascitic fluid procalcitonin is elevated significantly in cardiac cirrhosis/miscellaneous subgroup compared to the hepatitis C virus and alcoholic cirrhosis subgroups.
CONCLUSION: Procalcitonin in the ascitic fluid, but not in the serum, differentiates between cirrhotic subgroup reflecting the dynamic interplay of ascites, bacterial translocation and the peri-peritoneal cytokine.
PMCID: PMC3942841  PMID: 24605035
Ascites; Bacterial translocation; Inflammatory markers; Procalcitonin; Cirrhosis
3.  The prosurvival activity of ascites against TRAIL is associated with a shorter disease-free interval in patients with ovarian cancer 
The production of ascites is a common complication of ovarian cancer. Ascites constitute a unique tumor microenvironment that may affect disease progression. In this context, we recently showed that ovarian cancer ascites may protect tumor cells from TRAIL-induced apoptosis. In this study, we sought to determine whether the prosurvival effect of ascites affects disease-free intervals.
Peritoneal fluids were obtained from 54 women undergoing intra-abdominal surgery for suspected ovarian cancer (44 cancers and 10 benign diseases). The ability of peritoneal fluids to protect from TRAIL was assessed in the ovarian cancer cell line CaOV3, and IC50 were determined. The anti-apoptotic activity of 6 ascites against cisplatin, paclitaxel, doxorubicin, etoposide and vinorelbine was also assessed in CaOV3 cells, and the prosurvival activity of two ascites was assessed in 9 primary ovarian cancer cultures.
Among the 54 peritoneal fluids tested, inhibition of TRAIL cytotoxicity was variable. Fluids originating from ovarian cancer were generally more protective than fluids from non-malignant diseases. Most of the 44 ovarian cancer ascites increased TRAIL IC50 and this inhibitory effect did not correlate strongly with the protein concentration in these ascites or the levels of serum CA125, a tumor antigen which is used in the clinic as a marker of tumor burden. The effect of ascites on cisplatin- and paclitaxel-induced cell death was assessed with 4 ascites having inhibitory effect on TRAIL-induced cell death and 2 that do not. The four ascites with prosurvival activity against TRAIL had some inhibitory on cisplatin and/or paclitaxel. Two ovarian cancer ascites, OVC346 and OVC509, also inhibited TRAIL cytotoxicity in 9 primary cultures of ovarian tumor and induced Akt activation in three of these primary cultures. Among a cohort of 35 patients with ascites, a threshold of TRAIL IC50 with ascites/IC50 without ascites > 2 was associated with shorter disease-free interval.
The prosurvival activity of ascites against TRAIL is associated with shorter disease-free interval, which may be explained, at least in part, by ascites-induced cisplatin/paclitaxel resistance. Our findings suggest that ascites may contain prosurvival factors that protect against TRAIL and chemotherapy and consequently affect disease progression.
PMCID: PMC2821314  PMID: 20157422
4.  Role of malignant ascites on human mesothelial cells and their gene expression profiles 
BMC Cancer  2014;14:288.
Malignant ascites is often present at diagnostic in women with advanced ovarian cancer (OC) and its presence is associated with a worse outcome. Human peritoneal mesothelial cells (HPMCs) are key components of malignant ascites. Although the interplay between HPMCs and OC cells is believed to be critical for tumor progression, it has not been well characterized. The purpose of this study was to assess the effect of ascites on HPMCs and clarify the role of HPMCs in OC progression.
Human OC ascites and benign peritoneal fluids were assessed for their ability to stimulate HPMC proliferation. Conditioned medium from ascites- and benign fluid-stimulated HPMCs were compared for their ability to attenuate apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). We conducted a comparative analysis of global expression changes in ascites-stimulated HPMCs using Agilent oligonucleotide microarrays.
As compared to benign peritoneal fluids, malignant ascites stimulated the proliferation of HPMCs. TRAIL-induced apoptosis was attenuated in OC cells exposed to conditioned medium from ascites-stimulated HPMCs as compared to OC cells exposed to conditioned medium from benign fluid-stimulated HPMCs. A total of 649 genes were differentially expressed in ascites-stimulated HPMCs. Based on a ratio of more than 1.5-fold and a P < 0.05, 484 genes were up-regulated and 165 genes were down-regulated in ascites-exposed HPMCs. Stimulation of HPMCs with OC ascites resulted in differential expression of genes mainly associated with the regulation of cell growth and proliferation, cell death, cell cycle and cell assembly and organization, compared to benign peritoneal fluids. Top networks up-regulated by OC ascites included Akt and NF-κB survival pathways whereas vascular endothelial growth factor (VEGF) pathway was down-regulated.
