Exosomes Derived From Pancreatic Cancer Cells Promote Its Own Recruitment On Cancer Associated Fibroblasts And The Underlying Mechanism

Background and objective:Pancreatic cancer is the most invasive solid tumor with a five-year survive rate merely 8%.Most patients suffer from advancement or distant metastasis when first diagnosed and lost the opportunities for radical resection.Accordingly,it is of tremendous significance of exploring the metastatic mechanism of pancreatic cancer,understanding the biological behaviors and exploring the therapeutic targets of pancreatic cancer.In the history of research on tumor metastasis,the "seed and soil"hypothesis proposed by British doctor Stephen Paget has a far-reaching influence.The important implication lies on the fact that Stephen Paget recognized only the metastatic host organ(soil)matching with tumor cells(seed),the metastatic site occurs,which laid the foundation for the concept of metastatic microenvironment.Cancer associated fibroblasts(CAFs)are key members of tumor microenvironment which secret various signal protein regulating the proliferation of cancer cells,inducing angiogenesis and promoting invasion and metastasis.Moreover,circulating tumor cells siting in the host organs tend to recruite CAFs and participates in the construction of tumor microenvironment which promotes the occurrence of metastatic nodules.Exosomes are extracellular membranous vesicles(EVs)with diameters range from 30-100nm derived from various kinds of cells both in physiological and pathological status.Exosomes loaded with proteins,bioactive lipids,and nucleic acid act on the target cells through the paracrine and telecine pathways and realizes intracellular communication.Lin28B is a RNA binding protein expressing both in the nucleus and the cytoplasm.The protein is highly expressed in tremendous cancer cell lines which is closely related to tumorigenesis,rapid progression and metastasis.In the pancreatic cancer,activation of Lin28B/let-7 pathway will promote the expression of the downstream gene including HMGA2,c-Myc and facilitates pancreatic cancer progression and metastasis.Hence,our research aims to explore the existence of the phenomenon that pancreatic cancer derived exosomes promote the pancreatic cells' recruitment effect on CAFs and the underlying mechanism.We hope the results will benefit the discovery of new diagnostic and therapeutic targets.Method:1 PANC-1 and MIA PaCa-2 cell lines were selected from all the pancreatic cancer cell lines based on the metastatic ability.2 pancreatic cancer derived exosomes were harvested from the PANC-1 and MIA PaCa-2 cell through the following steps:cells culture,cell passage,conditioned medium collection,ultracentrifugation.Identification of the exosomes and the internalization assay verifys the crosstalk between exosomes and pancreatic cancer cells.3 pancreatic cancer cells were treated with exosomes,then Transwell assay was conducted to observe pancreatic cancer cells' recruitment effect on pancreatic stellate cell(PSC).Moreover,additional Transwell assay was conducted to eliminate the confounding factors on the recruitment effect.4 The expression level of recruitment associated protein PDGFB was measured using the western blotting assay.Meanwhile,the PDGFR? expression level was also measured to make sure the effect of exosomes on the expression of receptor protein.5 By means of reviewing the literature,key metastasis-related protein Lin28B came into our sight.Western blotting and PCR techniques were applied to verify the expression level of Lin28B in pancreatic cancer derived exosomes and the role of Lin28B in the recruitment effect driven by exosomes,Meanwhile,finding out the underlying potential downstream pathways.6 The metastatic tumor model was established by injection of the pancreatic cancer cell-PANC-1 in the spleen of nude mice,and the fluorescence imaging experiment in vivo was used to further verify whether the pancreatic cancer derived exosomes promote the recruitment of PSC by pancreatic cancer cells.Result:1 Pancreatic cancer derived exosomes readily enter to the PANC-1 and MIA PaCa-2 cells and promote the pancreatic cancer cells' recruitment effect on PSC.2 Exosomes-treated group(pancreatic cancer treated with exosomes)shows a significant up-regulating of the expression level of PDGFB.However,PSC treated with exosomes has no impact on the expression level of PDGFR?.3 Protein Lin28B is rich in the pancreatic cancer derived exosomes.Additionally,pancreatic cancer derived exosomes will upregulating the protein Lin28B,which results in the downregulating of the tumor suppressor-let-7,leading to the over-expression of oncogenes-HMGA2?c-MYC.Conclusion:The research suggests that pancreatic cancer derived exosomes promote the pancreatic cancer cells' recruitment effect on CAFs and the Lin28B/let-7 pathway plays important role in the pathological courses.