Biological Characteristics Of Ovarian Cancer Cell Line OVCAR4 And Validation Of CDC50A As Ovarian Cancer Stem Cell Marker Protein

BackgroundOvarian cancer(OC)is the most lethal gynecological genital tract cancer,the standard treatment for advanced OC is primary debulking surgery followed by the platinum-based chemotherapy.However,the survival rate of advanced ovarian cancer is only 29%.The drug resistance to platinum drugs caused by tumor recurrence is one of the main reasons that the survival rate of ovarian cancer cannot be further improved.Epithelial ovarian cancer(EOC)is a group of highly heterogeneous tumors with a variety of histological types,accounting for 95%of ovarian malignant tumors.In terms of histological types,EOC is mainly divided into serous cancer,mucinous cancer,clear cell cancer,endometrioid cancer and so on[4].Among them,high-grade serous ovarian cancer(HGSOC)accounts for about 60%-70%.It is a kind of histologic type that can best reflect the biological and clinical characteristics of ovarian cancer with strong invasion and rapid progress of disease,and thus becomes the core type of basic and clinical research on ovarian cancer.Since the establishment of HeLa cell line in 1951,cell culture and establishment of tumor cell line has become an important means of tumor research,and its use has greatly promoted human understanding of tumor biology.The advantage of cell line is that it can provide a relatively homogeneous cell group,and can reproduce and propagate without restriction.In vitro cell line and cell line xenotransplantation model become an important method to study ovarian cancer.In 2011,the Cancer Genome Atlas(TCGA)used a second-generation sequencing method to describe the genome and expression map of various tumors.In 2012,the broad Novartis cancer cell line Encyclopedia(CCLE)detailed the origin and gene map of about 1000 tumor cell lines.On this basis,Domke et al.analyzed DNA copy number changes(CNAs),mutation types and mRNA expression levels of 47 ovarian cancer cell lines and ovarian cancer tissues,and focused on HGSOC to find the most suitable ovarian cancer cells as in vitro models.At present,the most commonly used HGSOC cell lines in vitro include SKOV3,A2780,igrov1,OVCAR3,and CAOV3.SKOV3 and A2780 are wild-type p53 with KRAS gene mutations,indicating that these two strains may be low-grade epithelial ovarian cancer.IGROV1 cell line was found to be MLH1,MSH3 and MSH6 gene mutations.It is a common mutation gene in Lynch syndrome.Its origin and genome have the characteristics of endometrioid carcinoma.Both OVCAR3 and CAOV3 have p53 mutation and high CNAs value,but the correlation between CNAs and HGSOC in TCGA is low.Domke et al.recommended some cell lines that can better simulate HGSOC genomic characteristics,but are rarely used in previous studies,including KURAMOCHI and OVCAR4.Among them,OVCAR4 has p53 mutation,CNAs has strong correlation with HGSOC,and has low mutation frequency in key oncogenes and tumor suppressors,which is considered to be a good representative of HGSOC cell line.This study has changed people's cognition of HGSOC cell line,and has been verified and supported by many later studies.With the development of tumor research,the theory of cancer stem cells(CSCs)was put forward around 1970s.CSCs are considered to be a small group of stem cells with the ability of self-renewal and differentiation in tumor cells,which can in a resting state for a long time in the tumor,can escape the role of chemotherapy drugs,and mediate the drug resistance and recurrence of the tumor.In recent years,monoclonal antibodies against CSCs surface specific marker proteins have become a hot topic in the field of tumor therapy.It was found that monoclonal antibody targeting to kill CSCs,combined with conventional anti-tumor therapy such as surgery,radiotherapy and chemotherapy,can achieve the purpose of curing tumor.The most prominent feature of epithelial ovarian cancer is recurrence and drug resistance,which highly suggests that there may be CSCs cells in the tumor.Therefore,how to identify the stem cells through cell surface specific marker protein and then target to kill them becomes the key to the treatment of epithelial ovarian cancer.Our research group has been devoted to the study of the surface marker proteins of ovarian cancer stem cells for a long time,and has been supported by the National Natural Science Foundation twice.Through the quantitative proteomics technology,comparative