The Experimental Study Of CCR7 Regulating Na(?)ve CD8~+ T Cell Activation To Affect The Tumor Microenvironment For Promoting Proliferation Of Oral Squamous Cell Carcinoma

Objective:Head and neck cancer(HNC)is the sixth most widespread cancer worldwide,and oral squamous cell carcinoma(OSCC)is an important branch of HNC.The prognosis for OSCC patients is poor,and the underlying causes of invasion and metastasis remain unclear,with poorly understood mechanisms.The tumor microenvironment(TME)is the immediate surrounding where tumor cells reside,and immune cells play a pivotal role in the TME,influencing tumor survival and progression.CD8~+T cells are vital components of adaptive immunity.CC chemokine receptor 7(CCR7)and its ligands CC chemokine ligand 19(CCL19)and CC chemokine ligand 21(CCL21),have been implicated in promoting the progression of various malignancies.Previous findings from our group demonstrated significant overexpression of CCR7 in OSCC patients and its involvement in multiple signaling pathways that regulate the malignant behavior of OSCC cells.However,the effect of CCR7 on the OSCC TME and its potential mechanisms are still unclear.Therefore,this study aims to utilize molecular biology techniques to explore the role of CCR7 in regulating the OSCC TME,further enhancing our understanding of related biological mechanisms,and providing a theoretical basis for an advanced comprehension of OSCC’s development process and the formulation of clinical treatment approaches.Materials and methods:1.Experimental materials:C57BL/6 mouse oral squamous carcinoma cell line 2(MOC2)and primary extracted na(?)ve CD8~+T cells.2.The experimental methods employed were as follows:1)The expression of CCR7 in oral squamous cell carcinoma patients was detected by bioinformatics;2)Subcutaneous tumorigenesis assay conducted in mice to validate the biological impact of CCR7 on the tumorigenesis of oral squamous carcinoma cells;3)Evaluation of CD8~+T cell distribution in tumors and spleens of two mice;4)In vitro extraction of na(?)ve CD8~+T cells from mice and division into different groups based on various sources and treatments,including the WT group,KO group,WT+CCL19/21 group,and KO+CCL19 group;5)Implementation of real-time PCR,flow cytometry,immunohistochemistry,and immunofluorescence to measure the expression levels of CD8~+T cell surface markers under different grouping and treatment conditions;6)Annexin V/PI apoptosis assay and CCK-8 cytotoxicity assay were used to investigate the effects of CD8~+T cells on the biological behavior of oral squamous cell carcinoma under different grouping treatment conditions;7)Statistical analysis conducted using SPSS 23.0 and Graph Pad Prism 8.0.All experiments were independently repeated at least three times,and the results were presented as mean±standard deviation(SD).The experimental data were first assessed for normality,and if they met the normal distribution,the differences between two groups were determined using Student’s t-test(independent or paired samples),while differences among more than two groups were analyzed using one-way analysis of variance(one-way ANOVA)and Dunnett’s multiple comparison test.In cases where normal distribution criteria were not satisfied,the Mann-Whitney rank sum test was employed for comparing two groups,and the Kruskal-Wallis test was used to compare differences among more than two groups.A two-sided P<0.05was considered statistically significant.Results:1.The analysis using bioinformatics methods revealed overexpression of CCR7in oral squamous cell carcinoma tissues.2.Subcutaneous tumorigenesis assays showed that CCR7 deficiency resulted in the inhibition of OSCC tumor growth.3.Single-cell RNA sequencing of tumor tissues indicated that CCR7 had an impact on the heterogeneity of CD8~+T cells.4.Flow cytometry,immunohistochemistry,and immunofluorescence analyses conducted on dissociated tumor tissues in vivo demonstrated that CCR7influenced the heterogeneity of CD8~+T cell infiltration proportions within the tumor tissues.5.The results of flow cytometry after dissociation of spleen tissues in vivo showed that CCR7 affected the heterogeneity of the proportion of CD8~+T cell distribution in spleen tissues of mice in different states.6.In vitro experiments utilizing flow cytometry,real-time fluorescence quantitative PCR,and CFSE cell proliferation assay showed that CCR7inhibited the activation and proliferation of na(?)ve CD8~+T cells.7.The results of Annexin V/PI apoptosis assay and CCK-8 cytotoxicity assay demonstrated that CD8~+T cells in different activated states influenced the apoptosis and viability of tumor cells.Conclusion:1.CCR7 deficiency inhibited OSCC tumor growth.2.CCR7 had an impact on the heterogeneity of CD8~+T cells in both tumor tissues and spleens.3.CCR7 inhibited na(?)ve CD8~+T cell activation to promote the proliferation of oral squamous carcinoma cells.