Isolation And Identification Of Cancer Stem Cells From Human Ovarian Carcinoma And Screening Of Its Specific MicroRNAs

BackgroundCancer stem cells (CSCs) play an important role in the recurrence and drug resistance of cancer. Isolation and characterization of cancer stem cell from ovarian cancer samples may help to provide novel diagnostic and therapeutic targets in management of recurrent disease and drug resistance in ovarian cancer. microRNA is related to tumorigeness of various cancer types, many microRNAs can regulate self-renewal and differentiation ability of stem cells. Identification of specific microRNAs of ovarian cancer stem cells may help to kill ovarian cancer stem cells.This study was undertaken to identify ovarian cancer stem cells and to screen its specific microRNAs.Methods1. We developed a xenograft model in which cells from 14 samples of human ovarian serous adenocarcinoma tissue or ascites were implanted in immunodeficient mice to test the tumorigenic potential of different populations of ovarian cancer cells. CD117+Lineage-ovarian cancer cells were identified and isolated by flow cytometry. The characteristics of CD117+Lineage-ovarian cancer cells were investigated. Immunohistochemistry analysis of 25 patients with advanced ovarian serous adenocarcinoma was conducted to analyze the distribution of CD117-positive cases according to the clinical response to chemotherapy. 2. We examined the SP phenotype in six human ovarian cancer cell lines (HO-8910, OVCAR-3,3A0, ES-2, A2780 and SK-OV-3) and three xenograft tumors by staining them with Hoechst 33342 dye. SP and non-SP (NSP) cells were isolated from HO-8910, OVCAR-3,3A0, and one xenograft tumor using flow cytometry. Self-renewal, differentiation, and tumorigenic ability of SP and NSP cells were compared in vitro and in vivo. The mRNA expressions of"stemness"markers (0ct4, Klf4, and Nanog) and ATP-binding cassette (ABC) transporters (ABCG2, ABCB1, and ABCC2) were measured using a real-time PCR assay. The response of the SP and NSP cells to Taxol, Cisplatin, Mitoxantrone, and Doxorubicin was investigated using a drug sensitivity assay.3. miRNA was isolated from HO-8910 SP and NSP cell. Hybridization was carried Out on Affymetrix GeneChip miRNA 2.0 Arrays. We compared miRNA expression profiles between SP and NSP cells. Screening of specific miRNA which can regulate self-renewal and multiple drug resistant ability was undertaken using reverse screening assay.Results1. As few as 103 cells with the CD117+Lineage-phenotype, which comprise<2% of the xenograft tumor cells, were able to regenerate tumors in a mouse model, a 100-fold increase in tumorigenic potential compared to CD117-Lineage- cells. The tumors that arose from purified CD117+Lineage-cells reproduced the original tumor heterogeneity and could be serially generated, demonstrating the ability to self-renew and to differentiate, two defining properties of stem cells. Furthermore, immunohistochemistry analysis revealed that positive immunostaining for CD117 in 40%(10 of 25) of patients. CD117 expression was statistically correlated with resistance to conventional chemotherapy (P=0.027).2. SP phenotype was detected in HO-8910 (10.81±2.36%), OVCAR-3 (1.22±0.19%),3AO (6.47±0.33) cell lines and three xenograft tumors (0.53±0.3,0.32±0.05, and 0.37±0.12, respectively), but not in ES-2, A2780, and SK-OV-3 cell lines. SP cells have enhanced self-renewal, differentiation, and tumor-initiating capacity compared to NSP cells. The mRNA expression of""stemness" markers and ABC transporters were markedly elevated in SP cells. SP cells also showed resistance to multiple chemotherapeutic drugs and were enriched by Cisplatin treatment. 3.26 miRNAs were found to be differently expressed in HO-8910 SP cells compared with NSP cells.11 miRNAs were upregulated and 15 miRNAs were downregulated in SP cells.11 downregulated miRNAs were related to abilities of self-renewal and multiple drug resistance.Conclusion1. Human ovarian cancer cells with the CD117+ phenotype possess the unique properties of CSCs, including self-renewal, differentiation, a high tumorigenic potential, and chemoresistance. Future studies designed to target CD117+cancer cells may identify more attractive and effective therapies for treatment of ovarian cancer.2. SP is an enriched source of CSCs and a useful tool to investigate tumorigenesis and chemoresistance of ovarian cancer.3. Some specific mirRNAs which were related to abilities of self-renewal and multiple drug resistance were found in HO-8910 SP cells.