| Ovarian cancer is the most lethal of all gynaecological malignancies among women worldwide. In the United States, there was about 22,240 new cases of ovarian cancer patients,with whose deaths were 14,030 cases. Epithelial ovarian cancer(EOC) accounts for 90% of ovarian cancer and above 60% of patients develop terminal disease to die. In the past 25 years, there was only a little of improvement in EOC overall mortality rates. One hand, the high mortality is mainly due to a lack of effective progress in the disease treatment.The other hand due to, in large part, to the frequent diagnosis of the disease at advanced stages and the almost inevitable emergence of chemoresistant disease. One theory that has emerged by Hamburger and Salmon to explain EOC is a Cancer Stem Cell disease, Cancer Stem Cells(CSCs)is the main factor causing relapse. CSC Theory states that only some cells from a heterogeneous tumor are capable of tumorigenesis. These cells have been collectively termed as CSCs due to their stem cell-like properties of self-renewal(SR), differentiation and tumorigenesis(malignant tissuegenesis).EOC represent ~90% of all ovarian cancer cases and are thought to arise from ovarian surface epithelium stem cells or fallopian tube epithelium. As such, the majority of ovarian CSC research has focused on EOC. The commonly methods to identify and isolate CSCs based on Hoechst Side Population(SP)assay, ovarian CSCs markers such as enzyme Aldehyde Dehydrogenase 1(ALDH1), CD133+,CD24+,CD44 and CD117,etc. According to a latest study, Lgr5 is one of ovarian surface epithelial cells(epithelial stem cells) markers. In the future, it may help to be treated as a biomarker for early detection of ovarian cancer disease. However, the existing markers are not be used as ovarian cancer stem cell markers successfully. The collated reports are yet to culminate in description of a consensus CSC model for ovarian cancer. In recent years, researchers tend to assume that most malignant tumors, including ovarian cancer, are CSC model diseases. CSC model diseases, some reports called it as CSC hierarchy. In this CSC model(or CSC hierarchy) only a small part of the tumor cells has CSC-like characteristic and these parts of cells sit in a dormant(quiescent)state at the apex of the hierarchy. The traditional chemotherapy, maximum tolerated dose chemotherapy(MTD), can’t be effective for killing this small fraction of ovarian cancer stem cells which has the most “stemness”. By Contrast, MTD model can enrich the “stemness” of ovarian CSC which in a active proliferation. The active state will lead to new mass and recurrent disease.The first part of the study is to test the expression of Lgr5 and ALDH1 in EOC patients by immunohistochemical method. To investigate their relationships with age, tumor stage, pathological classification, lymphatic metastasis and prognosis in EOC patients and also to discuss the correlation between the expression of Lgr5 and ALDH. To provide the theoretical and experimental basis on ovarian CSCs in subsequent tests. The second part of the study is to explore the difference in two chemotherapy models. To investigate the impact of LDM and MTD administration of cisplatin in Balb/c nude mice bearing SKOV3 ovarian cancer xenografts, the solid tumor after chemotherapy is formed by grinding of suspension. We use Fluorescence Activated Cell Sorter(FACS) to isolate and identify CD133+, ALDH1+, ALDH1+CD133+cells by putative ovarian stem cell markers. After FACS,we use Western Blot method to test the expression of Lgr5, BCRP and Lef1 protein.Valid the CSC stemness by assay of clone froming and tumorigenic ability.. Given above all, we design this protocol which aims to explore how the different DDP administration to affect the hierarchy in EOC. Part One High expressions of Lgr5 and ALDH1 in primary epithelial ovarian cancers correlate with advanced tumor stage and grade, as well as poor clinical outcome of the patientsObjectives: To investigate their relationships with clinical pathological parameters by selecting Lgr5 and ALDH1 as putative stem