The Effects Of Pancreatic Cancer Cell-derived Migrasomes On The Phenotype And Function Of Cancer Cells And Related Immune Cells In Tumor Microenvironment

BackgroundPancreatic cancer is one of the malignant digestive tumors,with a high degree of malignancy and a poor prognosis.Pancreatic ductal adenocarcinoma is the most common pathological type.Epidemiological data show that pancreatic cancer is the sixth leading cause of cancer-related death in China with rising incidence.Most patients lose the opportunity of surgery due to local invasion or distant metastasis of the tumor at the initial diagnosis.Tumor microenvironment(TME)of pancreatic cancer is characterized by massive infiltrations of inflammatory cells and extensive fibrosis.It is a hot topic to explore the effects of TME on pancreatic cancer progression.Tumor associated macrophages(TAMs)in TME promoted the development of pancreatic cancer and the immune escape of complement.Extracellular vesicles(EV)are involved in the signal transmission and material exchange between different cells in TME,and are a key medium for regulating TAM differentiation and functional changes in pancreatic cancer.Migrasome is a kind of EV-like structure that is newly discovered in recent years and dependent on cell movement,in which many signal mediators,chemokines,cytokines and other regulatory proteins are enriched,which is of great significance for organism development and signal transduction There is a lack of research of the effect of pancreatic cancer cell-derived migrasomes on the phenotype and functions of TAM in TME in pancreatic cancer.ObjectiveTo identify the protein components in PCCDM that could regulate the tumor immune microenvironment,and to explore the effects of these migrasomes on the proportion of immune cells in TME and the influence of such changes on the growth of pancreatic cancer To explore the effects of PCCDM on the regulation of phenotype and function of TAMs.To investigate the effects of TAMs induced by PCCDM on pancreatic cancer cell function To investigate the effects of TAMs induced by PCCDM on T lymphocytes.To investigate the effects of TAMs induced by PCCDM on immune escape of pancreatic cancer complement.Some key proteins enriched in PCCDM were screened to preliminarily explore the expression of the key protein in PCCDM that regulate the immune microenvironment in pancreatic cancer tissues and explore their relationship with immune cell infiltration and prognosis.Methods1.PCCDMs were purified by differential centrifugation and density gradient centrifugation,and the ultrastructure and morphology observation of migrasomes was identified by electron microscopy.The cytokines and chemokines and other regulation proteins enriched in migrasomes were detected by protein spectrum and protein chip,and the production process of migrasomes and the concentration of the key proteins were observed by confocal microscopy.A peritoneal model of pancreatic cancer in immunologically mice was constructed,and the immune microenvironment was constructed by intraperitoneal injection of migrasomes.Then explore the changes in the proportion of immune cells in the immune microenvironment induced by migrasome and the influence of such changes on the growth of pancreatic cancer.2.Using murine macrophage cell line RAW264.7 and bone marrow-derived macrophages(BMDMs)as macrophage models,the effects of PCCDM on the morphology and chemotaxis of macrophages and the changes of phenotype and function of the migrasome-induced macrophages were investigated by the co-culture system.The functional phenotypes of macrophages induced by migrasomes derived from pancreatic cancer cells were identified and analyzed by protein chip and transcriptome sequencing 3.The cultured supernatant of macrophages induced by migrasomes and macrophages in the control group was used to prepare the conditioned medium,and the pancreatic cancer cells were stimulated by the conditioned medium.The effects of the conditioned medium on the proliferation ability of pancreatic cancer cells were detected by CCK8 method,and the effects of the conditioned medium on the invasion and migration ability of pancreatic cancer cells were detected by Traswell method4.ELISA was used to detect the secretion of the key protein that could regulate the tumor immunity in RAW264.7 and BMDM macrophage models and mice peritoneal lavage fluid after PCCDM education.CD4+and CD8+T lymphocytes were purified from the spleen of mice,and the effects of macrophages induced by PCCDM on the proliferation ability of T cells was investigated by using the method of conditioned medium.The effects of the key protein inhibitor on the proliferation ability of T cells in the macrophage cultured supernatant was investigated.5.Using human macrophage line THP-1 as the macrophage model,the effects of pancreatic cancer on THP-1 phenotype was investigated by the stimulation of conditioned medium from pancreatic cancer.Using human