Female Lewis strain rats aged 4-6 weeks (purchased from Charles River Breeding Laboratories, Inc., Wilmington, MA) were kept in sterile micro-isolator cages and fed food and water ad libitum. The institutional guidelines of Johns Hopkins University concerning the care and use of research animals were followed. The animals were challenged intraperitoneally with graded numbers of FNAR cells and monitored daily for abdominal swelling. At various intervals after tumor challenge or when animals appeared moribund (pallor, lethargy, and marked abdominal distension), the animals were sacrificed by CO₂ asphyxiation and the cells within the peritoneal cavity harvested by flushing the abdomen with 35 milliliters of sterile phosphate buffered saline (PBS, Grand Island Biological Co., Gibco BRL, Grand Island, NY). At sacrifice, the animals were examined for tumor growth and tissues taken for histological examination.

A cell line (FNAR) that grows in vitro as an adherent monolayer was established by culture in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum in 30 ml tissue culture flasks (Corning Flask 3056, Corning Inc., Corning NY). Cells used for experiments were low passage and maintained in culture for one to three months. The doubling time of the cell line was measured by plating 10⁴ cells into macrotiter wells then harvesting and counting at 19.5, 43.5, and 115.5 hours.

Flow cytometry was utilized to assess in vitro FNAR cells for expression of known phenotypic markers. Briefly, 5 × 10⁵ tumor cells were incubated in polystyrene tubes. Analysis of the intracellular antigens estrogen receptor α, progesterone receptor, and androgen receptor first required fixation in 2% formaldehyde (Polysciences, Warrington, PA) in phosphate buffered saline (PBS, Gibco Invitrogen, Carlsbad, CA) for 15 minutes at 4°C followed by permeabilization with 0.1% Triton-X-100 (Sigma-Aldrich, St. Louis, MO) in PBS for 15 minutes at 4°C. The cells were then incubated for 30 minutes at 4°C with commercially purchased murine monoclonal antibodies. The concentrations of antibodies used are as follows: estrogen receptor (ER) α at 8 μg/10⁶ cells (Abcam, Cambridge, MA), progesterone receptor (PR) at 16 μg/10⁶ cells (Affinity Bioreagents, Golden, CO), or androgen receptor (AR) at 2 μg/10⁶ cells (Pharmingen, San Diego, CA). The cells were washed and counterstained with phycoerythrin (PE) rat anti-mouse IgG₁ (Becton Dickinson, San Jose, CA) at 125 ng/10⁶ cells for 30 minutes at 4°C. Commercially purchased murine monoclonal antibody to the rat c-neu oncogene product (Calbiochem, San Diego, CA) was used at 1 μg/10⁶ cells and was counterstained with PE rat anti-mouse IgG_2a+b (Becton Dickinson, San Jose, CA) at 30 ng/10⁶ cells for 30 minutes at 4°C. Tumor cells incubated with secondary antibody alone served as a negative control. Epithelial cell adhesion molecule (EPCAM) expression was analyzed using a PE-conjugated antibody (Santa Cruz, Santa Cruz, CA) at 1 μg/10⁶ cells with mouse IgG₁-PE as a negative control (Becton Dickinson, San Jose, CA). A commercially available rabbit polyclonal antibody to CA125 (Abbiotec, San Diego, CA) was used at 2 μg/10⁶ cells and counterstained with 1 μg/10⁶ cells APC goat anti-rabbit IgG (Invitrogen Molecular Probes, Carlsbad, CA). The cells were analyzed on a Becton-Dickinson FACSCalibur flow cytometer and data was analyzed using FlowJo (Tree Star, Inc, Ashland, OR).

FNAR cells were plated onto four-well CultureSlides (BD Falcon, San Jose, CA). Cells were fixed in 2% formaldehyde in PBS for 20 minutes followed by permeabilization in 0.5% Triton X-100 in PBS for 10 minutes. Cells were then incubated with a mouse monoclonal antibody to beta-catenin conjugated to Cy3 (Abcam, Cambridge, MA) at 6 μg/ml for one hour and counterstained with 500 ng/ml DAPI for five minutes (Invitrogen Molecular Probes, Carlsbad, CA). Images were captured using the Nikon Eclipse E800 (Tokyo, Japan) at 200× magnification with standard filters for DAPI and Cy3, the DS-QiMc digital camera (Nikon, Tokyo, Japan), and the Advanced Research Elements AR 3.0 software (Nikon, Tokyo, Japan).

