Compound design of a patient-derived 3D cell culture system modelling early peritoneal endometriosis.

Study question Can an in vitro patient-derived multicellular model of superficial endometriosis be generated to support studies exploring associated fibrosis? Summary answer The three-dimensional (3D) co-culture of primary peritoneal cells and endometrial spheroids is a promising mimic of superficial endometriosis to support studies exploring early endometriosis formation. What is known already Peritoneal endometriosis causes adhesions and scars that can interfere with ovulation and conception, resulting in infertility. However, the underlying mechanisms that drive endometriosis-associated scar formation are not fully understood. Endometriotic lesions are proposed to arise from menstrual debris attachment to the peritoneum during retrograde menstruation, creating a new endometrial-peritoneal microenvironment. However, it is difficult to explore the process of early endometriosis formation in situ from patient biopsies due to the typically late diagnosis of endometriosis. A 3D co-culture approach provides unique opportunities to explore endometriosis lesion initiation and progression. Study design, size, duration For primary cell isolation, peritoneal washes, fallopian tube mesentery and endometrial tissue biopsies were collected over a two-year period from 10 patients undergoing laparoscopic surgery. Primary peritoneal mesothelial cells and fibroblast were co-cultured for 3 days to model the peritoneum before incorporation of an endometrium-derived construct to mimic superficial lesion formation. The superficial endometriosis model was co-cultured for a maximum of 10-days. Participants/materials, setting, methods A 3D peritoneal model was assembled by embedding human peritoneal fibroblasts (HPFs) in a bipartite Matrigel-collagen I matrix (2,000 cells/µl) before seeding human peritoneal mesothelial cells (HPMCs) (125 cells/µl). Following endometrial digestion, endometrial epithelial organoids (EEOs) were expanded in Matrigel, then co-cultured with endometrial stromal cells (ESCc) in rat-tail collagen I (4,000 cells/µl) to form endometrial spheroids. The 3D peritoneal model and endometrial spheroid were co-cultured and were characterised using immunostaining and ELISA measurements. Main results and the role of chance HPMCs were successfully isolated from peritoneal washes and exhibited a polygonal morphology up to passage 5, expressing dual positivity for pan-cytokeratin (pCK) and vimentin with 98.2% purity (n = 6). HPFs isolated from fallopian tube mesentery explants grew in a spindle shape appearance up to passage 4, with localisation of vimentin and lack of pCK that supported fibroblast characteristics and showed 81.4% purity (n = 3). Optimisation of hydrogel matrices demonstrated that a Matrigel-collagen I matrix at 1:1 ratio (v/v) supported the establishment of a 3D peritoneal in vitro model with minimal contraction. Histological investigation confirmed successful establishment of a simple peritoneum structure comprising a mesothelial monolayer with basal secretion of a collagen IV basement membrane and underlying fibroblasts. Secretion of tissue plasminogen activator (tPA) following 3- to 10-day culture demonstrated physiological functioning of the mesothelial element. Co-culture of EEOs and ESCs formed a compact endometrial spheroid comparable to in vivo endometrial tissue architecture. Endometrial spheroids adhered to the peritoneal 3D model, thus mimicking early endometriotic lesion formation. Histological analysis demonstrated direct cell-cell contacts between HPMC, HPF and ESC at the endometrial-peritoneal interface, suggesting the involvement of those cell types in lesion initiation and early endometriosis scar formation. Limitations, reasons for caution Characterisation of HPMC and HPF was performed using relatively few donors. Other important cell types present in endometriotic lesions, such as endothelial and immune cells, have not yet been incorporated into the model. Wider implications of the findings Our modifiable 3D endometriosis model enables further studies to explore the involvement of other cells and substances, such as the impact of immune cells and ovarian hormones, on superficial endometriosis initiation. This platform shows promising features to explore the underlying molecular mechanism involved in endometriotic lesion formation and scarring. Trial registration number No

