The Role And Molecular Mechanism Of SRC Family Kinases In The Immune Escape Of Oral Squamous Cell Carcinoma

Head and neck squamous cell carcinoma(HNSCC)is a deadly malignancy worldwide with ranking 6th among all cancer types.Oral squamous cell carcinoma(OSCC)accounts for 90%of oral and oropharyngeal malignancies.For the past three decades,the 5-year overall survival rate of patients remains 40.0-61.7%despite emerged advances in the early diagnosis,reconstructive surgery and adjuvant radio-chemotherapy.One of the major reasons of the difficulty in the OSCC therapy is the activation of aberrant signaling pathways and biological heterogeneity of the disease.Recent study suggests that the occurrence and progress of tumors not only depend on the effects intrinsic to the tumor itself,but also require support from the mesenchymal cells and immune cells in the tumor microenvironment.The complex signal transduction between cancer cells and stroma play an important role in the process of the tumor occurrence,development and invasion.Therefore,it shows important clinical significance to explore and investigate the cross-talk between cancer cells and stroma for tailoring existing treatment strategies.SRC family kinases(SFKs)are a member of non-receptor tyrosine kinases,which intermediate various biological processes,such as cell growth,differentiation,metabolism,immune responses and signal transduction.SFKs comprise nine family members that share similar structure and function.Recent studies have shown a different level of activation of SFKs in the development of multiple types of human tumors.And the SFKs are central mediators in multiple signaling pathways of oncogenesis.However,the relationship between SFKs and their clinical implications in OSCC patients remains unclear.Accumulating evidence suggests that the high infiltration of immature myeloid cells in OSCC microenvironment,including myeloid-derived suppressor cells(MDSCs)and tumor associated macrophages(TAMs),fosters potent immunosuppressive tumor microenvironment activity by dampening T cell activation.The investigation of the functions and mechanism of these immunosuppressive cells will help us to better understanding the interaction of tumor cells and stromal cells.Collectively,this study is aimed to investigate the expression of SFKs in OSCC and the role of SFKs in the anti-tumor immune responses.In addition,this study also performed preliminary exploration on the combination of dasatinib and CTLA-4 antibody as an application prospect in OSCC.Part oneInvestigating the regulatory function of SFKs in the immune escape of OSCCObjective:Immune escape plays a major role in the process of development,invasion and metastasis in OSCC.This section is aimed at exploring the expression of two major members of SFKs,SRC and LYN,in HNSCC tissues,and their functions in immunosuppressive cells,and the correlation between LYN and immunosuppressive cells biomarkers(CD11b,CD33,CD68,CD163).Methods:Firstly,we used the public available cancer microarray database Oncomine to assess SRC and LYN gene expression levels in human HNSCC.Immunohistochemistry(IHC)and Western blot assay were used to analyze the protein level of SRC and LYN in Tgfbr1/Pten 2cKO mouse tissues.Secondly,to investigate the effect of SRF inhibition on MDSCs,we used SFKs inhibitor Dasatinib in immunocompetent Tgfbr1/Pten 2cKO mouse model and detected the MDSCs by flow cytometry,and assessed the effect on SKFs by IHC and Western blot assay.Thirdly,we used OSCC tissue microarrays to study the LYN expression and its correlation with immunosuppressive cells biomarkers(CD11b,CD33,CD68,CD 163)by IHC,with quantified by Aperio Scanscope as well as analyzed by Pearson correlation and hierarchical cluster.In addition,survival analysis was performed later by Kaplan-Meier analysis followed by long-rank test and Cox hazard multivariant analysis.Results:SRC and LYN were significantly activated in Tgfbr1/Pten 2cKO mouse model.In vivo experiments indicated that Dasatinib could effectively inhibit the tumor growth and decrease the population of MDSCs in peripheral immune organs and the tumor microenvironment.Molecular mechanism research suggested that LYN kinase may display a predominant role in this progress.It was verified that LYN protein expression significantly increased in human OSCC and was correlated with MDSCs.And the dual positive epithelial and stromal expression of LYN indicated a poor prognosis of OSCC patients(p = 0.0005).Conclusions:SRC and LYN were over expressed in OSCC.The expression of LYN was correlated with immunosuppressive cells and indicated a poor survival in OSCC.Targeting SFKs by dasatinib could effectively reduce tumor size and attenuated the MDSCs to enhance the anti-tumour immunity.Part twoExploring the combinational effect of Dasatinib with anti-CTLA-4 monoclonal antibody in mice OSCC:immunotherapy resistanceObjective:Application of anti-CTLA-4 monoclonal antibody(anti-CTLA-4 mAb)provided effective inhibition of tumour growth,but its drug resistance also gradually revealed.This section focused on the possible mechanism of anti-CTLA-4 drug resistance in mouse model,and the anti-tumour effect of combination Dasatinib with anti-CTLA-4 mAb in mouse model.Methods:Chemopreventive and chemotherapeutic experimental studies of anti-CTLA-4 mAb were performed in Tgfbrl/Pten 2cKO mouse model.The alternative populations of MDSCs,Tregs,CD4+T cells and CD8+T cells in the peripheral immune organ and tumor microenvironment were analyzed by flow cytometry.Immunohistofluorescence were used to detect the CD4+T cells and CD8+T cells in tumour tissues.SFKs signaling activation was investigated in mouse model tumour by IHC and Western blot.Chemotherapeutic experimental studies of combination Dasatinib with anti-CTLA-4 mAb were performed in Tgfbrl/Pten 2cKO mouse model.The cell populations of MDSCs,Tregs,CD4+ and CD8+T were analyzed accordingly.Results:In vivo chemotherapeutic experiments indicated that anti-CTLA-4 mAb could induce the activation of SFKs in the OSCC,and resulted in immunotherapy resistance.Combination of dasatinib and anti-CTLA-4 mAb could effectively reduce the tumour growth,lower the populations of MDSCs and Tregs in circulation and tumour microenvironment,and enhanced the anti-tumour response by activating the effector T cells.Conclusions:anti-CTLA-4 mAb may combine with Dasatinib to overcome immunotherapy resistance and promoting anti-tumour immune effect.