| Background:Pancreatic cancer has a high degree of malignancy and poor prognosis,even if surgery combined with radiotherapy and chemotherapy,the effect is not satisfactory,and the 5-year survival rate is less than 10%.In recent years,immunotherapy has been widely used in the treatment of a variety of tumors and has achieved significant results.However,the effect of immunotherapy on pancreatic cancer is not ideal due to the high immunosuppressive tumor microenvironment(TME).Therefore,it is of great significance to find effective molecular targets and explore the composition and function of the immune microenvironment of pancreatic cancer to improve the therapeutic effect of pancreatic cancer.The TME of pancreatic cancer is highly heterogeneous.The proportion of tumor cells is relatively small and most of them are composed of stroma,which contains a variety of immunosuppressive cells,such as tumor-infiltrating T lymphocytes,macrophages,myeloid-derived suppressor cells(MDSC),etc.Immune cells interact with tumor cells to maintain the immunosuppression of TME through their own functions or expression of inhibitory molecules.Tumor-associated macrophages(TAMs)are the most abundant immune cells in TME.They play a crucial role in promoting tumor invasion and metastasis by secreting a variety of cytokines.TAMs often indicate a poor prognosis of pancreatic cancer,which can be roughly divided into two cell types:anti-tumor M1 type and pro-tumor M2 type.In pancreatic cancer,macrophages mainly show the characteristics of M2 type.M2 macrophages release several derived factors in the tumor stroma,such as epidermal growth factor(EGF),cytokines such as IL-6,tumor necrosis factor(TNF),and tumor necrosis factor(TNF)and chemokines such as CXCR17,CCL22,CCL24,etc.These derived factors have the ability to directly promote tumor growth and metastasis.At the same time,M2-type TAMs also produce some immunosuppressive factors such as IL-10 and PGE2,which inhibit anti-tumor immune response and promote tumor immune escape.Therefore,focusing on the molecular mechanisms that affect the phenotype and function of TAMs is expected to provide new ideas for the immunotherapy of pancreatic cancer.Tumor invasion and distant metastasis are closely related to the process of tumor cells breaking through the Extra Cellular matrix(ECM)barrier and basement membrane of blood vessel wall.Malignant tumors’ destruction of these structures necessitates the involvement of a variety of enzymes,with Matrix metalloproteinase(MMP)being a pivotal enzyme in this process.This enzyme has been a major focus of research in recent years due to its significant role in tumor invasion and metastasis.MMP14,a member of the MMP family,is mainly found on the membrane of tumor cells,and is known as matrix metalloproteinase 14(MMP 14).It can lead to tumor invasion and metastasis by directly degrading ECM or indirectly activating MMP2.It has been shown that MMP14 is widely expressed in 30 different tumors,with the most prominent expression in pancreatic cancer.In addition,pathway enrichment analysis showed that MMP 14 was enriched in immune-related pathways,suggesting that MMP 14 may be associated with the suppressive immune microenvironment of pancreatic cancer.In this study,we analyzed the correlation between the expression level of MMP14 and the surrounding infiltrating immune cells in pancreatic cancer tissues in TCGA database,and found that the expression of macrophages was positively correlated with the expression of MMP 14,and the correlation was the highest.We speculated that MMP 14 may regulate the biological behavior of pancreatic cancer by inducing the phenotypic transformation of macrophages in the tumor microenvironment.The Wnt/β-catenin pathway is considered to be one of the key drivers of cancer initiation.More and more studies have found that Wnt/β-catenin signaling pathway is closely related to the immune response in the tumor microenvironment,and it is one of the important signaling pathways related to immune escape.Wnt signaling pathway is closely related to macrophages.On the one hand,macrophages are one of the main cells secreting Wnt ligands in the microenvironment,which can regulate Wnt signaling in neighboring cells through the paracrine pathway to affect their functions.On the other hand,Wnt signaling in macrophages can be activated in different ways and directly affect the homeostasis of surrounding tissues and organs by changing the state of autoimmune activation.Studies have shown that MMP 14 can affect the occurrence and development of a variety of diseases by regulating Wnt/β-catenin signaling pathway,including gastric cancer and non-small cell lung cancer.In