Spontaneous Transformation of Murine Oviductal Epithelial Cells: A Model System to Investigate the Onset of Fallopian-Derived Tumors

High-grade serous carcinoma (HGSC) is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs) are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOELOW) was developed and continuously passaged in culture to mimic cellular aging (MOEHIGH). The MOEHIGH cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOEHIGH cells proliferated significantly faster than MOELOW, and the MOEHIGH cells produced more 2D foci and 3D soft agar colonies as compared to MOELOW. MOEHIGH were xenografted into athymic female nude mice both in the subcutaneous and the intraperiteonal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOEHIGH and MOELOW were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOEHIGH had enhanced protein expression of c-myc, Cyclin E, p53 and FOXM1 with reduced expression of p21. MOEHIGH were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans

In vivo tumor growth of high-grade serous ovarian cancer cell lines

OBJECTIVE: Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. METHODS: To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119 and UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. RESULTS: Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. CONCLUSIONS: Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community

Multimodal, Biomaterial-Focused Anticoagulation via Superlow Fouling Zwitterionic Functional Groups Coupled with Anti-Platelet Nitric Oxide Release

The functions of anti-fouling, zwitterionic polycarboxybetaine (pCB) and anti-platelet nitric oxide (NO) release replicate key anticoagulant properties of the endothelium. The two approaches, only tested separately thus far, were paired on gas permeable polydimethylsiloxane (PDMS) membranes and evaluated for anti-coagulation. Uncoated PDMS (control) and PDMS coated with pCB were screened for fibrinogen (Fg) fouling followed by platelet adsorption testing to evaluate the effects of coating and/or NO using bioreactors. Bare or coated PDMS membranes separated sheep plasma (108 platelets/ml) and gas flow chambers within the bioreactors. Either 100 or 0 ppm of NO/N2 flowed through the gas chamber for NO release at the plasma/biomaterial interface. Surface-adsorbed platelets were quantified using a lactate dehydrogenase assay after 8 hrs of plasma recirculation. Fg fouling and platelet adsorption on pCB-coated PDMS were 10.40 ± 3.0% of control (

Investigation of the effect of surface released nitric oxide on fibrinogen adsorption, An

2014 Fall.The search for improved biomaterials is a continually ongoing effort to prevent the failure of medical devices due to blood clotting. Each group of researchers has their own set of methods to create the ideal material for biological systems. In the pursuit of materials to prevent blood clot formation, these attempts have been focused on alterations in surface properties, pre-adsorption of proteins, and release of drugs. In this work I took a high-throughput approach to the prevention of device failure by investigating a model material system. Starting with a nitric oxide (NO) releasing material, a sample preparation method was developed to ensure that surface properties could be compared to a non-NO releasing control. With this material, the effect of the NO release on fibrinogen adsorption to these surfaces could be isolated. Fibrinogen is instrumental in the formation of blood clots. Determining the effect that NO has on this protein will help determine why NO has been previously found to prevent clotting in blood-contacting systems. Once the model system was developed, further investigation into changes in the fibrinogen resulting from its interaction with the released NO could be undertaken. A full investigation was completed on control non-NO releasing, low NO flux, and high NO flux materials. A qualitative assessment of the fibrinogen adsorption shows that the high NO releasing material exhibits significantly higher fibrinogen adsorption compared to both the control and low NO flux materials. Quantitative assessment of fibrinogen adsorption was attempted through a variety of methods, which indicate that conformational changes are happening upon adsorption of fibrinogen to all materials. To this end, FTIR spectra from the adsorbed fibrinogen and native fibrinogen were compared to elucidate changes in the protein's conformation. Control and low NO flux materials had too little protein to gain insight into these changes. For the high NO flux material, the fibrinogen had a significant decrease in α-helices and an increase in random chains compared to native fibrinogen. To begin understanding the effect that these changes will have on blood clot formation, these materials were further analyzed for platelet adhesion. A comparison of the control, low NO flux, and high NO flux materials with and without fibrinogen adsorbed to the material surface shows that the fibrinogen has a distinct effect on platelet adhesion and aggregation. The high NO flux materials exhibited less aggregation and full activation of platelets when fibrinogen was adsorbed prior to incubation with platelets than if fibrinogen was not present before incubation. Overall, the effect of NO on fibrinogen adsorption can be seen through these measurements. Nitric oxide release causes an increase in fibrinogen adsorption, as well as protein reorganization. Surprisingly, we see that this adsorbed fibrinogen actually improves the viability of platelets. Further study must be done using whole blood and in vivo measurements to fully understand what effect the adsorbed fibrinogen will have on the device. Despite this we can say that the adsorption of fibrinogen onto these NO releasing materials helps to improve the biocompatibility of this biomaterial due to its bulk adsorption and conformational changes