RNA was extracted and purified from cell lysates of 1-5 × 10⁵ in vitro FNAR tumor cells and the REH cell line of normal rat endothelial cells, as a control, with 500 μl Trizol reagent (Invitrogen, Carlsbad, CA). Tissue samples were frozen in liquid nitrogen and pulverized with a mortar and pestle. The powder was dissolved in Trizol and centrifuged. Purified RNA was dissolved in 20 μl diethyl-pyrocarbonate-treated distilled water. The resulting RNA was analyzed at the Johns Hopkins microarray core. RNA from control and experimental samples was processed using the RNA amplification protocol described by Affymetrix (Affymetrix Expression Manual). Briefly, 5 μg of total RNA was used to synthesize first strand cDNA using the SuperScript Choice System (Invitrogen, Carlsbad, California) and oligonucleotide primers with 24 oligo-dT plus the T7 promoter (Proligo LLC, Boulder, Colorado). Following the double stranded cDNA synthesis, the product was purified by phenol-chloroform extraction and biotinilated anti-sense cRNA was generated through in vitro transcription using the BioArray RNA High Yield Transcript Labeling kit (ENZO Life Sciences Inc., Farmingdale, New York). Fifteen μg of the biotinilated cRNA was fragmented at 94°C for 35 minutes in buffer (100 mM Tris-acetate, pH 8.2, 500 mM potassium acetate, and 150 mM magnesium acetate), and 10 μg of total fragmented cRNA was hybridized to the Affymetrix GeneChip rat 230 2.0 array (Santa Clara, CA) for 16 hours at 45°C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was then used to wash and stain the chips with a streptavidin-phycoerythrin conjugate. The staining was then amplified as follows: blocking was performed using goat IgG, then a biotinilated anti-streptavidin antibody (goat) was bound to the initial staining, and amplification was completed by the addition of a streptavidin-phycoerythrin conjugate. Fluorescence was detected using the Affymetrix 3000 7G GeneArray Scanner and image analysis of each GeneChip was done through the GeneChip Operating System 1.4.0 (GCOS) software from Affymetrix using the standard default settings. For comparison between different chips, global scaling was used to scale all probesets to a user defined target intensity (TGT) of 150.

Quantitative RT-PCR (Taqman, Applied Biosystems, ABI, Foster City, CA) was utilized to assess levels of cytokine mRNA transcripts of in vitro FNAR cells as previously described 20. The oligonucleotide primers and fluoresceinated probes for the cytokine genes (IL-6, IL-12, and IL-18), ER, PR, and stathmin were purchased from ABI. Data were analyzed in real-time with Sequencer Detection version 1.6 software, with the results normalized against mRNA transcripts for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GADPH).

Examination of a normal female Lewis rat sacrificed for harvesting normal splenic T cells showed a spontaneously occurring tumor (approximately 0.5 cm³) derived from the left ovary and attached to and invading the abdominal wall (Figure 1A). In addition, tumor studding was observed at several sites on the wall of the peritoneum, and ascites was present. Histologic evaluation revealed an epithelial neoplasm with features most consistent with an adenocarcinoma (Figure 1B). The tumor was composed of nests displaying admixed cribriform and solid architecture. The tumor cells had modest amounts of amphophilic/eosinophilic cytoplasm and relatively uniform, moderately atypical oval nuclei that were predominantly vesicular to modestly hyperchromatic with small nucleoli. Occasional mitotic figures and apoptotic bodies were noted, as was focal necrosis. Based on analogy to human ovarian epithelial tumors, this tumor most closely resembled a moderately differentiated endometrioid carcinoma (a cribriform variant of that subtype, with cells being less columnar than the classical human endometrioid carcinoma), with disease distribution paralleling a typical high-stage (human FIGO stage IIIB) ovarian carcinoma. Lymphocyte infiltration into the tumor mass was minimal at best, although numerous lymphocytes were present in the peritoneal fluid. The tumor was excised and pushed through a 100 micron wire mesh screen to obtain a single cell suspension.

