| Objective:Epithelial ovarian cancer is one of serious threaten to the lives of women. As the disease advances gradually, the majority of patients have been in advanced stage in diagnosis. Although the long time survival rate of patients may be improved by cytoreductive surgery followed by chemotherapy of plain-based regimen, but it may impact the patients quality of life for the basis of tumor invasion, metastasis and recidivism. To study the pathogenetic and tumor recurrence of ovarian cancer is becoming investigative emphasis and hot spot.Cancer stem cells are a kind of reproducible tumor cells, and its amounts are rare. These cells are characteristic of marked proliferation, self-renewal and tumorigenicity. They can xenotransplantation successfully and has the same phenotype of the original tumor cells. Cancer stem cells have the crucial role in the tumor invasion, metastasis and recidivism. CD133, CD117, CD24, CD44 and ALDH have been widely used as CSCs markers in lots of carcinoma. Because cancer stem cells of epithelial ovarian cancer have not been isolated and identificated, we call the sorted cells from ovarian cancer as cancer stem-like cells. Up till now, vasculogenic mimicry(VM) has more than 10 years’ history. This structure are composed of the shaped tumor cells and Periodic acid Schiff positive staining of the material. The blood flow in VM, so it can provide the nutrition for the tumor growth. VM is another vital structuer besides the blood vessles. CSCs maybe participate in not only vasculogenesis but also the formation of VM by such as transduction pathway and the characteristics of transversal differentiation, and it may influence occurrence, invasion and metastasis of tumor.The first part of the study was colleceted a total of 120 paraffin samples of epithelia ovarian cancer with complete clinicopathologic data. The CD31/Periodic acid Schiff(PAS) dual staining was performed to identify the structure of VM. The expression of CD133 was detected by SP immunohistochemical method. The correlation of clinical pathologic data of EOC patients with CD133 expression and VM formation were studied retrospectively. The impacts of CD133 expression and VM formation on the survial time of patient with EOC were also evaluated. To investigate the relationships of CD133 expression in epithelia ovarian cancer tissues with vascularization mimicry with the clinicopathologic features and prognosis of patients. In the second part of this study, SKOV3 cells were cultured and passaged to the 7th generation by suspension culture in serum free medium. The percentage of CD133 positive espression cells of the 1st, 2rd, 5th and 7th generation was analysed by flow cytometry separately. The proliferation ability of the sorted cells(the 7th generation, similarly hereinafter) was detected by colony formation assay. Three-dimensional cell culture model was established to observe the ability of VM formation between the sorted cells and the parental SKOV3 cells. The expression levels of matrix metalloproteinases-2, 9(MMP-2, MMP-9) in two groups were deteted by RT-PCR. And in the third part of the study, to investigate the impact of low-dose metronomic chemotherapy(LDM) versus maximum tolerated dose chemotherapy(MTD) administration of cisplatin over an 18-day period to Balb/c nude mice bearing the 7th sorted cells and SKOV3 ovarian cancer xenografts. The number of tumor microvascular and VM was calculated through the CD31/Periodic acid Schiff(PAS) dual staining. And to explore the influence of the two chemotherapy models on the expression level of Eph A2, PI3 K and LN-5γ2 on transduction pathway. And it could provide a new idea for clinic therapy of targeted epithelia ovarian cancer. Part One Expression of CD117 in Vasculogenic Mimicry of Epithelia Ovarian Cancer and its Significance Methods: A total of 120 paraffin-embedded samples of EOC with complete clinicopathologic data were collected. The CD31/Periodic acid Schiff(PAS) dual staining was performed to identify the structure of VM. The expression of CD133 was detected by SP immunohistochemical method. The correlation of clinical pathologic data of epithelia ovarian cancer patients with CD133 expression and VM formation were studied retrospectively. The impacts of CD133 expression and VM formation on the survial time of patient with epithelia ovarian cancer were also evaluated. Results: 1 The clinical pathology characteristic of 120 patients with epithelia ovarian cancerThe study included 120 epithelia ovarian cancer patients, 78 cases of patients(65.0%) under 60 years old verse 42 cases(35.0%) above 60 years. According to the histological classification,there were 74 cases(61.7%) of serous carcinoma, 35 cases(29.2%) of Mucinous carcinoma, 11 cases(29.2%) of endometrioid ovarian carcinomas. According to the FIGO surgical pathological staging(2009), there were 28 cases(23.3%) in Ⅰstage, 10 cases(8.3%) in stage, 28 cases(50.0%) in stage, 28 cases(18.4%) in Ⅱ Ⅲ Ⅳstage. According to the degree of tissue differentiation, these patients included 17 cases(14.2%) in high differentiation, 59 cases(49.2%) in middle differentiation and 44 cases(36.6%) in low differentiation. 