Accumulation Of Ascitic Fluid Research Articles

Ovarian cancer ascites proteomic profile reflects metabolic changes during disease progression

Ovarian cancer (OC) patients develop ascites, an accumulation of ascitic fluid in the peritoneal cavity anda sign of tumour dissemination within the peritoneal cavity. This body fluid is under-researched, mainly regarding the ascites formed during tumour progression that have no diagnostic value and, therefore, are discarded. We performed a discovery proteomics study to identify new biomarkers in the ascites supernatant of OC patients. In this preliminary study, we analyzed a small amount of OC ascites to highlight the importance of not discarding such biological material during treatment, which could be valuable for OC management. Our findings reveal that OC malignant ascitic fluid (MAF) displays a proliferative environment that promotes the growth of OC cells that shift the metabolic pathway using alternative sources of nutrients, such as the cholesterol pathway. Also, OC ascites drained from patients during treatment showed an immunosuppressive environment, with up-regulation of proteins from the signaling pathways of IL-4 and IL-13 and down-regulation from the MHC-II. This preliminary study pinpointed a new protein (Transmembrane Protein 132A) in the OC context that deserves to be better explored in a more extensive cohort of patients’ samples. The proteomic profile of MAF from OC patients provides a unique insight into the metabolic kinetics of cancer cells during disease progression, and this information can be used to develop more effective treatment strategies.

Abstract A105: Untangling the effect of ascites in shaping high grade serous ovarian cancer progression

Abstract High Grade Serous Ovarian Cancer (HGSOC) is the eighth cause of cancer-related mortality worldwide. Progress in eradicating this disease has been hampered by several factors, including the uncertainty about its tissues of origin, late diagnosis coupled to early dissemination through accumulation of ascitic fluid and extensive inter-patient variability. While being histopathologically homogeneous, HGSOC is characterized by several layers of heterogeneity that require the development of ad hoc research strategies and to focus on specific scales in order to deconvolve HGSOC pathogenetic mechanisms. In this context, our lab has previously shown that HGSOC can be derived both from the distal tract of the fallopian tube (fallopian tube epithelium, FTE) and the ovarian surface epithelium (OSE), with a prognostic impact for the tissue of origin and origin-specific molecular phenotypes, including an immune-suppressive response in the more aggressive OSE-derived subtype (Lo Riso, Villa et al. 2020), pointing at the role of tumor microenvironment in shaping therapeutical response. Moreover, we have also shown the role of ascitic fluid in sustaining the generation of single cell-derived metastatic ovarian cancer spheroids (sMOCS), that enrich for cancer stem-like cells, and allowing to identify a consistent intra-patient variability in response to carboplatin treatment (Velletri, Villa, Cilli, Barzaghi et al. 2022). To understand the specific impact of the tumor microenvironment in shaping HGSOC complexity, here we show an experimental framework aimed at the identification of the downstream effect of ascites in shaping cellular and molecular features of HGSOC. Here we leverage a newly generated single cell transcriptomic atlas, encompassing 1.5 million cells from 79 patients derived from primary tumors, ascites and metastatic samples, to identify specific expression networks activated during the metastatization process. In parallel, we derive from bulk transcriptomic data the set of genes that are induced upon ascitic fluid supplementation in 2D and 3D in vitro models, that are coupled to model-specific phenotypes, including cell proliferation, stemness and invasiveness. These genes are then integrated in the single-cell derived metastasis-specific expression networks to identify the upstream regulators imputable to ascitic fluid exposure. The linked regulators and downstream effectors are then used to identify in the atlas cell populations whose expression landscape is directly affected by ascitic fluid. The identification of such cell populations will allow to highlight cell-to-cell interactions existing between i) different tumor subpopulations; ii) tumor and tumor-associated compartments, in both primary and metastatic samples, that can be ascribed to the presence of ascitic fluid. Moreover, the molecular characterization of these interacting populations will allow to identify novel targets, paving the way to the identification of novel therapeutical avenues for HGSOC patients. Citation Format: Pietro Lo Riso, Carlo Emanuele Villa, Bianca Barzaghi, Marta R. Sallese, Giulia Picco, Alessia Gatto, Ugo Cavallaro, Giuseppe Testa. Untangling the effect of ascites in shaping high grade serous ovarian cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A105.

BBT-877, a Novel Autotaxin Inhibitor, Abrogates Drug Resistance in Epithelial Ovarian Cancer Stem Cells.