Normal Lewis rats were given either intraperitoneal (IP) or subcutaneous injection of graded numbers (5 × 10⁴, 1 × 10⁵, 5 × 10⁵, or 1 × 10⁶) of tumor cells. The animals were monitored daily for overall general health as well as degree of abdominal extension. The tumor repeatedly failed to grow subcutaneously, even with the administration of systemic immunosuppression (Cyclosporine, 10 mg/kg/d) or passage into thymectomized animals. However, all rats became moribund at 150-160 days after IP injection with 5 × 10⁵ or 1 × 10⁶ cells (Table 1). Rats injected with 1 × 10⁵ cells became moribund around 175 days. Rats receiving IP injections of 5 × 10⁴ cells generally did not appear ill by 6 months, but tumor cells were detected in the peritoneal cavity when sacrificed on day 175. Tumor growth recapitulated that seen in the initial rat with IP tumoral masses adhering to all of the visceral organs and the abdominal wall. Histologically, the tumors appeared to be of epithelial origin. Affected rats also showed enlargement of the ovaries and fallopian tubes, with a marked increase in vascularization. Successful serial passage was conducted by IP challenge with 1 × 10⁵ tumor cells harvested by flushing of the peritoneal cavity.

The doubling time of the FNAR cell line was measured by plating 10⁴ cells into macrotiter wells then harvesting and counting at 19.5, 43.5, and 115.5 hours (Figure 2). The slope of the line of log number of tumor cells versus hours estimates a doubling time of 22.9 hours.

ER is detected in 60-90% of ovarian carcinomas 2122232425, 25-50% express PR 2123242526, and 45% expressed both 2325. AR is expressed in 50-70% of ovarian carcinomas 2426. Accordingly, in the appropriate clinical and pathologic setting, sex hormone receptor expression is characteristic of ovarian carcinoma 2527. The tumor expressed ER, PR, and AR by flow cytometry (Figure 3A-C), with ER and PR confirmed by PCR (data not shown). The tumor also expressed her-2/neu (Figure 3D), which is expressed in 25-35% of ovarian carcinomas 2829. The epithelial origin of this carcinoma was confirmed by its expression of EPCAM (Figure 3E). Consistent with previous reports of endometrioid carcinoma, FNAR cells display cell-surface expression of CA125 (MUC16, data not shown) 30. FNAR cells also show nuclear staining of β-catenin (Figure 4), which is strongly associated with the endometrioid subtype 31.

Gene expression profiling demonstrated that FNAR gene expression was similar to that reported for human ovarian carcinoma (Table 2). Metallothioneins are generally not found at immunohistochemically detectable levels in normal cells, but their expression increases in ovarian carcinoma with increasing grade 323334. Metallothionein I was overexpressed 11.38-fold in FNAR cells when compared to endothelial cells, and metallothionein II showed 3.56-fold increased expression. Thioredoxin expression correlates with cis-diaminedichloroplatinum resistance 35 and is expressed in FNAR cells 3.07-fold higher than in endothelial cells. Stathmin regulates microtubules during the formation of the mitotic spindle and is not expressed at detectable levels in normal cells; however, high-level expression is generally seen in ovarian carcinoma 363738. Accordingly, stathmin expression was 3.23-fold higher in FNAR cells than in endothelial cells. This data was confirmed by PCR (data not shown). A nuclear factor that it is involved in cell cycle progression, b-myb, is also highly expressed in both FNAR cells (3.33-fold) and human ovarian carcinoma 39.

High levels of interleukin-6 (IL-6), a proinflammatory cytokine and hematopoietic growth factor, are found in both normal ovarian epithelium and human ovarian carcinoma 4041. Interleukin-18 (IL-18) is a proinflammatory cytokine that stimulates interferon-γ production. Ovarian carcinoma expresses IL-18, but it is predominantly the pro-IL-18 form 42. Interleukin-12 (IL-12) is a cytokine that encourages a T_h1 immune response. IL-12 has been detected in ascites fluid and serum of ovarian cancer patients 43, although no reports have examined the expression of IL-12 by the ovarian carcinoma cells themselves. Expression of all three cytokines by FNAR cells was detected by real time RT-PCR (Figure 5).