| Background and objective:Osteosarcoma is one of the most common malignant tumors,which originates from bone and has a high tendency to metastasize.The worldwide incidence of osteosarcoma is 2-3/million/year,and the five-year survival rate of osteosarcoma patients has remained 60%in the last decades.This rate merely reached 25%for those who have metastatic or relapsed issues.Abundant studies demonstrate that tumor progression is associated with abnormal immune response,yet the interaction between them is poorly understood.Currently,it has been proved that immune evasion is a key cause of tumor progression,while its molecular mechanism in osteosarcoma is still obscure.As an important part of immune system,increasing evidences indicate that immune checkpoints are crucial for maintaining and adjusting self-tolerance.However,these immune checkpoints could also allow the immune tolerance for tumors,and therefore contribute to immune evasion of tumors.Tumor cells can preferentially up-regulate the expression of inhibitory immune checkpoint,therefore suppress the anti-tumor activity and complete immune evasion.Immune checkpoints CTLA-4 or PD-1/PD-L1 have shown co-inhibitory impact on immune system,their overexpression in malignant tumors usually correlate with poor prognosis.To date,blockade inhibitors targeting these immune checkpoints have been utilized in immune treatments against many tumors and showed clinical efficacy.Nonetheless,such alternatives didn't show promising results in osteosarcoma treatments,indicating the need of investigating novel immune checkpoints.B7-H3 is aberrantly overexpressed in multiple malignant tumors,and promotes the proliferation and metastasis of tumor cells.By investigating the impact of B7-H3 on osteosarcoma and the correlations between B7-H3 and clinical features of patients,it might be able to help understand its role in progression and immune evasion of osteosarcoma.By conducting this study,we hope to find more factors affecting the progression and prognosis of osteosarcoma,and the possible mechanism of osteosarcoma progression,in order to provide evidence to support the development of immunotherapy against osteosarcoma.Methods:The mRNA expression of B7-H3 and some common immune checkpoints in human osteosarcoma tissues was analyzed by using the data of GSE87437 chip,and the result was confirmed by performing immunohistochemistry staining in 104 clinical specimens.Correlations between B7-H3 and clinical features including tumor stages and pulmonary metastasis incidence were analyzed.Kaplan-Meier analysis was used to assess the relationship between B7-H3 expression and overall survival of osteosarcoma patients.The stable overexpression cell lines were constructed by infecting with recombinant lentivirus.Cell proliferation,colony formation,adhesion and wound healing assays were conducted to assess the involvement of B7-H3 in vitro.By constructing the subcutaneous tumor model and pulmonary metastasis model on immune competent BALB/c mouse,the impact of B7-H3 on osteosarcoma was verified in vivo.The correlations between CD4,CD8,FOXP3 expression and B7-H3 were analyzed by using the data of GSE87437.The interaction between B7-H3 and tumor-infiltrating T cells was assessed by immunohistochemistry and immunofluorescent staining in clinical specimens and mouse osteosarcoma tissues.Results:Analysis of data from GSE87437 chip showed that B7-H3 had a significantly higher mRNA expression level than other common immune checkpoints in human osteosarcoma tissues.Results of immunohistochemistry staining confirmed that B7-H3 was expressed in 89.4%(93/104)of the clinical specimens,and tumor tissues had significantly higher B7-H3 expression than adjacent areas.High expression level of B7-H3 was significantly correlated with advanced Enneking stage and pulmonary metastasis.Patients with higher B7-H3 expression exhibited shorter survival time than those of lower level.Overexpressing B7-H3 in osteosarcoma cells partly enhanced cell proliferation and colony formation in vitro,but didn't show apparent effect on migration or adhesion.However,B7-H3 dramatically promoted tumor growth in vivo.Analysis of the data from GSE87437 demonstrated that,in human osteosarcoma tissues,CD4 expression had a significantly positive correlation with B7-H3 expression,while CD8 and FOXP3 expression did not.Immunohistochemistry and immunofluorescent staining in human and mouse osteosarcoma tissues indicated that B7-H3 could enhance the infiltration of CD4+T cells in tumor microenvironment,but had no significant effect on CD8+T cells.Conclusion:Aberrantly high expression of B7-H3 in osteosarcoma is significantly associated with poor prognosis of patients.B7-H3 contributes to osteosarcoma progression,and promotes the tumor-infiltrating CD4+T cells,while had no distinct effect on CD8+T cells.This study preliminarily reveals the impact of B7-H3 on osteosarcoma and its possible molecular mechanism,with a hope to provide novel evidence for immunotherapy against osteosarcoma. |
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