Ovarian Cancer Spheroid Cells with Stem Cell-Like Properties Contribute to Tumor Generation,Metastasis and Chemotherapy Resistance through Hypoxia-Resistant Metabolism

Cells with sphere forming capacity, spheroid cells, are present in the malignant ascites of patients with epithelial ovarian cancer (EOC) and represent a significant impediment to efficacious treatment due to their putative role in progression, metastasis and chemotherapy resistance. The exact mechanisms that underlie EOC metastasis and drug resistance are not clear. Understanding the biology of sphere forming cells may contribute to the identification of novel therapeutic opportunities for metastatic EOC. Here we generated spheroid cells from human ovarian cancer cell lines and primary ovarian cancer. Xenoengraftment of as few as 2000 dissociated spheroid cells into immune-deficient mice allowed full recapitulation of the original tumor, whereas >10⁵ parent tumor cells remained non-tumorigenic. The spheroid cells were found to be enriched for cells with cancer stem cell-like characteristics such as upregulation of stem cell genes, self-renewal, high proliferative and differentiation potential, and high aldehyde dehydrogenase (ALDH) activity. Furthermore, spheroid cells were more aggressive in growth, migration, invasion, scratch recovery, clonogenic survival, anchorage-independent growth, and more resistant to chemotherapy in vitro. ¹³C-glucose metabolic studies revealed that spheroid cells route glucose predominantly to anaerobic glycolysis and pentose cycle to the detriment of re-routing glucose for anabolic purposes. These metabolic properties of sphere forming cells appear to confer increased resistance to apoptosis and contribute to more aggressive tumor growth. Collectively, we demonstrated that spheroid cells with cancer stem cell-like characteristics contributed to tumor generation, progression and chemotherapy resistance. This study provides insight into the relationship between tumor dissemination and metabolic attributes of human cancer stem cells and has clinical implications for cancer therapy.     

TRPC3对上皮性卵巢癌细胞株迁移和侵袭的影响

目的:探讨瞬时受体电位通道C3(TRPC3)对人卵巢癌细胞迁移、侵袭能力的影响。方法:采用蛋白免疫印迹法和实时荧光定量PCR法分别检测卵巢癌细胞株SKOV3、ES-2和HEY-T30中TRPC3蛋白和m RNA的表达水平。通过Transwell迁移实验(不含Matrigel胶的Transwell小室)和Transwell侵袭实验分别检测卵巢癌细胞株SKOV3、ES-2和HEY-T30的迁移、侵袭能力。结果:在SKOV3、ES-2和HEY-T30三种卵巢癌细胞株中,ES-2中TRPC3的蛋白和m RNA表达均显著高于其他两株(P0.05)。Transwell迁移实验和Transwell侵袭实验显示卵巢癌细胞株ES-2的迁移、侵袭能力均显著高于其他两种细胞株(P0.05)。结论:瞬时受体电位通道C3(TRPC3)在ES-2人卵巢癌细胞中高表达,并可能促进人卵巢癌细胞的迁移、侵袭。     

Fluid-Flow Induced Wall Shear Stress and Epithelial Ovarian Cancer Peritoneal Spreading

Epithelial ovarian cancer (EOC) is usually discovered after extensive metastasis have developed in the peritoneal cavity. The ovarian surface is exposed to peritoneal fluid pressures and shear forces due to the continuous peristaltic motions of the gastro-intestinal system, creating a mechanical micro-environment for the cells. An in vitro experimental model was developed to expose EOC cells to steady fluid flow induced wall shear stresses (WSS). The EOC cells were cultured from OVCAR-3 cell line on denuded amniotic membranes in special wells. Wall shear stresses of 0.5, 1.0 and 1.5 dyne/cm² were applied on the surface of the cells under conditions that mimic the physiological environment, followed by fluorescent stains of actin and β-tubulin fibers. The cytoskeleton response to WSS included cell elongation, stress fibers formation and generation of microtubules. More cytoskeletal components were produced by the cells and arranged in a denser and more organized structure within the cytoplasm. This suggests that WSS may have a significant role in the mechanical regulation of EOC peritoneal spreading.     