Continuous ambulatory peritoneal dialysis (CAPD) is a major treatment modality for end-stage renal failure. The peritoneal membrane exhibits pathological changes that correlate with the duration of dialysis. These changes are due to the exposure of the peritoneum to non-physiologic peritoneal dialysis solution (PDS) with a high glucose content, and containing potentially toxic substances including glucose degradation products (GDP) and advanced glycation end products (AGE). Connective tissue growth factor (CTGF/CCN2) is one of the determinants of progressive fibrosis and peritoneal membrane dysfunction in CAPD. In this study, we examined the CCN2 expression and its regulation in peritoneal resident cells using a cell culture model. The expression of transforming growth factor-beta (TGF-beta), CCN2 and vascular endothelial growth factor (VEGF) in human peritoneal mesothelial cells (HPMC), human peritoneal fibroblasts (HPF) or endothelial cell line EA.hy926 (EC) cultured with various PDS and their components was examined by quantitative PCR (qPCR). The modulation of CCN2 synthesis under the crosstalk between HPMC and HPF or EC was examined using a conditioned medium transfer system in which HPMC was exposed to conditioned media obtained from HPF or EC incubated with PDS and their components. The differential effects of TGF-beta, CCN2 and VEGF in inducing the expression of transcriptional factors as well as interleukin-6 (IL-6), matrix metallopeptidase 9 (MMP-9) and collagen I were examined by electrophoretic mobility-shift assay (EMSA) and qPCR. PDS and their components differentially modulated the expression of TGF-beta, CCN2 and VEGF in HPMC, HPF and EC. The expression of CCN2 by HPMC was significantly increased after cultured with a HPF-conditioned medium and an EC-conditioned medium. Neutralizing anti-TGF-beta antibodies reduced but not completely abolished the CCN2 synthesis in HPMC cultured with the HPF- or EC-conditioned medium. CCN2, TGF-beta and VEGF activated distinct transcriptional factors in HPMC, which resulted in divergent biological responses in terms of IL-6, MMP-9 and collagen I mRNA expression. AGE and GDPs in PDS differentially regulate the synthesis of CCN2 by peritoneal resident cells. The CCN2 synthesis by HPMC can be further amplified by TGF-beta released from HPF or EC. The differential activation of different transcriptional factors and diverse response of HPMC towards CCN2, TGF-beta and VEGF suggest that these cytokines/growth factors have an overlapping and distinct role on HPMC.

Animal experiments have suggested that a high intraperitoneal pressure (IPP) might adversely affect the surgical peritoneal environment. The present experimental study investigates the impact of IPP of a CO(2) pneumoperitoneum on human peritoneum. Patients undergoing laparoscopic surgery were subjected to either low (8 mmHg) or standard (12 mmHg) IPP. Normal peritoneum was collected from the parietal wall at the beginning of surgery and every 60 min thereafter. Expression levels of 168 genes that encode extracellular matrix proteins, adhesion molecules or inflammatory cytokine signaling molecules were measured in peritoneal tissues using real-time polymerase chain reaction (PCR)-based assay panels. Human peritoneal mesothelial cells (HPMCs) and human peritoneal fibroblasts (HPFBs) were incubated in a CO(2) insufflation chamber for 1 h at 12 or 8 mmHg. Hyaluronan (HA) synthesis and mRNA expression levels of hyaluronic acid synthases (HAS) and hyaluronidases (Hyal) in HPMCs and HPFBs were measured at 0, 4, 8, 12, 24 and 48 h after CO(2) gas exposure by ELISA and real-time PCR, respectively. Expression levels of connective tissue growth factor (CTGF), matrix metalloproteinase-9, E-selectin, chemokine (C-X-C motif) ligand 2 (CXCL-2), Hyal-1 and Hyal-2 were significantly higher and those of HAS-1, HAS-3, thrombospondin-2 (TSP-2) and interleukin-10 were significantly lower in the 12 mmHg group compared with the 8 mmHg group. HA synthesis was significantly lower in the 12 mmHg group compared with the 8 mmHg group in HPMCs and HPFBs throughout the time course. A low IPP (8 mmHg) may be better than the standard IPP (12 mmHg) to minimize the adverse impact on the surgical peritoneal environment during a CO(2) pneumoperitoneum.