Kaempferol Exhibits Progestogenic Effects in Ovariectomized Rats

OBJECTIVE: Progesterone (P(4)) plays a central role in women's health. Synthetic progestins are used clinically in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Unfortunately, synthetic progestins are associated with side effects, including cardiovascular disease and breast cancer. Botanical dietary supplements are widely consumed for the alleviation of a variety of gynecological issues, but very few studies have characterized natural compounds in terms of their ability to bind to and activate progesterone receptors (PR). Kaempferol is a flavonoid that functions as a non-steroidal selective progesterone receptor modulator (SPRM) in vitro. This study investigated the molecular and physiological effects of kaempferol in the ovariectomized rat uteri. METHODS: Since genistein is a phytoestrogen that was previously demonstrated to increase uterine weight and proliferation, the ability of kaempferol to block genistein action in the uterus was investigated. Analyses of proliferation, steroid receptor expression, and induction of well-established PR-regulated targets Areg and Hand2 were completed using histological analysis and qPCR gene induction experiments. In addition, kaempferol in silico binding analysis was completed for PR. The activation of estrogen and androgen receptor signalling was determined in vitro. RESULTS: Molecular docking analysis confirmed that kaempferol adopts poses that are consistent with occupying the ligand-binding pocket of PRA. Kaempferol induced expression of PR regulated transcriptional targets in the ovariectomized rat uteri, including Hand2 and Areg. Consistent with progesterone-l ke activity, kaempferol attenuated genistein-induced uterine luminal epithelial proliferation without increasing uterine weight. Kaempferol signalled without down regulating PR expression in vitro and in vivo and without activating estrogen and androgen receptors. CONCLUSION: Taken together, these data suggest that kaempferol is a unique natural PR modulator that activates PR signaling in vitro and in vivo without triggering PR degradation

Tumorigenesis and peritoneal colonization from fallopian tube epithelium

Ovarian cancer is the most lethal gynecological malignancy, primarily because its origin and initiation factors are unknown. A secretory murine oviductal epithelial (MOE) model was generated to address the hypothesis that the fallopian tube is an origin for high-grade serous cancer. MOE cells were stably altered to express mutation in p53, silence PTEN, activate AKT, and amplify KRAS alone and in combination, to define if this cell type gives rise to tumors and what genetic alterations are required to drive malignancy. Cell lines were characterized in vitro and allografted into mice. Silencing PTEN formed high-grade carcinoma with wide spread tumor explants including metastasis into the ovary. Addition of p53 mutation to PTEN silencing did not enhance this phenotype, whereas addition of KRAS mutation reduced survival. Interestingly, PTEN silencing and KRAS mutation originating from ovarian surface epithelium generated endometrioid carcinoma, suggesting that different cellular origins with identical genetic manipulations can give rise to distinct cancer histotypes. Defining the roles of specific signaling modifications in tumorigenesis from the fallopian tube/oviduct is essential for early detection and development of targeted therapeutics. Further, syngeneic MOE allografts provide an ideal model for pre-clinical testing in an in vivo environment with an intact immune system

Nitric Oxide Releasing Tygon Materials: Studies in Donor Leaching and Localized Nitric Oxide Release at a Polymer-Buffer Interface