Establishment,Characterization and Downstream Application of Primary Ovarian Cancer Cells Derived from Solid Tumors

Ovarian cancer is the deadliest of the gynecological diseases and the fifth cause of cancer death among American women. This is mainly due to the lack of prognostic tools capable of detecting early stages of ovarian cancer and to the high rate of resistance to the current chemotherapeutic regimens. In this scenario the overall 5-year survival rate for ovarian cancer patients diagnosed at late stage is less than 25%. Abnormalities associated with the malignant phenotype and the mechanisms of tumor progression are not clearly understood. In vitro studies are necessary, yet have been hampered due to the limitations accompanied with the use of ovarian cancer cell lines and the heterogeneity of the ovarian cancer cell population derived from ascites fluids. In this study we present a simple, rapid and reproducible method for the isolation and characterization of ovarian cancer cells from solid tumor tissue and show that enzymatic digestion for 30 minutes with dispase II results in the most effective recovery of viable epithelial ovarian cancer (EOC) cells. The resulting cancer (EOC) cell preparations demonstrate a significant yield, high levels of viability and are fibroblast-free. They grow for up to six passages and retain the capacity of forming spheroids-like structures in agarose. In addition, they can be genetically manipulated and used for drug screening, thus rendering them highly suitable for downstream applications. Notably, isolation of ovarian cancer cells from solid specimens using this method has the advantage of allowing for isolation of cancer cells from early stages of ovarian cancer as well as obtaining cells from defined either primary and/or metastatic ovarian cancer sites. Thus, these cells are highly suitable for investigations aimed at understanding ovarian cancer.     

Tetramethoxychalcone,a Chalcone Derivative,Suppresses Proliferation,Blocks Cell Cycle Progression,and Induces Apoptosis of Human Ovarian Cancer Cells

In the present study, we investigated the in vitro antitumor functions of a synthetic chalcone derivative 4,3′,4′,5′- tetramethoxychalcone (TMOC) in ovarian cancer cells. We found that TMOC inhibited the proliferation and colony formation of cisplatin sensitive cell line A2780 and resistant cell line A2780/CDDP, as well as ovarian cancer cell line SKOV3 in a time- and dose-dependent manner. Treatment of A2780 cells with TMOC resulted in G₀/G₁ cell cycle arrest through the down-regulation of cyclin D1 and CDK4, and the up-regulation of p16, p21 and p27 proteins. We demonstrated that TMOC might induce cell apoptosis through suppressing Bcl-2 and Bcl-xL, but enhancing the expression of Bax and the cleavage of PARP-1. Treatment of TMOC also reduced the invasion and migration of A2780 cells. Finally, we found that TMOC inhibited the constitutive activation of STAT3 signaling pathway and induced the expression of the tumor suppressor PTEN regardless of the p53 status in cell lines. These data suggest that TMOC may be developed as a potential chemotherapeutic agent to effectively treat certain cancers including ovarian cancer.     

IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. The insulin-like growth factor (IGF) system plays a key role in regulating growth and invasiveness in several malignancies, including ovarian cancer. IGF1R targeting showed antiproliferative activity of EOC cells. However, clinical studies failed to show significant benefit. EOC cells suppress antitumor immune responses by inducing dendritic cell (DC) dysfunction. The IGF1 axis can regulate DC maturation.The current study evaluated involvement of the IGF1 axis in DC differentiation in EOC. Studies were conducted on EOC and on a human monocyte cell line. Tissue microarray analysis (TMA) was performed on 36 paraffin blocks from EOC patients. Expression of IGF1R, p53, Ki67, BRCA1, and DC markers was evaluated using immunohistochemistry. Co-culture of EOC cells with DC pretreated with IGF1R inhibitor blocked cancer cell migration. TMA demonstrated higher rate of IGF1R protein expression in patients with advanced (76.9%) as compared to early (40%) EOC. A negative correlation between IGF1R protein expression and the CD1c marker was found. These findings provide evidence that IGF1R axis inhibition could be a therapeutic strategy for ovarian cancer by restoring DC-mediated antitumor immunity.     

FAP和Rho家族蛋白在卵巢癌侵袭迁移中的作用

目的：明确FAP是否通过RhoA／ROCK、Racl-GTP通路发挥促增殖、侵袭和迁移作用。方法：用MTT实验,Transwell实验和迁移实验检测FAP、RhoA／ROCK、Racl-GTP对卵巢癌细胞系HO-8910PM的增殖,侵袭和迁移的影响。结果：1、MTT法,迁移和侵袭实验证实用Y-27632抑制RhoA／ROCK途径能够促进卵巢癌细胞的增殖、迁移和侵袭,与FAP联合作用时促进作用增强。2、MTT法,迁移和侵袭实验证实NSC23766抑制Racl途径能够抑制卵巢癌细胞的增殖、迁移和侵袭,与FAP联合作用使FAP的促进作用减弱。结论：l、RhoA／ROCK通路抑制HO一8910PM细胞增殖、迁移和侵袭;Racl-GTP促进H0—8910PM细胞增殖、迁移和侵袭。2、FAP不是通过RhoA／ROCK而是通过Racl—GTP信号通路在HO．8910PM细胞发挥促增殖、迁移和侵袭作用的。     