The results of this study not only provide evidence supporting the importance of the interplay between cancer cells and HPMCs but also define the role that the tumor environment plays in these interactions.
PMCID: PMC4008408  PMID: 24761768
5.  Prognostic significance of IL-6 and IL-8 ascites levels in ovarian cancer patients 
BMC Cancer  2011;11:210.
The acellular fraction of epithelial ovarian cancer (EOC) ascites promotes de novo resistance of tumor cells and thus supports the idea that tumor cells may survive in the surrounding protective microenvironment contributing to disease recurrence. Levels of the pro-inflammatory cytokines IL-6 and IL-8 are elevated in EOC ascites suggesting that they could play a role in tumor progression.
We measured IL-6 and IL-8 levels in the ascites of 39 patients with newly diagnosed EOC. Commercially available enzyme-linked immunosorbent assay (ELISA) was used to determine IL-6 and IL-8 ascites levels. Ascites cytokine levels were correlated with clinicopathological parameters and progression-free survival.
Mean ascites levels for IL-6 and IL-8 were 6419 pg/ml (SEM: 1409 pg/ml) and 1408 pg/ml (SEM: 437 pg/ml) respectively. The levels of IL-6 and IL-8 in ascites were significantly lower in patients that have received prior chemotherapy before the surgery (Mann-Whitney U test, P = 0.037 for IL-6 and P = 0.008 for IL-8). Univariate analysis revealed that high IL-6 ascites levels (P = 0.021), serum CA125 levels (P = 0.04) and stage IV (P = 0.009) were significantly correlated with shorter progression-free survival. Including these variables in a multivariate analysis revealed that elevated IL-6 levels (P = 0.033) was an independent predictor of shorter progression-free survival.
Elevated IL-6, but not IL-8, ascites level is an independent predictor of shorter progression-free survival.
PMCID: PMC3118896  PMID: 21619709
6.  Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis 
Molecular Cancer  2012;11:84.
Ascites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.
Using human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.
In the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.
These results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.
PMCID: PMC3526430  PMID: 23158473
Ovarian cancer; Resistance; Mcl-1; ERK1/2; TRAIL; Elk-1
7.  Patients with spontaneous bacterial peritonitis, and malignant and cirrhotic ascites. 
BACKGROUND: Cytokines play a key role in the regulation of cells of the immune system and also have been implicated in the pathogenesis of malignant diseases. METHOD AND PATIENTS: We studied tumor necrosis factor-alpha, tumor necrosis factor receptor and C-reactive protein levels in both ascitic fluid and serum in patients with spontaneous bacterial peritonitis (SBP) (n = 22), and in the malignant (n = 38) and cirrhotic (n = 32) ascites. RESULTS: C-reactive protein, tumor necrosis factor-alpha and tumor necrosis factor receptor levels in the ascitic fluid were found to be elevated in the SBP (p < 0.001) and malignant groups (p < 0.005) when compared with the sterile cirrhotic group. C-reactive protein levels in the serum were found to be elevated in the SBP group when compared with the sterile cirrhotic (p < 0.001) and malignant group (p < 0.005). Tumor necrosis factor-alpha in the serum was significantly elevated in the SBP when compared with the cirrhotic (p < 0.005) and malignant ascites (p < 0.001). Sensitivity and specificity of ascitic fluid CRP in discriminating malignant 84% and 67% and SBP from sterile ascites were 90% and 76%, respectively. Sensitivity and specificity of ascitic fluid TNF-alpha in discriminating malignant 77% and 60% and SBP from sterile ascites were 82% and 66%, respectively. Sensitivity and specificity of TNF-r p60 in discriminating malignant 74% and 70% and SBP from sterile ascites were 80% and 76%, respectively. CONCLUSION: The sensitivity and specificity of ascitic fluid CRP, TNF-alpha and TNF-r values were found to be similar. Ascitic fluid Creactive protein to differentiate SBP and malignant ascitic from cirrhotic ascites are cheap, practical and safe tests used in the differential diagnosis of ascites.
PMCID: PMC2568775  PMID: 15712792
8.  Expression and significance of cyclooxygenase-2 mRNA in benign and malignant ascites 
AIM: To investigate the mRNA expression of cyclooxygensae-2 (COX-2) in benign and malignant ascites, and to explore the difference in COX-2 mRNA expression among different diseases.
