B7­H3 promotes malignant progression of muscle­invasive bladder cancer.

The objective of the present study was to investigate the expression of B7 homologue 3 (B7­H3) in muscle­invasive bladder cancer (MIBC) tissues, evaluate its correlation with patient clinicopathological characteristics, and to explore the effect of B7­H3 on MIBC cells. B7­H3 expression levels in tumor tissues from 115 patients undergoing radical cystectomy for MIBC were detected by immunohistochemical staining, followed by analysis of the association with clinicopathological characteristics and survival. A B7­H3­silenced cell line was established by RNA interference (RNAi). Alterations in cell proliferation, cell cycle, migration and invasion were analyzed in vitro. The proteins associated with cancer cell behavior were detected by western blot analysis. In addition, we utilized a xenograft tumor assay in nude mice to test the inhibitory effect of B7­H3 shRNA on MIBC in vivo. The results revealed that, among the 115 patients, the B7­H3 expression level was significantly associated with an increased incidence of distant metastasis (P=0.014) and vascular invasion (P=0.031), whereas it was not statistically associated with sex, age, pathologic grade, tumor stage, recurrence and lymphatic metastasis. Overall survival (OS) and progression­free survival (PFS) were significantly worse for patients with high B7­H3 expression (P<0.001 and P<0.001, respectively) among the 115 MIBC patients. Suppression of B7­H3 significantly inhibited the proliferation, caused G2 phase arrest, as well as declined migration and invasion abilities in vitro. The protein expression of Ki67, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP2) and MMP9 were decreased in the T24/B7­H3 shRNA group compared with the control (P<0.05, respectively). Finally, we were able to inhibit tumor development by decreasing B7­H3 expression in vivo. In conclusion, a high expression level of B7­H3 in MIBC tissues is associated with a poor clinicopathological status and poor prognosis, and promotes the development of MIBC in vitro and in vivo. Thus, B7­H3 may be a potential novel biomarker for the poor prognosis of MIBC patients.

High expression of B7-H3 and CD163 in cancer tissues indicates malignant clinicopathological status and poor prognosis of patients with urothelial cell carcinoma of the bladder.

The objective of the present study was to investigate the association of B7-H3 expression and cluster of differentiation (CD)163+ tumor-associated macrophage (TAM) infiltration with clinicopathological parameters in urothelial cell carcinoma of the bladder (UCB), and to investigate their potential conjoint effects on progression of UCB. B7-H3 expression and CD163+ TAM infiltration in tumor specimens from 134 consecutive patients that underwent radical cystectomy for UCB were tested using immunohistochemistry, followed by statistical analysis. In these 134 patients, B7-H3 expression and CD163+ TAM infiltration in the bladder carcinoma tissues were significantly associated with an increased ratio of vascular invasion (P=0.009; P=0.012) and distant metastasis (P=0.015; P=0.038); however, they were not associated with gender, age, pathologic grade, tumor stage, recurrence or lymphatic metastasis. The results of χ2 test analysis indicated that CD163+ TAM infiltration and B7-H3 expression were positively correlated (χ2=20.714; P<0.001). Overall survival (OS) and progression-free survival (PFS) rates were significantly worsened by high B7-H3 expression (P=0.002; P=0.020). However, CD163+ TAM infiltration was not associated with OS or PFS rate. Notably, the OS and PFS rates in patients with high B7-H3 expression or high CD163+ TAM infiltration were significantly poorer than the patients with low B7-H3 expression (P<0.001; P<0.001) or low CD163+ TAM infiltration (P=0.022; P=0.017) in the subgroup of 115 patients with muscle-invasive bladder cancer. The results of the present study indicate that B7-H3 expression level could be used as an independent prognostic indicator following radical cystectomy for UCB and patients with high B7-H3 expression and high CD163+ TAM infiltration experience a poorer prognosis.

Increased B7-H4 expression during esophageal squamous cell carcinogenesis is associated with IL-6/STAT3 signaling pathway activation in mice.