YAP Promotes Ovarian Cancer Cell Tumorigenesis and Is Indicative of a Poor Prognosis for Ovarian Cancer Patients

YAP is a key component of the Hippo signaling pathway and plays a critical role in the development and progression of multiple cancer types, including ovarian cancer. However, the effects of YAP on ovarian cancer development in vivo and its downstream effectors remain uncertain. In this study we found that strong YAP expression was associated with poor ovarian cancer patient survival. Specifically, we showed for the first time that high YAP expression levels were positively correlated with TEAD4 gene expression, and their co-expression was a prognostic marker for poor ovarian cancer survival. Hyperactivation of YAP by mutating its five inhibitory phosphorylation sites (YAP-5SA) increased ovarian cancer cell proliferation, resistance to chemotherapeutic drugs, cell migration, and anchorage-independent growth. In contrast, expression of a dominant negative YAP mutant reversed these phenotypes in ovarian cancer cells both in vitro and in vivo. Our results suggested that YAP caused these effects by promoting an epithelial-to-mesenchymal transition. Thus, YAP promotes ovarian cancer cell growth and tumorigenesis both in vitro and in vivo. Further, high YAP and TEAD4 expression is a prognostic marker for ovarian cancer progression and a potential target for ovarian cancer treatment.     

FAP和Rho 家族蛋白在卵巢癌侵袭迁移中的作用

目的：明确FAP 是否通过RhoA/ROCK、Rac1-GTP 通路发挥促增殖、侵袭和迁移作用。方法：用MTT 实验,Transwell 实验 和迁移实验检测FAP、RhoA/ROCK、Rac1-GTP 对卵巢癌细胞系HO-8910PM 的增殖,侵袭和迁移的影响。结果：1、MTT 法,迁移和 侵袭实验证实用Y-27632 抑制RhoA/ROCK 途径能够促进卵巢癌细胞的增殖、迁移和侵袭,与FAP 联合作用时促进作用增强。 2、MTT 法, 迁移和侵袭实验证实NSC23766 抑制Rac1 途径能够抑制卵巢癌细胞的增殖、迁移和侵袭,与FAP 联合作用使FAP 的 促进作用减弱。结论：1、RhoA/ROCK 通路抑制HO-8910PM 细胞增殖、迁移和侵袭;Rac1-GTP 促进HO-8910PM 细胞增殖、迁移 和侵袭。2、FAP不是通过RhoA/ROCK而是通过Rac1-GTP 信号通路在HO-8910PM细胞发挥促增殖、迁移和侵袭作用的。     

Circulating Exosomal miR-1290 for Diagnosis of Epithelial Ovarian Cancer

The aim of the study was to develop a new diagnostic biomarker for identifying serum exosomal miRNAs specific to epithelial ovarian cancer (EOC) and to find out target gene of the miRNA for exploring the molecular mechanisms in EOC. A total of 84 cases of ovarian masses and sera were enrolled, comprising EOC (n = 71), benign ovarian neoplasms (n = 13). We detected expression of candidate miRNAs in the serum and tissue of both benign ovarian neoplasm group and EOC group using real-time polymerase chain reaction. Immunohistochemistry were constructed using formalin fixed paraffin embedded (FFPE) tissue to detect expression level of suppressor of cytokine signaling 4 (SOCS4). In the EOC group, miRNA-1290 was significantly overexpressed in serum exosomes and tissues as compared to benign ovarian neoplasm group (fold change ≥ 2, p < 0.05). We observed area under the receiver operating characteristic curve (AUC) for miR-1290, using a cut-off of 0.73, the exosomal miR-1290 from serum had AUC, sensitivity, and specificity values of 0.794, 69.2 and 87.3, respectively. In immunohistochemical study, expression of SOCS4 in EOC was lower than that in benign ovarian neoplasm. Serum exosomal miR-1290 could be considered as a biomarker for differential diagnosis of EOC from benign ovarian neoplasm and SOCS4 might be potential target gene of miR-1290 in EOC.     

Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion

Background

Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC.

Methodology/Principal Findings

A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration.

