Clinical Significance And Contribution Of The Co-stimulatory Molecules B7-H1 And B7-H3 In Human Esophageal Cancer

In recent years, the study of the tumor microenvironment and immune escape demonstrated that a cohort of important molecules involved in immune regulation, namely immune checkpoints, such as the B7 family of negative co-stimulatory molecules, B7-H1, B7-H3, B7-H4, CTLA-4 and Tim-3, and etc., could be abnormally expressed in numerous human cancers as well as infiltrating immune cells, those molecules constitute an important part of tumor microenviroment, and play an important role in tumor immune escape and showed close associations with patients’ clinical parameters and postoperative prognoses. On one hand, they can negatively regulate the T cell mediated anti-tumor immunity, and on the other hand, these molecules expressed by the cancer cells themselves can regulate the biological function of the cancer cells, thereby contribute to the oncogenesis and cancer progression. As of know, the clinical significance and contribution of the abnormal expression of costimulatory molecules B7-H1 and B7-H3 in esophageal cancer still remains to be studied. Firstly, we collected the esophageal cancer tissues samples and performed the retrospective study to investigate the expression levels of B7-H1 and B7-H3 in cancer tissues as well as in human esophageal cancer cells lines, respectively, and to analyze the associations with patients’ clinical parameters, prognoses, EMT status and T-cell infiltration. Secondly, we further investigated the abnormal expression of B7-H3 in esophageal cancer cells and its contribution to the biological features of cancer cells themselves.Our present results showed that:(1) The co-stimulatory molecule B7-H1 could be highly expressed in esophageal cancer tissues as well as esophageal cacner cell lines, while it was weakly expressed in adjacent normal tissues. The immunostaining of B7-H1 molecule was mainly located on the cytoplasm and membrane of the cancer cells.(2) The expression level of B7-H1 in esophageal cancer tissues was positively and significantly assocated with tumor invasion(P=0.0261). The overall survival rate of the cancer patients with higher B7-H1 was significantly poorer than that of the cancer patients with lower B7-H1 expression(HR=2.157, 95% CI: 1.017-4.577, P=0.0452).(3) The immunostaining of B7-H1 could also be found in the nuclei of esophageal cacner cells in cancer tissues, and its expression level was positively and significantly associated with tumor invasion(P=0.0331), while it was not significantly associated with the postoperative prognoses of the cancer patients(P=0.6755).(4) The combination of E-cadherin and Vimentin expression was used to evaluate the EMT status, and we found that in T(3+4) subgroup, the incidence of EMT was significantly higher than that in T(1+2) subgroup(P=0.0097), and in the M1 subgroup, the incidence of EMT was significantly higher than that in T(1+2) subgroup(P=0.0253), the survival analysis showed that the overall survival rate of the patients in the EMT group was significantly poorer than that in the wide type group(HR=2.470, 95% CI: 1.971-2.970, P=0.0278). When we selected gender, age, tumor size, TNM stage and EMT status in the COX model analysis, we could found that the EMT status was an independent prognostic factor for predicting prognosis of esophageal cacner patients(HR=2.306, 95% CI: 1.103-4.824, P=0.026).(5) We also found that in EMT subgroup, the ratio of higher B7-H1 expression was significantly higher than that in wide type subgroup(P=0.0314), the overall survival rate of the patients with B7-H1 high expression and EMT status, was siginificantly poorer than that with B7-H1 low expression and without EMT(HR=2.876, 95% CI: 1.182-6.997, P=0.0199).(6) B7-H3 was highly expressed on the esophageal cancer cell lines as well as cancer tissues, while it was weakly expressed in normal esophageal tissues, the immunostaining of B7-H3 was mainly located on the cytoplasm and membrane of esophageal cancer cells.(7) The expression level of B7-H3 in human esophageal cancer tissues was positively and significantly associated with tumor invasion depth(P=0.0250), the overall survival rate of the patients with high B7-H3 expression was significantly poorer than that of the patients with low B7-H3 expression(HR=1.564, 95%CI: 1.040-2.351, P=0.0318).(8) The lentivirus transfection and RNA interference technology, we constructed the B7-H3-knockdown model in Eca-109 cells, and confirmed by using PCR and Western blot at m RNA and protein levels respectively.(9) We further used colony formation study, CCK-8 assay, scrape assay and Transwell assay to reveal that the knockdown expression of B7-H3 in Eca-109 cells could significantly inhibit the colony formation ability, cell proliferation, migration and invasion.In summary, we demonstrated the clinical significance of negative costimulatory molecules B7-H1 and B7-H3 expression in human esophageal cancer and its biological function. These two molecules could be involved in the esophageal cancer progression via negatively regulating the T lymphocytes infiltration in cancer tissues, and promoting the cancer cell proliferation, migration and invasion. The negative costimulatory molecules B7-H1 and B7-H3 can be used as important biological marker for the diagnosis and the prognostic prediction for human esophageal cancer. Our present study provided the experimental basis for investigating the tumor immunity in the progression of human esophageal cancer, and provided a potential target for the immunotherapy of this malignancy. The value of the clinical application of these two molecules in esophageal cancer merits further investigation.