Barbara Grun

Three-dimensional in vitro cell biology models of ovarian and endometrial cancer

Objectives:  This study aims to establish three‐dimensional (3D) cell culture models of human ovarian and endometrial cancers and to compare biological and morphological characteristics of these models with those of two‐dimensional (2D) models of the same cell lines and the primary tumours.

Chromosomes 6 and 18 induce neoplastic suppression in epithelial ovarian cancer cells

Metaphase comparative genomic hybridisation (CGH) studies indicate that chromosomes 4, 5, 6, 13, 14, 15 and 18 are frequently deleted in primary ovarian cancers (OCs). Therefore we used microcell‐mediated chromosome transfer (MMCT) to establish the functional effects of transferring normal copies of these chromosomes into 2 epithelial OC cell lines (TOV112D and TOV21G). The in vitro neoplastic phenotype (measured as anchorage dependent and independent growth and invasion) was compared between recipient OC cell lines and multiple MMCT hybrids. Chromosomes 6 and 18 showed strong evidence of functional, neoplastic suppression for multiple hybrids in both cell lines. We also found evidence in 1 cancer cell line suggesting that chromosomes 4, 13 and 14 may also cause functional suppression. Array CGH and microsatellite analyses were used to characterise the extent of genomic transfer in chromosome 6 and 18 hybrids. A 36 MB deletion on chromosome 6 in 2 hybrids from 1 cell line mapped the candidate region proximal to 6q15 and distal to 6q22.2; and an ∼10 MB candidate region spanning the centromere on chromosome 18 was identified in 2 hybrids from the other cell line. These data support reported functional effects of chromosome 6 in OC cell lines; but to our knowledge, this is the first time that functional suppression for chromosome 18 has been reported. This suggests that these chromosomes may harbour tumour suppressor‐“like” genes. The future identification of these genes may have a significant impact on the understanding and treatment of the disease and the identification of novel therapeutic targets.

NPPB is a novel candidate biomarker expressed by cancer‐associated fibroblasts in epithelial ovarian cancer

Most solid tumors contain cancer‐associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF‐like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF‐specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n = 145) but not in the stroma of normal ovaries (n = 4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n = 8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC.

Abstract 1415: Stromal and epithelial interactions in three dimensional models of epithelial ovarian cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC In vitro cell biology models of disease are a fundamental tool for the understanding of disease development in vivo. For cancer, two dimensional (2D) monolayer cell cultures have been used to establish in vitro models. However, it has become increasingly evident that these models fail to address important characteristics of tumours in vivo. The three dimensional (3D) architecture and the resulting interactions with the tumour microenvironment cannot be recreated in standard 2D cultures. We have shown that homotypic 3D models of normal ovarian surface epithelial (OSE) cells and epithelial ovarian cancers (EOCs) represent good models of tissues in vivo. However, heterotypic models including two or more cell types are likely to be even more relevant. Therefore we have created 3D models of ovarian stromal and epithelial cell interactions for EOC and the normal ovary. We established an immortalised ovarian fibroblast cell line from a normal ovary to represent the microenvironment in normal ovarian tissues. To recreate the stroma of a normal ovary in a postmenopausal woman we induced senescence in the normal ovarian fibroblasts (NOFs). Morphological and histocytochemical examination of spheroids with senescent ovarian fibroblasts (SOFs) showed increased numbers of mitosis and staining for Mib-1, suggesting that SOFs can promote epithelial cell proliferation. To model the stroma of a malignant tumour we used mesenchymal stem cells (MSC) which can differentiate into cancer associated fibroblasts (CAFs) after being recruited into the tumour stroma. We induced differentiation of MSC into a ‘CAF -like’ phenotype using conditioned medium from EOC cell line cultures. Differentiation of MSCs was evident one week after conditioning and was verified by staining for the CAF markers aSMA, FSP, Vimentin and FAP. Stromal and epithelial cells were labelled using eGFP and the far red fluorescent protein mKate2 respectively, to enable discrimination between both cell types. Heterotypic 3D cultures were set-up by co-culturing the stromal cells with transformed ovarian epithelial cells using polyHEMA coated tissue culture plastics. Fluorescent labelling has enabled us to study the 3D invasive properties and the influence of the changing microenvironment on the invasive ability of transformed epithelial cells. In conclusion, we have established 3D spheroid models of stromal and epithelial interactions representing both normal ovarian tissues and EOCs. These models will serve as valuable reagents in our understanding of stromal-epithelial interactions and the development of ovarian cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1415.