In patients on long-term continuous ambulatory peritoneal dialysis (CAPD), peritoneal dysfunction may occur due to loss of peritoneal mesothelial cells, peritoneal fibrosis and neovascularization. Lactate, long used as a buffer in peritoneal dialysates, has been substituted by bicarbonate in recent years. However, their effects on the peritoneum of CAPD patients are unknown. This study investigated the influence of lactate and bicarbonate on peritoneal dysfunction in CAPD patients. The mitochondrial activity of human peritoneal mesothelial cells (HPMCs) and their expression of basic fibroblast growth factor (bFGF) were studied after culture under various conditions. We also assessed the mitochondrial-activating effect of the supernatant of those cultures on human peritoneal fibroblasts (HPFBs) and human umbilical vein endothelial cells (HUVECs) and the effect of recombinant human bFGF on the mitochondrial activity of HPFBs and HUVECs. We used the WST-1 assay to determine mitochondrial activity in HPMC. At pH 7.4, the mitochondrial activity of HPMCs was lowest in a medium containing 40 mM (Lac), intermediate in a lactate (15 mM) plus bicarbonate (25 mM) medium (Lac/Bic), and highest in a 40 mM bicarbonate medium (Bic). In culture supernatant, the increase of bFGF was: Lac > Lac/Bic > Bic. Mitochondrial activation of HPFBs and HUVECs was stimulated by HPMC culture supernatants in the following decreasing order: Lac > Lac/Bic > Bic. The effects of these supernatants were suppressed by a bFGF-neutralizing antibody, while recombinant bFGF caused concentration-dependent mitochondrial activation in HPFBs and HUVECs. The role of bFGF in peritoneal fibrosis and neovascularization may be important. A bicarbonate-containing medium is better than a lactate-containing medium for preserving cell viability in HPMCs and preventing bFGF expression by these cells.

Biocompatibility and buffers: Effect of bicarbonate-buffered peritoneal dialysis fluids on peritoneal cell function

This paper demonstrates an in vitro model to simulate the microenvironment of endometriosis. We used microfluidic channels with cover slips to pattern and release endometrial stromal cells (ESCs) and human peritoneal mesothelial cells (HPMCs) in a way that mimicked the pathophysiology of peritoneal endometriosis. This approach enabled observation in real time interactions between ESCs and HPMCs both in their normal and pathological states. HPMCs from control individuals were able to resist the invasion of ESCs from both control and endometriotic individuals. By contrast, HPMCs from endometriotic individuals were unable to resist the invasion of ESCs from both normal and endometriotic individuals. We further analyzed the dynamics between HPMCs and ESCs from endometriotic individuals. HPMCs from endometriotic individuals relaxed their adhesion to each other at the beginning of invasion of ESCs, lose their adhesion to the substrate and apoptosed when surrounded by ESCs. These data implicate that the peritoneal physiology may play an important role in endometriosis.

Bystander senescence in human peritoneal mesothelium and fibroblasts is related to thrombospondin-1-dependent activation of transforming growth factor-β1

Ovarian cancer cells metastasize to organs in the abdominal cavity, such as the omentum, which are covered by a single layer of mesothelial cells. Thus, the cell-cell connection between ovarian cancer cells and mesothelial cells is the crucial step of metastasis. Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions through transfer of proteins, mRNAs and microRNAs. However, whether exosome-mediated transfer plays any role in ovarian cancer cell invasion remains poorly understood. Thus, the aim of this study is to identify the functional role of ovarian cancer-derived exosomes during the process of ovarian cancer metastasis. Methods: Exosomes were isolated from two ovarian cancer cell lines (HeyA8 and TYK-NU) and immortalized normal ovarian epithelial cell line (IOSE) using differential centrifugation. Human peritoneal mesothelial cells (HPMCs) were isolated from normal omentum of patients undergoing gynecologic surgery. The isolation of exosomes was confirmed by electron microscope and the transfer of exosomes into HPMCs was confirmed by fluorescent-labeling exosomes. The effects of exosome transfer from ovarian cancer cells to mesothelial cells were analyzed in vitro 3D culture model, morphological assessment, Western Blotting and gelatin zymography. We found that CD44 was enriched in cancer derived exosome. Thus, gain or loss of function of CD44 was analyzed. CD44 expression of ovarian cancer omental metastasis and surrounding organs in clinical samples was assessed by immunohistochemistry. Results: Fluorescent-labeled exosomes were evidently transferred into HPMCs. Exosome-treated HPMCs changed in cellular morphology to spindle phenotype. Ovarian cancer invasion was significantly promoted in the presence of exosome-treated HPMCs. In exosome-treated HPMCs, MMP-9 secretion was up-regulated and E-cadherin expression was down-regulated. The clearance of mesothelial barrier was increased in exosome-treated HPMC monolayer. By Western Blotting, we confirmed CD44 was enriched in exosomes and exosome-treated HPMCs display high-level of CD44. When CD44 expression was knocked down by siRNA in ovarian cancer cells, these effects to HPMCs were significantly attenuated. In contrast, the enforced expression of CD44 into HPMCs promoted cancer invasion by secreting MMP-9 and increasing mesothelial clearance. In human omentum with microscopic metastasis of ovarian cancer, positive CD44 expression was confirmed in a mesothelial cell layer when cancer cells are attaching onto it, while CD44 expression was generally negative in normal mesothelial cells. Conclusion: Herein, we revealed that ovarian cancer-derived exosomes transfer CD44 to HPMCs, which can facilitate ovarian cancer invasion by up-regulating of MMP-9 secretion and increasing clearance of HPMCs. Citation Format: Koji Nakamura, Kenjiro Sawada, Yasuto Kinose, Akihiko Yoshimura, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Exosome transfer from ovarian cancer cells to mesothelial cells promotes cell invasion by upregulating MMP-9 secretion and increasing clearance of mesothelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5060. doi:10.1158/1538-7445.AM2015-5060

