The Expression Of Negative Costimulatory Molecules B7-H1,B7-H3 And B7-H4 On Gallbladder Carcinoma Tissues As Well As Tumor Cells And Its Clinical Significance

Gallbladder carcinoma is a malignant tumor that originates in the gallbladder epithelium.Comparing with other digestive system malignancies,the incidence of gallbladder carcinoma is not high,but it is characterized by high degree of malignancy and rapid progress.Gallbladder carcinoma is often secondary to chronic cholecystitis or gallstones.Early symptoms are often be neglected and are easily covered by the primary disease.When the clinical symptoms are obvious,the carcinoma often progresses to the middle and late stages.Most patients have lost the opportunity for radical surgery at this time.Gallbladder carcinoma not sensitive to radiotherapy and chemotherapy.For the patients who are not suitable for the radical surgery,palliative surgery are mostly used.Its purpose is only to reduce or relieve the symptoms of jaundice or duodenal obstruction in patients,and it can not delay the progression of the carcinoma.These patients usually have a poor prognosis,as the overall survival are short and the 3-year survival rate is less than 20%.The establishment of tumor immunology closely combines the clinical diagnosis and treatment of tumors with basic immune research,which is of great significance for guiding immunotherapy of tumors.With the gradual deepening of research on the relationship between immune function andis carcinoma development,a series of tumor immunotherapy methods have entered the clinic.Antibodies,targeting EGFR/HER,such as cetuximab and trastuzumab,have achieved certain results in the immunotherapy of gallbladder carcinoma,but still have the characteristics of poor sensitivity and high drug resistance rate.To a certain extent,it restricts the promotion of immunotiherapy in patients with gallbladder carcinoma.Immunomodulators targeting negative costimulatory molecules are expected to improve this dilemma.Negative co-stimulatory molecules,also known as immunological checkpoints,down-regulate immune responses primarily by inhibiting immune cell function.The B7 family is considered to be a key regulator of tumor immune escape.Previous studies have indicated that B7 family members,such as PD-1,B7-H1,B7-H3,B7-H4 and B7-H7 and other immune checkpoint molecules,are abnormally expressed in vanous tumor tissues as the expression are associated with the prognosis.For this reason,the study of the expression pattern of B7 family molecules in gallbladder carcinoma tissues and its clinical significance are of great significance for immunotherapy of gallbladder carcinoma.In this study,the expressions of B7-H1,B7-H3 and B7-H4 in gallbladder carcinoma and adjacent normal tissues were detected by the tumor and adjacent tissue pathological sections of patients with gallbladder carcinoma.The expressions of the three molecules in gallbladder carcinoma were up-regulated.On this basis,the correlations between the three molecules and the corresponding clinicopathological features were analyzed.The correlations between the three molecules were also be analyzed.The clinical significance of the expression of the three molecules in gallbladder carcinoma was discussed.Then,a co-culture system was constructed using PBMC and the gallbladder carcinoma cell line GBC-SD and the blocking antibody was added to block the three molecules separately or in combination.Preliminary study on the negative regulation of B7-H1,B7-H3 and B7-H4 on immune cells in gallbladder cancer cells.Part ? The clinical significance of the expression of negative costimulatory molecules B7-H1,B7-H3 and B7-H4 in gallbladder carcinomaObjective:To compare the inhibition and blocking effects of B7-H1,B7-H3 and B7-H4 expressed by gallbladder carcinoma cells on PBMC in vitro.Methods:A total of 90 cases of gallbladder carcinoma pathological paraffin sections were collected from the first hospital affiliated to Soochow University from October 2014 to June 2017.The clinical data of age,sex,clinical stage and pathological grade were recorded.20 cases of adjacent tissues were collected as controls at the same time.Immunohistochemical SP method was used to detect the expression of B7-H1,B7-H3 and B7-H4 in gallbladder carcinoma tissues and the adjacent tissues.The differences in expression of B7-H1,B7-H3 and B7-H4 on gallbladder carcinoma and adjacent tissues were compared,and the correlation between the three and clinical pathological parameters was analyzed.The differences between the expressions of B7-H1,B7-H3 and B7-H4 in gallbladder carcinoma and that of adjacent tissues were eompared.The eorrelation between the three molecules and clinical pathological parameters was analyzed.Results:(1)The positive high expression rates of B7-H1,B7-H3 and B7-H4 in gallbladder carcinoma were 68.8%(62/90),40.0%(36/90)and 34.4%(31/90)respectively.The positive high expression rates in adjacent tissues were 30%(6/20),5.0%(1/20),5.0%(1/20).The level of B7-H1 expression was associated with distant metastasis of gallbladder carcinoma.The expression level of B7-H3 was correlated with infiltration depth and Nevin stage.It was also significantly correlated with lymph node metastasis,clinical stage and serum CA125 level.B7-H4 1s involved in the growth of gallbladder carcinoma in situ.(2)There was a statistical association between B7-H1 and B7-H4 expression.The expression of B7-H3 correlated with the expression of B7-H4.Spearman rank correlation analysis showed that the correlation intensity of B7-H3 and B7-H4 was higher than that of B7-H1 and B7-H4.Conclusions:(1)Negative costimulatory molecules B7-H1,B7-H3 and B7-H4 abnormally expressed in gallbladder carcinoma tissues.B7-H1 was involved in distant metastasis of gallbladder carcinoma.B7-H3 had a certain correlation in the progression of gallbladder carcinoma as B7-H4 increased the invasiveness of gallbladder carcinoma cells.(2)B7-H1 and B7-H4,B7-H3 and B7-H4 were positively correlated in expression.Comprehensive detection of the three expressions had certain clinical significance for assessing the prognosis of patients.Part ? Preliminary analysis of the inhibitory effects of B7-H1,B7-H3 and B7-H4 expressed in gallbladder carcinoma cells on peripheral blood lymphocytesObjective:To compare the inhibitory effects of B7-H1,B7-H3 and B7-H4 on PBMC in gallbladder carcinoma as well as their blocking effects.Methods:Immunohistochemical SP method and flow cytometry were used to detect the expression of B7-H1,B7-H3 and B7-H4 in gallbladder carcinoma cell line GBC-SD.GBC-SD which were treated with mitomycin and then were co-cultured with PBMC to simulate the immune microenvironment of gallbladder carcinoma.Single-molecule blocking groups,two-molecules joint blocking groups and three-molecules joint blocking group were established.The B7-H1 blocking antibody,the B7-H3 blocking antibody or the B7-H4 blocking antibody were added in the co-culture environment separately or in combination according to the groups.The absorbance of each group was measured by the CCK-8 method.The effects of B7-H1,B7-H3 and B7-H4 molecular blockade were compared and analyzed through the changes of PBMCs mass in each group.Results:(1)Both cellular immunohistochemistry and single-label flow cytometry showed that B7-H1,B7-H3 and B7-H4 were expressed in GBC-SD cells.(2)Blocking any of B7-H1,B7-H3 or B7-H4 increased the number of PBMC cells in the co-culture environment..The comparisons between the groups showed that the number of PBMC cells increased most significantly after blocking B7-H3,blocking B7-H3 and B7-H4 or blocking B7-H1,B7-H3 and B7-H4.Conclusions:(1)B7-H1,B7-H3 and B7-H4 played the immunoregulatory role in gallbladder carcinoma.(2)Vitro co-culture experiments showed that B7-H3 molecular blockade,B7-H3 and B7-H4 blockade as well as B7-H1,B7-H3 and B7-H4 combined blockade were significant.B7-H3 may be an effective target for the immunotherapy of gallbladder carcinoma.