Tygon is a proprietary plasticized poly­(vinyl chloride) polymer that is used widely in bioapplications, specifically as extracorporeal circuits. To overcome issues with blood clot formation and infection associated with the failure of these medical devices upon blood contact, we consider a Tygon coating with the ability to release the natural anticlotting and antibiotic agent, nitric oxide (NO), under simulated physiological conditions. These coatings are prepared by incorporating 20 w/w% <i>S</i>-nitrosoglutathione (GSNO) donor into a Tygon matrix. These films release NO on the order of 0.64 ± 0.5 × 10^–10 mol NO cm^–2 min^–1, which mimics the lower end of natural endothelium NO flux. We use a combination of assays to quantify the amount of GSNO that is found intact at different time points throughout the film soak, as well as monitor the total thiol content in the soaking solution due to any analyte that has leached from the polymer film. We find that a burst of GSNO is released from the material surface within 5 min to 1 h of soaking, which only represents 0.25% of the total GSNO contained in the film. After 1 h of film soak, no additional GSNO is detected in the soaking solution. By further considering the total thiol content in solution relative to the intact GSNO, we demonstrate that the amount of GSNO leached from the material into the buffer soaking solution does not contribute significantly to the total NO released from the GSNO-incorporated Tygon film (<10% total NO). Further surface analysis using SEM-EDS traces the elemental S on the material surface, demonstrating that within 5 min −1 h soaking time, 90% of the surface S is removed from the material. Surface wettability and roughness measurements indicate no changes between the GSNO-incorporated films pre- to postsoak that will be significant toward the adsorption of biological components, such as proteins, relative to the presoaked donor-incorporated film. Overall, we demonstrate that, for a 20 w/w% GSNO-incorporated Tygon film, relatively minimal GSNO leaching is experienced, and the lost GSNO is from the material surface. Varying the donor concentration from 5 to 30 w/w% GSNO within the film does not result in significantly different NO release profiles. Additionally, the steady NO flux associated with the system is predominantly due to localized release from the material, and not donor lost to soaking solution. The surface properties of these materials generally imply that they are useful for blood-contacting applications

Intrabursal injection of AdCreGFP, but not intraoviductal injection, modifies ovarian phenotype.

<p>Fluid-filled bursal cysts and degenerate ovaries were observed in <i>p53</i>^flox/flox mice 6 months after intrabursal injection of AdCreGFP (A-C). Black arrow indicates ovarian tissue and blue arrow indicates oviductal tissue. The TEC showed normal expression of CK8 (D), PAX8 (E), and OVGP1 (F) both three and six months after intraoviductal viral injection. Scale bar represents 100 µm.</p

Conditional Inactivation of <i>p53</i> in Mouse Ovarian Surface Epithelium Does Not Alter MIS Driven Smad2-Dominant Negative Epithelium-Lined Inclusion Cysts or Teratomas

<div><p>Epithelial ovarian cancer is the most lethal gynecological malignancy among US women. The etiology of this disease, although poorly understood, may involve the ovarian surface epithelium or the epithelium of the fallopian tube fimbriae as the progenitor cell. Disruptions in the transforming growth factor beta (TGFβ) pathway and p53 are frequently found in chemotherapy-resistant serous ovarian tumors. Transgenic mice expressing a dominant negative form of Smad2 (Smad2DN), a downstream transcription factor of the TGFβ signaling pathway, targeted to tissues of the reproductive tract were created on a FVB background. These mice developed epithelium-lined inclusion cysts, a potential precursor lesion to ovarian cancer, which morphologically resembled oviductal epithelium but exhibited protein expression more closely resembling the ovarian surface epithelium. An additional genetic “hit” of <i>p53</i> deletion was predicted to result in ovarian tumors. Tissue specific deletion of <i>p53</i> in the ovaries and oviducts alone was attempted through intrabursal or intraoviductal injection of Cre-recombinase expressing adenovirus (AdCreGFP) into <i>p53</i>^flox/flox mice. Ovarian bursal cysts were detected in some mice 6 months after intrabursal injection. No pathological abnormalities were detected in mice with intraoviductal injections, which may be related to decreased infectivity of the oviductal epithelium with adenovirus as compared to the ovarian surface epithelium. Bitransgenic mice, expressing both the Smad2DN transgene and <i>p53</i>^flox/flox, were then exposed to AdCreGFP in the bursa and oviductal lumen. These mice did not develop any additional phenotypes. Exposure to AdCreGFP is not an effective methodology for conditional deletion of floxed genes in oviductal epithelium and tissue specific promoters should be employed in future mouse models of the disease. In addition, a novel phenotype was observed in mice with high expression of the Smad2DN transgene as validated through qPCR analysis, characterized by teratoma-like lesions implicating Smad signaling in teratoma development.</p></div