This research was performed at the Keck School of Medicine, University of California, USA, and University College London, UK. KL is funded by National Institute of Health grant 5 U19 CA148112-02. BG was funded by a project grant from the Eve Appeal gynecological oncology charity (UK). Some of this work undertaken at UCLH/UCL was partly funding from the Department of Health's NIHR Biomedical Research Centre funding scheme.

Figure 1 illustrates an overview of the workflow described below.
1. Isolation of Normal Ovarian Fibroblasts and Extension of in vitro Lifespan by Overexpression of the Catalytic Subunit of the hTERT Holoenzyme
<ol>
 <li>Ovarian tissues can be collected with informed patient consent and approval of the Institutional Review Board (for US institutions). Normal ovarian tissues can be collected following total abdominal hysterectomy or total laparoscopic hyster...

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The biology of early-stage epithelial ovarian cancer (EOC) is poorly understood. Perhaps one of the main impediments in this field for many years has been the lack of understanding of the tissue specific origins of the disease, and of the significance of the role of the microenvironment in EOC development. Over the past few years, is has become clear that EOC is a heterogeneous disease with multiple distinct histophathological subtypes, probably with different cellular origins for different subtypes. For example, ...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Supplier</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>PolyHEMA, suitable for cell culture</td>
 <td>Sigma Aldrich</td>
 <td>P3932</td>
 </tr>
 <tr>
 <td>Molecular biology grade ethanol</td>
 <td>Sigma Aldrich</td>
 <td>E7023</td>
 </tr>
 <tr>
 <td>Sterile water for cell culture</td>
 <td>VWR</td>
 <td>12001-356</td>
 </tr>
 <tr>
 <td>MCDB105</td>
 <td>Sigma Aldrich</td>
 <td>M6395</td>
 </tr>
 <tr>
 <td>Medium 199</td>
 <td>Sigma Aldrich</td>
 <td>M2154</td>
 </tr>
 <tr>
 <td>Hyclone Fetal bovine serum</td>
 <td>Thermo Scientific</td>
 <td>SH30088.03</td>
 </tr>
 <tr>
 <td>Gentamicin</td>
 <td>Sigma Aldrich</td>
 <td>G1397</td>
 </tr>
 <tr>
 <td>Amphotericin B</td>
 <td>Sigma Aldrich</td>
 <td>A2942</td>
 </tr>
 <tr>
 <td>pBABE-hygro-hTERT</td>
 <td>Addgene</td>
 <td>1773</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>VWR</td>
 <td>12001-766</td>
 </tr>
 <tr>
 <td>0.25% trypsin-EDTA</td>
 <td>Invitrogen</td>
 <td>25200-072</td>
 </tr>
 <tr>
 <td>Cell strainer (40 or 70 &mu;m)</td>
 <td>VWR</td>
 <td>21008-949 
 21008-952 </td>
 </tr>
 <tr>
 <td>Anti-fibroblast surface protein antibody, clone 1B10</td>
 <td>Sigma</td>
 <td>F4771 </td>
 </tr>
 <tr>
 <td>Anti-pan-cytokeratin antibody (C11)</td>
 <td>Santa Cruz</td>
 <td>sc-8018 </td>
 </tr>
 <tr>
 <td>Polybrene (hexadimethrine bromide)</td>
 <td>Sigma</td>
 <td>107689</td>
 </tr>
 <tr>
 <td>TeloTAGGG Telomerase PCR ELISAPLUS</td>
 <td>Roche</td>
 <td>12013789001</td>
 </tr>
 <tr>
 <td>TeloTAGGG Telomere Length Assay</td>
 <td>Roche</td>
 <td>12209136001</td>
 </tr>
 </tbody>
 </table>
Table 1. Reagents and Equipment Referred to in this study.

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 &gt;85%, survival rates for stage III/IV disease are &lt;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)&nbsp;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.

Watch this Scientific Journal Video about Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression at JoVE.com

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

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

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 ...

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14.9K visualizações.  University of Southern California. Descrevemos metodologias para estabelecer In vitro Heterotípicas modelos tridimensionais compreendendo fibroblastos de ovário e de superfície do ovário normal ou células ovarianas cancerosas epiteliais. Discute-se o uso desses modelos para estudar estromal-epiteliais interações que ocorrem durante o desenvolvimento do câncer de ovário.