conclusion,MMP14 is closely related to the occurrence and development of pancreatic cancer.MMP14 may regulate the polarization direction of tumor macrophages by activating the downstream Wnt/β-catenin signaling pathway.Furthermore,it regulates the proliferation,invasion,migration,apoptosis and Epithelial-mesenchymal transition(EMT)of pancreatic cancer cells.This study can further understand the changes of immune microenvironment and molecular events in the occurrence and development of pancreatic cancer,and provide theoretical research basis for exploring the targets for the diagnosis and treatment of pancreatic cancer and the development of anti-tumor new drugs.Objective:The aim of this study is to explore the role of MMP14 in the occurrence and development of pancreatic cancer,and to explore the potential molecular mechanism of MMP14 in the phenotypic transformation of macrophages in the tumor microenvironment,which provide theoretical basis for further research on the immune escape of pancreatic cancer.Methods:1.Bioinformatics analysis.The expression of MMP14 in pancreatic cancer tissues and its correlation with clinical characteristics and prognosis were analyzed in TCGA database and GTEx database.A study was conducted to assess the relationship between the amount of MMP14 expressed in pancreatic cancer tissues and the amount of different immune cells present in the tumor microenvironment.2.Studies based on clinical data and samples.Staining of the MMP14 expression in pancreatic cancer tissues and those adjacent to it was done using immunohistochemical methods.Using RT-qPCR and Western Blot,the expression of MMP14 was determined in 3 pancreatic cancer cases and their neighboring tissues.A summary of 38 patients’ clinical data,including gender,tumor location,magnitude,TNM stage,and lymphatic metastasis,was documented.The relationship between MMP14 expression and clinicopathological features of pancreatic cancer was analyzed combined with immunohistochemical results.3.Immunofluorescence assay was used to analyze the infiltration abundance of M0,M1 and M2 polarization macrophages,CD4+T cells and CD8+T cells in pancreatic cancer tissues and adjacent non-tumor tissues,as well as the main cells expressing MMP14,and the correlation between MMP14 and these cells was analyzed.4.Correlation between MMP14 and macrophage phenotypes.The human monocytic cell line U937 was induced into macrophage-like cells.The expression of MMP14 mRNA and protein in four pancreatic cancer cell lines were detected by RT-qPCR and Western Blot,and PANC-1 and CAPAN-2 cell lines with higher expression levels were screened.sh-NC control group and sh-MMP14 knockdown group of pancreatic cancer cells were constructed using shRNA lentivirus infection.They were co-cultured with macrophages in vitro.There were four experimental groups:the first group:sh-NC PANC-1+TAM;Group 2:sh-MMP14 PANC-1+TAM;Group 3:sh-NC CAPAN-2+TAM;Group 4:sh-MMP14 CAPAN-2+TAM.Using ELISA,the markers of M1 and M2 macrophages in the cell supernatant of each group were identified.By means of immunofluorescence,the surface protein markers of M1 and M2 macrophages in cells were identified.5.Cell phenotype experiments.Pancreatic cancer cells in the above groups were detected by CCK8 and monoclonal formation assay.Flow cytometry was employed to ascertain cell apoptosis.A Transwell assay was employed to ascertain the alterations in cell infiltration.Scratch test revealed the capacity of cells to migrate.6.Animal experiments.Twelve nude mice aged 5-6 weeks were weighed and then divided into 4 groups with 3 mice in each group.From each group of the cell phenotype experiment,1×105 pancreatic cancer cells were collected and subcutaneously injected into each nude mouse.Every 5 days,the tumor diameter was gauged and the tumor volume was determined(recorded until day 35).The experiment consisted of four groups:group 1:sh-NC PANC-1+TAM;Group 2:sh-MMP14 PANC-1+TAM;Group 3:sh-NC CAPAN-2+TAM;Group 4:sh-MMP14 CAPAN-2+TAM.Immunohistochemistry was employed to measure the expression of Ki-67 in the tumor tissues of nude mice that had been removed.RT-qPCR was employed to ascertain mRNA expression of molecules associated with EMT(N-cadherin,E-cadherin,Vimentin)in tissues.Western Blot was used to verify the expression of EMT-related proteins.7.Pancreatic cancer cell lines PANC-1 and CAPAN-2 were constructed by sh-RNA lentivirus infection and divided into two groups:sh-NC group and sh-MMP14 group.They were co-cultured with macrophages in vitro.RT-qPCR was employed to identify mRNA expression of the essential β-catenin protein in the Wnt signaling pathway,as well as its downstream target genes c-Myc and Cyclin D1.Western Blot