cell markers and to discuss the correlation between the expression of Lgr5 and ALDH1. To provide the theoretical and experimental basis on ovarian CSCs in subsequent tests.Methods: One-hundred primary ovarian epithelial tumor samples were obtained for immunohistochemistry analysis of ALDH1 and Lgr5 expressions. Tissues from 20 normal ovaries and 30 benign ovarian neoplansms were collected as controls. Correlation analysis was performed between ALDH1 or Lgr5 and clinical factors, as well as ALDH1 and Lgr5 expressions in the tumor samples. Survival analysis between the groups was performed using the Kaplan-Meier survival method. Results:1 Clinical pathology characteristicThe stydy include 100 EOC patients, 34 cases of women patients under 50 years old verse 66 case of women patients above 50 years old. 67% patients were diagnosed with lymphatic metastasis. More patients(59%) were diagnosed in FIGO Ⅲand Ⅳ,in which high differentiation accounted for 58%,low-middle differentiation accounted for 27%. Serous ovarian cancer is the most common ovarian cancer, account for 50% of EOC and the least is mucinous mucinous carcinomas accounting for 10%. The survival day of patients ranged from nine months to 79 months, the median surial was 36.89 months.2 The expression of Lgr5 and ALDH1 in normal ovarian, benign ovarian tumor and EOC tissueImmunohistochemistry analysis Results: Lgr5 was mainly expressed in the cytoplasm and shown yellow or yellow brown granules. Lrg5 protein expression was detected in 93 ovarian epithelial cancer tissues, and 55 specimens of cancer tissues were found to be highly positively stained for Lrg5. In the benign ovarian tumor tissues and normal ovarian tissues, Lrg5 was negatively or only weakly positive stained(5/20 and 20/30, respectively). ALDH1 protein expression was detected in 90 of 100 ovarian epithelial cancer tissues, and the high expression of ALDH1 was detected in 71 ovarian epithelial cancers. By contrast, ALDH1 showed only weakly positive staining in 17 of 30(56.7%) of benign ovarian tumor tissues, and normal ovarian tissue was only weakly positive for ALDH1(3/20, 15%).Forty-six ovarian epithelial cancer tissues were positively stained for both Lrg5 and ALDH1, while twenty cases showed low expressions of Lrg5 and ALDH1. The expressions of both Lrg5 and ALDH1 were significantly higher in the ovarian epithelial cancer tissues that that in the benign ovarian tumor tissues and normal ovarian tissues(P <0.05).3 Correlation analysis of Lgr5 expression with the clinicopathologic parametersAssociations between Lgr5 or ALDH1 expression profile and the patients’ clinicopathologic parameters are presented in Table 1. High expressions of both Lgr5 and ALDH1 were strongly associated with advanced FIGO cancer stages(P =0.009 and P =0.029, respectively), and higher GOG grades(P=0.034 and P=0.005,respectively). However, no significant association was observed between Lgr5 or ALDH1 expression profile and age, lymphatic metastasis, and histology of carcinomas(P >0.05).4 Correlation analysis of Lgr5 and ALDH1 expression in ovarian epithelial cancerResults of Spearman correlation analysis showed that there was a significant positive correlation between Lgr5 and ALDH1 protein expressions in the ovarian epithelial cancer tissues(r=0.385, P <0.001).5 Correlation analysis of Lgr5 or ALDH1 expression with the survival of ovarian epithelial cancer patientsKaplan-Meier analysis showed that both Lgr5 and ALDH1 expressions displayed a significant inverse association with progression-free survival of patients with ovarian epithelial cancerConclusions:1 In conclusion, our study indicated that both Lgr5 and ALDH1 were highly expressed in the tumor sample of EOC patients.2High expressions of both Lgr5 and ALDH1 were not only correlated with different FIGO operation-pathological stage and tumor grades, clinical outcome of the patients, but also associated with each other. Part Two Influence of different chemotherapy models on stemness of CSC in ovarian carcinoma xenograft tumorsObjectives: To establish nude mouse xenograft modelsl and