A Mechanistic Study Of Macrophage-Derived MMP14 Inducing A Suppressive Tumor Microenvironment In Colon Cancer Through Regulation Of SEMA4D Shedding From Tumor Cells

Part Ⅰ:Expression Characteristics of SEMA4D in Colon Cancer Patients and Its Relationship with Patient PrognosisObjective:SEMA4D is highly expressed in multiple types of tumors,promoting tumor growth.In colon cancer,SEMA4D may not only directly affects tumor cells but also potentially participates in immune regulation within the tumor microenvironment.By analyzing the expression characteristics of SEMA4D in colon cancer and its impact on patient prognosis,we aim to provide insights into the mechanisms through which SEMA4D may be involved in the progression of colon cancer.Methods:(1)A retrospective study included 89 patients with colon cancer,evaluating SEMA4D expression through immunohistochemical staining of tumor and adjacent tissues.(2)Based on SEMA4D expression levels,patients were divided into high and low expression groups,and survival analysis was performed to investigate the influence of SEMA4D expression on patient prognosis.(3)ELISA was used to measure free SEMA4D levels in the blood of 23 patients with colon cancer before and after surgery,assessing SEMA4D expression changes.(4)SEMA4D levels in tumor tissues from 23 patients with colon cancer were measured to analyze the relationship between tumor tissue and free SEMA4D expression.Results:(1)SEMA4D expression was significantly higher in tumor tissues than in adjacent tissues.(2)Colon cancer patients with high SEMA4D expression had a worse prognosis compared to those with low expression.(3)Free SEMA4D expression levels decreased significantly after surgery.(4)Preoperative free SEMA4D levels showed a positive correlation with SEMA4D expression in tumor tissues.Conclusion:SEMA4D plays a significant role in colon cancer.High expression of SEMA4D within tumor tissue can be shed as a soluble form and enter the peripheral blood.The levels of peripheral soluble SEMA4D decrease as tumor burden decreases after surgery.The expression level of SEMA4D holds profound significance in the diagnosis and postoperative prognosis prediction for colon cancer patients.Part Ⅱ:Investigating the role of tumor-derived SEMA4D in modulating the suppressive tumor immune microenvironment in colon cancerObjective:SEMA4D is the first reported signaling molecule that plays a crucial role in immune responses.It can influence a wide range of immune cell populations within the tumor microenvironment and exert immunoregulatory effects.In this study,we aim to further elucidate the key regulatory role of SEMA4D in shaping the immunosuppressive microenvironment of colon cancer.Methods:(1)Employing bioinformatics analysis to investigate the relationship between SEMA4D and immune cell infiltration as well as immune checkpoint expression in the tumor microenvironment.(2)To clarify the effect of SEMA4D knockdown on macrophage polarization and on macrophage cytokine secretion by coculture model of tumor cells and mouse macrophages.(3)To investigate the effects of SEMA4D knockdown on CD8+T cell proliferation and effector functions.(4)Validating the effects of SEMA4D knockout on tumor growth through animal experiments and exploring its impact on the tumor tissue immune microenvironment.Results:(1)SEMA4D in the tumor microenvironment is associated with various immune cell infiltrations and exhibits extensive correlations with immune checkpoints.(2)Knocking out SEMA4D in the co-culture model significantly induces more M1 macrophages,as evidenced by increased expression of M1 macrophage activation markers and decreased presence of M2 macrophages.Antibody cytokine arrays reveal that SEMA4D knockout can induce the upregulation of various cytokines and chemokines secreted by macrophages.(3)The co-culture system with SEMA4D knockout promotes the proliferation and secretion of effector factors in T cells.(4)Knocking out SEMA4D in tumor cells improves the tumor immune microenvironment and inhibits tumor growth in mice.Conclusion:In the tumor microenvironment of colon cancer,tumor cell-derived SEMA4D extensively modulates the anti-tumor immune response.SEMA4D induces an increase in immunosuppressive M2 tumor-associated macrophages,suppresses the function of effector T cells,resulting in the formation of an immunosuppressive microenvironment,and subsequently promotes tumor progression.Part Ⅲ:Investigating the Mechanism of Macrophage MMP14 in Controlling Tumor Cell SEMA4D Shedding to Induce an Immunoregulatory MicroenvironmentObjective:SEMA4D is able to affect the immune microenvironment of colon cancer extensively,and soluble SEMA4D may be the primary that exerts immunosuppression,while the exact mechanism of SEMA4D shedding in the immune microenvironment and its effect on immunosuppression have not been clarified.We hope to clarify the key molecular mechanisms affecting the SEMA4D-mediated immunosuppressive effect by exploring the key sites and exact mechanisms mediating SEMA4D shedding from tumor cells.Methods:(1)In order to elucidate the mechanisms by which SEMA4D induces an immunosuppressive microenvironment,we conducted co-cultures of extracellular vesicles derived from SEMA4D-deficient tumor cells with macrophages.(2)Subsequent to administering MMP14 inhibitors to tumor-bearing mice to inhibit the functional activity of MMP14,we conducted a comparative analysis of the levels of circulating SEMA4D and the genetic alterations of SEMA4D in the peripheral blood of the mice,aiming to determine the influence of MMP14 on the shedding of SEMA4D.(3)Analyzing the changes in tumor growth and the immune microenvironment in mice after inhibiting MMP 14,(4)Analyzing the enrichment sites of MMP 14 in the immune microenvironment using single-cell databases.Generating macrophage-specific MMP 14 knockout mice to observe the effects of MMP 14 knockout on SEMA4D shedding.(5)Investigating the effects of macrophage-specific MMP 14 knockout on the immunosuppressive effects mediated by SEMA4D overexpression through animal experiments.(6)Co-culturing macrophages derived from macrophage-specific MMP 14 knockout mice with tumor cells to investigate the effects of MMP 14 knockout on SEMA4D-induced polarization of M2 macrophages and T cell functional suppression.Results:(1)Knockout of SEMA4D in tumor cells did not significantly affect the polarization phenotype of tumor-associated macrophages induced by secreted extracellular vesicles.(2)Inhibiting MMP 14 resulted in decreased levelsConclusion:In the tumor immune microenvironment,soluble SEMA4D plays a crucial role in mediating immunosuppression and is the primary form through which an immunosuppressive microenvironment is formed.Macrophage-derived MMP 14 controls the shedding of SEMA4D from tumor cells,influencing the reshaping of the immune microenvironment.By inhibiting macrophage MMP14,the immunosuppressive effects mediated by SEMA4D are weakened,leading to the promotion of anti-tumor immune responses.