Expression And Clinical Significance Of Costimulatory Molecule B7-H3 In Lung Cancer

Primary lung cancer is the leading cause of cancer deaths in most industrialized countries, and 75-80% of lung cancer is non-small cell lung cancer (NSCLC). Although there is development of diagnosis and treatment in recent years, the five-year survival rate of patients still remains poor. The exploration of new methods in diagnosis and therapy has become a first priority. It might be a relatively new strategy to identify and correct abnormal immune status of patients. In the process of interaction between tumor cells and the immune system cells, tumor cells with more immunogenicity will be inhibited or killed, while tumor cells with weaker immunogenicity will be given the opportunity of proliferation. The result is that tumor cells with weaker immunogenicity will grow and diffuse out of control. As we all know, T cell activation requires dual signals, in addition to T cell antigen receptor (TCR) recognize the first signal provided by MHC-antigen complex on antigen presenting cells (APC), the second signal (i.e. the co-stimulatory signals) is expressed by interactions between T cells and APC costimulatory moleculars must also be catched. With the first signal only, T cells will remain no response or immune tolerance status, and even into the programmed death. These years the co-stimulatory signal in tumor immunity has become a hot topic. B7 co-stimulatory molecule family are the only co-stimulatory molecules that can send signals from APCs to T cells. B7-H3 is a new member of the B7 family. Although the receptor has not been identified and it may play contradictory role in immune response. Therefore, it is realistic to investigate whether B7-H3 is a new tumor marker or new therapeutic target in the diagnosis and treatment of lung cancer. Expression of CD133 was identified in various solid tumors such as glioma. breast cancer, melanoma and colon cancer, and expanding evidence highlights the roles of CD133 as a marker of cancer stem cells (CSCs) in these human tumours. The CD 133 positive tumour cells were capable of self-renewal, proliferation, and multi-lineage differentiation in vitro to recapitulate the original tumour phenotype, consistent with CSC properties. Many different studies showed that CD 133 positive cells were associated with the tumor immune escape, chemoresistance and radioresistance. therefore CD 133+cells maybe possible targets for immunotherapy. However, the role of CD133 playing in the cancer stem cells of lung cancer remains unclear.In the present study. B7-H3 expression in lung cancer was detected at mRNA and protein levels, then soluble B7-H3 in serum was detected and its diagnostic value was analyzed. The possible mechanism of antitumor effect of B7-H3 in vivo was approached by establishing a mouse tumor model. The relevance of B7-H3 and CD 133, B7-H4 in NSCLC was analyzed and whether CD 133 and B7-H3 was an independent risk factor of NSCLC patients was evaluated.Part I Expression and clinical significance of co-stimulatory molecule B7-H3 in non-small cell lung cancerObjective:To investigate the expression and clinical significance of co-signal molecule B7-H3 in five cell lines and tissues of NSCLC.Methods:We evaluated B7-H3 expression by flow cytometry, RT-PCR and western blot in five NSCLC cell lines. Specimens from 102 participants were stained immunohistochemically by mouse anti-human B7-H3 monoclonal antibody. Staining was quantified using Image-Pro Plus software, and mean integrated optical density (mIOD) was measured on each section.Results:We found the constitutive expression of B7-H3 in all five cell lines. The mIOD of B7-H3 in 102 NSCLC tissues was higher than that in 25 adjacent non-cancer (P<0.001) and in 24 benign lesion samples (P<0.001). The sensitivity and the specificity were 68.63% and 91.49% respectively. B7-H3 expression in well differentiated NSCLC was higher than those in the moderately and poorly differentiated tumors (P<0.05). The expression of B7-H3 was not related to gender, age, smoking history, histology and TNM staging (P>0.05).Conclusion:B7-H3 expression was identified both in mRNA and protein level in NSCLC tissue. Detection of B7-H3 protein may be helpful in the diagnosis of NSCLCPart II Detection and Quantitation of Circulating B7-H3, a Novel Marker for non-small Cell Lung CarcinomaObjective:Previous studies have suggested that membrane B7-H3 is aberrantly expressed in tumor cells. The aim of this study was to determine the expression level of soluble B7-H3 (sB7-H3), and subsequently evaluate the clinical significance of circulating B7-H3 in patients with NSCLC.Methods:Circulating B7-H3 was determined with ELISA, and the relationship to the clinical data was analyzed. Receiver operating characteristic curve analysis was performed to compare the sensitivity and specificity in the diagnosis in NSCLC. Kaplan-Meier analysis was performed to examine the relationship between the level of sB7-H3 and survival using SPSS software. Survival curves from the time of diagnosis were obtained by the method and compared by the Log rank test.Result:Circulating