The Study Of CD47-SIRPα Signal Axis In The Immune Escape Of Head And Neck Squamous Cell Carcinoma

Background: Head and neck squamous cell carcinoma（HNSCC）is one of the top ten most common cancers in the world,with approximately 48,000 new cases each year in China.To date,there have been many important risk factors for squamous cell carcinoma of the head and neck identified,including smoking,drinking,chewing betel nut,and immune status,and smoking and drinking have a synergistic induction effect.Despite the progress of conventional treatments,the mortality rate of HNSCC is still about 50%,which has not improved significantly.Recent research evidence suggests that the poor clinical prognosis of patients with head and neck squamous cell carcinoma is mainly due to tumor recurrence,cervical lymph node metastasis,and resistance to chemoradiotherapy.These evidences indicate that the current surgical treatment of head and neck squamous cell carcinoma combined with radiotherapy and chemotherapy still has certain limitations.Therefore,new treatment methods for HNSCC need to be explored.In recent years,immunotherapy as a new and effective cancer treatment has made significant progress in a variety of tumor types,such as advanced melanoma and non-small-cell lung cancer.Similar to many types of cancer,head and neck squamous cell carcinoma is also an immunosuppressive disease.Many immunosuppressive cells are recruited in the tumor microenvironment,including regulatory T cells（Tregs）,myeloid-derived suppressor cells（MDSCs）and tumor-associated macrophages（TAMs）.These immunosuppressive microenvironments provide a rationale for the use of immunotherapy in head and neck squamous cell carcinoma.Among these immunotherapy methods,immune checkpoint blocking therapy is a promising treatment.The latest clinical research on cancer immune checkpoint therapy shows that blocking programmed cell death protein 1（PD-1）and cytotoxic T lymphocyte-associated antigen-4（CTLA-4）has achieved excellent therapeutic effects in multiple cancer types.These clinical results have prompted us to look for more new potential immune checkpoint molecular proteins.The CD47 protein molecule is considered to be a promising tumor immune checkpoint molecule after PD-1/PD-L1 research.CD47 protein is also known as integrin-associated protein（IAP）,a protein molecule that binds to αvβ3 integrin and was originally purified on the surface of human placenta and platelet cells.It is widely expressed on the cell surface of various tissues.Studies have shown that the CD47 protein is highly expressed on the surface of a variety of cancer cells.It binds to signal regulatory protein-α（SIRPα）molecules expressed on the surface of macrophages and dendritic cells,and is mainly used as an anti-phagocytic or “don’t eat me” signal,which enables tumor cells with high expression of the CD47 protein molecule to escape the phagocytosis of macrophages,thereby avoiding elimination.Therefore,blocking the CD47/SIRPα signaling pathway can relieve inhibition of phagocytes of tumor cells,thereby inducing tumor cells to be phagocytosed and eliminated.Recent research shows that blocking the CD47/SIRPα signaling pathway can also activate effector T cells through dendritic cells to achieve an anti-tumor immune response.The important role of CD47 protein in the treatment of many types of cancer has led us to explore the role of CD47/SIRPα signaling pathway in the immunotherapy of head and neck squamous cell carcinoma.Part one Study of CD47 expression in head and neck squamous cell carcinomaObjective: To explore the expression of CD47 protein molecule in human head and neck squamous cell carcinoma tissue,and to explore the correlation between the expression level of CD47 protein and clinicopathological parameters.At the same time,explore the possibility of CD47 protein molecule as a clinical prognostic indicator for patients with head and neck squamous cell carcinoma.The relationship between the CD47 protein molecule and the immunosuppressive microenvironment of head and neck squamous cell carcinoma was clarified.The immunocompetent transgenic HNSCC mouse model was used to explore the expression of CD47 on tumor cells of tumor-bearing mice so as to lay a solid foundation for studying the role of CD47 in immune escape of head and neck squamous cell carcinoma in this mouse model.Methods: The expression of CD47 protein in head and neck squamous cell carcinoma tissues was determined by immunohistochemical staining using tissue microarray.The relevance between CD47 protein and pathological grade,tumor size,lymph node status and HNSCC patients with TPF were explored by immunohistochemical analysis.Kaplan-Meier survival analysis was