proteomics analysis was carried out on the membrane proteins of side population(SP)and non SP cells of ovarian epithelial cancer,and the candidate protein CDC50A for the surface marker of ovarian cancer stem cells was screened out,further through in vivo and in vitro research we confirmed that CDC50A could be used as the surface marker protein of ovarian cancer stem cells.In the previous in vitro study,our research group mainly focused on SKOV3 cell line,which is the most commonly used in the literature.Now,in order to further study the expression and characteristics of CDC50A in HGSOC,we selected OVCAR4 cell line with HGSOC genomic characteristics as the research object,and compared its biological characteristics with SKOV3 cell line.The intraperitoneal and ovary in situ tumorigenesis model of OVCAR4 cell line in NSG mice was constructed and the growth of tumor cells was observed under the dynamic monitoring of the in vivo imaging system,to further verify the stem cell characteristics of CDC50A positive cells in ovcar4 cell line,and to preliminarily explore the feasibility of CDC50A as the treatment target of ovarian epithelial cancer stem cells in OVCAR4 and SKOV3 cell lines.Methods1.Comparison of biological characteristics between ovcar4 cell line and SKOV3 cell line.The comparison of cell morphology of OVCAR4 andSKOV3 was performed by Giemsa staining;The cell growth curve of those two cell lines was sketching by cell counting in 7 days;Cell cycles of those cell lines were analyzed by flow cytometry;The half maximal inhibitory concentration of the two cell lines was compared with cck-8 method;The expression of p53 in the two cell lines was detected by immunohistochemistry;The subcutaneous tumor models of OVCAR4 and SKOV3 cell lines were established to compare the subcutaneous tumorigenic ability of the two cell lines.2.The establishment and dynamic observation of ovarian cancer cell line OVCAR4 mouse model of intraperitoneal and ovarian tumorigenesis in situ.OVCAR4-luciferase cell line was established by lentivirus transfection technology;Puromycin was used to screen OVCAR4 cell line stably expressing luciferase,and its cell morphology and population doubling time were no different from OVCAR4 cell line;OVCAR4-luciferase cells(1 × 107/100 ?L)were injected into the abdominal cavity of 4-6 week old female NSG mice by intraperitoneal injection,5 mice in total;The right ovary was dissected in the back of mice,and OVCAR4-luciferase cells(5 X 106/40 ? L/mouse)were injected into the right ovary by microinjection,5 mice in total;Mice were anesthetized regularly,and D-luciferin was injected intraperitoneally.The fluorescence signal was detected by the living imaging instrument,and the tumorigenesis was continuously observed in the living state,and the mice were dissected timely to monitor the tumorigenesis.3.In vitro validation of CDC50A as a surface marker protein of epithelial ovarian cancer stem cells.The proportion of CDC50A positive cells in the ovarian cancer cell lines OVCAR4 were analyzed by flow cytometry;OVCAR4 CDC50A positive and CDC50A negative cells were sorted by Moflo fluorescent activated cell stream sorter and the expression of CDC50A gene was tested by qPCR;The sphere formation ability of the OVCAR4-CDC50A positive and CDC50A negative cells were tested by serum-free suspension culture technology;The enrichment of CDC50A positive cells in OVCAR4 spheres were tested by immunofluorescence and flow cytometry;OVCAR4 CDC50A positive and SKOV3 CDC50A positive cells were sorted by Moflo fluorescent activated cell stream sorter and cultured in serum-free suspension culture technology,both kinds of cells were divided into antibody group,Ig-G control group,PBS group and blank group.CDC50A antibody,Ig-G and PBS were added to cell culture medium regularly to compare the formation of cell sphere;The Cisplatin resistance ability of the OVCAR4-CDC50A positive and CDC50A negative cells were tested by CCK-8 method.Results1.Under light microscope OVCAR4 cells were in multilateral shape,with obvious nucleoli,obvious cell atypia,small clusters of cells growing in clusters,which were in sharp contrast with the morphology of SKOV3 cells.SKOV3 cells were spindle shaped,dendritic raised,mostly scattered and distributed,with large and deeply stained nuclei and relatively uniform cytoplasm;The population doubling time(PDT)of cell line SKOV3 was shortest for 32.54 hours,while OVCAR4 was longest for 49.2 hours;The proportion