The Study Of Epithelial Ovarian Cancer Tumoregenesis And Early Detection

BackgroundOvarian cancer has the highest mortality of all the gynecologic malignancies worldwide. With no adequate screening tests, detection at an early stage remains the most significant prognostic factor. There are no gold standard screening methods for the early detection of ovarian cancer exist. Approximately 85% of patients with ovarian cancer are diagnosed at a late stage of the dieases. Epithelial ovarian cancer (EOC), the most common subgroup of ovarian cancer, is the deadliest gynecological cancer, accounting for more deaths than all other gynecological cancers combined. The high mortality rate for EOC is a result of technical obstacles to early detection of the disease and a high prevalence of distal metastasis at late stages of the disease, seventy percent of the cases occur distal metastasis. It is important for enlonging the survival time to have the early detection. However,the explaination of the etiology of epithelial ovarian cancer, is still doubted. The development of specific histotypes of human ovarian cancer is thought to be influenced by multiple factors, including genetic and epigenetic changes in epithelial cells.HER2/neu gene is an oncogene, which encodes a 185 kd trans-membrane protein partially homologous to epidermal growth factor receptor with intrinsic tyrosine kinase activity. This oncogene has been studied mainly in breast cancer where it has prognostic, predictive and therapeutic target value. The expression of HER2/neu in epithelial ovarian cancer has been less studied. Amplication of this gene has been found in 25-30% breast and ovarian cancer and correlate with poor prognosis. HER2/neu has been shown to be involved in the progression of ovarian cancer through multiple mechanisms. High expression of HER2/neu is associated with advanced cancer stages, higher frequency of surgical residual larger tumor, poor histological grade, higher recurrence frequency, shorter survival time and lower sensitivity to platinumbased chemotherapy.In this study, we generated T29Nt and T80Nt cell models using genetic modification of human ovarian surface epithelial cell lines in combination with a peritoneal microenvironment to describe the development of a novel murine model of high-grade papillary serous carcinoma. This murine model should be useful for studying the mechanisms involved in the development and histologic differentiation of human ovarian carcinoma. It may also help us to generate other ovarian epithelial cancer models with additional or different genetic modifications. This model should be helpful for defining pathways and identifying novel targets to improve the treatment and early detection of ovarian cancer. ObjectiveTo harvest the retrovirus-containing HER2/neu and infect two ovarian epithelial cell lines, T29 and T80, and select the cells. To design the resulting cells overexpressing HER2/neu as T29N and T80N cells. To observe the characters of theT29N and T80N cell lines and test if HER2/neu overexpression can increase the number of anchorage independent colonies that grew in soft agar in both the T29N and T80N cell lines.Methods1. HER2/neu was transfected into the Phoenix amphotropic cell line, and the retrovirus-containing HER2/neu cells were harvested.2. After a 24-h recovery from virus infection, the cells were selected by growing them in a medium containing neomycin then grown in the medium without neomycin,and obtained the T29N and T80N cells.3. Observe the T29, T29Nt, T80 and T80Nt cells by phase-contrast microscopy to compare the morphologic characteristics.4. HER2/neu expression was detected using Western blot analysis.5. We use Anchorage-independent cell growth assay to compare the cells growth character in vitro. Cells were suspended in 2 mL of growth medium with 0.35% agarose (Invitrogen), and the suspension was placed on 5 mL of solidified 0.7% agarose. Triplicate cultures of each cell type were maintained for 14 days at 37℃in a 5% CO2 atmosphere, and fresh medium was added at 7 days. The number of colonies that were larger than 50μm in diameter was counted on the 14th day. These experiments were repeated twice.Results1. We Harvested the retrovirus-containing HER2/neu cells were harvested for the experiments.2. We infected the retrovirus-containing HER2/neu into two immortalized nontumorigenic human ovarian surface epithelial cell lines T29 and T80. The cells were selected in medium containing neomycin (1 mg/ml) after recovery from virus infection.3. The resulted cells increased expression of the HER2/neu protein.4. The T29N cells have mesenchymal morphologic characteristics, and the T80N cells have an epithelial phenotype.5. HER2/neu overexpression increased the number of anchorage independent colonies that grew in soft agar in both the T29N and T80N cell lines.Conclusion1. The introduction of HER2/neu into two immortalized nontumorigenic human ovarian surface epithelial cell lines (T29 and T80) resulted in increased expression of the HER2/neu protein.2. The T29N cells showed mesenchymal morphologic