to discusses the influenceof different chemotherapy models on stemness of CSC inovarian carcinoma xenograft tumors. Using FACS to isolate and identify the putative CSC characteristics markers: CD133+, ALDH1+ and ALDH1+CD133+. To discuss the enrich influence of MTD-DDP chemotherapy and LDM-DDP chemotherapy in xenograft tumor and to prepare for the validation of ovarian cancer stem cells in subsequent experiments.Methods: To set up the abdominal transplantation tumor model of Balb/c nude mice bearing SKOV3 ovarian cancer cell. The nude mice were given by LDM and MTD administration of cisplatin. To observe the influence of different chemotherapy models on nude mouse,such as living condition,side effects and xenografts growth. Then the solid tumor after chemotherapy is formed by grinding of suspension. We use Fluorescence Activated Cell Sorter(FACS)to isolate and identify CD133+,ALDH1+, ALDH1+CD133+cells by putative ovarian stem cell markers.In the end, we dicuss Whether MTD-DDP would enrich CSCs or not, which can provide a new strategy for clinical treatment.Results: 1The abdominal transplantation tumor model of Balb/c nude mice bearing SKOV3 ovarian cancer cells is established.Tumor formation rate: SKOV3 cells(1×107) were injected into the nude mice abdomen. Two weeks later xenograrfs were formation. The total tumor formation rate is 100%. 2The general situation and side effects assessment in nude mice 2.1 Food intake: The mice were randomly divided into three groups a: control group, MTD-DDP group and LDM-DDP group. There was no one died in 18 days chemotherapy. Each group of nude mice were in good condition, without physical activity decrease and movement slowly. Compared with control group, food-intake of nude mice in MTD-DDP group and LDM-DDP group decline significantly. On 9th day, food-intake of nude mice in MTD-DDP group and LDM-DDP group were less than control group(P=0.000,P=0.000) and there is no statistical difference in MTD-DDP group and LDM-DDP group(P=0.877);On 18 th day, food-intake of nude mice in MTD-DDP group and LDM-DDP group were less than control group(P=0.000,P=0.000)and there is no statistical difference in MTD-DDP group and LDM-DDP group(P=0.740). 2.2 Body weight: There was a delcline in MTD-DDP group and LDM-DDP group from 1th day to 18 th of the chemotherapy. On 9th day, body weight of nude mice in MTD-DDP group and LDM-DDP group were lower than control group(P=0.000,P=0.000) and there is no statistical difference in MTD-DDP group and LDM-DDP group(P=0.458);On 18 th day, body weight of nude mice in MTD-DDP group and LDM-DDP group were lower than control group(P=0.000,P=0.000)and there is no statistical difference in MTD-DDP group and LDM-DDP group(P=0.581). 2.3 Abdominal diameter and ascites formation: From 1th day to 18 th of the chemotherapy, there were two nude mice had ascites in MTD-DDP group. The abdominal diameter were all increased in control group,MTD-DDP group and LDM-DDP group. On 12 th day, the abdominal diameter of nude mice in control group were smaller than LDM-DDP group(P =0.025)and there is no statistical difference in MTD-DDP group and LDM-DDP group(P =0.246);On 15 th day, the abdominal diameter of nude mice in control group were smaller than MTD-DDP group and LDM-DDP(P =0.000,P =0.000);the abdominal diameter of nude mice in MTD-DDP were bigger than LDM-DDP group and the result has statistical difference.(P=0.037). 2.4 white blood cells: From the 1th day to 18 th of the chemotherapy, there was a constantly decline tend of white blood cells in MTD-DDP group. On 6th day, it showed no statistical differences among the three groups.(F=0.194,P=0.824); On 12 th day, white blood cells in MTD-DDP group were lower than control group(P=0.018),while there was no significantly difference between LDM-DDP group and control group(P=0.099);On 18 th day, there was no significantly difference between LDM-DDP group and control group(P=0.050); white blood cells in MTD-DDP were lower than control group and LDM-DDP group.