The Study On Characteristics Of Cancer Stem Cell In Epithelial Ovarian Cancer Dormant SKOV3Cell Group

Objective: We established xenotransplanted tumor model with epithelial ovarian cancer SKOV3cells and sorted the dormant cells from the transplanted tumor by PKH26dyes labeling technique and flow cytometry. Meanwhile,we analyzed the expression of stem cell markers,tumorigenicity in vivo, potential of clone formation in vitro and cell cycle of thedormant cells and investigated the stem cell characteristic of ovarian cancer dormant cells in PKH^hisubset to provid theoretical basis for ovariancancer.Methods:The establishment of xenotransplanted tumor model: PKH26-labeled4×106SKOV3cells were injected s.c. into the20thigh of4-6-wk-old female nude mice.10nude mice with PKH26-labeled cells were separatelyraised for3and6weeks.Such transplanted tumors were finely minced and subjected to collagenase digestion to obtain single-cellsuspensions named SKOV3-P.The other10nude mice injected with PKH-labeled SKOV3cells for2weeks were subsequently administered by cisplatin treatmentfor5times,20mg/Kg,once every six days. The single-cell suspensions ofsuch tumor were called SKOV3-R.The detailed experimental method were as follows:1To observe the labeled efficiency of PKH26for SKOV3cells underfluorescence microscope.2Flow cytometry was used for analyzing the distribution of PKH26-label-retaining subsets in SKOV3-P and SKOV3-R cells.3Real-time PCR was applied to analyze the Mrna expression of Oct-4andNestin in3PKH26-label-retaining subsets from SKOV3-P and SKOV3-R cells. 4The expression of CD44protein was detected in the3PKH26–labelretaining subsets from SKOV3-P and SKOV3-R cells by flow cytometry.5Plate clone formation test aimed at3PKH26-label-retaining subsets fromSKOV3-P and SKOV3-R cells was used to detect clonality in vitro.63PKH26-label-retaining subsets from SKOV3-P and SKOV3-R cells wereseparately injected s.c. into the thigh of nude mice to observe tumorigenicityin vivo.7Making use of flow cytometry to analyze cell cycle in the3PKH26-label-retaining subsets from SKOV3-P and SKOV3-R cells.8Statistical analysis: Data were evaluated using SPSS13.0statisticalsoftware, each assay was performed in triplicate. The measurement data wereexpressed as mean±standard deviation. For Homogeneity of variance test,single factor analysis of variance （One-Way ANOVA）, LSD and t-test wereused for data analysis. α=0.05was size of test. When P＜0.05, the differencehad statistical significance; when P＞0.05, there was no statisticallysignificant difference.Results:1The PKH26-labeled SKOV3cells took on red under fluorescencemicroscope and the labeled efficiency could achieve to over90%.It wascontented to experiment requirement.2Analyses of3-and6-week-old SKOV3-P cells for distribution of PKH26intensity revealed a continuous gradient of cells with fluorochromePKH26retention ranging from high (PKH26^hicells), low (PKH26^locells),to total label quenching (PKH26^negcells) according to PKH26-label-retaining intensity;the proportion of the PKH26^hicells in3,6-week-old SKOV3-P cells and SKOV3-R cells were （7.06±0.14）%,（2.65±0.39）%,（6.31±0.38）%.The PKH26^hisubset of6-week-old SKOV3-P cells were less than thatin3-week-old SKOV3-P cells （P <0.01）; the PKH26^hisubset in SKOV3-R cells were obviously more than that in6-week-old SKOV3-P cells（P <0.01）.3Real-time PCR indicated that Oct-4mRNA and Nestin mRNA were expressed in PKH26^hiand PKH26^losubsets from SKOV3-P and SKOV3-Rcells with being higher in the PKH26^hisubset（P <0.01）；Oct-4mRNA andNestin mRNA expressions in PKH26^hisubset from SKOV3-R cells were higherthan that in SKOV3-P cells（P <0.05）.4Flow cytometry displayed expression of CD44protein in PKH26^hisubsetsfrom SKOV3-P and SKOV3-R cells with higher than that in PKH26^losubset（P<0.01）；the expression of CD44protein in PKH^hisubsets from SKOV3-R cellswas higher than that in PKH26^hisubsets from SKOV3-Pcells，however, therewas no statistically significant difference （P>0.05）.5Plate clone formation test showed that the cloning efficiency in PKH26^hisubsets from SKOV3-P and SKOV3-R cells was obviously higher thanPKH26^losubsets （P<0.05），while there was no cloning efficiency in PKH26^negsubsets from SKOV3-P and SKOV3-R cells. Cloning efficiency in PKH26^hisubsets from SKOV3-R cells was evidently higher than that in SKOV3-P cells,there was statistically significant difference （P<0.05）.6The tumorigenic rates of5000PKH26^hicells from SKOV3-P andSKOV3-R cells were60%（3/5）,80%（4/5）; the tumorigenic rates of of10000PKH26^hi,locells from SKOV3-P cells were80%（4/5）,20%（1/5）and thetumorigenic rates of10000PKH26^hi,locells from SKOV3-R cells were100%（5/5）,20%（1/5）. Whereas the PKH26^negcells failed to displaytumorigenicity injected5000or10000cells.7Cell cycle analysis indicated that aneuploid cells in PKH26^hisubsets from SKOV3-P cells and SKOV3-R cells tended to accumulate in G0/G1phase. Aneuploid cells in PKH26^hisubsets from SKOV3-R cells in G0/G1phase were more than SKOV3-R cells,and the difference was statistically significant（P=0.02<0.05）.The aneuploid cells in PKH26^losubsets from SKOV3-P cells in S phase were few,however aneuploid cells in PKH26losubsets from SKOV3-R cells in S phase were obviously more than SKOV3-P cells（P=0.01<0.05）. Aneuploid cells in PKH26^losubsets from SKOV3-P cells in G0/G1phase was significantly higher than that from SKOV3-R cells（P<0.05）. Conclusion:1The SKOV3-P-PKH^hisubset and SKOV3-R-PKH^hisubset over expressstem cells markers Oct-4and Nestin, possesing tumorigenicity in vivo andpotential of clone formation in vitro，furthermore,the latter is higher than theformer.It indicates that the dormant PKH^hicells in tumor befor and afterchemotherapy are stem-like cells and the the characteristic of stem cells isobvious after treatment.It suggests that there is a relation between dormant cellsof stem-like cells and cancer resistance.2Chemotherapy coaxes aneuploid cells from SKOV3-P-PKH^losubsets out of their quiesent states due to selective pressure.They re-enter into Sphase and form SKOV3-R-PKH^losubsets with proliferative potential. It indicates that dormant aneuploid cells have a relation with cancer relapse.