Cancer stem cells (CSCs) contribute significantly to the poor prognosis of patients with epithelial ovarian cancer (EOC) due to their roles in drug resistance and tumor metastasis. Autotaxin (ATX) plays a pivotal role in the maintenance of the CSC-like properties of EOC tumors. BBT-877 is a novel ATX inhibitor used in clinical treatment of idiopathic pulmonary fibrosis. However, the effects of BBT-877 on drug resistance and metastasis in ovarian CSCs remain unknown. In this study, we aimed to investigate the effects of BBT-877 on drug resistance and intraperitoneal metastasis of EOC. Spheroid-forming CSCs, which were isolated from two EOC cell lines, A2780 and SKOV3, were investigated by cell viability, western blot, PCR, Spheroid-forming assay, and in vivo experiments. Spheroid-forming CSCs exhibited increased CSC-like properties and paclitaxel (PTX) resistance. BBT-877 treatment inhibited the viability of spheroid-forming CSCs more potently than that of adherent ovarian cancer cell lines. Combinatorial treatment with BBT-877 and PTX significantly attenuated the viability of spheroid-forming CSCs. In a SKOV3 cells-derived intraperitoneal metastasis model, BBT-877 treatment reduced the number of metastatic tumor nodes, while combinatorial treatment with BBT-877 and PTX more potently attenuated the formation of metastatic nodes and accumulation of ascitic fluid. These results suggest that BBT-877 can be combined with conventional anticancer drugs for the treatment of patients with recurrent or drug-resistant EOC.

Environmental physical factors produce mitotic aberration and multicellularity in the ovarian cancer cell line SKOV3

The spread of ovarian cancer (OC) to the coelomic cavity triggers the secretion and accumulation of ascitic fluid (AF). Although its biochemical composition has been well studied, less is known about the implications of physical factors such as the pH and the mechanical properties of the AF for the malignancy of tumor cells. In this work, we investigated the effect of pH and the mechanical properties of AF on cell proliferation and mitotic morphology. We employed biopsies from patients with OC and the SKOV3 cell line as an in vitro model of OC with HeLa cells as controls. Sections of each tumor were stained with HE, analyzed, and related to clinical data. AF from patients with OC exhibited an alkaline pH (ranging from 7.3 to 7.8). Compared to control conditions, the 3 AFs significantly enhanced the proliferation of SKOV3 and HeLa cells. These effects were more pronounced at a more alkaline pH. In addition, we found that AFs have different densities that correlated with a significant increase in multinucleated tumor cells and severe morphological defects in cells undergoing mitosis. In agreement with these data, we found that higher concentrations of soft agar provoked significantly higher numbers of multinucleated and morphologically abnormal SKOV3 cells with no effect on HeLa cells.We conclude that an alkaline pH and greater rigidity could enhance the metastatic potential of OC cells. We propose that these two physical factors could be parameters of clinical importance as predictors of malignancy.

The Impact of Mitochondria in Ovarian Cancer Cell Metabolism, Proliferation, and Metastasis.

Mitochondrial dysfunctions are significantly implicated in cancer initiation, progression, and metastasis, which have been shown for several cancers including ovarian cancer.An increase in mitochondrial dysfunction is also associated with drug resistance along with cancer progression, which in part is related to its specific microenvironment that is characterized by ascites, low glucose levels, and hypoxia that causes ovarian cancer cells to switch to mitochondrial respiration to enable their survival. Peritoneal ascitic fluid accumulation is a specific feature of ovarian cancer, and it is a major cause of its metastatic spread that also presents challenges for effective treatment. Among the treatment difficulties for ovarian cancer is the mutation rate and frequency of mtDNA in ovarian cancer tissue that can affect the efficiency of chemotherapeutic drugs. The varied and multiple mutations of different types enable metabolic reprogramming, cancer cell proliferation, and drug resistance.New specific information on mechanisms underlying several of the mitochondrial dysfunctions has led to proposing various mitochondrial determinants as targets for ovarian cancer therapy, which include targeting specific mitochondrial proteins and phosphoproteins as well as reactive oxygen species (ROS) that accumulate abnormally in cancer cells. Because of the genetically and histologically heterogeneous nature of the disease, combination therapy approaches will be necessary to combat the disease and achieve progress in effective treatment of ovarian cancer. This chapter will address (1) mitochondrial vulnerabilities underlying dysfunction and disease; (2) mitochondrial dysfunction in ovarian cancer; (3) present treatment difficulties for ovarian cancer and new potential treatment strategies to target ovarian cancer mitochondrial metabolism; and (4) biobehavioral factors influencing ovarian cancer development.