51 cases(42.5%) were diagnosed with lymphatic metastasis. And 39 cases(32.5%) produced platinum resistance. 2 VM formation and CD133 expression in ovarian cancer tissueA total of 120 primary ovarian carcinoma samples were analyzed for identification of VM and CD133 expression in our study. The results of CD31/ PAS double-staining indicated VM-positive cells in 52(43%) of 120 ovarian carcinoma tissues, while immunohistochemical staining for CD133 indicated CD133-positive cells in 56(47%) cases(Table 1). In addition, ovarian carcinoma tissues with both VM and CD133 expression were found in 38 cases(32%), with only VM were found in 14 cases(11%), with only CD133 expression in 18 cases(15%) and without both markers were in 50 cases(42%). 3 Association of VM formation or CD133 expression with patients’ clinical and pathological characteristics Both VM formation and CD133 expression were associated with age, the histological classification and lymphatic metastasis(P=0.395, 0.436, 0.479) Both VM formation and CD133 expression were associated with tumor grade(P<0.001), FIGO stage(P=0.003) and response to chemotherapy(VM formation: P <0.001; CD133 expression: P = 0.001). VM seemed to form more usually in ovarian carcinoma tissues in patients with high-grade ovarian carcinoma, late-stage disease and non-response to chemotherapy, and carcinoma tissues from these patients always combined with CD133 expression. 4 Association of VM formation combined with CD133 expression with patients’ clinical and pathological variables We also analyzed the potential correlation factors associated with the combined marker between VM formation and CD133 expression in patients with ovarian carcinoma. VM formation and CD133 expression always co-occurred in ovarian carcinoma tissues from patients with high-grade ovarian carcinoma(P<0.001), late-stage disease(P = 0.002) and non-response to chemotherapy(P<0.001). 5 The relationship between VM formation and the expression of CD133 in epithelia ovarian cancerSepearman correlation analysis revealed that VM formation was positively related with the expression of CD133 in epithelia ovarian cancer(r=0.463,P =<0.001). 6 Association of VM formation and CD133 expression with patients’ clinical and pathological variables We also analyzed the potential correlation factors associated with the combined marker between VM formation and CD133 expression in patients with ovarian carcinoma. As shown in Table 3, VM formation and CD133 expression always co-occurred in ovarian carcinoma tissues from patients with high-grade ovarian carcinoma(P<0.001), late-stage disease(P=0.002) and non-response to chemotherapy(P<0.001). As shown in Figure 1, VM formation and CD133 expression were both associated with overall survival. There was significant difference of median survival between cases with VM formation and without VM(VM+ vs VM- = 25 months vs 63 months, P< 0.001). In addition, patients who had ovarian carcinoma with CD133 expression had shorter median overall survival than those without(CD133+ vs CD133- =25 months vs 63 months, P<0.001). We also analyzed the association between VM formation and CD133 expression with overall survival. As shown in Figure 2, significant difference of median overall survival existed among four groups(P<0.001) and patients who had ovarian carcinoma with both VM formation and CD133 expression had shortest median overall survival of 22 months, while patients who had ovarian carcinoma neither with VM formation nor with CD133 expression had longest median overall survival(63 months). Part Two The Effect of Ovarian Tumor Stem-like Cells Sorted from SKOV3 Cells on Formation Vasculogenic Mimicry Mechanisms Methods: SKOV3 cells were cultured and passaged to the 7th generation by suspension culture in serum free medium. The percentage of CD133 positive espression cells of the 1st, 3rd, 5th and 7th generation was analysed by flow cytometry separately. The proliferation ability of the sorted cells(the 7th generation, similarly hereinafter) was detected by colony formation assay. Three-dimensional cell culture model was established to observe the ability of VM formation between the sorted cells and the parental SKOV3 cells. The expression levels of matrix metalloproteinases-2, 9(MMP-2, MMP-9) in two groups were deteted by Western Blotting. Results:1 Morphological observation of human ovarian cancer SKOV3 cells by suspension culture in serum free