METHODS: A total of 36 samples were collected from the Fifth Affiliated Hospital of Sun Yat-Sen University and divided into two experimental groups: benign ascites (n = 21) and malignant ascites (n = 15). Benign ascites included cirrhotic ascites (n = 10) and tuberculous ascites (n = 5). Malignant ascites included oophoroma (n = 7), cancer of colon (n = 5), cancer of the liver (n = 6), gastric cancer (n = 2), and bladder carcinoma (n = 1). The mRNA expression of COX-2 in ascites was examined with reverse transcriptase polymerase chain reaction (RT-PCR) technology, and the positive rate of COX-2 mRNA was compared between different diseases.
RESULTS: The positive rate of COX-2 mRNA in malignant ascites was 42.9% (9/21), which was significantly higher than in benign ascites, 6.7% (1/15), difference being significant between these two groups (χ2 = 4.051, P = 0.044). The proportion of the positive rate in the malignant ascites was as follows: ovarian cancers 57.1% (4/7), colon cancer 40.0% (2/5), liver cancer 33.3% (2/6), gastric cancer 50.0% (1/2), and bladder cancer 0.00% (0/1). However, there was no significant difference in COX-2 mRNA expression among various tumors with malignant ascites (χ2 = 1.614, P = 0.806). Among the benign ascites, COX-2 mRNA levels were different between the tuberculous ascites (0/5) and cirrhotic ascites (1/10), but there was no significant difference (P = 1.000).
CONCLUSION: COX-2 mRNA, detected by RT-PCR, is useful in the differential diagnosis of benign and malignant ascites, which also has potential value in the clinical diagnosis of tumors.
PMCID: PMC3812489  PMID: 24187465
Ascites; Cyclooxygensae-2 mRNA; Reverse transcriptase polymerase chain reaction; Malignant tumor
9.  TGF-β blockade controls ascites by preventing abnormalization of lymphatic vessels in orthotopic human ovarian carcinoma models 
Ovarian cancer patients with malignant ascites have poor prognosis. The accumulation of ascites is caused by an imbalance between fluid extravasation from the blood vessels and reabsorption by lymphatic vessels. Whereas, the role of Transforming Growth Factor beta (TGF-β) in tumor progression has been well studied, the role of TGF-β in lymphatic vessel function is far from understood. Here, we sought to dissect the role of TGF-β blockade in the formation of ascites.
Experimental Design
We used soluble TGF-β Receptor II (sTβRII) to block TGF-β signaling in two orthotopic human ovarian carcinoma models: SKOV3ip1 and Hey-A8. We measured tumor proliferation, apoptosis, lymphangiogenesis and angiogenesis by immunohistochemical staining, and examined diaphragm lymphatic vessel network by intraperitoneal injection of a fluorescent dye. Diaphragm lymphatic vessel function was assessed by tracking fluorescent beads in the diaphragm and measuring their drainage rate.
TGF-β blockade impaired tumor growth in both models, accompanied by a decreased tumor cell proliferation and angiogenesis. More strikingly, TGF-β blockade almost completely abolished ascites formation. TGF-β blockade significantly inhibited the expression of VEGF, which is the major contributor to ascites formation. At the same time, TGF-β blockade prevent ‘abnormalization’ of diaphragm lymphatic vessels and improved ascites drainage.
TGF-β blockade decreased ascites by both inhibiting ascites formation and improving ascites drainage. Based on our finding, it is reasonable to consider the use of TGF-β blockade as a palliative treatment for symptomatic ascites.
PMCID: PMC3060297  PMID: 21278244
10.  Effect of Ovarian Cancer Ascites on Cell Migration and Gene Expression in an Epithelial Ovarian Cancer In Vitro Model1 
Translational Oncology  2010;3(4):230-238.
A third of patients with epithelial ovarian cancer (EOC) present ascites. The cellular fraction of ascites often consists of EOC cells, lymphocytes, and mesothelial cells, whereas the acellular fraction contains cytokines and angiogenic factors. Clinically, the presence of ascites correlates with intraperitoneal and retroperitoneal tumor spread. We have used OV-90, a tumorigenic EOC cell line derived from the malignant ascites of a chemonaive ovarian cancer patient, as a model to assess the effect of ascites on migration potential using an in vitro wound-healing assay. A recent report of an invasion assay described the effect of ascites on the invasion potential of the OV-90 cell line. Ascites sampled from 31 ovarian cancer patients were tested and compared with either 5% fetal bovine serum or no serum for their nonstimulatory or stimulatory effect on the migration potential of the OV-90 cell line. A supervised analysis of data generated by the Affymetrix HG-U133A GeneChip identified differentially expressed genes from OV-90 cells exposed to ascites that had either a nonstimulatory or a stimulatory effect on migration. Ten genes (IRS2, CTSD, NRAS, MLXIP, HMGCR, LAMP1, ETS2, NID1, SMARCD1, and CD44) were upregulated in OV-90 cells exposed to ascites, allowing a nonstimulatory effect on cell migration. These findings were validated by quantitative polymerase chain reaction. In addition, the gene expression of IRS2 and MLXIP each correlated with prognosis when their expression was assessed in an independent set of primary cultures established from ovarian ascites. This study revealed novel candidates that may play a role in ovarian cancer cell migration.
