Ovarian normal and tumor-associated fibroblasts retain in vivo stromal characteristics in a 3-D matrix-dependent manner

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Abstract

Objective

Due to a lack of experimental systems, little is known about ovarian stroma. Here, we introduce an in vivo-like 3-D system of mesenchymal stromal progression during ovarian tumorigenesis to support the study of stroma permissiveness in human ovarian neoplasias.

Methods

To sort 3-D cultures into ‘normal,’ ‘primed’ and ‘activated’ stromagenic stages, 29 fibroblastic cell lines from 5 ovarian tumor samples (tumor ovarian fibroblasts, TOFs) and 14 cell lines from normal prophylactic oophorectomy samples (normal ovarian fibroblasts, NOFs) were harvested and characterized for their morphological, biochemical and 3-D culture features.

Results

Under 2-D conditions, cells displayed three distinct morphologies: spread, spindle, and intermediate. We found that spread and spindle cells have similar levels of α-SMA, a desmoplastic marker, and consistent ratios of pFAKY397/totalFAK. In 3-D intermediate cultures, α-SMA levels were virtually undetectable while pFAKY397/totalFAK ratios were low. In addition, we used confocal microscopy to assess in vivo-like extracellular matrix topography, nuclei morphology and α-SMA features in the 3-D cultures. We found that all NOFs presented ‘normal’ characteristics, while TOFs presented both ‘primed’ and ‘activated’ features. Moreover, immunohistochemistry analyses confirmed that the 3-D matrix-dependent characteristics are reminiscent of those observed in in vivo stromal counterparts.

Conclusions

We conclude that primary human ovarian fibroblasts maintain in vivo-like (staged) stromal characteristics in a 3-D matrix-dependent manner. Therefore, our stromal 3-D system offers a tool that can enhance the understanding of both stromal progression and stroma-induced ovarian tumorigenesis. In the future, this system could also be used to develop ovarian stroma-targeted therapies.

Section snippets

Background

Ovarian cancer is the most lethal among gynecological malignancies in the United States. About 22,430 new cases and 15,280 deaths from ovarian cancer were estimated for 2007 [1]. More than 70% of patients with epithelial ovarian cancer are diagnosed at an advanced stage when the disease has already spread throughout the peritoneal cavity. Although there has been an improvement in the five-year survival rate for patients diagnosed with advanced, stage III disease, the long-term survival rate is

Antibodies

Mouse anti-α-SMA (1:4000, Western blot; 1:300, immunofluorescence; 1:200, immunohistochemistry) and rabbit anti-human fibronectin (1:100, immunofluorescence) were obtained from Sigma-Aldrich (St. Louis, MO), mouse anti-total FAK (1:1000, Western blot) from Millipore (Billerica, MA) and rabbit anti-pFAKY397 (1:500, Western blot; 1:50, immunohistochemistry) from Invitrogen (Carlsbad, CA). Rabbit anti-Vimentin (1:3000, Western blot) was obtained from Biovision (Mountain View, CA), mouse anti-pan

Primary ovarian fibroblasts cultured under 2-D conditions, present three distinct morphological phenotypes

Table 1 lists 14 normal ovarian fibroblast (NOF) cell lines derived from 14 postmenopausal non-cancerous ovaries (from patients aged 55.7 +/− 8 years) that were incidentally resected during laparotomies from patients with high risks of developing ovarian cancers (i.e., based on BRCA1/2 mutational status). In addition, Table 1 also lists 29 tumor ovarian fibroblast (TOF) cell lines from 5 distinct surgical ovarian tumor samples (from patients aged 57.4 +/− 17 years), which were harvested as

Discussion

It is increasingly recognized that interactions between cancer cells and their surrounding stroma are critical for promoting the growth and invasiveness of tumors [10], [11], [61], [62], [63]. For example, cancer cells alter the topography and molecular composition of stromal ECM by paracrine regulation of fibroblastic stromal cells [10,11,64] during early tumor development. These physical and biochemical alterations of the stroma, in turn, profoundly affect the properties of the cancer cells

Conflict of interest statement

No conflict of interest.

Acknowledgments

We thank Dr. A. Klein-Szanto for assistance in pathological observations of immunohistochemistry assays, Ms. C. M. Slater for her expertise in tissue culture, Mrs. O. Villamar for assistance on immunohistochemistry techniques, Drs. R. Castelló-Cros and G. P. Adams, for critical comments, Ms. R. Roth for technical assistance and Mrs. K. Buchheit for proofreading. This study was assisted by the following Fox Chase Cancer Center facilities: Biosample Repository, Histopathology Facility, Cell

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