| Objective:Esophageal carcinoma is the occurrence of esophageal epithelial malignancies and the world’s seventh largest cancer. The incidence and mortality of esophageal carcinoma also make one of the higher countries in the world. Looking for early biomarkers of esophageal carcinoma become hot spot. Tumor immunotherapy has been proven to be the fourth effective anti-tumor strategy following surgery, radiotherapy, chemotherapy for malignancies. In recent years, B7-H3, as a new member of B7 immunoregulatory superfamily, overexpressed in multiple tumor types, is significantly correlated with biological characateristics of tumor and considered to be a new tumor marker and potential theraputic target. TAM plays an important role in tumorigenesis, growth, invasion and metastasis in tumor microenvironment. Specific tumor microenvironment forced the tumor macrophage phenotype toward the direction that conducive to the evolution of tumor development, named M2 TAM. Based on the important role of M2 TAM in tumor progression and metastasis, it has become a new target for anti-cancer therapy. Our study aims at examing the expression of B7-H3, CD68 and CD163 protein in human esophageal carcinoma tissues and the corresponding and determinng its correlations with clinicopathological data in paitients. In addtion, we analyzed the expression of B7-H3 in esophageal carcinoma cells lines of Eca-109, TE-1, TE-13, KYSE-170. RNA interference was used to investigate the effect of B7-H3 in Eca-109 on the reversal of macrophages polarization.Methods:1 Real-time PCR was used to detect the expression of HLA-DRa and CD163 m RNA in esophageal carcinoma tissues and the corresponding adjacent tissues.2 Immunohistochemical staining was used to detect the expression of B7-H3, CD68 and CD163 protein in esophageal carcinoma tissues and the clinical factors of esophageal carcinoma were analyzed.3 Real-time PCR and Western blot were used to detect the m RNA and protein expression of B7-H3 in esophageal carcinoma cell lines of Eca-109, TE-1, TE-13, KYSE-170.4 Real-time PCR and Western blot were used to detect the expression of B7-H3 m RNA and protein in Eca-109/B7-H3 and Eca-109/B7-H3 si RNA.5 Real-time PCR was used to detect the expression of HLA-DRa and CD163 m RNA in macrophages co-cultured with Eca-109/B7-H3 and Eca-109/B7-H3 si RNA.6 Flow cytometry was used to detect the expression of HLA-DR, CD86, CD163 and CD206 protein in macrophages co-cultured with Eca-109/B7-H3 and Eca-109/B7-H3 si RNA.7 ELISA was used to detect the secretion of IL-6 and M-CSF in Eca-109/B7-H3 and Eca-109/B7-H3 si RNA.8 Western blot was used to detect the expression of P-STAT3, STAT3, P-P65, and P65 protein in macrophages co-cultured with Eca-109/B7-H3 and Eca-109/B7-H3 si RNA.9 Real-time PCR was used to detect the expression of HLA-DRa and CD163 m RNA in macrophages adding AG490 or PDTC before co-cultured with Eca-109/B7-H3 and Eca-109/B7-H3 si RNA.10 Flow cytometry was used to detect the expression of HLA-DR, CD86, CD163 and CD206 protein in macrophages adding AG490 or PDTC before co-cultured with Eca-109/B7-H3 and Eca-109/B7-H3 si RNA.Results:1 The results of real-time PCR showed that, in 30 cases of esophageal carcinoma tissues, the expression of HLA-DRa(2.128±0.127) and CD163(5.696±0.136) m RNA was significantly higher than the corresponding adjacent tissues(1.000±0.000)(P<0.01), the expression of CD163 m RNA in esophageal carcinoma tissues was significantly higher than HLA-DRa(P<0.001).2 Immunohistochemical staining discovered that, in 70 cases of esophageal carcinoma tissues, B7-H3 protein was mainly localized in the cytoplasm and membrane of tumor cells, the positive rate was 98.6%(69/70), significantly higher than the corresponding adjacent tissues 7.1%(5/70), χ2 =117.41, P < 0.001; CD68 and CD163 protein mostly gathered in the cytoplasm and cell membrane of macrophages, and was spotty or patchy distribution. The positive rate of CD68 was 91.4%(64/70), significantly higher than the corresponding adjacent tissues of 18.6%(13/70), χ2 =75.07, P<0.001. The expression of CD163 protein was found in 84.3%(59/70), while it was almost negative in adjacent tissues. The abnormal expression of