Conclusion/Significance

Our work shows that claudin-7 is significantly upregulated in EOC and that it may be functionally involved in ovarian carcinoma invasion. CLDN7 may therefore represent potential marker for ovarian cancer detection and a target for therapy.     

Interleukin 6 Receptor Is an Independent Prognostic Factor and a Potential Therapeutic Target of Ovarian Cancer

Ovarian cancer remains the most lethal gynecologic cancer and new targeted molecular therapies against this miserable disease continue to be challenging. In this study, we analyzed the expressional patterns of Interleukin-6 (IL-6) and its receptor (IL-6R) expression in ovarian cancer tissues, evaluated the impact of these expressions on clinical outcomes of patients, and found that a high-level of IL-6R expression but not IL-6 expression in cancer cells is an independent prognostic factor. In in vitro analyses using ovarian cell lines, while six (RMUG-S, RMG-1, OVISE, A2780, SKOV3ip1 and OVCAR-3) of seven overexpressed IL-6R compared with a primary normal ovarian surface epithelium, only two (RMG-1, OVISE) of seven cell lines overexpressed IL-6, suggesting that IL-6/IL-6R signaling exerts in a paracrine manner in certain types of ovarian cancer cells. Ovarian cancer ascites were collected from patients, and we found that primary CD11b⁺CD14⁺ cells, which were predominantly M2-polarized macrophages, are the major source of IL-6 production in an ovarian cancer microenvironment. When CD11b⁺CD14⁺ cells were co-cultured with cancer cells, both the invasion and the proliferation of cancer cells were robustly promoted and these promotions were almost completely inhibited by pretreatment with anti-IL-6R antibody (tocilizumab). The data presented herein suggest a rationale for anti-IL-6/IL-6R therapy to suppress the peritoneal spread of ovarian cancer, and represent evidence of the therapeutic potential of anti-IL-6R therapy for ovarian cancer treatment.     

Inhibition of Ovarian Epithelial Carcinoma Tumorigenesis and Progression by microRNA 106b Mediated through the RhoC Pathway

Epithelial ovarian cancer (EOC) is the most lethal of the gynecological malignancies. Exploring the molecular mechanisms and major factors of invasion and metastasis could have great significance for the treatment and prognosis of EOC. Studies have demonstrated that microRNA 106b (miR-106b) may be a promising therapeutic target for inhibiting breast cancer bone metastasis, but the role of miR-106b in EOC is largely unknown. In this work, miRNA-106b expression was quantified in various ovarian tissues and tumors. Ovarian carcinoma cell lines were transfected with miR-106b, after which, cell phenotype and expression of relevant molecules was assayed. Dual-luciferase reporter assays and xenograft mouse models were also used to investigate miR-106b and its target gene. MiR-106b mRNA expression was found to be significantly higher in normal ovarian tissues and benign tumors than in ovarian carcinomas and borderline tumors (p < 0.01), and was negatively associated with differentiation (Well vs. Por & Mod) and the International Federation of Gynecology and Obstetrics (FIGO) staging (stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05). MiR-106b transfection reduced cell proliferation; promoted G1 or S arrest and apoptosis (p < 0.05); suppressed cell migration and invasion (p < 0.05); reduced Ras homolog gene family member C (RhoC), P70 ribosomal S6 kinase (P70S6K), Bcl-xL, Matrix metallopeptidase 2 (MMP2), MMP9 mRNA and protein expression; and induced p53 expression (p < 0.05). Dual-luciferase reporter assays indicated that miR-106b directly targets RhoC by binding its 3’UTR. MiR-106b transfection also suppressed tumor development and RhoC expression in vivo in xenograft mouse models. This is the first demonstration that miR-106b may inhibit tumorigenesis and progression of EOC by targeting RhoC. The involvement of miR-106b-mediated RhoC downregulation in EOC aggression may give extended insights into molecular mechanisms underlying cancer aggression. Approaches aimed at overexpressing miR-106b may serve as promising therapeutic strategies for treating EOC patients.     

Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1)

Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted.     

