Identification of gene signature in ascitic fluid-isolated mesothelial cells from high grade serous ovarian cancer patients
ABSTRACT: RNA-sequencing analysis was carried out on ascitic fluid-isolated mesothelial cells from ovarian cancer patients compared to control human peritoneal mesothelial cells. Overall design: Three control human peritoneal mesothelial cell samples isolated from omentum obtained from non-oncologic patients undergoing abdominal surgery and four ascitic fluid-isolated mesothelial cell samples obtained from the peritoneal effucsions of stage III/IV
Project description:RNA-sequencing analysis was carried out on ascetic fluid-isolated mesothelial cells from ovarian cancer patients compared to control human peritoneal mesothelial cells to identify a mesothelial-mesenchymal gene signature. Overall design: Three control human peritoneal mesothelial cell samples isolated from omentum obtained from non-oncologic patients undergoing abdominal surgery and three ascitic fluid-isolated mesothelial cell samples obtained from the peritoneal effucsions of stage III/IV ovarian serous carcinoma patients
Project description:Organ-specific colonization suggests that specific cell-cell recognition is essential. Yet, very little is known about this particular interaction. Moreover, tumor cell lodgement requires binding under shear stress, but not static, conditions. Here, we successfully isolate the metastatic populations of cancer stem/tumor-initiating cells (M-CSCs). We show that the M-CSCs tether more and roll slower than the non-metastatic (NM)-CSCs, thus resulting in the preferential binding to the peritoneal mesothelium under ascitic fluid shear stress. Mechanistically, this interaction is mediated by P-selectin expressed by the peritoneal mesothelium. Insulin-like growth factor receptor-1 carrying an uncommon non-sulfated sialyl-Lewisx (sLex) epitope serves as a distinct P-selectin binding determinant. Several glycosyltransferases, particularly α1,3-fucosyltransferase with rate-limiting activity for sLex synthesis, are highly expressed in M-CSCs. Tumor xenografts and clinical samples corroborate the relevance of these findings. These data advance our understanding on the molecular regulation of peritoneal metastasis and support the therapeutic potential of targeting the sLex-P-selectin cascade.
Project description:The human omentum has been long regarded as a healing patch, used by surgeons for its ability to immunomodulate, repair and vascularise injured tissues. A major component of the omentum are mesothelial cells, which display some of the characteristics of mesenchymal stem/stromal cells. For instance, lineage tracing studies have shown that mesothelial cells give rise to adipocytes and vascular smooth muscle cells, and human and rat mesothelial cells have been shown to differentiate into osteoblast- and adipocyte-like cells in vitro, indicating that they have considerable plasticity. However, so far, long-term cultures of mesothelial cells have not been successfully established due to early senescence. Here, we demonstrate that mesothelial cells isolated from the mouse omentum could be cultured for more than 30 passages. While epithelial markers were downregulated over passages in the mesothelial cells, their mesenchymal profile remained unchanged. Early passage mesothelial cells displayed clonogenicitiy, expressed several stem cell markers, and up to passage 5 and 13, respectively, could differentiate along the adipogenic and osteogenic lineages, demonstrating stem/progenitor characteristics and differentiation potential.
Project description:Patients undergoing continuous ambulatory peritoneal dialysis are classified according to their peritoneal permeability as low transporter (low solute permeability) or High transporter (high solute permeability). Factors that determine the differences in permeability between them have not been fully disclosed. We investigated morphological features of cultured human peritoneal mesothelial cells from low or high transporter patients and its response to All trans retinoic Acid (ATRA, vitamin A active metabolite), as compared to non-uremic human peritoneal mesothelial cells. Control cells were isolated from human omentum. High or low transporter cells were obtained from dialysis effluents. Cells were cultured in media containing ATRA (0, 50, 100 or 200 nM). We studied length and distribution of microvilli and cilia (scanning electron microscopy), epithelial (cytokeratin, claudin-1, ZO-1 and occludin) and mesenchymal (vimentin and ?-smooth muscle actin) transition markers by immunofluorescence and Western blot, and transforming growth factor ?1 expression by Western blot. Low and high transporter exhibited hypertrophic cells, reduction in claudin-1, occludin and ZO-1 expression, cytokeratin and vimentin disorganization and positive ?-smooth muscle actin label. Vimentin, ?-smooth muscle actin and transforming growth factor-?1 were overexpressed in low transporter. Ciliated cells were diminished in low and high transporters. Microvilli number and length were severely reduced in high transporter. ATRA reduced hypertrophic cells number in low transporter. It also improved cytokeratin and vimentin organization, decreased vimentin and ?-smooth muscle actin expression, and increased claudin 1, occludin and ZO-1 expression, in low and high transporter. In low transporter, ATRA reduced transforming growth factor-?1 expression. ATRA augmented percentage of ciliated cells in low and high transporter. It also augmented cilia length in high transporter. Alterations in structure, epithelial mesenchymal markers and transforming growth factor-?1 expression were differential between low and high transporter. Beneficial effects of ATRA were improved human peritoneal mesothelial cells morphology tending to normalize structures.