fresh serum to construct complement microenvironment,flow cytometry was used to detect the effects of M2-type macrophages on the complement immune escape of pancreatic cancer cells.Transcriptome sequencing was used to investigate the gene expression differences in macrophages induced by PCCDM and pancreatic cancer cells,as well as the mechanism on complement immune escape.6.Key proteins in PCCDM that regulate tumor immunity were screened.Immunohistochemical staining and database methods were used to explore the relationship between the expression of this protein in pancreatic cancer tissues and the clinical characteristics and prognosis of patients,as well as the relationship between the expression of this protein and the infiltration of immune cells.Results1.Pancreatic cancer cells could produce migrasome in large quantities during migration,and it was identified that migrasome is rich in CXCL5,TGF-?1,integrin pathway proteins,Rab family proteins and other factors that could regulate immune microenvironment.The infiltration of immune cells increased in the immune microenvironment of mice induced by PCCDM,in which the proportion of M2-type macrophages increased and the proportion of T cells significantly decreased,which had a promoting effect on the growth of pancreatic cancer cells in mice.2.PCCDM could be actively swallowed by macrophages and induced macrophages to migrate to migrasomes.The proportion of CD206+macrophages in macrophages induced by migrasome was increased,and the expression of ARG1,IL-10,and TGF-?1 in M2-type macrophages was significantly increased.Protein-chip analysis showed that the expression levels of immunosuppressive factors(TGF-?1,IL-4,IL-10,CSF1,CSF3,CTLA4 and MMP9)as well as chemokines and receptors(CXCL5,CXCL9,CXCL12,CCL4,CCL7,CCL21,CCR3 and CCR7)that play important roles in tumor were significantly higher in the migrasome-induced macrophages than that in the control group Transcriptome sequencing results showed that the genes up-regulated in migrasome-induced macrophages were associated with tumor promotion or immunosuppression,including IL-6,MMP9,MET,MYC,EGFR,CCL7,CSF3,CCL12,CXCL5,ITGA9,CDH5,and CD274(namely PD-L1).Among the down-regulated genes are numerous genes that regulate the proliferation and activation of immune cells,including CCL24,CX3CL1,and MHC-? proteins(H2-OA,H2-OB,and H2-Q6)3.In the RAW264.7 and BMDM macrophage models,the macrophages induced by PCCDM exerted a promoting effect on the proliferation,invasion and migration of pancreatic cancer cells4.After the induction of PCCDM,the contents of-ARG1 in RAW264.7 and BMDM culture supernatant and peritoneal perfusion fluid in mice were increased significantly Macrophages induced by PCCDM had an inhibitory effect on T cell proliferation,which was partially reversed by ARG1 inhibitor.5.Pancreatic cancer induced THP-1 differentiation into M2-type macrophages,and the differentiated macrophages up-regulated the expression of CD59 in pancreatic cancer cells by secreting IL-6 and promoted the occurrence of complement immune escape in pancreatic cancer cells.IL-6 was secreted by macrophages after induction of PCCDM PCCDM is involved in the regulation of CD59 expression and complement immunity by macrophages in pancreatic cancer.6.High expression of CXCL5 was observed both in pancreatic cancer tissue and cell lines.High expression of CXCL5 in pancreatic cancer tissues was correlated with T3 stage,N2 stage and poor differentiation status.Patients with high expression of CXCL5 had relatively a poor prognosis.The CXCL5 expression level was an independent risk factor in patients with high CA242.The infiltration of M2-type macrophages,neutrophils and IgG+ plasma cells were increased in the pancreatic cancer tissues with high expression of CXCL5.ConclusionPancreatic cancer cells produced migrasomes rich in regulatory proteins such as CXCL5 and TGF-?1.Migrasome induced immunesuppressive microenvironment to promote tumor growth.The proportion of M2-type macrophages was increased and that of T cells was decreased in the microenvironment induced by migrasomes derived from pancreatic cancer.Migrasome could promote the chemotaxis of macrophages and induced their differentiation into M2-type macrophages,and these macrophages could promote the proliferation,invasion and migration of pancreatic cancer,and inhibit the proliferation of T cells by secreting ARG1.The macrophages induced by pancreatic cancer migrasome secreted IL-6 to promote the high expression of CD59 in pancreatic cancer cells,which led to the complement immune escape.Migrasomes derived from pancreatic cancer were rich in CXCL5.Patients with high expression of CXCL5 had a poor prognosis,and their tissues present an immunosuppressive environment,with increased infiltration of M2-type macrophages,neutrophils and IgG+plasma cells.The study of the treatment strategy for migrasomes may provide a new idea for the comprehensive treatment of pancreatic cancer.