| Objective: Death rates from ovarian cancer are higher than any other female reproductive system tumor. Among the newly diagnosed cases, about70 percent were at the late stage, who tolerated abdominal and pelvic organs metastasis, such as omentum, liver, mesenteric root, and so on. Abdominal and pelvic extensive planting, disseminated and massive ascites are the main clinic features of advanced ovarian cancer. There are lots of immune cells, especially macrophages, in normal peritoneal microenvironment. Macrophages, as the main cells of innate immunity, play an important role in maintaining normal abdominal microenvironment. During ovarian cancer cultivation spreading,macrophage cells and tumor cells carry out a certain interaction with each other, which can affect the malignant phenotype and poor outcome.We intend to discuss the effect of abdominal macrophages on ovarian cancer by detecting different macrophages subtypes of ovarian cancer patients at different stages and building macrophages model in vitro. Then we will go on to describe the effects of different macrophages subtypes on ovarian cancer malignant phenotype; and also to explain the immune mechanism of ovarian cancer abdominal metastasis. We will provide theoretical reference for the effects of abdominal immune microenvironment on the ovarian cancer stage and outcome; besides, we will also provide new molecular target for ovarian cancer immune treatment, as well as new clues to explain the occurrence and development of ovarian cancer, and furthermore we will come up with a theoretical foundation for the intervention strategies based on immunotherapy for ovarian cancer.Methods:1 To mark macrophages(M0), M1 subtype macrophages and M2 subtype macrophages with CD68, CD64 and CD163, and then test M2/M0 ratio ofascites or peritoneal washings of 10 patients with ovarian cancer and 17 patients with ovarian benign tumor.2 To separate peripheral blood mononuclear cells from healthy adults with the Ficoll density gradient method, and induce them to macrophages(M0)by GM-CSF. The study group was cultured in SKOV3 ovarian cancer cells supernatant, and the control group in 1640. On the 7th and 14 th day, the morphological changes of macrophages were observed under microscope respectively, and the expression of CD64-M1 and CD163-M2 was detected by flow cytometry.3 To separate peripheral blood mononuclear cells from healthy adults with the Ficoll density gradient method, and induce them to macrophages(M0)by GM-CSF. M0 was cultured in SKOV3 ovarian cancer cells supernatant. On the 14 th day, the subtypes changes of macrophages and the expression of CD80?CD86 and CD40 was detected by flow cytometry.4 To build mice orthotopic ovarian cancer model: to inoculate 5BALB/c A-nu mice's left kidney fat pad with SKOV3 cell(with luciferase marker) suspension. At the 2th and 4th week, we test M2/M1 ratio of 5 mice's ascites respectively, and observe the growth and metastasis of tumors. Before observation, 5 mice were injected with substrate(1.5mg/10g), and anesthetized with 1% pentobarbital. Then,we put them in fluorescence imaging system camera. Slide Book 4.0 software is used for analysis.Results:1 M2/M1 ratio was(0.75±0.31) vs.(2.14±0.37) vs.(3.01±0.45) in control group, I+II stage and III+IV stage respectively. III+IV stage M2/M1 ratio was higher than I+II stage; I+II stage M2/M1 ratio was higher than control group.There was statistically significant difference(P<0.05) in two groups.2 Macrophage cell morphology changed significantly in SKOV3 cell supernatant on the 7th day as cellular bulge increased and cells became elongated. The macrophages gradually restored on the 14 th day.3 The CD64-M1 macrophages were(24.2 + 2.9) % vs.(15.4±2.3) %respectively in study group and control group on the 7th day detected by flowcytometry. The CD163-M2 macrophages were(1.41±0.5)% vs.(2.05±0.7)%.The CD64-M1 macrophages were(1.88±0.9) % vs.(12.10±0.9) %respectively in study group and control group on the 14 th day. The CD163-M2 macrophages were(11.5±1.4) % vs.