Identification And Characterization Of Ovarian Cancer-Initiating Cells And Their Role In The Chemoresistance

Background and ObjectiveOvarian cancer is the most lethal malignancy of the female reproductive system. Greater than 70% of ovarian cancers were diagnosed in late stage and had broad metastasis. Whereas standard therapy, cytoreductive surgery followed by cisplatin, results in complete response in 80% of patients,20% will relapse within 6 months with chemoresistance disease and bad prognosis. The 5-year survival rate is about 30% and the mortality is the highest in all malignant gynecologist tumors. Thus, improved targeted therapies and chemosensitization strategies to cisplatin are essential for reducing the mortality of this devastating malignancy.Recently, with the development of the theory about cancer-initiating cells (CICs) in the solid tumors, most researchers realized that in most cancers, the initial source is possibly CICs, also named cancer stem cells (CSCs). They are self-renewal, and can generate a caricature of the tissue from which they derive. They are the reason of cancer metastasis and recurrence. CICs have been identified in many kinds of tumors including melanoma, lung, and prostate and so on, and the identification methods have developed at the same time, but no one method was agreed by all researchers. In solid tumors, only the markers CD24-CD44+in breast cancers were considered as the precise ones. There are still many controversies about the CIC markers in other tumors. As for ovarian cancer, the public reports are not too many. Besides, they come from different research teams and lack of latter more precise data. In this sense, ovarian CICs (OCICs), which play an important role in the recurrence of ovarian cancers, should be paid more attention to. In this study, we will use all the reported methods that can be used to identify OCICs, including immunophenotype, side population and serum-free medium. We will characterize and verify the OCICs isolated by all methods, and their specific targeting is highly valuable for therapy of recurrent, chemoresistant disease.Methods1. The methods for identifying CICs:(1) Immunophenotype, (2)Side population phenotype, (3)Spheroid by serum-free medium.2. The characterization of OCICs:(1)The expression detection of stem cell markers in ovarian cancer cells and the isolated OCICs by RT-PCR.(2)The best serum-free medium for OCICs spheroids formation.(3) How to transfer the spheroids to the next generation. (4) The cell cycle detection by Flow CytoMeter (FCM). (5) In vitro migration and invasion assays by transwell chamber. (6) In vivo xenograft experiments for the tumorigenesis and metastasis.3. The relationship of OCICs and chemoresistance:(1) the change of side population BY FCM and the drug resistance-related protein by RT-PCR when the ovarian cancer cells were induced by different doses of cisplatin; (2) The apoptosis ratio changes to cisplatin by AnnexinV-PI of FCM.Results1. The several ways for isolating OCICs have different effects. As for immunophenotype, we could detect just CD44+subpopulation but no CD117+ and CD133+ one. By serum-free medium, OCICs could be isolated but they were not stable for further and long time study. Side population phenotype was a better way than the former two to isolate OCICs, but its ratio is low and it was different to get enough numbers of OCICs. In our study we chose side population phenotype to get OCICs. 2. Side population was expressed stem cell marker, such as Oct4, Sox2, and Bim1 et al in higher level than non-SP. It also has stronger spheroid formation and metastasis ability. In cell cycle assays, the G0 arrest was found in SP cells. In vivo,500 SP cells could form xenografts in NOD/SCID mice, and it is 100 times as the non-SP cells. Besides, the mice of injected SP cells have higher mortality and metastasis ability than the ones of non-SP. Lung surface is the metastasis place.3. The side population is in direct proportion to the chemoresistant extent. The ratio of side population, the G0 arrest and the expression of drug resistance-related protein increased after the ovarian cancer cells were induced by cisplatin. Meanwhile, the sensitivity of SP cells to cisplatin is lower than non-SP cells. SP cells are high drug resistant.Conclusion1. There are OCICs in ovarian cancer cells. CD44 will be one of the CIC markers but the CD117 and CD133 should be further confirmed.2. Side population phenotype is one of the best ways to isolate OCICs, but only a small numbers of the OCICs can be available.3. The side population shows stem-cell like characters and high tumorigenic, and it is just the enrichment of OCICs.4. The side population was high resistant to cisplatin in vitro and it may play an important part in the formation of chemoresistance of ovarian cancers.