Abstract 4654: Differential immunomodulation following checkpoint blockade in the orthotopic ID8-luc ovarian model

Abstract Syngeneic tumor models for ovarian cancer are limited in preclinical research. One of these models, ID8-luc, is established intraperitoneally with disease progression resulting in the accumulation of ascites fluid. Treatment efficacy can be determined by non-invasive bioluminescence imaging (BLI), but solid ID8-luc tumors do not form in the peritoneal cavity, making assessment of immunomodulation challenging. To this end, we have developed the ID8-luc model as a tool to investigate immuno-modulatory drug candidates by utilizing tissues in the peritoneal cavity of diseased animals for ex vivo analyses. Specifically, flow cytometry of diseased ovaries was employed to examine the effects of checkpoint inhibition on T cell phenotypes and NK cell infiltration. To identify tumor deposits in organs from mice bearing ID8-luc disease for analysis tumor infiltrate, the model was established in untreated C57BL/6 albino mice, and ovaries, liver, peritoneal wall, pancreas, and spleen were evaluated pathologically. Progressive establishment of tumor deposits were seen in the ovaries, pancreas, and peritoneal wall of untreated mice, but not in naïve mice. Further, immune infiltration in ovaries were markedly higher compared to naïve mice. To assess immunomodulation, disease was established in a second cohort of mice, and mice were treated with anti-mPD-1, anti-mPD-L1, and anti-mCTLA-4. Disease progression was monitored by BLI, and following treatment, ovaries containing tumor deposits were collected and lymphoid infiltrate was analyzed by flow cytometry. Checkpoint blockade did not result in efficacy under the conditions tested. However, differences in T cell infiltration were notable with treatment of anti-PD-1 and anti-mPD-L1. Particularly, a marked increase of five to ten fold CD8+ and CD4+ T cells by absolute counts was seen with anti-mPD-1 and anti-mPD-L1 treatment compared to control. Further, the percentage of CD8+ T cells expressing the activation markers CD69 and ICOS as well as PD-1, TIM-3, and LAG-3 checkpoint proteins increased with anti-mPD-1 treatment. Less prominent yet similar trends in this biomarker expression were seen with anti-mPD-L1 treatment. NK and NKT infiltration also increased five to eight fold in ovaries following anti-mPD-1 treatment. Interestingly, anti-mCTLA-4 treatment did not result in any remarkable changes in infiltration or activation marker dynamics. Taken together, these findings are suggestive of a mounting immune response following PD-1 and PD-L1 blockade, but not CTLA-4 blockade, characterized by changes in the lymphocyte compartment in the ovaries. This response is seen in the absence of efficacy, suggesting the potential that this immunomodulation can be exploited in rational combination strategies with checkpoint blockade for immune-oncology drug candidates using the ID8-luc ovarian tumor model. Citation Format: Sheri Barnes, Anita Zaitouna, Lauren Kucharczyk, Scott Wise. Differential immunomodulation following checkpoint blockade in the orthotopic ID8-luc ovarian model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4654.

Deletion of Slc6a14 reduces cancer growth and metastatic spread and improves survival in KPC mouse model of spontaneous pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal. There is a dire need for better therapeutic targets. Cancer cells have increased demand for sugars, amino acids, and lipids and therefore up-regulate various nutrient transporters to meet this demand. In PDAC, SLC6A14 (an amino acid transporter (AAT)) is up-regulated, affecting overall patient survival. Previously we have shown using in vitro cell culture models and in vivo xenograft mouse models that pharmacological inhibition of SLC6A14 with α-methyl-l-tryptophan (α-MLT) attenuates PDAC growth. Mechanistically, blockade of SLC6A14-mediated amino acid transport with α-MLT leads to amino acid deprivation, eventually inhibiting mTORC1 signaling pathway, in tumor cells. Here, we report on the effect of Slc6a14 deletion on various parameters of PDAC in KPC mice, a model for spontaneous PDAC. Pancreatic tumors in KPC mice show evidence of Slc6a14 up-regulation. Deletion of Slc6a14 in this mouse attenuates PDAC growth, decreases the metastatic spread of the tumor, reduces ascites fluid accumulation, and improves overall survival. At the molecular level, we show lower proliferation index and reduced desmoplastic reaction following Slc6a14 deletion. Furthermore, we find that deletion of Slc6a14 does not lead to compensatory up-regulation in any of the other amino transporters. In fact, some of the AATs are actually down-regulated in response to Slc6a14 deletion, most likely related to altered mTORC1 signaling. Taken together, these results underscore the positive role SLC6A14 plays in PDAC growth and metastasis. Therefore, SLC6A14 is a viable drug target for the treatment of PDAC and also for any other cancer that overexpresses this transporter.