used to explore the correlation between CD47 protein expression and clinical prognosis in patients with head and neck squamous cell carcinoma.Correlation analysis and hierarchical cluster analysis were used to determine the correlation between CD47 protein expression and tumor immunosuppressive status.In an immunocompetent transgenic HNSCC mouse model,immunohistochemistry,immunofluorescence,and western blot were used to explore the expression of CD47 protein in tumor-bearing mice.Results: The results of tissue microarray immunohistochemistry showed that the expression of CD47 protein was significantly increased in human head and neck squamous cell carcinoma tumor tissue and dysplasia tissue.Analysis of the results of immunohistochemistry showed that the expression level of CD47 was closely related to pathological grade,lymph node status and metastatic lymph nodes,and had no significant correlation with tumor size and HNSCC patients with TPF chemotherapy.Kaplan-Meier survival analysis showed that CD47 protein expression can predict the prognosis of patients with head and neck squamous cell carcinoma.Correlation analysis showed that the expression of CD47 protein was correlated with PD-1/PD-L1 and immunosuppressive cell surface markers CD11 b,CD33 and Foxp3.Cluster analysis showed that CD47 protein expression was closely related to PD-1.CD47 protein expression in tumor-bearing mice was significantly higher than that in wild-type mice using an immunocompetent transgenic Tgfbr1/Pten 2c KO HNSCC mouse model.Conclusions: We found that the expression level of CD47 protein in head and neck squamous cell carcinoma tumor tissues was significantly increased,and that the CD47 protein molecule can be used as a clinical prognostic indicator for head and neck squamous cell carcinoma patients.The expression level of CD47 protein is related to the immunosuppressive status of human head and neck squamous cell carcinoma.At the same time,our immunocompetent transgenic Tgfbr1/Pten 2c KO HNSCC mouse model also shows that CD47 is highly expressed on tumor cells of tumor-bearing mice,which provides an early basis for the subsequent exploration of CD47 protein function in this mouse model.Part two Research on CD47 antibody therapy enhances the antitumor immunity of head and neck squamous cell carcinomaObjective: In order to further explore the role of CD47 in immune escape of head and neck squamous cell carcinoma,we use immunocompetent transgenic HNSCC mouse model to explore the effect of CD47 antibody treatment on tumor growth in mice,and to explore the regulatory effect of CD47 antibody treatment on tumor immunosuppression microenvironment.Methods: The immunocompetent transgenic HNSCC mouse model of head and neck squamous cell carcinoma was used to explore the effect of CD47 antibody blocking treatment on tumor growth.The effect of CD47 antibody treatment on tumor exhausted T cells and the effect on the population of MDSCs and Tregs compared with the isotype control group were analyzed by flow cytometry.The regulatory effects of CD47 antibody treatment on CD11b⁺Ly6G⁺Ly6CloMDSCs,CD11b⁺Ly6G-Ly6 ChiMDSCs and CD4⁺CD25^hiTregs inhibitory functions were explored by cell co-culture and CFSE staining flow cytometry analysis.Western blot and immunohistochemical staining were used to analyze the possible cellular pathways of CD47 antibody treatment on tumor proliferation.Results: Using immunocompetent transgenic HNSCC mouse model of head and neck squamous cell carcinoma,it was found that CD47 antibody blocking treatment significantly delayed the growth of mouse tumors.Flow cytometry results showed that CD47 antibody treatment reduced the expression of PD-1 on effector T cells and increased the secretion of IFN-γ in a mouse model of head and neck squamous cell carcinoma,thereby improving the T cell exhausted status.Further analysis showed that CD47 monoclonal antibody treatment reduced the number of Treg cells and MDSC cell populations.The results of co-culture experiments showed that the CD47 monoclonal antibody treatment could regulate the immunosuppressive function of CD11b⁺Ly6G⁺MDSCs in a mouse model.Analysis by western blot and immunohistochemical staining showed that the inhibition of tumor proliferation by CD47 antibody treatment in mouse models may affect Akt-dependent cellular pathways.Conclusion: We found that CD47 monoclonal antibody treatment can effectively delay the progression of tumors in an immunocompetent transgenic HNSCC mouse model.At the same time,this treatment effectively improves the tumor immunosuppressive microenvironment.Our research shows that targeting CD47 antibody therapy may be an effective tumor immunotherapy in future clinical applications.