The Establishment Of Human Ascites Derived Ovarian Cancer Cell Line And The Screen And Biological Properties Study Of The Side Population Cells

Aims:Ovarian cancer(OC)is the most common gynecological fatal cancer type. The 5-year survival rate is around 30%. Ovarian cancer was always diagnosed at late stage. Although OC is sensitive to the first-line chemotherapy such as platinum-based drugs, the high rate of recurrence and drug resistance usually leads to poor outcomes. These properties are supposed to result from the existence of cancer stem cells(CSCs). Therefore, we plan to establish an OC cell line which is originated from an ovarian serous cystadenocarcinoma patient’s ascites. Then we investigated biological characteristics of the side population(SP) cells. The differences of transcriptome between SP and NSP(none SP) were enriched by GO, KEGG and PPI. Furthermore, we analyzed the gene function and the regulation of the pathway. Thus, the works will facilitate the consequent study that how CSCs run the key function in tumorigenesis, progression, invasion and drug resistance.Methods: 1.OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient(stage IIIc, grade 3). 2.SP and non-SP(NSP) cells were isolated by fluorescence-activated cell sorting(FACS), and cultured in serum-free medium, serum-contained medium and soft agar to compare their self-renewal, differentiation and colony formation capacities. Moreover, SP and NSP cells were stained by immunocytochemistry to compare their expression of stem cell markers.and stem cell genes. At last SP and NSP cells tumorigenesis was examined by subcutaneous and intraperitoneal injection of these cells to NOD-SCID mice. 3.Invasion ability was examined by wound healing assay and boydon chamber.The drug resistance was assessed by the survival rate of SP and NSP under a series concentration of cis-platinum. 4. The enrichment of the differences of transcriptome between SP and NSP. The method of GO, KEGG and PPI enrichment were used after the test of transcriptome chip, then the differences were proved by q PCR.Results: 1.OC cell line was established from ascites of an ovarian serous cystadenocarcinoma. The progeny which underwent 70 times continuous passage perform the same phenotype of its ancestor. 2. We obtained(0.38±0.05)% SP cells by FACS. In vitro, SP cells and non-SP cells were cultured in same condition, SP ratios were always higher in SP cells when compared with non-SP cells. And SP cells abilities of tumorsphere and colonies forming were also more prominent. In addition, SP cells expressed more CSCs surface markers and stem cell genes than NSP cells.In vivo, few SP cells(1.0×103)could lead to tumorigenesis subcutaneously or in enterocoelia within NOD-SCID mice. The xenograph showed same histological features as primary tumor, such as the expression of ovarian serous cystadenocarcinoma biomarker CA125. 3.SP cells were more migratory and invasive which assessed by wound healing assay and boydon assay. After cisplatinum treatment, SP cells IC50 value was significantly higher than non-SP cells and non-sorting cells. Moreover, cisplatinum administration elevate SP cells proportion in OC cells. However, verapamil could counteract the SP cells resistance.4. There are 18 genes were up-regulate and 3 were down-regulate. Three pathway and four key molecular were enriched in total.Conclusion: 1.In this study, we established an OC cell line from a high stage ovarian serous cystadenocarcinoma sample. The cell line maintained the features of the primary tumor tissue. 2.The cell line can be separated to a small subgroup of SP cells. SP cells performe the function of self-renewal, multipotent differentiation, expression of CSCs markers, impressive abilities of colony formation and tumorigenesis in vivo. These are consisted with the characteristics of cancer stem cells, and the cell line can be studied as an alternative model of CSCs. 3.SP cells of the OC cell line showed dramatically metastasis and invasion, it can also resisted cisplatinum treatment which may be partly result from the activity of ABC transporter. The subsequently mechanism research and related drug development could execute on the basis of our study. 4. The results of the enrichment indicate that 4 key molecular and 3 pathway have potential impact on the biological characters for SP cell.