Фенотипическое разнообразие макрофагов при раке яичников

Фенотипическое разнообразие макрофагов при раке яичников

Mesothelial-to-Mesenchymal Transition and Exosomes in Peritoneal Metastasis of Ovarian Cancer.

Most patients with ovarian cancer (OvCA) present peritoneal disseminated disease at the time of diagnosis. During peritoneal metastasis, cancer cells detach from the primary tumor and disseminate through the intraperitoneal fluid. The peritoneal mesothelial cell (PMC) monolayer that lines the abdominal cavity is the first barrier encountered by OvCA cells. Subsequent progression of tumors through the peritoneum leads to the accumulation into the peritoneal stroma of a sizeable population of carcinoma-associated fibroblasts (CAFs), which is mainly originated from a mesothelial-to-mesenchymal transition (MMT) process. A common characteristic of OvCA patients is the intraperitoneal accumulation of ascitic fluid, which is composed of cytokines, chemokines, growth factors, miRNAs, and proteins contained in exosomes, as well as tumor and mesothelial suspended cells, among other components that vary in proportion between patients. Exosomes are small extracellular vesicles that have been shown to mediate peritoneal metastasis by educating a pre-metastatic niche, promoting the accumulation of CAFs via MMT, and inducing tumor growth and chemoresistance. This review summarizes and discusses the pivotal role of exosomes and MMT as mediators of OvCA peritoneal colonization and as emerging diagnostic and therapeutic targets.

A Case of Rapid Decline in Alcohol-Associated Liver Disease.

This case demonstrates a rapid clinical decline in a patient with acute‐on‐chronic liver failure affecting the liver, kidneys, and brain. 1 Our patient presented with decompensated alcohol‐associated cirrhosis with accumulation of ascitic fluid and had a variceal bleed on presentation to the emergency department with superimposed ischemic hepatopathy in the setting of hypotension and rapid development of grade IV hepatic encephalopathy, which was unable to be reversed, with eventual development of multiorgan failure. This case highlights the importance of rapid recognition and response to complications of decompensated liver disease, including hepatic encephalopathy. Role of Ammonia According to the most recent American Association for the Study of Liver Diseases practice guidelines, a diagnosis of hepatic encephalopathy requires an evaluation for precipitating factors, which are listed in Table ​Table22. 2 High blood ammonia alone does not add any diagnostic value; however, a normal ammonia level may suggest alternative causes of encephalopathy. 2 Extremely high ammonia level may be associated with cerebral edema even in patients with cirrhosis. 3 Brain imaging is typically indicated for first‐time hepatic encephalopathy given the high risk for intracerebral hemorrhage, estimated at nearly a 5‐fold increased risk for those diagnosed with alcohol‐associated liver disease and cirrhosis. 4 However, brain imaging may not always consistently detect early cerebral edema. New evidence also indicates a prognostic role in ammonia levels, with lack of improvement of ammonia levels within 5 days being associated with higher mortality and multiorgan failure. 5 TABLE 2 Precipitating Factors for Hepatic Encephalopathy