Purpose: The peritoneum and organs in the peritoneal cavity are covered by a single layer of mesothelial cells. Therefore, ovarian cancer cells, which metastasize within the peritoneal cavity, directly encounter mesothelial cells as the initial step of metastasis. This contact has been found to involve cell-cell communication that affects cancer progression. Possible actors in this cell-cell communication are exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, and microRNAs. Here, we aim to identify the functional roles of ovarian cancer-derived exosomes in this metastatic process. Methods: Exosomes were isolated from two ovarian cancer cell lines (HeyA8 and TYK-NU) and immortalized normal ovarian epithelial cell line (IOSE) using differential centrifugation. Human peritoneal mesothelial cells (HPMCs) were isolated from normal omentum of patients undergoing gynecologic surgery. The isolation of exosomes was confirmed by electron microscope, nanoparticle tracking analysis, Western blotting and electrophoresis of RNA. The transfer of exosomes into HPMCs was confirmed by fluorescent-labeled exosomes. The effects of exosome transfer from ovarian cancer cells to mesothelial cells in cancer invasion were analyzed in vitro 3D culture model, morphological assessment, Western blotting and gelatin zymography. CD-44 was enriched in cancer derived exosome. Thus, gain or loss of function of CD-44 was analyzed. CD-44 expression in ovarian cancer omental metastasis and surrounding organs was assessed by immunohistochemistry using clinical samples. Results: Fluorescent-labeled exosomes were evidently transferred into HPMCs. Exosome-treated HPMCs changed in cellular morphology to spindle phenotype. Ovarian cancer invasion was significantly promoted in the presence of exosome-treated HPMCs. In exosome-treated HPMCs, MMP-9 secretion was up-regulated and E-cadherin expression down-regulated. The clearance of mesothelial barrier was increased in exosome-treated HPMC monolayer. CD-44 was enriched in cancer-derived exosomes and exosome-treated HPMCs display high-level of CD-44. When CD-44 expression was knocked down by siRNA in ovarian cancer cells, these effects to HPMCs were significantly attenuated. In contrast, the enforced expression of CD-44 into HPMCs promoted cancer invasion. In human omentum with microscopic metastasis of ovarian cancer, positive CD-44 expression was confirmed in a mesothelial cell layer when cancer cells are attaching onto it, while CD-44 expression was generally negative in normal mesothelial cells. Conclusion: Ovarian cancer-derived exosomes transfer CD-44 to HPMCs, which can facilitate ovarian cancer invasion by up-regulating of MMP-9 secretion and increasing mesothelial clearance. Citation Format: Koji Nakamura, Kenjiro Sawada, Yasuto Kinose, Akihiko Yoshimura, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Exosomes promote ovarian cancer invasion through CD-44 transfer to mesothelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1579.

PCBs enhance collagen I expression from human peritoneal fibroblasts

Matrix metalloproteinases are elevated in the urine of patients with endometriosis

IL-6 secretion by human peritoneal mesothelial and ovarian cancer cells

Biomarkers of endometriosis

We studied the effect of lipoprotein(a) [Lp(a)], low-density lipoprotein (LDL), and high-density lipoprotein (HDL) on tissue plasminogen activator (TPA) secretion from human endothelial cells. At 1 mumol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetate- and histamine-enhanced TPA secretion by 40%. LDL and HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were also examined and found to change in parallel with antigen secretion. In contrast to TPA, plasminogen activator inhibitor type-1 secretion and mRNA levels were not affected by any of the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may interfere with the proper production and/or secretion of TPA.

Changes in the human peritoneal mesothelial cells during aging