B7-H3 and CD68 was correlated with tumor invasion depth(P < 0.05). The expression of CD163 was correlated with tumor size and tumor invasion depth(P<0.05).3 Spearman correlation analysis showed that, in 70 cases of esophageal carcinoma tissues, the expression of B7-H3 protein was positively correlated with CD68(R=0.300, P < 0.05)and CD163(R=0.288, P < 0.05). Kaplan-Meier survival analysis showed that, the survival rate with high expression of B7-H3, CD68 and CD163 in esophageal carcinoma patients was significantly lower than that in patients with low expression(P<0.05).4 The results of real-time PCR showed that, Eca-109(0.449±0.037), TE-1(0.488±0.027), TE-13(1.000±0.000) and KYSE-170(0.297±0.016) all expressed B7-H3 m RNA, the highest expression was in TE-13. Western blot analysis showed that, B7-H3 protein was moderated expression in TE-1(0.865±0.023), low expression in KYSE-170(0.443±0.018), and high expression in Eca-109(1.129±0.010) and TE-13(1.146±0.017), while the expression of Eca-109 and TE-13 was not different significantly(P>0.05).5 The results of real-time PCR showed that, the expression of B7-H3 m RNA in Eca-109/B7-H3 was significantly higher than Eca-109/control [(1.624±0.055)vs(0.932±0.037), P<0.01] and Eca-109 group [(1.624±0.055) vs(1.000±0.000), P<0.01]. The expression of B7-H3 m RNA in Eca-109/ B7-H3 si RNA was significantly lower than Eca-109/control si RNA [(0.295± 0.067)vs(0.929±0.037), P<0.01] and Eca-109 group [(0.295±0.067)vs(1.000± 0.000), P<0.01]. Western blot analysis showed that, the expression of B7-H3 protein in Eca-109/B7-H3 was significantly higher than Eca-109/control [(0.164±0.007)vs(0.092±0.002), P<0.001] and Eca-109 group [(0.164±0.007) vs(0.091±0.001), P<0.001]. However, the expression of B7-H3 protein in Eca-109/B7-H3 si RNA was significantly lower than Eca-109/control si RNA [(0.028±0.001)vs(0.091±0.001), P < 0.001] and Eca-109 group [(0.028± 0.001)vs(0.091±0.001), P<0.001].6 The results of real-time PCR showed that, compared with macrophages cultured alone, the expression of CD163 m RNA increased in macrophages that co-cultured with Eca-109 for 48 h [(1.645±0.062)vs(1.000±0.000), P<0.001], and the expression of HLA-DRa m RNA decreased [(0.865±0.065) vs(1.000±0.000), P<0.05], indicating that macrophages were induced to M2 type after Eca-109 cells co-cultured with macrophages. After Eca-109/B7-H3 co-cultured with macrophages for 48 h, the expression of CD163 m RNA in macrophages was significantly higher than normal co-culture group [(2.606±0.128)vs(1.645±0.062), P < 0.001], the expression of HLA-DRa m RNA was significantly lower than normal co-culture group [(0.537±0.083)vs(0.865±0.065), P<0.01], indicating that Eca-109/ B7-H3 can promote macrophages to M2 type. After Eca-109/B7-H3 si RNA co-cultured with macrophages for 48 h, the expression of HLA-DRa m RNA in macrophages was significantly higher than normal co-culture group [(1.753± 0.073)vs(0.865±0.065), P<0.001], the expression of CD163 m RNA was significantly lower than normal co-culture group [(0.544±0.049)vs(1.645± 0.062), P < 0.001], indicating that Eca-109/B7-H3 si RNA can reverse macrophages to M1 type.7 The results of flow cytometry showed that, compared with macrophages cultured alone, the expression of CD163 [(36.400±1.353)vs(23.367±1.201),P<0.001]and CD206[(26.367±1.210)vs(23.733±0.971),P<0.05]protein increased in macrophages that co-cultured with Eca-109 for48h,and the expression of HLA-DR[(20.333±0.833)vs(23.400±0.755),P<0.01]and CD86[(18.500±0.557)vs(21.633±0.833),P<0.01]protein decreased,indicating that macrophages were induced to M2 type after they co-cultured with Eca-109 cells.After Eca-109/B7-H3 co-cultured with macrophages for48h,the expression of CD163[(630.333±1.528)vs(36.400±1.353),P<0.001]and CD206[(36.200±1.114)vs(26.367±1.210),P<0.001]protein in macrophages was significantly higher than normal co-culture group,the expression of HLA-DR[(11.167±0.850)vs(20.333±0.833),P<0.001]and CD86[(10.867±0.611)vs(18.500±0.557),P<0.001]protein was significantly lower than normal co-culture group,indicating that Eca-109/B7-H3 can promote macrophages to M2 type.After Eca-109/B7-H3 si RNA co-cultured with macrophages for 48h,the expression