PMCID: PMC2915414  PMID: 20689764
11.  Disease dependent qualitative and quantitative differences in the inflammatory response to ascites occurring in cirrhotics 
World Journal of Hepatology  2014;6(2):85-91.
AIM: To assess differing patterns and levels of ascitic fluid cyctokine and growth factors exist between those with a high risk and low risk of spontaneous bacterial peritonitis (SBP).
METHODS: A total of 57 consecutive patients with ascites requiring a large volume paracentesis were studied. Their age, gender, specific underlying disease conditions were recorded after a review of their clinical records. Each underwent a routine assessment prior to their paracentesis consisting of a complete blood count, complete metabolic profile and prothrombin time/international normalized ratio (INR) determination. The ascitic fluid was cultured and a complete cell count and albumin determination was obtained on the fluid. In addition, blood and ascitic fluid was assessed for the levels of interleukin interleukin (IL)-1A, IL-1B, IL-2, IL-4, IL-8, IL-10, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) utilizing the Randox Biochip platforms (Boston, MA). A serum-ascites gradient, for each cytokine and growth factor was calculated. The results are reported as mean ± SEM between disease groups with statistical analysis consisting of the student t-test (two tailed) with a P value of 0.05 defining significance.
RESULTS: No clinically important demographic or biochemical differences between the 4 groups studied were evident. In contrast, marked difference in the cytokine and growth factors levels and pattern were evident between the 4 disease groups. Individuals with alcoholic cirrhosis had the highest levels of IL-1A, IL-1B, IL-4, IFNγ. Those with malignant disease had the highest levels of IL-2. Those with hepatitis C virus (HCV) associated cirrhosis had the highest value for IL-6, IL-8, IL-10, MCP-1 and VEGF. Those with cardiac disease had the highest level of TNF-α and EGF. The calculated serum- ascites gradients for the cardiac and malignant disease groups had a greater frequency of negative values signifying greater levels of IL-8, IL-10 and MCP-1 in ascites than did those with alcohol or HCV disease.
CONCLUSION: These data document important differences in the cytokine and growth factor levels in plasma, ascitic fluid and the calculated plasma - ascites fluid gradients in cirrhotics requiring a large volume paracentesis. These differences may be important in determining the risk for bacterial peritonitis.
PMCID: PMC3934639  PMID: 24575167
Ascites; Cirrhosis; Growth factors; Inflammation; Procalcitonin
12.  Kinetics of Host Cell Recruitment During Dissemination of Diffuse Malignant Peritoneal Mesothelioma 
Cancer Microenvironment  2010;4(1):39-50.
Diffuse malignant mesothelioma is an aggressive tumor which displays a median survival of 11.2 months and a 5-year survival of less than 5% emphasizing the need for more effective treatments. This study uses an orthotopic model of malignant mesothelioma established in syngeneic, immunocompetent C57Bl/6 mice which produce malignant ascites and solid tumors that accurately replicate the histopathology of the human disease. Host stromal and immune cell accumulation within malignant ascites and solid tumors was determined using immunofluorescent labeling with confocal microscopy and fluorescence-activated cell sorting. An expression profile of cytokines and chemokines was produced using quantitative real-time PCR arrays. Tumor spheroids and solid tumors show progressive growth and infiltration with host stromal and immune cells including macrophages, endothelial cells, CD4+ and CD8+ lymphocytes, and a novel cell type, myeloid derived suppressor cells (MDSCs). The kinetics of host cell accumulation and inflammatory mediator expression within the tumor ascites divides tumor progression into two distinct phases. The first phase is characterized by progressive macrophage and T lymphocyte recruitment, with a cytokine profile consistent with regulatory T lymphocytes differentiation and suppression of T cell function. The second phase is characterized by decreased expression of macrophage chemotactic and T-cell regulating factors, an increase in MDSCs, and increased expression of several cytokines which stimulate differentiation of MDSCs. This cellular and expression profile suggests a mechanism by which host immune cells promote diffuse malignant mesothelioma progression.
Electronic supplementary material
The online version of this article (doi:10.1007/s12307-010-0048-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3047623  PMID: 21505561
Malignant mesothelioma; Orthotopic model; Murine; Expression profile; Tumor microenvironment
13.  Kinetics of Host Cell Recruitment During Dissemination of Diffuse Malignant Peritoneal Mesothelioma 
Cancer Microenvironment  2010;4(1):39-50.