mediumWhen SKOV3 cells were inoculated into suspension culture in serum free medium, most of them deposited on the bottom of the culture plate, and the others were able to grow adhering to the wall. The initial form of these cells were in erery shape such as round or ellipsoid and were bright. With time, the adherent cells were distinctly increased. This cells group to form “islets”. And some cells formed short pseudopodia. With the bright reduced gradually, most of the adherent cells were dead. Meanwhile, a small number of cells could grow in serum free medium. Floating spheres could be observed 2-4 days later. The floating spheres consisted of 3-8 cells loosely, with the irregular of appearance but the similar size. The number of floating spheres increased gradually to dozens. The size of floating spheres increased, and they presented globular appearance. The tight junctions between these cells were demostrated. The boundary was not clear. As the cells repeatedly passaged, the account and volume of floating spheres were increasing. 2 The results of flow cytometryThe result of flow cytometry showed that the percentage of CD133 and CD117 positive espression cells increased with the increasing generation. The percentage of CD133 positive espression cells in 1st,3th,5th,7th generation were 40.41%, 70.00%, 84.69% and 91.58% respectively. And the percentage of CD117 positive espression cells in 1st,3th,5th,7th generation were 14.96%, 25.14%, 37.73% and 49.58% respectively. 3 Clone formation experimentThe 7th generation sorted cells cultured in serum free medium and the parental SKOV3 cells both had the ability of clone formation. The colony-forming efficiency of the 7th generation suspension cells(50.33%) were significantly higher than those of the maternal HO8910 cells(5.33%),( t=15.093,P<0.001). The 7th generation sorted cells showed more superior clone formation ability. 4 The nude mouse transplantation tumor experimentThe 7th generation sorted cells and the parental SKOV3 cells were inoculated into the six nude mouses subcutaneous tissues of right and left leg respectively. The 7th generation sorted cells cultured in serum free medium could form the tumor after inoculation. The six nude mouse could be visibly to the naked eye. But the parental SKOV3 cells could not form the tumor. The results of tumor formation in nude mice revealed that the rate of tumor formation in the 7th generation suspension cells(6/6)were significantly higher than maternal cells(0/6). The 7th generation sorted cells showed more superior formation tumor ability. 5 The ability of formation for VM of the 7th generation sorted cells and the parental SKOV3 cellsthe 7th generation sorted cells could form the VM structure in Matrigel. Some cells tended to be long and stringy, fomed protuberances, tightly connected, and were built to map the grid structure. After that, the concection of cells increased, and ducts and pipes crisscrossed. The regular grid structure formed after 4 hours, and they could last for a long time, loosened and faded after 30 hours; However, the parental SKOV3 cells began to form the VM tubular structure. The amout of protuberances were less than the 7th generation sorted cells. The connection of cells formed slowly, the ducts and pipes peaked after 6 hours. But the most of ducts and pipes were irregular. 6 The result of the expression levels of Metalloproteinases-2(MMP-2) and Metalloproteinases-9(MMP-9) in both of them were detected by real-time PCR(RT-PCR) methodReal-time fluorescent quantitative PCR method was used to detected the expression of MMP-2 and MMP-9 on the sorted cells and the parental SKOV3 cells, using GAPDH as internal standard control. Normalization was performed fistly. According to the melting curve, because no obvious primer dimers formation in the process of gene amplification, it proved that the primer had high specificity. According to the amplification curve, Fluorescent signal had obvious logarithmic phase, and the amplification curve were smooth. Ct values of the target gene were between 20 and 30, which were of a normal range. RT-PCR test showed the expression levels of MMP-2 and MMP-9 gene in the 7th generation suspension cells were higher than those of the maternal cells(t=-21.96, P<0.001; t=-6.94, P=0.016). Part Three The Influence of different chemotherapy models on Signaling Pathway of Vasculogenic Mimicry in Human Epithelial Ovarian Cancer Xenograft of Nude Mice Methods: The 7th generation sorted cells were impanted subcutaneously into nude mice respectively to establish a model of human epithelial ovarian cancer. the mice were randomly divided into three groups as follows: MTD-DDP group, LDM-DDP group(intraperitoneal injection of 1mg/kg cisplatin daily for eighteen days) and control group, nude mice were treated