Pulmonary Hypoplasia Associated with Ascites and Hydrothorax in a Newborn Pig

Background: Pulmonary hypoplasia is characterized by incomplete development of the lungs, owing to congenital defectsor the action of toxic substances. Moreover, it has rarely been described in pigs. Ascites or hydroperitoneum is characterized by the presence of fluid inside the abdominal cavity and does not generally cause changes in the abdominal organs. However, hydrothorax, characterized by the presence of fluid within the thoracic cavity, is responsible for the compression of thoracic organs and consequent heart and respiratory failure. This study aims to describe a case of congenital pulmonary hypoplasia associated with ascites and hydrothorax in a newborn pig.Case: A male neonate Landrace pig that died shortly after delivery was presented for necropsy with increased abdominal volume and bilateral extension of the pelvic limbs. The pig belonged to a litter of 13 piglets, four of which died shortly after birth. The rest of the piglets were poorly developed, but only one was presented for necropsy. Significant external changes, along with permanent distension, interpreted as arthrogriposis, were observed in the pelvic limbs. The skin of the ventral abdominal region was thin, with evidence of all blood vessels, interpreted as telangectasis. An internal examination revealed the presence of a slightly yellowish liquid in the thoracic and abdominal cavities, interpreted as hydrothorax and ascites, respectively. The lungs were reduced in size, indicating pulmonary hypoplasia. The liver had rounded edges, which were dark red and firm, with an irregular surface. Significant microscopic findings were observed in the lungs, which were divided by fibrous connective tissue and showed evidence of small and atrophied alveoli. Furthermore, connective tissue was observed around the peribronchiolar regions and underdeveloped cartilage around the airways. The liver showed dilation of the centrilobular veins. Moreover, the sinusoids were filled with erythrocytes, indicating congestion.Discussion: Pulmonary hypoplasia is a rare birth defect in pigs and can be accompanied by malformations of other organs, including anasarca. Pulmonary hypoplasia is caused by conditions that compress the lungs, such as congenital diaphragmatic hernia, intrathoracic masses, and pleural effusion or malformations in the chest cavity. However, none of these conditions were observed in the current case. Instead, the histopathological changes observed in the lungs were characteristic of pulmonary hypoplasia. A microscopic examination revealed that the hypoplastic lungs showed a reduced number of alveoli, replacement of the peribronchiolar smooth muscle by the connective tissue, and underdeveloped cartilage around the airways, all of which are characteristic of pulmonary hypoplasia. Vascular telangiectasia is characterized by abdominal distention due to a marked ascitic condition. Invariably, ascitic fluid accumulation results from changes in hydrodynamic parameters. The hydrothorax observed in this animal was suggestive of changes in the hydrodynamic parameters, which consequently led to fluid accumulation in the chest cavity. This has been previously described in a newborn pig infected with Clostridium difficile. The permanently and bilaterally extended pelvic joints observed at birth in this animal were interpreted as arthrogriposis. This change is attributed to various factors, such as fetal paralysis in pigs that ingest toxic substances like alkaloids present in Conium maculatum and in the stalks of Nicotiana tabacum and Datura stramunium. The literature describes pelvic limb paralysis in Landrace pigs as a genetically inherited disorder. However, it was not possible for us to establish the cause of arthrogriposis for certain in this pig.

Inactivation of TRP53, PTEN, RB1, and/or CDH1 in the ovarian surface epithelium induces ovarian cancer transformation and metastasis.

Ovarian cancer (OvCa) remains the most common cause of death from gynecological malignancies. Genetically engineered mouse models have been used to study initiation, origin, progression, and/or mechanisms of OvCa. Based on the clinical features of OvCa, we examined a quadruple combination of pathway perturbations including PTEN, TRP53, RB1, and/or CDH1. To characterize the cancer-promoting events in the ovarian surface epithelium (OSE), Amhr2cre/+ mice were used to ablate floxed alleles of Pten, Trp53, and Cdh1, which were crossed with TgK19GT121 mice to inactivate RB1 in KRT19-expressing cells. Inactivation of PTEN, TRP53, and RB1 with or without CDH1 led to the development of type I low-grade OvCa with enlarged serous papillary carcinomas and some high-grade serous carcinomas (HGSCs) in older mice. Initiation of epithelial hyperplasia and micropapillary carcinoma started earlier at 1 month in the triple mutations of Trp53, Pten, and Rb1 mice as compared to 2 months in quadruple mutations of Trp53, Pten, Rb1, and Cdh1 mice, whereas both genotypes eventually developed enlarged proliferating tumors that invaded into the ovary at 3-4 months. Mice with triple and quadruple mutations developed HGSC and/or metastatic tumors, which disseminated into the peritoneal cavity at 4-6 months. In summary, inactivation of PTEN, TRP53, and RB1 initiates OvCa from the OSE. Additional ablation of CDH1 further increased persistence of tumor dissemination and ascites fluid accumulation enhancing peritoneal metastasis.

Pulmonary manifestations of chronic liver disease: a comprehensive review.

Hepatopulmonary syndrome (HPS) and porto-pulmonary hypertension (PoPH) represent relatively common pulmonary vascular complications of advanced liver disease. Despite distinct differences in their pathogenetic background, both clinical states are characterized by impaired arterial oxygenation and limited functional status, and are associated with increased pre-transplantation mortality. Accumulation of ascitic fluid in the pleural cavity, known as hepatic hydrothorax (HH), is another frequent manifestation of decompensated cirrhosis, which may cause severe respiratory dysfunction, depending on the volume of the effusion, the rapidity of its development and its resistance to therapeutic measures. Orthotopic liver transplantation constitutes the only effective treatment able to resolve the pulmonary complications of liver disease. A prioritization policy for liver transplantation has evolved over the past years regarding advanced stages of HPS, yielding favorable outcomes regarding post-transplantation survival and HPS resolution. In contrast, severe PoPH is associated with poor post-transplantation survival. Hence, liver transplantation is recommended only for patients with PoPH and an acceptable reduction in pulmonary pressure values, after receiving PoPH-targeted vasodilating therapy. This review focuses on basic pathogenetic and diagnostic principles and discusses the current therapeutic approaches regarding HPS, PoPH, and HH.