Chk1反义寡核苷酸联合顺铂抑制卵巢癌侵袭转移的分子机制

目的:探究Chk1反义寡核苷酸(CHK1-ASODN)单独或联合顺铂(DDP)对卵巢癌细胞系SKOV-3侵袭转移能力的影响,并阐明其可能的分子机制。方法:体外培养人卵巢癌细胞系SKOV-3,CHK1-ASODN单独或联合DDP处理48 h后,划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力;显微镜下观察细胞上皮或间质表型特征;Western blot及实时定量PCR技术分别检测上皮间质转化(EMT)特异性标志物(E-cadherin、N-cadherin)以及EMT关键调控分子ZEB1的蛋白及m RNA的表达水平。结果:与对照组相比较,CHK1-ASODN单独或联合DDP均能显著抑制SKOV-3细胞的迁移及侵袭(P0.05);细胞表现为间质化表型;E-cadherin的表达显著升高(P0.05),而N-cadherin的表达则显著降低(P0.05);ZEB1的表达显著降低(P0.05)。结论:CHK1-ASODN单独或联合DDP下调ZEB1的表达进而逆转EMT可能是其抑制卵巢癌侵袭转移的重要机制之一。     

Anti-Tumor Effect of Pinus massoniana Bark Proanthocyanidins on Ovarian Cancer through Induction of Cell Apoptosis and Inhibition of Cell Migration

Pinus massoniana bark proanthocyanidins (PMBPs), an active component isolated from Pinus massoniana bark, has been reported to possess a wide range of biochemical properties. Here, we investigated the anti-tumor effect of PMBPs on ovarian cancer. The results indicated that PMBPs significantly reduced the growth of ovarian cancer cells and induced dose-dependent apoptosis. The underlying mechanisms involved were elucidated to include the loss of mitochondrial membrane potential, down-regulation of the anti-apoptotic protein Bcl-2 and the activation of Caspase 3/9, suggesting that PMBPs triggered apoptosis through activation of mitochondria-associated apoptotic pathway. In addition, wound healing and transwell chamber assays revealed that PMBPs could suppress migration and invasion of ovarian cancer cells. PMBPs dramatically inhibited MMP-9 activity and expression, blocked the activity of NFκB and the activation of ERK1/2 and p38 MAPK. Our findings suggest that PMBPs has the potential to be developed as an anti-tumor drug for ovarian cancer treatment and/ or disease management.     

PGPIPN,a Therapeutic Hexapeptide,Suppressed Human Ovarian Cancer Growth by Targeting BCL2

Bioactive peptides, either derived from nature resources or synthesized by rational design, have been demonstrated potential for therapeutic agents against numerous human diseases, including cancer. However, the mechanism of therapeutic peptides against cancer has not been well elucidated. Here we show that PGPIPN, a hexapeptide derived from bovine β-casein, inhibited the proliferation of human ovarian cancer cells line SKOV₃ as well as the primary ovarian cancer cells in vitro. Consistently, PGPIPIN also decreased tumor growth rate in xenograft ovarian cancer model mice in a dose-dependent manner. Further study demonstrated that the anti-tumor effect of PGPIPN is partially through promoting cell apoptosis by inhibiting BCL2 pathway. Thus, our study suggests that PGPIPN is a potential therapeutic agent for the treatment of ovarian cancer or other types of cancer.     

miR-181a通过调控上皮间质转化过程影响卵巢癌细胞的迁移和侵袭

目的:探讨mi R-181a在卵巢癌细胞中的表达及对卵巢癌细胞的迁移和侵袭的影响和可能机制。方法:采用细胞免疫荧光检测卵巢癌细胞中抗波形蛋白和E-钙粘蛋白的表达,Western blotting检测mi R-181a对抗波形蛋白和E-钙粘蛋白的表达情况的调控;划痕愈合实验检测mi R-181a对卵巢癌细胞迁移能力的影响;Transwell侵袭实验检测mi R-181a对卵巢癌细胞侵袭能力的影响。结果:增加mi R-181a的表达后,EMT过程中的相关蛋白激活水平下调,mi R-181a一定程度上可以抑制卵巢癌细胞的EMT过程;过表达mi R-181a后可以明显影响Vimentin[D1(4.58±0.85)vs(0.29±0.02),P0.05;D5(4.16±0.79)vs(0.29±0.02),P0.05]和E-Cadherin[D1(4.75±0.41) vs.(4.56±0.38),P0.05;D5(2.19±0.18) vs.(4.56±0.38),P0.05]蛋白的表达;COC1细胞的迁移能力随mi R-181a的表达的增高而降低[(19.24±4.31)%vs.(25.95±6.02)%,P0.05;(51.25±8.75)%vs.(73.49±12.54)%,P0.05];过表达mi R-181a后可以一定程度上减弱卵巢癌COC1细胞的侵袭能力[(74.64±8.21)vs(231.98±21.72),P0.05]。结论:mi R-181a通过调控上皮间质转化过程影响卵巢癌细胞的迁移和侵袭行为。     

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades ^1,2. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages ^3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren''t specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.