Project description:Ascitic multicellular aggregates (MCAs) promote peritoneal metastasis of ovarian cancer. The aim of the present study was to elucidate the role of cancer?associated fibroblasts (CAFs) in MCA formation and metastasis in patients with high?grade serous ovarian cancer (HGSOC). Immunohistochemistry was used to identify the cell phenotypes and the presence of CAFs in ascitic MCAs. The role of CAFs in tumor?cell MCA formation was assessed by co?culture in suspension. Primary ascitic tumor cells and omental CAFs were used to generate ex vivo MCAs in hanging drops, and the invasiveness of MCAs was evaluated by mesothelial clearance and adhesion assays in vitro and in vivo. MCAs containing CAFs and tumor cells were identified in the ascitic fluid. CAFs facilitated tumor cell aggregation and compaction to form MCAs, and enhanced the mesothelial clearance and adhesion abilities of tumor?cell MCAs. These findings suggest that ascitic CAFs promote peritoneal metastasis by forming heterotypic aggregates with tumor cells, and that they may serve as potential targets for the treatment of HGSOC.
Project description:The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD.
Project description:BACKGROUND: Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization. METHODOLOGY: Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma. Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice. CONCLUSION: Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.
Project description:Peritoneal mesothelial cells are harmed by peritoneal dialysis fluids (PDF) used in renal replacement therapy with peritoneal dialysis. The mechanisms of the cellular damage are not yet described in detail. Primary human peritoneal mesothelial cells derived from omentum of five donors were independently exposed to peritoneal dialysis fluids. The extent of cell damage was assessed using lactate dehydrogenase (LDH) release in the cell culture supernatant and cells were lysed in order to extract mRNA and proteins. Transcriptional changes induced by PDF were analyzed using gene expression microarrays and changes of the proteome were analyzed using 2D-electrophoresis.
Project description:We investigated the cytoprotective role of the dietary polyphenols on putative damage induced by Amadori adducts in Human Peritoneal Mesothelial Cells (HPMCs). Increased accumulation of early products of non-enzymatic protein glycation-Amadori adducts-in the peritoneal dialysis fluid due to their high glucose, induces severe damage in mesothelial cells during peritoneal dialysis. Dietary polyphenols reportedly have numerous health benefits in various diseases and have been used as an efficient antioxidant in the context of several oxidative stress-related pathologies. HPMCs isolated from different patients were exposed to Amadori adducts (highly glycated haemoglobin, at physiological concentrations), and subsequently treated with several polyphenols, mostly presented in our Mediterranean diet. We studied several Amadori-induced effects in pro-apoptotic and oxidative stress markers, as well as the expression of several pro-inflammatory genes (nuclear factor-kappaB, NF-kB; inducible Nitric Oxide synthetase, iNOS), different caspase-activities, level of P53 protein or production of different reactive oxygen species in the presence of different polyphenols. In fact, cytoprotective agents such as dietary polyphenols may represent an alternate approach to protect mesothelial cells from the cytotoxicity of Amadori adducts. The interference with the Amadori adducts-triggered mechanisms could represent a therapeutic tool to reduce complications associated with peritoneal dialysis in the peritoneum, helping to maintain peritoneal membrane function longer in patients undergoing peritoneal dialysis.