with the equal saline solution. The CD31/Periodic acid Schiff(PAS) dual staining was performed to identify the structure of VM and microvascular. The number of VM and microvascular were calculated. Western Blotting was used to measure the expression levels of Eph A2 、 PI3 K 、 LN-5γ2 in Signaling Pathway of Vasculogenic Mimicry in three groups. Results: 1 Xenograft growth and mice general condition assessmentThe weight of 45 female nude mice was 18 to 20 g. The average weight was(18.96±0.62)g. Accroding the different chemotherapy methods, the average weight of MTD-DDP group was(19.03±0.67)g; The average weight of LDM-DDP group was(18.97±0.61)g; And the average weight of control group was(18.87±0.62)g. No statistical difference was observed in the three groups(F=0.263,P=0.770). But after chemotherapy, there was significant differrence in the three groups(F=32.697,P<0.001). Compared with the control group, the weight of MTD-DDP group was the lightest[(16.85±0.69)g], the weight of LSD-DDP group[(17.77±0.61)g] was between MTD-DDP group and control group[(18.65±0.51)g]. 2 The formation of human epithelial ovarian cancer model of xenograft of nude miceAfter the 7th generation sorted cells were impanted subcutaneously into nude mice respectively, all the mice(45/45, 100%) could form transplanted tumor in 10-14 d. With the growth of tumor, tumor volume can reach 150 mm3-200mm3. The tumor volume in MTD-DDP group was(176.27±15.46)mm3,(178.73±14.40)mm3 in LDM-DDP group and(174.60±16.02)mm3 in control group. No statistical difference was observed in the three groups(F=0.277,P=0.760). In the process of chemotherapy, the tumor in three groups was in a contimuous state. But after chemotherapy, there was significant differrence in the three groups(F=105.883, P<0.001). Compared with the control group, the tumor volume of LDM-DDP group was the smallest [(436.33±68.29) mm3], the tumor volume of MTD-DDP group[(650.93±69.60)mm3] was between LDM-DDP group and control group[(802.40±69.79)mm3]. 3 The comparison of the number of VM and microvasculars in three groups of nude mouse transplantation tumorAfter CD31/PAS staining, the microvasculars were consisted of endothelial cells. By immunohistochemical staining, the endothelial cell membrane was stained yellow and yellow or brown particles was seen in cytoplasm. PAS staining of extracellular matrix was positive. If vascular cavity was greater than eight red blood cell diameter, Vascular muscle layer thicker, this kind of vessles were considered as large vessle. VM was consisted of tumor cells, PAS staining of extracellular matrix was positive. Part of the red blood cells could be seen in this structure.The data were compared among three groups. There was significant difference(F=21.115, P<0.001) in comparison of the number of microvascular among the three groups. The number of microvasculars in LDM-DDP group were the least(10.87±3.70). The number of microvasculars in MTD-DDP group(17.33±4.44) was between LDM-DDP group and control group(21.20±5.23).There was also significant difference(F=10.551,P<0.001) in comparison of the number of VM among the three groups. The number of VM in LDM-DDP group were the least(13.53±3.96). The number of VM in control group(17.33±4.44) was between LDM-DDP group and MTD-DDP group(21.20±5.23). 4 The comparison of the expression of the proteins related to the signal transduction pathway for formation of VMThere was significant difference in comparison of the expression of Eph A2, PI3 K and LN-5γ2 among the three groups(F=66.130, 40.132, 32.921, all P values <0.001). The expression of Eph A2, PI3 K and LN-5γ2 in LDM-DDP group were the least, and their expression in control group were between LDM-DDP group and MTD-group. Conclusions: 1 The expression of CD133 in epithelia ovarian cancer tissues with VM is related to tumor malignancy degree. And it is an important index for the prognosis of epithelia ovarian cancer patients. 2 CD133+-marked cancer stem cells may be involved in the occurrence of VM in epithelia ovarian cancer. 3 Floating spheres sorted from SKOV3 cells could grow in serum free medium. 4 The 7th generation sorted cells through biological identification had the biological characteristics of cancer stem cells. 5 The 7th generation sorted cells had the greater ability on VM formation than the parental SKOV3 cells. And they could play an important role in the formation of VM formation. 6 Compared with MTD-DDP, LDM-DDP could kill the ovarian cancer cells effectively, reduced the formation of VM and microvasculars and suppressed gowth of the tumor. 7 Compared with MTD-DDP, LDM-DDP could inhibit the expression of Eph A2, PI3 K and LN-5γ2 related to the signal transduction pathway, and may influence the formation of VM. |
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