Diffuse malignant mesothelioma is an aggressive tumor which displays a median survival of 11.2 months and a 5-year survival of less than 5% emphasizing the need for more effective treatments. This study uses an orthotopic model of malignant mesothelioma established in syngeneic, immunocompetent C57Bl/6 mice which produce malignant ascites and solid tumors that accurately replicate the histopathology of the human disease. Host stromal and immune cell accumulation within malignant ascites and solid tumors was determined using immunofluorescent labeling with confocal microscopy and fluorescence-activated cell sorting. An expression profile of cytokines and chemokines was produced using quantitative real-time PCR arrays. Tumor spheroids and solid tumors show progressive growth and infiltration with host stromal and immune cells including macrophages, endothelial cells, CD4+ and CD8+ lymphocytes, and a novel cell type, myeloid derived suppressor cells (MDSCs). The kinetics of host cell accumulation and inflammatory mediator expression within the tumor ascites divides tumor progression into two distinct phases. The first phase is characterized by progressive macrophage and T lymphocyte recruitment, with a cytokine profile consistent with regulatory T lymphocytes differentiation and suppression of T cell function. The second phase is characterized by decreased expression of macrophage chemotactic and T-cell regulating factors, an increase in MDSCs, and increased expression of several cytokines which stimulate differentiation of MDSCs. This cellular and expression profile suggests a mechanism by which host immune cells promote diffuse malignant mesothelioma progression.
Electronic supplementary material
The online version of this article (doi:10.1007/s12307-010-0048-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3047623  PMID: 21505561
Malignant mesothelioma; Orthotopic model; Murine; Expression profile; Tumor microenvironment
14.  Mixed-polarization phenotype of ascites-associated macrophages in human ovarian carcinoma: Correlation of CD163 expression, cytokine levels and early relapse 
Ovarian cancer is typically accompanied by the occurrence of malignant ascites containing large number of macrophages. It has been suggested that these tumor-associated macrophages (TAMs) are skewed to alternative polarization (M2) and thereby play an essential role in therapy resistance and metastatic spread. In our study, we have investigated the nature, regulation and clinical correlations of TAM polarization in serous ovarian cancer. Macrophage polarization markers on TAMs and ascites cytokine levels were analyzed for 30 patients and associated with relapse-free survival (RFS) in a prospective study with 20 evaluable patients. Surface expression of the M2 marker CD163 on TAMs was inversely associated with RFS (p < 0.01). However, global gene expression profiles determined for 17 of these patients revealed a mixed-polarization phenotype unrelated to the M1/M2 classification. CD163 surface expression also correlated with the ascites levels of IL-6 and IL-10 (p < 0.05), both cytokines induced CD163 expression, and their ascites levels showed a clear inverse association with RFS (p < 0.01). These findings define a subgroup of patients with high CD163 expression, high IL-6 and/or IL-10 levels and poor clinical outcome.
PMCID: PMC4232932  PMID: 23784932
ovarian carcinoma; tumor-associated macrophages; CD163; IL-6; IL-10
15.  The peritoneal macrophage inflammatory profile in cirrhosis depends on the alcoholic or hepatitis C viral etiology and is related to ERK phosphorylation 
BMC Immunology  2012;13:42.
The development of ascites in cirrhotic patients generally heralds a deterioration in their clinical status. A differential gene expression profile between alcohol- and hepatitis C virus (HCV)-related cirrhosis has been described from liver biopsies, especially those associated with innate immune responses. The aim of this work was to identify functional differences in the inflammatory profile of monocyte-derived macrophages from ascites in cirrhotic patients of different etiologies in an attempt to extrapolate studies from liver biopsies to immune cells in ascites. To this end 45 patients with cirrhosis and non-infected ascites, distributed according to disease etiology, HCV (n = 15) or alcohol (n = 30) were studied. Cytokines and the cell content in ascites were assessed by ELISA and flow cytometry, respectively. Cytokines and ERK phosphorylation in peritoneal monocyte-derived macrophages isolated and stimulated in vitro were also determined.
A different pattern of leukocyte migration to the peritoneal cavity and differences in the primed status of macrophages in cirrhosis were observed depending on the viral or alcoholic etiology. Whereas no differences in peripheral blood cell subpopulations could be observed, T lymphocyte, monocyte and polymorphonuclear cell populations in ascites were more abundant in the HCV than the alcohol etiology. HCV-related cirrhosis etiology was associated with a decreased inflammatory profile in ascites compared with the alcoholic etiology. Higher levels of IL-10 and lower levels of IL-6 and IL-12 were observed in ascitic fluid from the HCV group. Isolated peritoneal monocyte-derived macrophages maintained their primed status in vitro throughout the 24 h culture period. The level of ERK1/2 phosphorylation was higher in ALC peritoneal macrophages at baseline than in HCV patients, although the addition of LPS induced a greater increase in ERK1/2 phosphorylation in HCV than in ALC patients.