Host and Tumor Factor XII Drive Ovarian Cancer Maintenance and Progression

Host and Tumor Factor XII Drive Ovarian Cancer Maintenance and Progression Introduction . Epithelial ovarian cancer (EOC) is the leading cause of cancer death in women.Effective strategies to treat this disease are lacking due to the complexity of pathways involved and the multitude of cells that contribute to EOC biology. To this end, coagulation factor XII (FXII) and its receptor urokinase plasminogen activator receptor (uPAR) represent very promising therapeutic targets because i) uPAR is overexpressed in more than 90% of ovarian cancer patients, ii) FXII has been shown to be upregulated in the peritoneum of EOC patients and promotes EOC dissemination and iii) FXII-uPAR signal and upregulate key neutrophil functions, recently linked to tumor growth and metastasis. Through analysis of the TCGA ovarian cancer patient cohort, we have found that FXII and uPAR are co-expressed in ovarian tumors and their overexpression is associated with decreased overall survival (Fig 1A-B). Given that FXII and uPAR can be produced by both the EOC tumor and the host, the contribution of each of these compartments which has not been examined to date needs to be explored. Thus, we asked if the FXII-uPAR axis in neutrophils and EOC cells synergistically influences tumor biology. Methods - Results. We utilized a tissue microarray to determine FXII and uPAR expression. Both FXII and uPAR were co-expressed in all major histologic subtypes of EOC tumors but not in normal ovarian epithelium (Fig C-D). We adopted a composite expression score system and found significantly increased expression of FXII and uPAR in high grade tumors compared to low grade (grade 1-2) tumors, independently of tumor subtype and stage (Fig 1C-D). Given prior reports that neutrophils are enriched in the ovarian tumor microenvironment (TME) and our findings that FXII-uPAR are integral to neutrophil activation, tumors were also examined for their neutrophil content. Invariably, as tumor grade advanced, Neutrophil Elastase expression significantly increased (data not shown). We next asked if neutrophils contribute to EOC tumor progression or their recruitment merely correlates with advanced disease. We found that in the presence of neutrophils, EOC cells migrate significantly faster (p<0.0001). Since enhanced tumor cell migration implies a pro-mesenchymal phenotype, we examined if neutrophils facilitate EOC cell epithelial-to-mesenchymal transition (EMT). Murine EOC cancer cells (ID8) were co-cultured with wild type (WT) neutrophils for 24 h; cells were harvested and used for immunoblotting and mRNA studies. We found that WT neutrophils significantly increased the expression of mesenchymal marker(s) vimentin and N-cadherin while they decreased the expression of E-cadherin, suggesting that neutrophils promote EMT of EOC cancer cells (Fig 1E). To investigate whether the FXII-uPAR axis contributed to neutrophil-induced EMT, we next co-cultured ID8 cells with neutrophils from tumor-bearing FXII deleted (F12-/-)mice. In ID8 cells co-cultured with F12-/-neutrophils, EOC induced EMT was blocked. Similarly, treatment with 2 peptide inhibitors that block the FXII-uPAR interaction (collectively termed, SMPAs), reversed the pro-invasive effects of both ID8 cells and WT neutrophils (Fig 1F). Next, in order to assess whether host FXII could contribute to EOC dissemination and progression we utilized WT, F12-/-, and uPAR deficient (Plaur-/-) mice for in vivo EOC tumor model. After orthotopic injection of ID8 EOC cells, WT mice exhibited faster rates of tumor development and ascitic fluid accumulation (13.4 ± 0.92 ml), relative to F12-/- (0.37 ± 0.26 ml) and Plaur-/- (0.5 ± 0.5 ml) mice (Fig 1G). Blinded examination of tumors showed significantly higher tumor burden in WT mice compared to F12-/-and Plaur-/- mice, with F12-/-mice exhibiting the highest degree of protection (Fig 1H). Conclusions . Our studies indicate three novel aspects: i) a direct crosstalk between ovarian cancer cells and neutrophils, that enhances tumor cell migration through FXII-uPAR signaling; ii) the presence of neutrophils in the TME induces EMT of cancer cells which is abolished when the FXII-uPAR interaction is inhibited and; iii) abrogation of the FXII-uPAR axis in EOC tumor cells and neutrophils, synergistically restricts the pro-invasive phenotype of ovarian tumor cells. Disclosures No relevant conflicts of interest to declare.