of HLA-DR[(241.667±1.528)vs(20.333±0.833),P<0.001]and CD86[(64.567±0.850)vs(18.500±0.557),P<0.001]protein in macrophages was significantly higher than normal co-culture group,the expression of CD163[(17.467±0.907)vs(36.400±1.353),P<0.01]and CD206[(13.533±1.069)vs(26.367±1.210),P<0.001]protein was significantly lower than normal co-culture group,indicating that Eca-109/B7-H3 si RNA can reverse macrophages to M1 type.8 The results of ELISA showed that, IL-6 [(38.713±1.991 pg/ml) vs(26.267±1.560 pg/ml), P < 0.01] and M-CSF [(85.380±6.052 pg/ml)vs(59.600±4.733 pg/ml), P < 0.01]secretion in Eca-109/B7-H3 was significantly higher than Eca-109 cells. However, IL-6 [(15.033±1.427 pg/ml)vs(26.267±1.560 pg/ml), P < 0.001] and M-CSF [(28.367±4.747 pg/ml)vs(59.600±4.733 pg/ml), P<0.01] secretion Eca-109/B7-H3 si RNA was was significantly lower than Eca-109 cells.9 Western blot analysis showed that, after Eca-109/B7-H3 co-cultured with macrophages for 48 h, the expression of P-STAT3 [(0.219±0.010) vs(0.097±0.008), P<0.001] and STAT3 [(1.539±0.026)vs(1.876±0.012), P<0.001] protein in macrophages was significantly higher than normal co-culture group. After Eca-109/B7-H3 si RNA co-cultured with macrophages for 48 h, the expression of P-P65 [(0.257±0.007)vs(0.173±0.002), P<0.001] and P65 [(0.347±0.007)vs(0.292±0.009), P < 0.01] protein in macrophages was significantly higher than normal co-culture group.10 The results of real-time PCR showed that, macrophages adding AG490 before co-cultured with Eca-109/B7-H3 for 48 h, the expression of CD163 m RNA was significantly lower than those without adding inhibitor group [(1.918±0.088)vs(2.606±0.128), P<0.01], the expression of HLA-DRa m RNA was slightly higher than those without adding inhibitor group [(0.624±0.079)vs(0.537±0.083), P>0.05], indicating that macrophages adding AG490 can suppress the effect of Eca-109/B7-H3 promoting macrophages to M2 type. The macrophages adding PDTC before co-cultured with Eca-109/B7-H3 si RNA for 48 h, the expression of HLA-DRa m RNA was significantly lower than those without adding inhibitor group [(1.310±0.056) vs(1.753±0.073), P<0.01], the expression of CD163 m RNA was significantly higher than those without adding inhibitor group [(1.185±0.134)vs(0.544±0.049), P < 0.01], indicating that macrophages adding PDTC can suppress the effect of Eca-109/B7-H3 si RNA promoting macrophages to M1 type.11 The results of flow cytometry showed that, macrophages adding AG490 before co-cultured with Eca-109/B7-H3 for 48 h, the expression of CD163 [(70.367±0.862)vs(159.333±2.517), P<0.001] and CD206 [(60.967± 1.301)vs(149.667±2.082), P<0.001] protein in macrophages was significantly lower than those without adding inhibitor group, the expression of HLA-DR [(60.433±1.026)vs(29.500±0.985), P<0.001] and CD86 [(24.767±0.551) vs(11.867±0.651), P < 0.001] protein was significantly higher than those without adding inhibitor group, indicating that macrophages adding AG490 can suppress the effect of Eca-109/B7-H3 promoting macrophages to M2 type. The macrophages adding PDTC before co-cultured with Eca-109/B7-H3 si RNA for 48 h, the expression of HLA-DR [(140.333±1.528) vs(238.667±3.512), P<0.001] and CD86 [(30.567±0.709)vs(49.433±0.709), P<0.001]protein in macrophages was significantly lower than those without adding inhibitor group, the expression of CD163 [(31.700±0.755)vs(12.700±0.819), P<0.001] and CD206 [(25.500±0.656)vs(13.100±0.557), P<0.001] protein was significantly higher than those without adding inhibitor group, indicating that macrophages adding PDTC can suppress the effect of Eca-109/B7-H3 si RNA promoting macrophages to M1 type.Conclusion:1 B7-H3 and M2 TAM are high expression in esophageal carcinoma tissues, and correlated with tumor progression and prognosis. They may become a promising indicator of monitoring esophageal carcinoma progress.2 Targeted interference B7-H3 gene can reverse M2 type polarization of macrophages in vitro, suggesting that B7-H3 gene may be involved in the regulation of esophageal carcinoma immune cells in the tumor microenvironment. It may serve as a potential therapeutic target for tumor immunotherapy. |
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