The macrophage inflammatory status is higher in ascites of alcohol-related cirrhotic patients than in HCV-related patients, which could be related with differences in bacterial translocation episodes or regulatory T cell populations. These findings should contribute to identifying potential prognostic and/or therapeutic targets for chronic liver diseases of different etiology.
PMCID: PMC3496568  PMID: 22866973
Ascites; Cirrhosis; Cytokines; Etiology; MAP kinases; HCV; Alcohol
16.  Ascites Specific Inhibition of CD1d-Mediated Activation of NKT cells 
Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate anti-tumor immunity. However, it has been shown that tumor associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor associated and other ascites on CD1d-mediated antigen presentation to NKT cells.
Experimental Design
To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-Ig-based artificial Antigen Presenting Cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classical CD8+ T cells.
Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the cells’ ability to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of α-GalCer loaded CD1d-Ig-based artificial Antigen Presenting Cells (aAPC) to activate NKT cells. In addition, our data demonstrate that treatment with ascites does not inhibit HLA-A2 mediated activation of classical CD8+ T cells.
Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system.
PMCID: PMC2676584  PMID: 19047090
NKT cells; CD1d1; ascites; ovarian cancer
17.  Molecular Profiling and Clinical Outcome of High-Grade Serous Ovarian Cancer Presenting with Low- versus High-Volume Ascites 
BioMed Research International  2014;2014:367103.
Epithelial ovarian cancer consists of multiple histotypes differing in etiology and clinical course. The most prevalent histotype is high-grade serous ovarian cancer (HGSOC), which often presents at an advanced stage frequently accompanied with high-volume ascites. While some studies suggest that ascites is associated with poor clinical outcome, most reports have not differentiated between histological subtypes or tumor grade. We compared genome-wide gene expression profiles from a discovery cohort of ten patients diagnosed with stages III-IV HGSOC with high-volume ascites and nine patients with low-volume ascites. An upregulation of immune response genes was detected in tumors from patients presenting with low-volume ascites relative to those with high-volume ascites. Immunohistochemical studies performed on tissue microarrays confirmed higher expression of proteins encoded by immune response genes and increased tumorinfiltrating cells in tumors associated with low-volume ascites. Comparison of 149 advanced-stage HGSOC cases with differential ascites volume at time of primary surgery indicated low-volume ascites correlated with better surgical outcome and longer overall survival. These findings suggest that advanced stage HGSOC presenting with low-volume ascites reflects a unique subgroup of HGSOC, which is associated with upregulation of immune related genes, more abundant tumor infiltrating cells and better clinical outcomes.
PMCID: PMC4055662  PMID: 24982872
18.  Polyfunctional T-Cell Responses Are Disrupted by the Ovarian Cancer Ascites Environment and Only Partially Restored by Clinically Relevant Cytokines 
PLoS ONE  2010;5(12):e15625.
Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.
Methodology/Principal Findings
Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.
The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.
PMCID: PMC3008736  PMID: 21203522
19.  Increased intestinal macromolecular permeability and urine nitrite excretion associated with liver cirrhosis with ascites 
AIM: To determine intestinal permeability, the serum tumor necrosis factor (TNF)-α level and urine nitric oxide (NO) metabolites are altered in liver cirrhosis (LC) with or without ascites.
METHODS: Fifty-three patients with LC and 26 healthy control subjects were enrolled in the study. The intestinal permeability value is expressed as the percentage of polyethylene glycol (PEG) 400 and 3350 retrieval in 8-h urine samples as determined by high performance liquid chromatography. Serum TNF-α concentrations and urine NO metabolites were determined using an enzyme-linked immunosorbent assay (ELISA) and Greiss reaction method, respectively.
RESULTS: The intestinal permeability index was significantly higher in patients with LC with ascites than in healthy control subjects or patients with LC without ascites (0.88 ± 0.12 vs 0.52 ± 0.05 or 0.53 ± 0.03, P < 0.05) and correlated with urine nitrite excretion (r = 0.98). Interestingly, the serum TNF-α concentra-tion was significantly higher in LC without ascites than in control subjects or in LC with ascites (198.9 ± 55.8 pg/mL vs 40.9 ± 12.3 pg/mL or 32.1 ± 13.3 pg/mL, P < 0.05). Urine nitrite excretion was significantly higher in LC with ascites than in the control subjects or in LC without ascites (1170.9 ± 28.7 μmol/L vs 903.1 ± 55.1 μmol/L or 956.7 ± 47.7 μmol/L, P < 0.05).
