The Correlation Between NK Cells And Angiogenesis In Ovarian Cancer

Research background and Objective:Epithelial ovarian cancer (hereinafter referred to as ovarian cancer) is a common malignant tumor in female reproductive system, which has the characteristics of high mortality and poor prognosis, the five-year survival rate is less than 30%. In the ovarian cancer, blood vessels is rich, tumor growth is rapid, the degree of malignancy is high. At present, anti-tumor angiogenesis therapy has been introduced conventional treatment for ovarian cancer. There were two kinds of drugs (recombinant human endostatin, bevacizumab), and they have achieved good effect. However, in the face of the current grim situation of ovarian cancer, it is the key to improve the survival by finding a reasonable and effective therapeutic target in the molecular level.Traditionally, natural killer cells can kill tumor cells directly without the identification of tumor-specific antigen. Thus, it has a important status in the anti-tumor immune therapy [1-3]. However, the study[4] reported that, according to the different expression of CD56 on the surface of cell membrane, NK cells could divided into two subgroups:(1) CD56dimCD16bright, accounting for 90 percent of NK cells, which main function is cytotoxic effect; (2) CD56brightCD16dim, accounting for 5-10 percent of NK cells, and its main function is proangiogenesis. It can secrete proangiogenesis cytokines (VEGF,PIGF, IL-8), and play its role in promoting tumor angiogenesis by these factors. In the process of the growth of the placenta, natural killer cells is CD56supeibrightCD16dim, which promot angiogenesis is more obvious, so it is known as Decidual natural killer cells (dNK).Therefore, the purpose of this study was to verify the correlation between ovarian cancer infiltrating NK cells and tumor angiogenesis, and to provide new ideas for the treatment of ovarian cancer.Methods:1. We got 31 ovarian cancer tissues and 30 normal ovarian tissues from the third affiliated hospital of Kunming Medical University (Tumor Hospital of Yunnan province) between January 2015 and January 2016. Those cases did not accept radiotherapy or chemotherapy. We collected clinical features (age, histopathological type, histological grade, stage). CD56brightCD16dimNK cells in ovarian cancer and normal ovarian tissues was tested by flow cytometry, and we detected VEGF, MVD, a-SMA by immunofluorescence. Then we analysised the relationship of NK cells,VEGF, MVD and a-SMA between clinical pathological features in ovarian cancer.2. On the other hand, we established a tumor-bearing model of ovarian cancer of SKOV3, then randomly divided into three groups. We used two methods to treat tumor(saline, recombinant human endostatin, bevacizumab). Two weeks later, the cancer tissue, adjacent tissue, peri cancerous tissue were texted by flow cytometry, and the VEGF, MVD and a-SMA were detected by immunofluorescence. Finally we analyzed the relationship between tumor-infiltrating NK cells and tumor angiogenesis.3. Statistical methods:experimental data was analysised by SPSS 17.0 software package. Data is represented by mean±standard deviation (x±s). The Kolmogorov Smirnov was used to test normality test, and Levene’s test was used to test homogeneity of variance. Multiple samples were compared by one factor analysis of variance, x2 test or Fisher exact probability method, and The t or t’test with different samples was adopted for comparison between groups. Pearson correlation analysis was used to compute the results. a=0.05(bilateral) as the test level, P<0.05 as a statistically significant.Results:1. Expressed of VEGF, MVD, and a-SMA in normal ovarian and ovarian carcer tissues1) Expressed of VEGF in normal ovarian and ovarian cancer tissuesVascular endothelial growth factor (VEGF) was mainly localized in cell plasma or cell membrane, which is red. In ovarian cancer tissues, VEGF positive expression rate was 87.1%(27/31). In normal ovarian tissues, VEGF positive expression rate was 23.33%(7/23). VEGF expression intensity in ovarian cancer tissues was significantly higher than that in normal ovarian tissues(P=0.000). The expression of VEGF in ovarian cancer was not related to the age of the patients, pathological types, histological grade, clinical stage, respectively(P>0.05).2) Expressed of a-SMA in normal ovarian and ovarian carcer tissuesa-SMA was mainly localized in interstitial cytoplasm, which is red. In ovarian cancer tissues, a-SMA positive expression rate was 74.19%(23/31). In normal ovarian tissues, a-SMA positive expression rate was 26.7%(8/30). a-SMA expression intensity in ovarian cancer tissues was significantly higher than that in normal ovarian tissues(P=0.000). The expression of a-SMA in ovarian cancer was not related to age, pathological types, histological grade, clinical stage, respectively(P>0.05).3) Expressed of MVD in normal ovarian and ovarian cancer tissuesMVD is mainly localized in cytoplasm, which is green. In ovarian cancer tissues, MVD value was 21.95±3.37. In normal ovarian tissues, MVD value was 8.82±1.38. MVD value intensity in ovarian cancer tissues was significantly higher than that in normal ovarian tissues(P=0.000). The expressed of MVD in ovarian cancer was not related to age, the pathologic type, clinical stage and lymph node metastasis, but it was related to the histological grade. MVD value was highest at the low differentiated tissues, and there was a difference between the middle differentiation and low or high differentiation, there was no differentiation between the high and low differentiation.2. NK cells percentage of lymphocytes cells in normal ovarian and ovarian cancer tissuesIn ovarian cancer tissues, CD56brightCD16dimNK cells was (71.88±11.76)%. In normal ovarian tissues, CD56brightCD16dimNK cell was (19.22±11.64)%, the difference between the two groups has statistical significance(P=0.000). CD56brightCD16dimNK cells in ovarian cancer tissues was significantly higher than that in normal ovarian tissues. CD56brightCD16dimNK cells was related to lymph node metastasis and clinical stage, CD56brightCD16dimNK cells ratio with lymph node metastasis or Ⅰ, Ⅱ phase is less than no lymph node metastasis or Ⅲ, Ⅳ phase, but it has no correlation with age, histological grade and histological type.CD56brightCD16dimNK cells and the expressed of MVD in ovarian cancer was correlated(r=0.621, P=0.022), and it was a positive correlation.3. The proportion of NK1.1+ cells of lymphocytes in tumor-bearing model of ovarian cancer.In the normal saline group, NK1.1+cell was (4.34±1.56)%. In the recombinant human endostatin group, NK1.1+cell was (6.22±0.29)%. In the bevacizumab group, NK1.1+cell was (6.08±1.45)%. Three groups were compared, there were difference (P=0.028、0.036) between the normal saline group and the recombinant human endostatin group or the bevacizumab group. There was not difference (P=0.883) between the recombinant human endostatin group and the bevacizumab group. Compared with the normal saline group, the number of NK1.1+cells increased significantly in the recombinant human endostatin group or the bevacizumab group.Conclusion(s):1. CD56brightCD16dimNK cells in ovarian cancer tissues is significantly higher than that in normal ovarian tissues. CD56brightCD16dimNK is related to lymph node metastasis and clinical stage, but it has no correlation with age, histological grade and histological type.2. There is a positive correlation between CD56brightCD16dimNK cells and the expression of MVD in ovarian cancer. It may be a new target for the biological treatment of ovarian cancer.3. bevacizumab and recombinant human endostatin can increase the proportion of NK1.1+ cells in lymphocyte.