The Isolation, Identification, And Chemotherapeutic Sensitivity Of Cancer Stem Cell-like Cells From Human Ovarian Carcinoma

Ovarian cancer is the most lethal malignancy in the female reproductive system. The high percentage of recurrence and chemoresistance is one of the main reasons for its high mortality rate. It has been assumed that a small subset of stem-like cancer cells, named the cancer stem cells (CSCs), constitute a reservoir of self-sustaining cells with the exclusive ability of multi-differentiation, unlimit proliferation and self-renewal to maintain the heterogeneous tumor in the CSCs theory. And CSCs may be the primary source leading to the genesis, development, invasion, metastasis, recurrence, and drug resistance of cancers. CSCs have been identified in hematological system tumors and series of solid tumors including breast, brain and etc. Since the research on ovarian CSCs was relatively late, the exact phenotypes and functional features of ovarian CSCs were still disputable, and the study on the character of drug resistance still needed to be further improved.In current study, we first used the human epithelial ovarian carcinoma cell line 3AO as a model system to isolate, identify cancer stem cell-like cells(CSC-LCs) and ascertain their phenotypes. Second, we investigated the sensitivity of ovarian CSC-LCs and non CSC-LCs to the chemotherapeutic drugs of Paclitaxol and Carboplatin. Finally, CSC-LCs were preliminarily studied in primary cultured ovarian cancer cells from the tissue and ascites samples of advanced adenocarcinoma patient. This study was performed to elucidate the genesis and drug resistance mechanism of ovarian carcinoma in the perspective of CSCs, and to provide some effective experimental evidence to explore the new targets for clinic diagnosis and treatment of ovarian cancer. Part I The isolation and identification of ovarian CSC-LCs in human epithelial ovarian cancer cell line 3AOObjective:To isolate and identify CSC-LCs from Chinese epithelial ovarian cancer cell line 3AO, ascertain their phenotypes, and study their chemotherapeutic sensitivity.Methods:The 3AO cells were placed in the serum-free medium on the Ultra Low Attachment plates to form spheroid cells. Then, the 3AO spheroid cells were analyzed the expression of those common stem cell markers, such as CD44/CD24, CD 133, and ABCG2 by fluorescence-activated cell sorting system (FACS) to ascertain the characteristic markers. Cells sorted by FACS based on the markers were collected to perform the identification-related experiments of tumorigenicity, differentiation, and immunofluorescence analysis of stem cell markers. Furthermore, the sensibility of Paclitaxol and Carboplatin were compared between CSC-LCs and non CSC-LCs.Results:1.A subpopulation of 3AO cells formed anchorage-independent, self-renewing spheres.2.CD44+CD24- cells were enriched in 3AO sphere-forming cells.3.Spheroid cells and CD44+CD24" cells were highly tumorigenic4.CD44+CD24-cells could differentiate into CD44+CD24+cells5.CD44+CD24- cells were highly resistant to Paclitaxol and Carboplatin.Conclusions:1.CD44+CD24- was the new phenotype characteristic of ovarian CSC-LCs; The ability of self-renewal, high tumorigenicity and differentiation into CD44+CD24+cells provided abundant evidence as ovarian CSC-LCs for CD44+CD24" cells.2.CD44+CD24- cells in ovarian cancer were markedly resistant to conventional chemotherapies, and should become the main target for ovarian cancer chemotherapy. Part II The preliminary study on CSC-LCs of primary cultured cancer cells from ovarian cancer tissue and ascites samplesObjective:To confirm the existence of ovarian CSC-LCs in primary cultured cancer cells from ovarian cancer tissue and ascites samplesMethods:Fresh tissue and ascites samples of advanced adenocarcinoma patient were collected, dissected, digested, filtered and isolated under sterile conditions, and the resulting single tumor cells were placed in the serum-free medium on the Ultra Low Attachment plates to form spheroid cells. The primary cultured ovarian cancer spheres were collected to analyze the phenotype characters, observe the differentiation phenomenon and verify the expression of stem cell markers by immunofluorescence. The sensibility of Paclitaxol and Carboplatin were compared between the primary cultured ovarian cancer spheres and their differentiated cells.Results:1.Primary cultured cancer cells from ovarian cancer tissue or ascites could form spheroid cells in vitro.2.CD44+CD24-cells were enriched in spheroid cells of the primary cultured ovarian cancer cells3.spheroid cells of the primary cultured ovarian cancer cells expressed stem cell markers.4.CD44+CD24" spheroid cells of the primary cultured ovarian cancer cells could differentiate into mature cancer cells.5.CD44+CD24- spheroid cells of the primary cultured ovarian cancer cells were highly resistant to Paclitaxol and Carboplatin. Conclusion:The spheroid cells of the primary cultured epithelial ovarian cancer cells enriched CD44+CD24- cells, expressed stem cell markers, possessed the ability of differentiation, and demonstrated distinguished drug resistance,suggesting the CD44+CD24- cells in the primary ovarian cancer cells were CSC-LCs.