CONCLUSION: Increased intestinal macromolecular permeability and NO is probably of importance in the pathophysiology and progression of LC with ascites, but the serum TNF-α concentration was not related to LC with ascites.
PMCID: PMC2721447  PMID: 18609714
Intestinal permeability; Tumor necrosis factor-α; Nitric oxide; Liver cirrhosis; Ascites
20.  Isolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors 
PLoS ONE  2012;7(10):e46858.
Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12–14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.
PMCID: PMC3466197  PMID: 23056490
21.  Leptin levels in the differential diagnosis between benign and malignant ascites 
AIM: To evaluate the role of leptin levels in the differential diagnosis of ascites.
METHODS: Ascitic leptin, TNFα and serum leptin levels were measured in 77 patients with ascites (35 with malignancies, 30 cirrhosis and 12 tuberculosis). Control serum samples were obtained from 20 healthy subjects. Leptin and TNFα levels were measured by ELISA. Body mass index (BMI) and percentage of body fat (BFM) by skin fold measurement were calculated for all patients and control groups. Peritoneal biopsy, ascites cytology and cultures or biochemical values were used for the diagnosis of patients.
RESULTS: In patients with malignancies, the mean serum and ascites leptin levels and their ratios were significantly decreased compared to the other patient groups and controls. In tuberculosis peritonitis, ascitic fluid TNFα levels were significantly higher than malignant ascites and cirrhotic sterile ascites. BMI and BFM values did not distinguish between patients and controls.
CONCLUSION: In patients with malignant ascites, levels of leptin and TNFα were significantly lower than in patients with tuberculous ascites.
PMCID: PMC4065894  PMID: 17230608
Leptin; Benign ascites; Malignant ascites; Tuberculosis; Cirrhosis
22.  Disaggregation and invasion of ovarian carcinoma ascites spheroids 
Malignant ascites often develops in advanced stages of ovarian carcinoma, consisting of single and aggregated tumor cells, or spheroids. Spheroids have commonly been used as tumor models to study drug efficacy, and have shown resistance to some chemotherapies and radiation. However, little is known about the adhesive or invasive capabilities of spheroids, and whether this particular cellular component of the ascites can contribute to dissemination of ovarian cancer. Here, we examined the invasive ability of ascites spheroids recovered from seven ovarian carcinoma patients and one primary peritoneal carcinoma (PPC) patient.
Ascites spheroids were isolated from patients, purified, and immunohistochemical analyses were performed by a pathologist to confirm diagnosis. In vitro assays were designed to quantify spheroid disaggregation on a variety of extracellular matrices and dissemination on and invasion into normal human mesothelial cell monolayers. Cell proliferation and viability were determined in each assay, and statistical significance demonstrated by the student's t-test.
Spheroids from all of the patients' ascites samples disaggregated on extracellular matrix components, with the PPC spheroids capable of complete disaggregation on type I collagen. Additionally, all of the ascites spheroid samples adhered to and disaggregated on live human mesothelial cell monolayers, typically without invading them. However, the PPC ascites spheroids and one ovarian carcinoma ascites spheroid sample occasionally formed invasive foci in the mesothelial cell monolayers, suggestive of a more invasive phenotype.
We present here in vitro assays using ascites spheroids that imitate the spread of ovarian cancer in vivo. Our results suggest that systematic studies of the ascites cellular content are necessary to understand the biology of ovarian carcinoma.
PMCID: PMC1397876  PMID: 16433903
23.  A ligand-free, soluble urokinase receptor is present in the ascitic fluid from patients with ovarian cancer. 
Journal of Clinical Investigation  1993;92(5):2160-2167.
We have identified a soluble form of the human urokinase plasminogen activator (uPA) receptor (uPAR) in the ascitic fluids from patients with ovarian cancer. After purification of uPAR from the ascitic fluids by ligand-affinity chromatography (pro-uPA Sepharose), the uPAR was initially identified by cross-linking to a radiolabeled amino-terminal fragment of human uPA. The uPAR purified from the ascitic fluid has no bound ligand (uPA), as similar amounts can be purified by ligand-affinity chromatography as by immuno-affinity chromatography. uPAR from ascitic fluids partitions in the water phase after a temperature-dependent phase separation of a detergent extract. It therefore lacks at least the lipid moiety of the glycophospholipid anchor present in cellular-bound uPARs. It is highly glycosylated and the deglycosylated form has the same electrophoretic mobility as previously characterized cellular uPAR from other sources. The immunoreactivity of the purified uPAR from the ascitic fluid is indistinguishable from that of characterized uPAR, demonstrated by Western blotting with three different anti-uPAR monoclonal antibodies. The uPAR was found in 11 of 11 ascitic fluids from patients with ovarian cancer and in elevated amounts in the plasma from 2 of 3 patients. The concentration of soluble uPAR in the ascitic fluid was estimated to range between 1 and 10 ng/ml. Human soluble uPAR, derived from the tumor cells, was also found in the ascitic fluid and serum from nude mice xenografted intraperitoneally with three different human ovarian carcinomas.