(3.30±1.3) %. At the early stage, M0 mainly distintegrated into CD64-M1 macrophages; later, M0 mainly distintegrated into CD163-M2 macrophages. This phenomenon seems not clear in the control group. There were statistically significant differences in two groups(P<0.05) at the same time in test group.4 With flow cytometry, the CD64-M1 macrophages percentage was M1/M0=(0.52±0.13) vs. M1/M0=(1.61±0.29) respectively on the 7th and14 th day in the study group, and the CD163-M2 macrophages percentage was M2/M0=(0.73±0.19) vs. M2/M0=(3.56±1.10). There were statistically significant differences(P<0.05).5 Detect the cytokine expression on macrophages surface by flow cytometry. 14 days after being cultured by SKOV3 cell culture supernatant,(1)CD80 was(7.50±1.10)% vs.(6.60±0.98)% on CD64-M1 macrophages and CD163-M2 macrophages respectively. There was no statistically significant difference(P>0.05) between the two groups.(2) CD86 was(10.53±1.52)% vs.(10.30±1.50)% on CD64-M1 macrophages and CD163- M2 macrophages respectively. There was no statistically significant difference(P>0.05)between the two groups.(3) CD40 was(10.22±1.10)% vs.(2.50±0.32) on CD64-M1 macrophages and CD163-M2 macrophages respectively. There was statistically significant difference(P<0.05) between the two groups.Expression of CD40 on CD163-M2 macrophage significantly reduced compared with CD64-M1 macrophage, while expression of CD80 and CD86 had no difference on CD64-M1 macrophages and CD163-M2 macrophages.6 Model of orthotopic ovarian cancer in mice: when BALB/c A-nu mice were injected with SKOV3 cell(with luciferase marker) suspension, all mice developed tumor in injection site; besides, mice tolerated significantly intraperitoneal dissemination and metastasis at the 4th week. 5 mice M2/M1 ratio were 1.45, 1.58, 1.66, 1.32, 1.78 respectively at the 2th week; 5 miceM2/M1 ratio were 3.25, 3.78, 2.68, 4.02, 3.17 respectively at 4th week. M2/M1 ratio was higher at the 4th week than the 2th week, and there was statistically significant difference(P<0.05). 5 mice fluorescence intensity(*10^6) were2.85, 1.86, 1.58, 2.31, 1.06 respectively at the 2th week; 5 mice fluorescence intensity(*10^6) were 6.32, 5.52, 4.87, 5.78, 3.88 respectively at the 4th week.The fluorescence intensity was significantly higher at the 4th week than at the2 th week, and there was statistically significant difference(P<0.05).Conclusion:1 There were macrophages in epithelial ovarian cancer patients' ascites;M1 subtype macrophages accounted for high proportion at the early stage,while M2 subtype macrophages accounted for high proportion at the terminal stage. It indicated that M0 macrophages mainly differentiate to M1 subtype macrophages initially, but with the changes of ovarian cancer patients' abdominal microenvironment, M0 macrophages mainly differentiate to M2 subtype macrophages. The high proportion of M2 subtype macrophages indicated that ovarian cancer patients were at the terminal stage.2 Macrophages immune response could be activated by SKOV3 cell culture supernatant, which was shown as the change of cell morphology, but the cell morphological recovery attributed to the occurrence of immune suppression with the passage of time.3 Macrophage differentiation could be promoted by SKOV3 cell culture supernatant; M0 macrophages mainly differentiate to CD64-M1 subtype macrophages initially at the early stage, but as time went by, M0 macrophages mainly differentiate to CD163-M2 subtype macrophages, while the common1640 medium could not promote this progress.4 SKOV3 cell culture supernatant could promote macrophages differentiation into different subtypes of macrophages. The expression of cytokines on cell surface of M2 subtype macrophages was down-regulation compared with M1 subtype macrophages, which suggested that cytokines played an important role in the process of activation and immune responses,and it seemed that cytokines was related to tumor immune escape.5 The model of Orthotopic ovarian cancer in mice proved that M1 subtype macrophages accounted for high proportion at the early stage, while M2 subtype macrophages accounted for high proportion at the terminal stage.M2 macrophages may create favorable conditions for ovarian cancer peritoneal dissemination and distant metastases. |
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