Enhanced efficacy and specificity of epithelial ovarian carcinogenesis by embedding a DMBA-coated cloth strip in the ovary of rat

Five-week-old female Wistar rats (purchased from the Experimental Animal Center, Chinese Academy of Science, SYXK (shanghai) 2004–0011) were housed five per cage and acclimated to the animal room for one wk before surgery and received food and tap water ad libitum. The room was controlled for a constant temperature (22 ± 2°C) and relative humidity (50 ± 20%) with a 12 h light/dark cycle. 7,12-dimethylbenz(a)anthracene (DMBA) (Sigma Chemical Co, St Louis, MO) was heated to 124°C, which is the fusion point of the carcinogen. A central portion of 3–0 silk suture or a piece of cloth strip (0.5 cm × 0.5 cm) was immersed in the melted DMBA. The sutures contained approximately 1 mg per centimeter of the carcinogen on average and the cloth strip contained approximately 1.5 mg of the carcinogen on average, as calibrated by a micro-chemical balance. Rats were anesthetized by intraperitoneal injection of chloral hydrate (Shanghai No.1 Biochemical & Pharmaceutical Co, Shanghai, China) at 50 mg/kg. Then both ovaries were surgically exposed, and a DMBA-coated cloth strip (CS group, 128 rats) or a DMBA-coated suture, as a control group (S group, 80 rats) was then packed or inserted into both ovaries and closed with the surround fatty substance (Figure 1A and 1B). An antibiotic (10⁵ units of benzylpenicillin potassium) was administered intraperitoneally for prophylaxis against infection before the abdominal wall was closed. Tumor number, size and volume were determined weekly after the first operation in both groups by palpating the abdominal wall of rat.

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Science and Technology Committee. All protocols were approved by the Biomedical Research Ethics Committee of Shanghai Institute for Biological Science of Chinese Academy of Sciences. Every effort was made to minimize suffering.

All rats were euthanized by CO₂ inhalation followed by cervical dislocation, necropsied and examined for gross abnormalities. Pathologically altered organs, entire reproductive tracts and representative specimens of multiple organs and tissues, including the brain, lung, liver, kidney, spleen, pancreas and intestine were removed at necropsy, fixed in 10% (v/v) neutral buffered formalin overnight, transferred to 70% ethanol and paraffin-embedded. In rats with evident tumor, specimens of the tumor tissue were also excised, snap frozen in liquid N2 and stored at −80°C. For histological analysis, 5 μm formalin fixed paraffin embedded tissue sections were cut for hematoxylin and eosin stain (H&E staining). Histopathological analysis was performed by a pathologist with expertise in human and murine malignancies. The tumors were subtyped according to the histologic characteristics of neoplastic cells (Table 1) and the results of immunostain.

Paraffin-embedded sections for immunohistochemistry were dewaxed, rehydrated and processed for antigen unmasking by heating to near boiling in citrate buffer (0.01 M, pH 6.0, 30 min) in a microwave oven, then allowed to cool for 20 min. Following this and all subsequent incubations except for the blocking serum, the slides were washed in phosphate-buffered saline (PBS, 3 × 5 min). Endogenous peroxidase activity was blocked by hydrogen peroxide incubation (0.3% in methanol) for 5 min. After incubation for 30 min in blocking serum (diluted 1:50 in PBS) appropriate to each secondary antibody, the slides were incubated with primary antibody at 4°C overnight (Primary antibodies were omitted as negative controls.) The primary antibodies used were as follows: polyclonal anti-rat Pan-CK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal anti-rat inhibin A (Santa Cruz Biotechnology, Santa Cruz, CA, USA),and polyclonal anti-rat vimentin (Abcam, ). Detection of primary antibody proceeded with the binding of biotinylated secondary antibody (diluted 1:200 for 10 min at room temperature) followed by avidin-biotin peroxidase complex (Vectastain Elite, Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB) chromagen substrate. Dark brown structures indicated positive immunostaining.

The comparison of distribution of variables between two groups was made using the t-test (SPSS Statistics v13.0, Chicago, IL), with significance set at a P value of less than 0.05.

Thirty-two wk post DMBA treatment, rats in CS group had developed significantly more advanced tumors (larger ovarian tumors) compared with that of S group. On an average of 32 wk post DMBA treatment, 75% of the rats in CS group (96/128) had developed tumors; whereas only 46.25% (37/80) of the rats in the S group had developed tumors (P < 0.001). Tumors in both groups were mainly solid in structure, although a few tumors had a cystic cavity containing an abscess; some were intraperitoneal spread or seeding of tumor, with or without ascites. As shown in Figure 2.

Morphologically, rats with tumors had an enlarged abdomen. The tumors presented as large masses with reddish color from the ovaries on both sides, and aggressively invaded surrounding organs including the spleen, intestine, kidney, and uterus, including some with bloody ascites. The mean greatest dimension of the tumors in CS group (3.63 ± 0.89 cm) was larger than that of S group (2.44 ± 1.89 cm) (*P < 0.05) (Figure 3).

The majority of tumors in both groups were adenocarcinoma. The adenocarcinoma could be categorized as a mixed carcinoma, in which the lining epithelial cells were composed of flattened or cuboidal cells resembling ovarian surface epithelium, hobnail cells, and columnar cells, often with pseudostratified nuclei. Histologically, 93.75% (90/96) of the ovarian tumors in CS group are adenocarcinomas; the remaining tumors (6/96) displayed typical features of ovarian sarcomas (Figure 4A). While in S group, the histological type of induced tumor included: adenocarcinoma (21/37), squamous carcinoma (3/37), granulosa cell tumor (3/37), sarcoma (4/37), undifferentiated carcinoma with no adeno character (2/37), benign ovarian tumor (2/37), and malignant teratoma (1/37) (Figure 4B). Representative and typical tumor samples are shown as adenocarcinoma, sarcoma, squamous carcinoma, and granulosa cell tumor in Figure 5.

To confirm histologic observations, we employed a number of immunohistochemical reagents for epithelial, grunulosa or mesenchymal cell types, such as pan-CK, vimentin and inhibin A (Figure 6). In adenocarcinoma, pan-CK staining of epithelial cells was most often diffuse but also appeared as localized to a luminal boundary. Epithelial cell staining of vimentin appeared as perinuclear, polar, or was absent from epithelial cells in more differentiated ductal structures and present only in the surrounding stroma. Inhibin A was strongly expressed in adenocarcinoma and weekly in sarcoma. There was no staining in the sarcoma cells for pan-CK while vimentin were positive.