| Metastasis of tumor is the main cause of death in patients.In terms of pathological staging,distant metastasis of tumor often represents that patients have developed to the advanced stage,often losing the opportunity for surgery and low survival rate.Therefore,in each stage of tumor metastasis,the search for some major molecules and cells will help to prevent and control tumor metastasis.Since Stephen Paget proposed the "seed-soil" theory of tumor metastasis for the specific organ of tumor metastasis in 1889,more and more evidence has confirmed that tumor metastasis requires coordination between tumor cells and microenvironment.Many treatments have also been developed for seeds(such as chemotherapy)and soils(such as EGFR inhibition).The tumor cells before metastasis are called circulating tumor cells,and the process of metastasis in the second organ is very complicated and difficult.The concept of pre-metastasis microenvironment,which is currently recognized,was proposed by professor Lyden in 2005.His study found that Hematopoietic progenitor cells(HPCs)positive for Vascular endothelial growth factor receptor 1(VEGFR1)were colonized in the activated areas of fibroblasts and fibroblasts under the mobilization of VEGF secreted by tumors.Promote the secretion of tumor metastasis related proteins and chemokines in target organs,promote the adhesion,colonization and growth of tumors,and form the pre-metastasis microenvironment.And they found that 80% of the cells in the liver that ingested exosomes were macrophages known as f4/8 + and CD11b+.As soon as the concept of pre-metastasis microenvironment was proposed,great attention was paid to it.Secreted substances TDSFs(g-csf,TGF-TGF,TNF-TNF,LOX,etc.)from various tumor sources and bone marrow source cells(tumor-related macrophages,neutrophils,mast cells,etc.)were proved to be involved in the formation of metastatic microenvironment.Tumor-related macrophages(TAMs)are the most common immune cells in the tumor microenvironment.These cells can not only prevent T cells from attacking tumor cells,but also secrete various cytokines to nourish tumor cells,promote tumor angiogenesis,and induce tumor cell colonization and growth.Macrophages can be divided into m1-type and m2-type macrophages.M1-type is currently considered to be an important part involved in inflammatory response and anti-tumor immunity.The M2 type ACTS as an anti-inflammatory and tumor-promoting agent.In the microenvironment before tumor metastasis,m1-type can be transformed into m2-type,while tumor-related macrophages are closer to the functional phenotype of m2-type macrophages.Macrophages promote tumor metastasis by regulating microenvironment.Kupffer cells are a group of immune cells located in the sinusoids of the liver.They are members of mononuclear macrophages,which are equivalent to macrophages that reside in the liver.In 2015,Costa Silva et al.proved that exosomes derived from pancreatic cancer could be selectively taken by liver cell Kupffer,which caused the secretion of TGF-1 and expression of fibronectin in hepatic stellate cells.Fibronectin deposition led to the formation of liver fibrosis microenvironment,promoted the recruitment of bone-marrow derived cells,and formed the microenvironment before liver metastasis.Pancreatic cancer is a highly aggressive disease with a near-death rate.Most patients with pancreatic cancer have no specific clinical symptoms in the early stage and lose the opportunity of surgical treatment when diagnosed.At present,the pathogenesis of pancreatic cancer has not yet been clarified.A large number of studies have shown that the microenvironment of pancreatic cancer can promote the tumor progression process and is closely related to tumor metastasis,drug resistance and immune tolerance.Exosomes are extracellular vesicles with a diameter of 30-100 nm,containing a variety of bioactive substances such as DNA,m RNA,micro RNA,proteins and lipids,which play an important role in intercellular communication.Previous studies have shown that a variety of cells in the tumor microenvironment can secrete exosomes,which are secreted by mesenchymal cells and tumor cells and are related to the development,invasion,metastasis and angiogenesis of tumors.At present,it is generally accepted that the proteins common to exosomes include heat shock proteins(HSPs),a group of highly conserved proteins,which can be divided into five families according to their molecular weight: HSP100 family,HSP90 family,HSP70 family,HSP60 family and small molecule HSP family.Studies have shown that heat shock protein is associated with pathological grade,tumor size,lymph node metastasis and distant metastasis of pancreatic cancer.For