Exosomal Zinc Transporter ZIP4 Promotes Cancer Growth And Is A Novel Diagnostic Biomarker For Pancreatic Cancer

Objective: Pancreatic cancer is one of the deadliest and most poorly prognostic gastrointestinal cancers.How to improve its overall survival rate is one of the hot spot in the field of cancer research.The early symptoms of pancreatic cancer are relatively hidden and the location of the disease is deep in the abdomen of the human body.These characteristics make it difficult to diagnose pancreatic cancer by routine physical examination,imaging examination or other clinical examination at the early phase of its disease progression.In addition,current clinically applied tumor diagnostic biomarkers such as CA19-9 have rather poor diagnostic efficiency for pancreatic cancer.This requires researchers to further elucidate the molecular mechanisms of pancreatic cancer's disease progression and develop novel diagnostic biomarkers.Exosomes are small extracellular vesicles that are secreted by cells to the extracellular space and act as message couriers in intercellular communication.Exosomes carry a large number of biologically active substances like proteins and nucleic acids,which can regulate the physiological functions of the recipient cells after being taken up.A large number of exosomes secreted by various tissue cells are enriched in the body fluids like blood.Substances carried in these exosomes are more stable for the protection of exosomal membrane.This favorable characteristic endows exosomal proteins and nucleic acids with good potential as diagnostic biomarkers.For patients with pancreatic cancer,cancer tissues release a large number of exosomes.These cancer derived exosomes are involved in all stages of disease progression and are significantly enriched in the body fluids like blood.At present,the role of pancreatic cancer cells derived exosomes in the disease progression and their potential as diagnostic biomarkers for pancreatic cancer have not been fully studied.In this study,we used two previously established isogenic hamster pancreatic cancer cell lines with different malignant phenotype(PC-1.0 cells as highly malignant phenotype,PC-1 cells as medium-low malignant phenotype)to isolate exosomes and further performed proteomic analysis.The results showed that zinc transporter ZIP4(ZIP4)was the most up-regulated protein in PC-1.0 derived exosomes.It has been reported that the expression level of ZIP4 in pancreatic cancer tissues and humanpancreatic cancer cell lines is higher than that of corresponding para-cancerous cancer tissues and human pancreatic cell lines.These published results and our own results suggested that ZIP4 may play a critical role in the progression of pancreatic cancer.However,studies on the role of exosomal ZIP4 in pancreatic cancer have not been reported so far.Therefore,in this study we focused on the role of exosomal ZIP4 in the progression of pancreatic cancer and its potential application as a diagnostic biomarker for early detection.Methods: In this study,we utilized 0.22 ?m sterilizing filter and 100 kd ultrafiltration tube to remove the cell debris and the unrelated supernatant protein contamination.The treated supernatant was further subjected to exosomes isolation using the SBI company's exosomes isolation kit.Isolated exosomes were validated by Western blotting for expression of exosomal markers and by direct observation with transmission electron microscopy.The exosomes derived from the PC-1.0 cells were fluorescently labeled and co-cultured with PC-1 cells.Uptake of fluorescently labeled exosomes by PC-1 cells was observed under a fluorescence microscope.Cell proliferation was analyzed by CCK-8 assay,cell migration was analyzed by wound healing assay,and cell invasion was analyzed by Transwell chamber assay to verify the effect of PC-1.0 derived exosomes on the phenotype of PC-1 cells.Differentially expressed exosomal proteins of PC-1.0 cells and PC-1 cells were analyzed using the isotope-labeled relative and absolute quantitation(iTRAQ)technique.Combined with identified fold change,literature mining,public database mining,candidate proteins for downstream experiments were screened and further verified by Western blotting with PC-1.0/PC-1 cells and human pancreatic cancer cell lines AsPC-1/Capan-2 cells.After selecting ZIP4 as the downstream research target,the expression level of ZIP4 in PC-1.0 derived exosomes was knocked down by short hairpin RNA(shRNA).The efficiency of shRNA transfection and knockdown was measured with observation of the fluorescence signal under a fluorescence microscope and Western blotting.Cell proliferation was analyzed by CCK-8 assay,cell cycle was analyzed by PI