PMCID: PMC288394  PMID: 8227331
24.  Screening and Identification of Biomarkers in Ascites Related to Intrinsic Chemoresistance of Serous Epithelial Ovarian Cancers 
PLoS ONE  2012;7(12):e51256.
The ability to predict responses to chemotherapy for serous epithelial ovarian cancer (EOC) would be valuable since intrinsically chemoresistant EOC patients (persistent or recurrent disease within 6 months) gain little benefit from standard chemotherapy. The aim of this study was to screen and identify distinctive biomarkers in ascites of serous EOC associated with intrinsic chemoresistance.
Protein samples from ascites of 12 chemosensitive and 7 intrinsically chemoresistant serous EOC patients were analyzed using two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Furthermore, the identified proteins were validated by ELISA in ascites samples from 19 chemosensitive and 9 intrinsically chemoresistant EOC patients.
The number of spots detected in all 2-D DIGE gels ranged from 1523–1711 using DeCyder software analysis. Thirty-four spots were differentially expressed based on the criteria of an average ratio of more than 1.5 and a student t-test P value <0.05. After MALDI-TOF/TOF MS analysis, 11 differentially expressed proteins, including 3 up-regulated and 8 down-regulated proteins, in ascites of chemoresistant tumors were successfully identified. Of the four selected proteins (ceruloplasmin, apoliprotein A-IV, transthyretin and haptoglobin) in ascites tested by ELISA, only ceruloplasmin was present at significantly different levels between the chemoresistant and chemosensitive ascites samples with average concentrations of 192.2 µg/ml and 157.5 µg/ml, respectively (P = 0.001).
The significantly up-regulated level of ceruloplasmin in the ascites fluid of intrinsic chemoresistant serous EOC patients suggests its potential as a prognostic biomarker for responses to chemotherapy. This finding prompts further investigation with a larger study in order to validate the clinical utility of ceruloplasmin.
PMCID: PMC3519621  PMID: 23251472
25.  Generation and Characterization of an Ascitogenic Mesothelin-Expressing Tumor Model 
Cancer  2007;110(2):420-431.
Intraperitoneal tumors expressing high amounts of mesothelin such as malignant mesothelioma and ovarian cancers tend to develop ascites and result in significant morbidity and mortality in the patient. A suitable preclinical intraperitoneal model will assist in the illustration of the mechanisms of molecular oncogenesis and facilitate in addressing issues related to early screening, diagnosis, and therapy for intraperitoneal tumors.
In the current study, an ascitogenic malignant tumor model (WF-3) was created. The mobility and proliferation of WF-3 and its precursor cells, WF-0, were characterized using transwell and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. In addition, the in vivo tumorgenicity of WF-3 and WF-0 was determined using intraperitoneal injection of the tumor cells. Microarray analysis was performed using WF-3 and WF-0. Northern blot analysis was used to characterize the expression of the mesothelin gene in WF-3 and WF-0. Furthermore, the mesothelin levels in serum and ascites were used to correlate with tumor load of WF-3 in tumor challenged mice.
The WF-3 tumor cells demonstrated relatively high proliferation and migration rates compared with the parental cell line, WF-0. The tumors from the WF-3 but not WF-0 were capable of forming ascites and peritoneal-based tumors after tumor challenge. The WF-3 tumor model was also capable of implanting into multiple organs including the diaphragm, intestines, and peritoneal wall. Furthermore, the WF-3 tumor expressed high levels of mesothelin, which is commonly observed in the majority of ovarian cancers, pancreatic cancer, and malignant mesothelioma. In addition, the authors found that the serum and ascites mesothelin levels correlated with tumor loads in tumor-challenged mice.
The data indicate that the WF-3 murine tumor model may potentially serve as a good model for understanding the molecular oncogenesis of peritoneal tumors. In addition, the preclinical model may potentially be useful for the development of diagnostic and therapeutic methods against intraperitoneal cancers.
PMCID: PMC3181493  PMID: 17559144
tumor model; animal model; microarray; mesothelin; ovarian cancer

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