example,tumor-derived exosomes can activate STAT3 through the membrane surface HSP72 protein to promote the immunosuppressive effect of myelo-derived inhibitory cells,thereby inhibiting the immune surveillance of tumors.Therefore,based on the above studies,we used the mouse model of pancreatic cancer liver metastasis to explore the molecular changes in the microenvironment before metastasis in the liver,and proposed the hypothesis that exosomes from pancreatic cancer had domesticated macrophages in the microenvironment before liver metastasis,namely kupffer cells.These domesticated tumor-related macrophages secrete cytokines that facilitate the colonization and growth of circulating tumor cells.Part 1.Tumor-derived exosomes regulate Kupffer cells to secrete chemokine to promote metastasisLiver metastases model is the most common way of modeling the spleen injection and intrahepatic plant directly,and in order to better simulate microenvironment before liver metastasis of pancreatic cancer,we choose the spleen injection of tumor cells to liver metastasis model,can be a good simulation of pancreatic cancer released from the primary tumor,invasive blood vessels,infringement of intravascular and domestication metastases microenvironment until a series of cascade process secondary tumor growth.Human pancreatic cancer cell lines including BXPC3,panc-1,and CFPAC were injected into the spleen of nude mice without thymus,and the pancreatic cancer cell lines of PAN02 mice were injected into the spleen of C57 mice to observe the liver metastasis of pancreatic cancer.The results showed that PAN02 cell line had the highest success rate,followed by panc-1.At the same time,in order to verify the tumorigenesis effect of pancreatic cancer cell line on mouse body weight,we observed the liver metastasis of tumor in the mouse pancreatic cancer cell line PAN02 with luciferase expression gene injected into the spleen.In vivo fluorescence imaging and HE imaging showed that the cancer cells could be transplanted into the liver of mice through a series of metastasis pathways.In addition,two mouse models,pan02-c57 and panc-1-nude mice,were used to isolate and purify kupffer cells from the liver of mice at different periods by gradient separation method for transcriptome sequencing.Sequencing results showed that the expression levels of various chemokines and receptors were significantly increased in domesticated Kupffer cells.These chemokines play an important role in the colonization of tumor cells.Meanwhile,q-pcr was used to verify the expression of different chemokines.Finally,we concluded that in the model of hepatic metastasis of pancreatic cancer,a large number of clustered Kupffer cells were domesticated and activated,resulting in the up-regulated expression of various chemokines and their receptors,which further promoted the metastasis of tumor cells.Part 2.Tumor-derived exosomes activate Kupffer cells via My D88-dependent TLR4 pathwayIn order to explore the activation of tumor derived exosomes on Kupffer cells in liver and the relevant mechanism,we asked,which active components in tumor derived exosomes activate Kupffer cells to make them tumor-related macrophages? What are the mechanisms and signaling pathways that activate Kupffer cells? We first cultured tumor tissue in vitro,then extracted exosomes by overspeed centrifugation,and identified them as vesicles with a diameter of 40-150 um by electron microscopy.Secondly,abdominal lavage was used to obtain peritoneal macrophages from mice,which were cultured in vitro,attached to the wall and rested.In order to explore the activation effect of exosomes on macrophages at different concentrations and at different times,the optimal stimulation concentration and time were obtained.Based on these results,we also investigated which active components in exosomes stimulate macrophages,DNA,RNA,or proteins? By adding DNA enzymes,RNA enzymes and proteases,we concluded that the proteins contained in exosomes were the most active to macrophages.Currently,TLRs are believed to be downstream receptors of exosome activity,and they can recognize endogenous or exogenous danger signals to initiate an immune response,including TLR2,TLR4,TLR7,and TLR8.To investigate the downstream signaling pathways activated by exosomes in macrophages,wild-type and defective mice were compared.We extracted macrophages from TLR2-/-,TLR4-/-,TLR9-/-,and related molecules myd-88-/-and rig-/-mice,stimulated them with the optimal exosomes,and then observed the activation of macrophages.Finally,TLR4 was found to be the main receptor of liver Kupffer cells to receive exosome stimulation. |
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