staining,cell migration was analyzed by wound healing assay,and cell invasion was analyzed by Transwell chamber assay to compare the different functions of normal exosomes and ZIP4 knockdown exosomes derived from PC-1.0 cells on PC-1 cells.By establishing a subcutaneous tumor model in nude mice,PC-1 cells were plantedinto the axilla of nude mice,and then normal exosomes or ZIP4 knockdown exosomes derived from PC-1.0 cells were injected regularly to further verify the experimental conclusions of the in vitro experiment.The expression level of ZIP4 was further verified by paraffin sectioning technique and immunohistochemical technique on the surgically resected tumor.Finally,serum samples from pancreatic cancer patients,benign pancreatic tumor patients,pancreatitis patients,gallbladder disease patients and healthy volunteers were collected from the clinic.Enzyme-linked immunosorbent assay(ELISA)was further used to analyze the expression level of ZIP4 for the exsomes isolated from serum by using a SBI kit.Finally,the receiver operating characteristic curve(ROC curve)was used to detect the diagnostic efficacy of serum-derived exosomal ZIP4 for pancreatic cancer,benign pancreatic tumor,pancreatitis,and gallbladder disease.The experimental results obtained in the study were statistically analyzed using SPSS 19.0 software.The quantifiable experimental data were expressed as mean and standard deviation,and the significance analysis was performed using the Student t test.p<0.05 was considered statistically significant.The results were visualized using GraphPad Prism 7 software.Results: 1.The Western blotting of exosomes markers and transmission electron microscopy confirmed that the 0.22 ?m sterilizing filter/100 kd ultrafiltration tube/exosome extraction kit method applied in this study could isolate exosomes from the supernatant with a satisfying purity.2.The results of fluorescent labeling experiments and in vitro functional experiments indicate that PC-1.0 cell-derived exosomes can promote the proliferation,migration and invasion of PC-1 cells after being taken up by PC-1 cells.3.The proteomic analysis Identified 154 up-regulated proteins in PC-1.0 cell-derived exosomes and 189 down-regulated(fold change >1.5 or fold change <0.67),in which ZIP4 was the most up-regulated exosomal protein with the highest fold change(fold change 7.152).Western blotting was used for fruther validation and the expression level of exosomal ZIP4 was higher in the pancreatic cancer cell lines with a highly malignant phenotype(PC-1.0 and AsPC-1 cells)than pancreatic cancer cell lines with a medium-low malignant phenotype(PC-1 cells and Capan-2 cells).4.In vitro and in vivo functional experiments showed that exosomal ZIP4 could promote the proliferation and migration of pancreatic cancer cells in vitro,but showed no obvious regulatory effect on invasive ability,and could promote the growth of implanted tumors in vivo.Theexpression level of ZIP4 was higher in the tumor with a faster growth rate.5.Results of ELISA assay showed that the expression level of serum exosomal ZIP4 in pancreatic cancer patients was higher than that in patients with benign pancreatic tumors,pancreatitis,gallbladder diseases and healthy controls.Results of ROC curve analysis suggested that the expression level of serum exosomal ZIP4 possesses a good diagnostic power for differential diagnosis of pancreatic cancer against benign pancreatic tumor,pancreatitis,gallbladder disease and healthy controls.Conclusion: 1.Exosomes derived from pancreatic cancer cell line with a highly malignant phenotype(PC-1.0)can promote the proliferation,migration and invasion ability after being taken up by pancreatic cancer cell line with a medium-low malignant phenotype(PC-1).2.The expression level of exosomal ZIP4 in pancreatic cancer cells with a highly malignant phenotype(PC-1.0,AsPC-1)is higher than that of pancreatic cancer cells with a medium-low malignant phenotype(PC-1,Capan-2).3.Exosomal ZIP4 could significantly promote the proliferation and migration of pancreatic cancer cells in vitro,but has no significant effect on the invasive ability.Exosomal ZIP4 could promote the growth of implanted tumors in the subcutaneous tumor model of nude mice and the expression level of ZIP4 was higher in the implanted tumors with a faster growth rate.5.The expression level of serum exosomal ZIP4 in patients with pancreatic cancer is higher than that in patients with benign pancreatic tumor,pancreatitis,gallbladder disease and healthy subjects.The expression level of serum exosomal ZIP4 has a good diagnostic power for differential diagnosis of pancreatic cancer and benign pancreatic tumor,pancreatitis,gallbladder disease and healthy controls.