Central Venous Catheter as Peritoneal Indwelling Catheter for the Management of Recurrent Malignant Ascites: A Case Series.

Background:Malignant ascites is an accumulation of fluid in the peritoneal cavity due to the manifestation of terminal metastatic malignancies. Accumulation of ascitic fluid could cause abdominal distention, early satiety, and shortness of breath. The onset and progression of these symptoms are not only distressing but also cause deterioration of the quality of life. Malignant ascites is associated with poor prognosis with anticipated life expectancy ranging from 1 to 4 months. Therapeutic paracentesis is the mainstay of palliation for malignant ascites. It only provides a temporary relief of symptoms, ascites re-accumulates, and paracentesis has to be repeated.Objectives:The aim of this case series was to evaluate our experiences when treating malignant ascites with placement of central venous catheter in the peritoneal cavity, with special emphasis on patient satisfaction, comfort of caregiver, technical success, and adverse events.Methods:Five patients with advanced disease and recurrent malignant ascites who required multiple paracentesis were selected for placement of 7-FG triple-port Central venous catheter in the peritoneal cavity. Maximum fluids that can be tapped were removed in ward. Patients and their relatives were assessed for satisfaction with the procedure.Results:Out of 5 patients, 3 were female and 2 male (age between 46 and 62 years). Two patients had carcinoma gallbladder and one each of carcinoma ovary, breast, and prostate. All patients were followed up till 1 month from catheter placement. Patients and their relatives were well satisfied in terms of frequent hospital visit for paracentesis.Conclusion:In summary, we suggest that central venous catheter may be used as indwelling peritoneal catheter for the symptom management of recurrent malignant ascites. It provides a relatively safe and cost-effective alternative to serial large-volume paracentesis that requires multiple hospital admissions.

Pseudoprogression manifesting as recurrent ascites with anti-PD-1 immunotherapy in urothelial bladder cancer

BackgroundImmunotherapies targeting the PD-1 checkpoint pathway have recently gained regulatory approval in numerous cancer types. With the widespread use of immune checkpoint therapies, varying patterns of responses and immune-related adverse events are being observed.Case PresentationIn this case, we highlight a patient who developed recurrent, large-volume ascites, while simultaneously having a 49% reduction in peritoneal tumor lesion size by RECIST criteria. Sampling of the fluid revealed high levels of IL-6 and IL-15. Cytology revealed no malignant cells on 4 separate paracenteses over a period of 6 weeks. Cell counts revealed that 45% of cells were lymphocytes, and further analysis was performed by fluorescence-activated cell sorting (FACS). The majority of lymphocytes were CD8+, of which 78% were PD-1+ and 43% were HLA-DR+ indicating an activated phenotype.ConclusionsIn summary, treatment with anti-PD-1 therapy may result in pseudoprogression manifested by ascitic fluid accumulation due to the influx of activated T cells. Since worsening of ascites is typically associated with disease progression, it is important to consider the possibility of pesudoprogression in such patients undergoing therapy with immune checkpoint inhibitors.

Mesothelial‐to‐mesenchymal transition as a possible therapeutic target in peritoneal metastasis of ovarian cancer

Peritoneal dissemination is the primary metastatic route of ovarian cancer (OvCa), and is often accompanied by the accumulation of ascitic fluid. The peritoneal cavity is lined by mesothelial cells (MCs), which can be converted into carcinoma‐associated fibroblasts (CAFs) through mesothelial‐to‐mesenchymal transition (MMT). Here, we demonstrate that MCs isolated from ascitic fluid (AFMCs) of OvCa patients with peritoneal implants also undergo MMT and promote subcutaneous tumour growth in mice. RNA sequencing of AFMCs revealed that MMT‐related pathways – including transforming growth factor (TGF)‐β signalling – are differentially regulated, and a gene signature was verified in peritoneal implants from OvCa patients. In a mouse model, pre‐induction of MMT resulted in increased peritoneal tumour growth, whereas interfering with the TGF‐β receptor reduced metastasis. MC‐derived CAFs showed activation of Smad‐dependent TGF‐β signalling, which was disrupted in OvCa cells, despite their elevated TGF‐β production. Accordingly, targeting Smad‐dependent signalling in the peritoneal pre‐metastatic niche in mice reduced tumour colonization, suggesting that Smad‐dependent MMT could be crucial in peritoneal carcinomatosis. Together, these results indicate that bidirectional communication between OvCa cells and MC‐derived CAFs, via TGF‐β‐mediated MMT, seems to be crucial to form a suitable metastatic niche. We suggest MMT as a possible target for therapeutic intervention and a potential source of biomarkers for improving OvCa diagnosis and/or prognosis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Novel pegylated interferon-β as strong suppressor of the malignant ascites in a peritoneal metastasis model of human cancer.