was used to detect its protein expression.8.Cell rescue experiments.Pancreatic cancer cells PANC-1 and CAPAN-2 with MMP14 knockdown were co-cultured with macrophages in vitro,and Wnt agonist BML284 was added to the co-culture system.They were divided into four groups:group 1:sh-MMP14 PANC-1+TAM;Group 2:sh-MMP14 PANC-1+TAM+BML284;Group 3:sh-MMP14 CAPAN-2+TAM;Group 4:sh-MMP14 CAPAN-2+TAM+BML284.ELISA was used to detect the markers of M1 macrophages and M2 macrophages in the cell supernatant of each group.Pancreatic cancer cells in each group were detected by related experiments:CCK8 cell viability assay was used to detect the change of cell proliferation;Cell apoptosis was determined by flow cytometry.The changes of pancreatic cell invasion were detected by shuttle chamber.The changes in cell migration ability were detected by scratch test.9.Animal experimental study.Twelve nude mice aged 5-6 weeks were weighed and then divided into 4 groups with 3 mice in each group.Pancreatic cancer cells from each group in the cell rescue experiment were collected,1 × 105 cells were subcutaneously injected,the tumor diameter was measured every 5 days,and the tumor volume was calculated(recorded until day 35).The experiment consisted of four groups,the first group:sh-MMP14 PANC-1+TAM;Group 2:sh-MMP14 PANC-1+TAM+BML284;Group 3:sh-MMP14 CAPAN-2+TAM;Group 4:sh-MMP14 CAPAN-2+TAM+BML284.The tumor tissues of nude mice were taken out,and the expression of Ki-67 in the tissues was measured by immunohistochemistry.Results:1.Analysis of bioinformatics demonstrated that MMP14 was expressed in pancreatic cancer tissues to a greater degree than normal pancreatic tissues;moreover,the expression of MMP14 was linked to both clinical stage and lymph node metastasis.Analysis of survival revealed that those with high MMP14 expression in pancreatic cancer had a poor DFS and OS,implying a bleak outlook.2.The expression of MMP14 in pancreatic cancer tissues was found to be higher than in adjacent tissues,a result that corroborated the database findings.The expression of MMP14 was correlated with clinical stage and lymph node metastasis.RT-qPCR and Western Blot results showed that the mRNA and protein expressions of MMP14 in pancreatic cancer tissues were higher than those in adjacent tissues.3.The infiltration of macrophages was increased,while the infiltration of CD4+T cells and CD8+T cells was decreased in pancreatic cancer tissues,and MMP14 was mainly expressed on immune cells.4.Pancreatic cancer cells with MMP14 knockdown were co-cultured with macrophages reduced M2 polarization of macrophages,inhibited the proliferation,invasion,and migration of pancreatic cancer cells,as well as promoting apoptosis.5.The results of tumor-bearing experiments in nude mice showed that compared with the sh-NC+TAM control group,the tumor growth of the sh-MMP14+TAM group was slow,and the tumor volume was smaller.Compared with the sh-NC+TAM control group,the level of Ki-67 in the tumor tissue of the sh-MMP14+TAM group was significantly reduced,and the EMT ability was decreased.6.The MMP14 knockdown group exhibited a marked decrease in the expression ofβ-catenin,c-Myc and Cyclin D1 when compared to the control group.7.The Wnt signaling pathway activator was augmented by MMP14 knockdown in pancreatic cancer cell-the macrophage co-culture system.It reduces the inhibitory effect on the proliferation,invasion,migration and metastasis of pancreatic cancer cells,and weakens the apoptotic ability of pancreatic cancer cells.8.The results of tumor-bearing experiments in nude mice showed that compared with sh-MMP14 control group,sh-MMP14+BML284 group had faster tumor growth and larger tumor volume at the same time.Ki-67 levels were significantly increased in tumor tissues of sh-MMP14+BML284 group compared with sh-MMP14 control group.Conclusions:1.The expression of MMP14 in pancreatic cancer was augmented when compared to neighboring tissues and typical pancreatic tissues.2.A poor prognosis for pancreatic cancer patients is linked to a heightened expression of MMP14.3.The expression of MMP14 in pancreatic cancer patients was correlated with TNM stage and lymph node metastasis.4.MMP14’s high expression in pancreatic cancer cells can cause the polarization of macrophages to the M2 direction,thus stimulating the proliferation,migration,invasion,and EMT of pancreatic cancer cells,and restraining apoptosis,encouraging the biological activity of pancreatic cancer cells.5.MMP14 in pancreatic cancer cells can regulate the phenotype of macrophages through Wnt/β-catenin signaling pathway and affect the biological behavior of pancreatic cancer cells. |
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