B7-homolog 4 (B7-H4), one of the costimulatory molecules of the B7 family, has been reported to be widely expressed in multiple types of cancer tissues, and to be important in tumor progression and poor prognosis. However, the role of B7-H4 in esophageal precancerous lesions has not been elucidated yet. In the present study, a model of esophageal squamous cell carcinogenesis was established in 208 C57BL/6 mice by 4-nitroquinoline-1-oxide (4NQO) drinking water of mice, and the changes in the expression of B7-H4 during the whole pathological process were investigated. Hematoxylin and eosin staining results demonstrated that the pathological stage was exacerbated with the increase in time of 4NQO-mediated carcinogenesis induction, and the pathological features were similar to those observed in humans. Immunohistochemistry results revealed that B7-H4 expression was upregulated and positively correlated with pathological stage (P<0.0001) as well as with infiltration of cluster of differentiation (CD) 11b+ macrophage cells (P=0.0002). In addition, B7-H4 messenger RNA expression increased in the esophagi of model mice compared with that of control mice, which was positively associated with the gene expression of interleukin (IL)-6 and signal transducer and activator of transcription 3 (STAT3), according to the results of reverse transcription-quantitative polymerase chain reaction analysis. Similarly, B7-H4 protein expression was upregulated in the esophageal tissues of model mice in comparison with that of control mice, and was positively associated with IL-6 expression and STAT3 phosphorylation. In conclusion, the present data suggested that B7-H4 expression increased during esophageal squamous cell carcinogenesis in mice in association with IL-6/STAT3 signaling pathway activation.

TIM-3 promotes the metastasis of esophageal squamous cell carcinoma by targeting epithelial-mesenchymal transition via the Akt/GSK-3ß/Snail signaling pathway.

T-cell immunoglobulin and mucin domain-con-taining protein-3 (TIM-3), a negative regulator of antitumor immune response, has been demonstrated to be involved in the onset and progression of several types of malignancies. The present study aimed to determine whether and how TIM­3 plays such a role in esophageal squamous cell carcinoma (ESCC). TIM-3 expression was analyzed by immunohistochemistry and real­time fluorescence quantitative PCR (qRT­PCR) in ESCC and matched adjacent normal tissues. Functional experiments in vitro were performed to elucidate the effect of TIM­3 knockdown on the proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) in Eca109 and TE­1 cell lines. Our data revealed that TIM­3 expression was significantly elevated at both the mRNA and protein levels in ESCC tissues compared with the levels in the matched adjacent normal tissues (both P<0.001). TIM­3 expression was significantly associated with lymph node metastasis (P=0.008), tumor­node­metastasis (TNM) stage (P=0.042) and depth of tumor invasion (P=0.042). In addition, we observed a strong correlation between high TIM­3 expression and a worse overall survival of ESCC patients (P=0.001). Functional study demonstrated that TIM­3 knockdown markedly inhibited proliferation, migration and invasion of ESCC cell lines without affecting apoptosis. In addition, TIM­3 depletion was associated with downregulation of matrix metalloproteinase (MMP)-9 and upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, and with reversion of EMT, as reflected by higher levels of the epithelial marker E­cadherin and lower levels of the mesenchymal markers N­cadherin and vimentin. Further study found that TIM­3 depletion suppressed the signaling pathway involving p­Akt, p­GSK­3ß and Snail. Taken together, these results suggest that TIM­3 is a novel therapeutic target and prognostic biomarker for ESCC and promotes metastasis of ESCC by inducing EMT via, at least partially, the Akt/GSK-3ß/Snail signaling pathway.

Roles of coinhibitory molecules B7-H3 and B7-H4 in esophageal squamous cell carcinoma.

The coinhibitory molecules, B7-H3 and B7-H4, have shown negative regulation in T cell activation and tumor-associated macrophage (TAM) polarization in tumor-specific immunity. Here, we investigated the expression of B7-H3 and B7-H4 in human and murine esophageal squamous cell carcinoma (ESCC) tissues to define their clinical significance and mechanism in a tumor microenvironment. In the present study, B7-H3 and B7-H4 were expressed in 90.6 and 92.7 % samples, respectively. High B7-H3 and B7-H4 expression was associated with advanced TNM stage and lymph node metastasis (p < 0.05, respectively). Patients with both B7-H3 and B7-H4 high-expressed tumors had the poorest prognosis (26.7 months), whereas those with both low-expressed tumors had the best survival (56.7 months). B7-H3 and B7-H4 expression were inclined to be positively related to the infiltration intensity of Treg cells and TAMs (p < 0.05, respectively), and B7-H3 expression is negatively associated with the intensity of CD8(+) T cells (p < 0.05). In 4-nitroquinoline 1-oxide (4-NQO)-induced murine models, high B7-H3 expression could only be detected at carcinoma stage, but abnormal B7-H4 expression appeared a little earlier at dysplasia stage. In vitro studies revealed that knockdown of B7-H3 on tumor cells suppressed ESCC cell migration and invasion, while knockdown of B7-H4 could inhibit ESCC cell growth. Overall, B7-H3 and B7-H4 are involved in ESCC progression and development and their coexpression could be valuable prognostic indicators. Interference of these negative regulatory molecules might be a new strategy for treating ESCC.