of G0/G1 phase cells in OVCAR4 cell line was slightly lower than that in SKOV3 cell line,but the proportion of S phase cells in OVCAR4 cell line was significantly higher than that in SKOV3 cell line;The IC50 of cisplatin in OVCAR4 cell line was significantly lower than that in SKOV3 cell line,and OVCAR4 was more sensitive to cisplatin;The p53 antibody was strongly positive in OVCAR4 cell line,but negative in SKOV3 cell line;The formation of transplanted tumor was observed in both cell lines of nude mice,the weight of transplanted tumor in SKOV3 group was significantly higher than that in OVCAR4 group(P<0.05);p53 staining was strongly positive in OVCAR4 and negative in SKOV3 cell line.2.A stable OVCAR4-luciferase cell line was established,the intraperitoneal and ovary in situ tumorigenesis model of OVCAR4 cell line in NSG mice was successfully constructed.After 10 days,fluorescence signals could be detected under the in vivo imaging system and the growth of tumor signals was good after regular monitoring.No massive tumor formation was found in the abdominal cavity after sacrifice of mouse anatomy.A little ascites was found in the abdominal cavity of 3 mice in the a intraperitoneal model and 5 mice in the ovary in situ model,military tumor tissue was found in the omentum and mesentery root of 3/3 mice,transplanted tumor was found in the right ovary of 3/5 mice,which were unilateral,normal appearance was found in the left ovary of 5/5 mice,no tumor tissue was found in the omentum and mesentery.3.CDC50A positive population were exist in OVCAR4 and the proportion of CDC50A positive cells were 1.03%;The results of qPCR showed that the expression of CDC50A gene in OVCAR4-CDC50A positive cells was significantly higher than that in OVCAR4-CDC50A negative cells,which further verified the specificity of CDC50A antibody and the correctness of sample selection;The sphere formation ability of OVCAR4-CDC50A positive cells was significantly higher than that of OVCAR4-CDC50A negative cells,the difference was statistically significant(P<0.01);The proportion of OVCAR4-CDC50A positive cells in OVCAR4 sphere was significantly higher than that of adherent OVCAR4 cells;In SKOV3-CDC50A positive cells and OVCAR4-CDC50A positive cells cultured in suspension medium,the number of sphere formation in CDC50A antibody group was significantly lower than that in ig-g group,PBS group and blank group,the difference was statistically significant(P<0.01);The activity of OVCAR4-CDC50A negative cells treated with cisplatin was significantly lower than that of OVCAR4-CDC50A positive cells(P<0.05).Conclusions1.OVCAR4 cell line has the biological characteristics of HGSOC.From the perspective of cell biological characteristics,OVCAR4 cell line has obvious cell atypia,the proportion of S phase cells is significantly higher than that of SKOV3 cell line,and is more sensitive to cisplatin treatment;p5 3 antibody is strongly positive in nuclear staining,with the biological characteristics of HGSOC,which can be used for cell experiments in vitro.OVCAR4 cell line can be transplanted subcutaneously in nude mice,and p53 antibody in paraffin section of transplanted tumor is strongly positive.SKOV3 cell line does not express p53 protein,which may not belong to HGSOC cell line.2.The intraperitoneal and ovary in situ tumorigenesis model of OVCAR4 cell line in NSG mice was successfully constructed and the growth of tumor cells was observed under the dynamic monitoring of the in vivo imaging system.OVCAR4 grew well after intraperitoneal injection in NSG mice and recapitulated military disease.3.CDC50A could be considered as a surface marker of ovarian cancer stem cells.The proportion of CDC50A positive cells in OVCAR4 cell line is in line with that of stem cells.The expression level of CDC50A gene in OVCAR4-CDC50A positive cells was significantly higher than that in OVCAR4-CDC50A negative cells.The sphere forming ability of OVCAR4-CDC50A positive cells is significantly higher than that of OVCAR4-CDC50A negative cells;The proportion of CDC50A positive cells in OVCAR4 sphere is significantly higher than that of adherent cells,and sphere has the function of enriching CDC50A positive cells;The sphere forming ability decreases significantly after the antibody blocked CDC50A positive cells;the anti-cisplatin treatment ability of OVCAR4-CDC50A negative cells is significantly lower than that of OVCAR4-CDC50A positive cells after different concentrations of cisplatin treatment.