Malignant ascites manifests as an end‐stage event during the progression of a number of cancers and lacks a generally accepted standard therapy. Interferon‐β (IFN‐β) has been used to treat several cancer indications; however, little is known about the efficacy of IFN‐β on malignant ascites. In the present study, we report on the development of a novel, engineered form of human and murine IFN‐β, each conjugated with a polyethylene glycol molecule (PEG‐hIFN‐β and PEG‐mIFN‐β, respectively). We provide evidence that these IFN‐β molecules retain anti‐viral potency comparable to unmodified IFN‐β in vitro and manifested improved pharmacokinetics in vivo. Interestingly, PEG‐mIFN‐β significantly inhibited the accumulation of ascites fluid and vascular permeability of the peritoneal membrane in models of ovarian cancer and gastric cancer cell xenograft mice. We further show that PEG‐hIFN‐β directly suppresses VEGF 165‐induced hyperpermeability in a monolayer of human vascular endothelial cells and that PEG‐mIFN‐β enhanced gene expression for a number of cell adhesion related molecules in mouse vascular endothelial cells. Taken together, these findings unveil a hitherto unrecognized potential of IFN‐β in maintaining vascular integrity, and provide proof‐of‐mechanism for a novel and long‐acting pegylated hIFN‐β for the therapeutic treatment of malignant ascites.

Development of Safe and Potent Oil-in-Water Emulsion of Paclitaxel to Treat Peritoneal Dissemination

To develop a safer and more potent paclitaxel (PTX) formulation, we prepared various oil-in-water emulsions by using egg phosphatidylcholine, Tween 80, and a mixture of triglycerides with different fatty acid chain lengths as the cosurfactant, surfactant, and oil phase component, respectively. The mean particle diameters of the PTX-emulsions prepared were around 100 nm. The PTX-emulsions did not provoke histamine release from rat mast cells and did not show any significant hemolytic activity, suggesting that PTX-emulsions are biocompatible. In vivo antitumor activity after single intraperitoneal injection of PTX-emulsions to ascites tumor-bearing mice revealed that the formulation containing tricaproin and triacetin (3:1, wt/wt, PTX-emulsion B) significantly prolonged the survival time and suppressed the accumulation of ascites fluid. Two distinct in vitro release studies showed that the release of PTX from emulsion B was significantly faster than those from other preparations. These results indicate that the adequately sustained PTX release would lead to potent in vivo antitumor activity and that PTX-emulsion B would offer an alternative approach to treat peritoneal dissemination.

Systemic effects of Subtilase cytotoxin produced by Escherichia coli O113:H21

Subtilase cytotoxin (SubAB) is a member of the AB5 cytotoxin family and is produced by certain strains of Shiga toxigenic Escherichia coli. The toxin is known to be lethal to mice, but the pathological mechanisms that contribute to Uremic Hemolytic Syndrome (HUS) are poorly understood. In this study we show that intraperitoneal injection of a sublethal dose of SubAB in rats triggers a systemic response, with ascitic fluid accumulation, heart hypertrophy and damage to the liver, colon and kidney. SubAB treated rats presented microalbuminuria 20 days post inoculation. At this time we found disruption of the glomerular filtration barrier and alteration of the protein reabsorption mechanisms of the proximal tubule. In the kidney, SubAB also triggered an epithelial to mesenchymal transition (Wuyts et al., 1996). These findings indicate that apart from direct cytotoxic effects on renal tissues, SubAB causes significant damage to the other organs, with potential consequences for HUS pathogenesis. ImportanceUremic Hemolytic Syndrome is an endemic disease in Argentina, with over 400 hundred new cases each year. We have previously described renal effects of Shiga Toxin and its ability to alter renal protein handling. Bearing in mind that Subtilase Cytotoxin is an emerging pathogenic factor, that it is not routinely searched for in patients with HUS, and that to the date its systemic effects have not been fully clarified we decided to study both its systemic effects, and its renal effects to assess whether SubAB could be contributing to pathology seen in children.