| Background and objectivesLung cancer is a kind of tumor with high morbidity and mortality in the world.according to the pathological types,lung cancer is divided into non-small cell lung cancer(non-small cell lung cancer,NSCLC)and small cell lung cancer(small cell lung cancer,SCLC),of which NSCLC accounts for about 85%.Traditional treatments,such as surgery,radiotherapy and chemotherapy,have not improved significantly after decades of development,and the 5-year survival rate is still less than 20%.In recent years,immunotherapy represented by CAR-T cells has attracted much attention for its effective control of hematological tumors,but its clinical effect in the treatment of NSCLC is not good.To investigate the reason,the infiltration of lethal T cells into the tumor site of NSCLC is the primary prerequisite for its function.however,the complex tumor microenvironment greatly restricts the ability of T cell infiltration.Therefore,to explore the potential mechanism of restricting immune cell infiltration in NSCLC microenvironment is very important to explore to enhance the ability of T cell infiltration,which will help to find more effective treatment strategies and provide an important reference for improving the clinical efficacy of NSCLC.Studies have confirmed that the immune system plays an important role in controlling the growth and metastasis of tumor cells,namely immune clearance,immune balance and immune escape.A number of studies have confirmed that regulating the ability of the immune system to monitor and clear is essential for controlling tumor progression.Therefore,to solve the problem of tumor treatment from the perspective of regulating the immune system is the key in the future.However,in the process of clinical treatment,the efficacy of tumor immunotherapy NSCLC varies greatly among different individuals.the reason is that the number of infiltrating immune cells in tumor tissue and the heterogeneity of groups are the main factors,among which CD8~+T cells are considered to be the main members of killing function.In recent years,researchers have gradually realized that it is the key to increase the number of cytotoxic CD8~+T cells(CTL)targeting tumor cells or enhance their anti-tumor function.Researchers have confirmed that the increase of cytotoxic CD8~+T cells in TME is positively associated with a good prognosis in many types of cancer.At present,most of the studies on T cell infiltration focus on the effects of external environmental factors on T cell infiltration,such as chemokines,tumor-induced vascular system,etc.,but there are few reports on the motor function of T cells.In fact,compared with the recruitment of T cells by chemotactic factors in the tumor microenvironment,it is also worth paying attention to the movement of T cells between tissues to reach cancer tissues.To explore the related inducing factors and mechanisms of regulating the spontaneous movement of CD8~+T cells is very important to improve the clinical immunotherapy.Therefore,taking NSCLC as the research object and taking the spontaneous movement of CD8~+T cells as the foothold,we will explore the key factors and potential mechanisms that affect the movement of CD8~+T cells to cancer tissues.In this study,we collected cancer tissues and identified the degree of infiltration of CD8~+T cells,and divided them into two groups:"high invasion"and"low invasion".After enriching CD8~+T cells by magnetic separation,RNA-seq sequencing was carried out to compare the difference of gene map between high and low infiltration subsets in order to screen the exercise-related differential genes ARHGEF1 which may be related to the different invasive ability of TIL in NSCLC patients.The ARHGEF family is the main functional component of Rho family guanosine triphosphatase(Rho GTPase),which has been proved to play an important role in mediating cell spontaneous movement.Only a few articles have reported that ARHGEF1,plays an important role in the migration of tumor cells and B cells.However,whether ARHGEF1 is involved in the movement of CD8~+T cells and its underlying mechanism have not been revealed.We also used cell experiments,gene knockout or overexpression to verify its effect on T cell movement,and analyzed its correlation with CD8~+T cell function in"high infiltration"group and"low infiltration"group.Finally,verification analysis was carried out in TCGA database.From the point of view of spontaneous movement of tumor infiltrating CD8~+T cells,this paper explores the important genes that regulate this process,and studies the biological process of their function and their effects on cell function,which may lay an important theoretical foundation for improving the clinical efficacy of immune cell therapy for NSCLC.Methods1.TCGA database was used to analyze the relationship between CD8~+T cell infiltration and prognosis in patients with lung cancer.2.Fresh NSCLC tissue samples were collected and the same quality tissue was digested with three enzymes to obtain single cell suspension.3.The proportion of CD8~+T cells was identified by flow cytometry and divided into"high infiltration"and"low infiltration"groups according to the proportion of infiltration.4.A few tissues were taken for embedding after dehydration,sliced and hydrated for immunohistochemical detection to detect the abundance of CD8~+T cell infiltration.5.CD8~+T cells from single cell suspension of tumor tissue were separated and enriched by magnetic separation,and RNAiso Plus was added to cleavage cells for RNA-seq sequencing.6.The RNA-seq sequencing data were normalized,and the significant differentially expressed genes were screened by volcano map,heat map and Wayne analysis.7.The up-regulated and down-regulated genes in"high infiltration"group were analyzed by GO and KEGG cluster analysis by DAVID platform,and the main enrichment pathways and key genes of differential genes were analyzed.8.q RT-PCR and western blot were used to detect the expression of ARHGEF1 in CD8~+T cells of high infiltration group and low infiltration group.9.The effect of ARHGF1 expression on the motor ability of CD8~+T cells was evaluated by 3D stromal glia cell migration assay.10.CD8~+T cells with ARHGEF1 knock-down and over-expression were constructed by lentivirus infection,and functional experiments were carried out to detect the regulation of ARHGEF1 expression on the motor ability of CD8~+T cells.11.The cytoskeleton protein F-actin was labeled with cyclopeptide and its fluorescence intensity was detected by flow cytometry.The correlation between ARHGEF1 and CD8~+T cell cytoskeleton was detected by cellular immunofluorescence.12.Download the single cell sequencing database of tumor infiltrating T lymphocytes from lung cancer patients in TCGA database,and analyze the correlation between the expression of ARHGEF1 and the expression of functional molecules of CD8~+T cells.13.The CD8~+T cells’function was detected by flow cytometry.Results1.The infiltration of CD8~+T cells in non-small cell lung cancer is positively correlated with clinical prognosis.2.RNA-seq results showed that there were significant differences in genes related to movement and function of CD8~+T cells between high infiltration group and low infiltration group.3.The expression of CD8~+T cell movement-related gene ARHGEF1 in the high infiltration group was higher than that in the low infiltration group.4.The results of cell experiment in vitro showed that the motility of CD8~+T cells in high infiltration group was stronger than that in low infiltration group.5.The results of knock-down and overexpression experiments showed that there was a positive correlation between ARHGEF1 and the motor ability of CD8~+T cells.6.The results of flow cytometry and immunofluorescence showed that the change of ARHGEF1 expression in CD8~+T cells resulted in the change of cytoskeleton F-actin content.7.TCGA database analysis showed that the expression of ARHGEF1 was positively correlated with the infiltration and function of CD8~+T cells in many kinds of solid tumors.8.RNA-seq data showed that the function of CD8~+T cells in the high infiltration group was stronger than that in the low infiltration group.9.The results of single cell sequencing of infiltrating T lymphocytes in lung cancer tissue from TCGA database showed that there was no significant correlation between the expression of ARHGEF1 and the expression of functional molecules of CD8~+T cells.ConclusionIn non-small cell lung cancer patients with higher TIL infiltration,the motor ability of tumor infiltrating CD8~+T lymphocytes was stronger and the expression of exercise-related gene ARHGEF1 was higher.Further studies found that the high expression of ARHGEF1 could change the content of cytoskeletal F-actin,and then promote the exercise ability of CD8~+T cells;finally,the expression of ARHGEF1 only affects the motor ability of CD8~+T cells,but does not participate in the regulation of immune-related functional molecules. |