(P=0.000,P=0.036); 3 Growth of abdominal transplantation tumor 3.1 Nude mices were euthanized at the end of chemotherapy. The total weights of xenografts were 1.83±0.12 g in control group, 1.53±0.20 g in MTD-DDP group and 1.02±0.10 g in LDM-DDP group. The total weights of xenografts showed statistical difference among three groups.(F=32.274, P =0.000) 3.2 The biggest diameter of xenograft is 1.75±0.24 cm in control group, 1.60±0.23 cm in MTD-DDP group and 1.33±0.16 cm in LDM-DDP group. The xenograft diameter showed statistical difference among three groups.(F=20.195, P =0.000) 4 CD133 positive cells(CD133+)expression in the primary cells of xenograft tumor in control group, MTD-DDP group and LDM-DDP group by fluorescence-activated cell sorting(FACS).By FACS, it showed the percentage of CD133+ cells is 0.77%±0.01% in the primary cells of xenograft tumor in LDM-DDP group,which was significantly lower than that in control group.(1.10%±0.13%,P=0.020);The percentage in MTD-DDP group is 3.17%±0.16%, which was significantly higher than that in control group.(P=0.000)There is statistical difference among control,MTD-DDP group and LDM-DDP group( F=94.441,P=0.000).The results showed that the CSC-like cells selected by CD133+ marker were enriched in MTD-DDP group, LDM-DDP chemotherapy reduced the percentage of CSC-like cells. 5 ALDH1 positive cells(ALDH1+)expression in the primary cells of xenograft tumor in control group, MTD-DDP group and LDM-DDP group by fluorescence-activated cell sorting(FACS).By FACS, it showed the percentage of ALDH1+ cells is 1.83%±0.06% in the primary cells of xenograft tumor in LDM-DDP group,which was significantly lower than that in control group.(3.10%±0.45%,P=0.000);The percentage in MTD-DDP group is 4.03%±0.10%,which was significantly higher than that in control group.(P=0.000)There is statistical difference among control,MTD-DDP group and LDM-DDP group( F=736.691,P=0.000).The results showed that the CSC-like cells selected by ALDH1+ marker were enriched in MTD-DDP group,LDM-DDP chemotherapy reduced the percentage of CSC-like cells. 6 ALDH1+CD133+ expression in the primary cells of xenograft tumor in control group, MTD-DDP group and LDM-DDP group by fluorescence-activated cell sorting(FACS).By FACS, it showed the percentage of ALDH1+CD133+cells is 0.21%±0.03% in the primary cells of xenograft tumor in LDM-DDP group, which was significantly lower than that in control group.(0.44%±0.01%,P=0.016);The percentage in MTD-DDP group is(0.65%±0.07%),which was significantly higher than that in control group.(P=0.006)There is statistical difference among control,MTD-DDP group and LDM-DDP group(F=736.691,P=0.000).The results showed that the CSC-like cells selected by ALDH1+CD133+ marker were enriched in MTD-DDP group, LDM-DDP chemotherapy reduced the percentage of CSC-like cells.Conclusions:1 LDM-DDP model resulted in less bone marrow suppression compared with MTD-DDP model.2 The results showed that the ALDH1+ cells, ALDH1+cells and ALDH1+CD133+cells were enriched in MTD-DDP group; however, LDM-DDP group reduced the percentage of positive cells among three groups. Compared with tradition chemotherapy MTD model, LDM model is more difficult to produce anti-chemotherapy and less recurrent rate. Part Three Validation of cancer stem cell in primary xenograft tumorsObjectives: To valid the CSC characteristic of ALDH1+ cells, CD133+ cells and ALDH1+CD133+ cells which were be sorted by FACS.Methods: ALDH1+ cells, CD133+ cells and ALDH1+CD133+cells were be isolatedn by FACS in part two. We used clonogenicity assays in vitro and xenograft assays in vivto to valid the CSC characteristic. And at the same time Western blot were used to analyze the expressions of Lrg5, LEF-1 and BCRP protein.Results:1 Clonogenic ability of the CD133+ cells and the CD133- cellsThe colony formation rate of CD133+ cells and CD133- cells is 54.14%±1.95%,3.26%±0.71%, there is statistical difference in CD133+ cells and CD133- cells(t=73.500,P =0.000). CD133+ cells demonstrated higher clonogenic capability, which can be used as isolation of ovarian CSCs marker.2 Clonogenic ability of the ALDH1+ cells and the ALDH1- cellsThe colony formation