characteristics, and the T80N cells have an epithelial phenotype.3. The HER2/neu overexpression T29N and T80N cell lines grew more anchorage independent colonies in soft agar than T29 and T80 cell lines. ObjectiveMice subcutaneously or intraperitoneally injected with T29, T80, T29N, T80N cells to compare the tumor development abilities of these defferent cell lines. To observe the characters of theT29Nt and T80Nt cell lines. Mice subcutaneously or intraperitoneally injected with T29Nt, T80Nt cells to compare the tumor development abilities of these defferent cell lines. To do the histopathologic examination and immunohistochemical analysis with the tumors grown.Methods1. Harvested, washed 5 x 10^6 cells from T29, T80, T29N, T80N cell lines twice and resuspended in 0.1 mL of saline. The T29, T80 and the T29-and T80-control cell suspensions were injected subcutaneously into 4-to 6-week-old BALB/c athymic nude mice. The mice were kept in a pathogen-free environment and checked every 2 days. The date on which grossly visible tumors first appeared and the size of the tumors were recorded. The mice were killed when tumors reached 1.5 cm in diameter. T29Nt and T80Nt cells were isolated from the T29N-and T80N-derived xenografts and cultured for the further experiments.2. Harvested, washed 5 x 10^6 cells from T29, T80, T29N, T80N cell lines twice and resuspended in 0.1 mL of saline. The T29, T80 and the T29-and T80-control cell suspensions were injected intro-peritoneally into 4-to 6-week-old BALB/c athymic nude mice. The mice were kept in a pathogen-free environment and checked every 2 days. The mice were observed for increased abdominal size, lethargy and jaundice and were killed when these signs occurred.3. Harvested, washed 5 x 10^6 cells from T29Nt, T80Nt cell lines twice and resuspended in 0.1 mL of saline. Each cell line was injected subcutaneously and introperitoneally into six 4-to 6-week-old BALB/c athymic nude mice individually. The mice were kept in a pathogen-free environment and checked every 2 days for 6 months. The date on which grossly visible tumors first appeared and the size of the tumors were recorded. The mice were killed when tumors reached 1.5 cm in diameter. The mice given intraperitoneal injections were observed for increased abdominal size, lethargy and jaundice and were killed when these signs occurred. Tumors from mice were fixed in 10% formalin and stained with hematoxylin and eosin. The histopathologic characteristics were examined by microscopy.4. The following antibodies were used for immunohistochemical analysis: monoclonal anti-HER2/neu, monoclonal anti-SV40 T antigen, monoclonal anti-p53 antigen, monoclonal anti-pan cytokeratin antigen, and mouse monoclonal anti-CA125, anti-WT-1 antigen. Secondary antibodies against each host were biotinylated.Results1. Four of the nine mice injected with the T29N cells developed subcutaneous tumors. Nine of the fourteen mice subcutaneously injected with the T80N cells developed subcutaneous tumors. Histopathologic examination of the tumors showed undifferentiated carcinomas.2. There were no tumors revealed in other mice either injected subcutaneously or intraperitoneally.3. The T29Nt and T80Nt both had an epithelial phenotype. Three of the six mice subcutaneously injected with the T29Nt and T80Nt cell lines respectively developed subcutaneous tumors. Histopathologic examination of these tumors revealed undifferentiated carcinomas. Three of the ten mice injected intraperitoneally with T29Nt and five of the nine mice injected with T80Nt developed tumors. Intraperitoneal inoculation of mice with T29Nt cells resulted in extensive tumor growth along the omentum. Interestingly, histologic examination revealed papillary carcinoma that was indistinguishable from human papillary serous ovarian carcinoma. These tumors also invaded the liver and pancreas. Tumors derived from the T80Nt cells grew along the peritoneal fat and the pancreas but remained undifferentiated.4. Immunohistochemical staining for large T antigen was positive, demonstrating that the tumors were derived from the immortalized T29 and T80 cells rather than having developed spontaneously in the mice. The tumor cells were also positive for cytokeratin, verifying their epithelial cell origin, and positive for p53, demonstrating that their p53 stability was increased and its function was disabled due to its binding by T antigen. The cells were also positive for CA125, WT-1, which are common markers expressed in human ovarian cancer.Conclusion1. We successfully isolated T29N-and T80N-derived xenografts and named them T29Nt and T80Nt, which have epithelial phenotype.2. we also found that the peritoneal microenvironment and the genetic background of the cells play a critical role in the differentiation of papillary carcinoma, genetic modifications alone are not sufficient for development of this carcinoma; rather, a specific ovarian epithelial cell type and an appropriate tumor microenvironment are required.3. HER2/neu can lead to papillary differentiation of immortalized human ovarian epithelial cells.