B7-H3 levels in pretreatment patients with NSCLC was higher than those in healthy volunteers (P<0.001), and decreased significantly after removal of tumor (P=0.039). Additionally, higher sB7-H3 was associated with higher stage (P=0.005), larger tumor size (P=0.001), distant metastasis (P=0.001) and lymph node metastasis (P=0.001). but not with sex (P=0.798), age (P=0.165) or histological subtype (P=0.334). An area under the curve (AUC) for all stages of NSCLC resulted from sB7-H3 (0.878) was higher than any one of the other tumor markers such as CA125 (0.657), CA153 9(0.628), CA199 (0.415) and CEA (0.627). Survival analysis showed that compared with sB7-H3low group(<30ng/ml), the sB7-H3high group (>30 ng/ml) had a shorter survival time in patients with NSCLC in stageⅢb or IV(P=0.007).Conclusions:Circulating B7-H3 was a highly sensitivity biomarker and elevated circulating B7-H3 indicated a poor prognostic clinical outcome in NSCLC. PartⅢInhibitive effect of B7-H3 on the growth of Lewis lung carcinomasObjective:To study the inhibitory mechanism of B7-H3 on the growth of Lewis lung carcinomas(LLC).Methods:Forty C57BL/6 mice were subcutaneously inoculated with LLC cells suspension (1×10’/mL) in the right armpit. The tumor-bearing mice were randomly divided into four groups:docetaxel. B7-H3 and docetaxel+B7-H3 were injected subcutaneously around the tumor, empty plasmid injected as control. Tumor sizes were valued at the first, third, seventh and fourteenth day after injection. B7-H3 expression was detected by immunohistochemistry and Western blot. The level of serum IL-12 was detected by ELISA.Results:Docetaxel and B7-H3 can inhibit tumor growth. The inhibitory rates in the groups of docetaxel, B7-H3 and docetaxel+B7-H3 were 32.98%,28.59% and 45.95% respectively. The apoptosis rate of tumor cells after treatment was significantly increased (P<0.01). B7-H3 expression in tumor tissue decreased in the groups of B7-H3 and docetaxel+B7-H3, although the difference was not significant (P> 0.05). The level of serum IL-12 increased in the groups of B7-H3 and docetaxel+B7-H3 than that in control group (P< 0.05).Conclusion:B7-H3 can inhibit the growth of murine Lewis lung cancer, and promote cytotoxic T cells induced by IL-12 leading to anti-tumor immune response.PartⅣExpression and clinical significance of B7-H3 and CD133 in non-small cell lung cancerObjective:To investigate the expression and clinical significance of B7-H3 and CD133 in tissues of NSCLC.Methods:Specimens from 102 participants were stained immunohistochemically by mouse anti-human B7-H3 and CD 133 monoclonal antibody. Staining was quantified using Image-Pro Plus software, and integrated optical density was measured on each section. Results:We found that the expression of B7-H3 or CD 133 was higher than that in benign lesions or non-cancer tissues respectively (P<0.001). The sensitivity and the specificity of B7-H3 expression in NSCLC were 68.63% and 91.49% respectively, and those of CD133 were 54.90% and 97.87%. CD133-positive was related to poor differentiation of tumor cells (P=0.02). B7-H3 positive or CD133 negative was significantly associated with survival by the COX regression analysis (p<0.001).Conclusion:B7-H3 is overexpressed in NSCLC tissue and may be an important target for diagnosis or therapy of the disease. CD133 was related to tumor cell differentiation, and the expression was negatively correlated with B7-H3 expression. CD 133 positive or B7-H3 negative was an independent risk factor of NSCLC patients.Part V Expression and clinical significance of B7-H3 and B7-H4 in non-small cell lung cancerObjective:Functions of B7-H3 and B7-H4 regarding T-cell activation have been reported recently. Little is known about the significance of B7-H3 and B7-H4 expression in human NSCLC. We investigate the expression and clinical significance of B7-H3 and B7-H4 in tissues of NSCLC in the present study.Methods:Specimens from 102 participants were stained immunohistochemically by mouse anti-human B7-H3 and B7-H4 monoclonal antibody. Staining was quantified using Image-Pro Plus software, and integrated optical density (IOD) and the number of tumor infiltrating lymphocytes (TILs) were measured on each section.Results:In tumor tissues, expression of B7-H3 and B7-H4 was found both on the cell membrane and in the cytoplasm. And the expression of B7-H3 or B7-H4 was higher than that in benign lesions or non-cancer tissues respectively (P<0.001). The sensitivity and the specificity of B7-H3 expression in NSCLC were 68.63% and 91.49% respectively, and those of B7-H4 were 67.60% and 93.60%. The mean number of TILs in B7-H3-positive group was 27.36±2.48, lower than that in B7-H3-negative group,34.09±6.12 (P=0.04). The mean TILs in B7-H4-positive group was 28.68±2.79, and it was lower than that in B7-H4-negative group,31.09±5.45 (P>0.05). B7-H3 positive contributed to survival of participants by COX regression analysis (P<0.01).Conclusion:Either B7-H3 or B7-H4 is overexpressed in NSCLC tissue and may be an important target for diagnosis or therapy of the disease. B7-H3 negative was an independent risk factor of NSCLC patients.