rate of ALDH1+ cells and ALDH1- cells is 54.14%±1.95%,3.26%±0.71%, there is statistical difference in ALDH1+ cells and ALDH1- cells(t=73.500,P =0.000). ALDH1+ cells demonstrated higher clonogenic capability, which can be used as isolation of ovarian CSCs marker.3 Tumorigenicity of the CD133+ cells and the CD133- cellsThe CD133+ cells and the CD133- cells were injected into the right leg of each nude mouse. The tumorigenic rate of 5,000 CD133+ cells was 50%(3/6)verse(0/6)in CD133- cells after 6 weeks. The tumorigenic rate of 10,000 CD133+ cells was 66.7%(4/6)verse(0/6)in CD133- cells after 6 weeks. The result showed only CD133+ cells had the tumorigenicity, whereas CD133-had little in tumorigenicity.4 Tumorigenicity of the ALDH1+ cells and the ALDH1- cellsThe ALDH1+ cells and the ALDH1- cells were injected into the right leg of each nude mouse. The tumorigenic rate of 5,000 ALDH1+ cells was 66.7%(4/6)verse(0/6)in ALDH1- cells after 6 weeks. The tumorigenic rate of 10,000 ALDH1+ cells was 83.3%(5/6)verse(0/6)in ALDH1- cells after 6 weeks. The result showed only ALDH1+ cells had the tumorigenicity, whereas ALDH1- had little in tumorigenicity.5 Tumorigenicity of the ALDH1+CH133+ cells and the ALDH1-CD133-cellsThe ALDH1+CH133+ cells and the ALDH1-CD133- cells were injected into the right leg of each nude mouse. The tumorigenic rate of 5,000 ALDH1+CH133+ cells was 66.7%(4/6)verse(0/6)in ALDH1-CD133- cells after 6 weeks. The tumorigenic rate of 10,000 ALDH1+CH133+ cells was 83.3%(5/6)verse(0/6)in ALDH1-CD133-cells after 6 weeks. The result showed only ALDH1+CH133+ cells had the tumorigenicity, whereas ALDH1-CD133- had little in tumorigenicity.6 The expression of Lgr5, BCRP and LEF-1 in ALDH1+ cells and the ALDH1- cells.The protein expression of Lgr5 in ALDH1+ cells was higher than it in ALDH1- cells. The MOD value was 0.61±0.17,025±0.13(P=0.000);The protein expression of BCRP in ALDH1+ cells was higher than it in ALDH1-cells. The MOD value was 0.57±0.17,0.23±0.11(P=0.000);The protein expression of LEF-1 in ALDH1+ cells was higher than it in ALDH1- cells. The MOD value was 0.57±0.17,0.23±0.11(P=0.000).7 The expression of Lgr5, BCRP and LEF-1 in CD133+ cells and the CD133- cells.The protein expression of Lgr5 in CD133+ cells was higher than it in CD133- cells. The MOD value was0.55±0.25,021±0.17(P=0.001);The protein expression of BCRP in CD133+ cells was higher than it in CD133-cells. The MOD value was 0.48±0.21,0.20±0.11(P=0.000);The protein expression of LEF-1 in CD133+ cells was higher than it in CD133- cells. The MOD value was 0.41±0.21,0.21±0.13(P=0.000);8 The expression of Lgr5, BCRP and LEF-1 in ALDH1+CD133+ cells and the ALDH1-CD133- cells.The protein expression of Lgr5 in ALDH1+CD133+ cells was higher than it in CD133-ALDH1- cells. The MOD value was 0.61±0.28,014±0.01(P=0.000);The protein expression of BCRP in ALDH1+CD133+ cells was higher than it in ALDH1-CD133-cells.The OD value was0.60±0.18,0.11±0.12(P=0.000);The protein expression of LEF-1 in ALDH1+CD133+ cells was higher than it in ALDH1-CD133- cells. The MOD value was0.47±0.15,0.18±0.12(P=0.000);9 The expression of Lgr5, BCRP and LEF-1 among three positive cellsThe protein expression of Lgr5 has no statistical difference in ALDH1+ cells and ALDH1+CD133+ cells(P=0.880)and it also has no statistical difference in CD133+ cells and ALDH1+CD133+ cells(P=0.140). The protein expression of BCRP has no statistical difference in ALDH1+ cells and ALDH1+CD133+ cells(P=0.190),however the result has statistical difference in CD133+cells and ALDH1+CD133+ cells(P=0.002);The protein expression of LEF-1 has no statistical difference in ALDH1+ cells and ALDH1+CD133+ cells(P=0.097),however the result has statistical difference in CD133+ cells and ALDH1+CD133+ cells(P=0.013)Conclusions:1 ALDH1+cells,CD133+cells and ALDH1+CD133+ cells showed obvious stem cell properties, the result is conformed to the the cancer stem cell hypothesis.2 Western Blot showed: Lgr5, LEF-1 and BCRP protein expression were high expression in ovarian cancer cells. |
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