IDO1-dependent Activation Of KYN/NFATC2/CXCL8 Pathway Increases Neutrophil Recruitment In EC

Background and objective:The latest cancer statistics in China(2015)show that the incidence of EC(esophageal cancer)rank the third among men,inferior only to lung cancer and gastric cancer,ranking the fifth among women with the fourth highest mortality.Since the advent of multidisciplinary treatment,the response of esophageal cancer patients has been greatly improved,but the 5-year survival rate still does not exceed 20%,One of the reasons for the poor prognosis of esophageal cancer is that many patients,in advanced stage once diagnosed,often have local infiltration and extensive lymph node metastasis.Infiltration into adjacent normal tissues and lymph node metastasis are the most important biological characteristics of EC and become the biggest obstacles for curing EC.Tumor combates the anti-tumor immune response through the formation of the suppressive TME(tumor microenvionment).TME is a complex system composed of tumor cells,lymphocytes,lymphoid endothelial cells and their secreted factors,which are closely related to tumor progression.Therefore,in this study,we will mainly explore mechanisms and clarify the relationship between tumor cells and immune cells in TME of EC,and provide new insights for the development of new immunotherapy for EC patients.Indoleamine 2,3-dioxygenase(IDO),belonging to heme-containing monomer redox enzymes,is rate-limiting enzyme mediating the metabolism of tryptophan(TRP)to kynurenine(KYN1).IDO1 is overexpressed in many kinds of tumor.It both induces Treg production and affects the proliferation and apoptosis of T cells,thereby allowing tumor cells to evade surveillance of immune system.More and more studies have found that the high expression of IDO is closely related to outcome of cancer patients.In hepatocellular carcinoma,non-small cell lung cancer,colon cancer and invasive cervical cancer,it has been reported that IDO is highly expressed and lead to poor prognosis.Therefore,a variety of small molecule inhibitors for IDO1 have been developed and shown promising results in a series of preclinical experiments and clinical trials.IDO1 inhibitors alone or in combination with other immunotherapy can restore T cell anti-tumor immune response and inhibit the tumor development.However,whether IDO1 is expressed in esophageal cancer,how it works in the esophageal cancer microenvironment,and whether IDO1 inhibitors can be used in esophageal cancer have not been reported.We first detected the expression of IDO1 in the tumor tissue and the normal tissue of esophageal cancer patients in part ?,collected the clinicopathological information of the patients,retrospectively analyzed the relationship between the expression of IDO1 and clinicopathological parameters,and found that higher IDO1 expression correlated significantly with poor prognosis of esophageal cancer patients.Then in part ? we screened the commonly used esophageal cancer cell lines for their IDO1 expression and selected EC1 as the cell line for follow-up experiments as it has the highest constitutive expression of IDO1.By stably knocking down IDO1 in EC1,we observed that IDO1 affected CXCL8 secretion most significantly and further found that IDO1 increased neutrophil recruitment by activating the KYN/CXCL8/CXCR1 pathway using transwell and other experiments in vitro.In order to investigate the mechanism how KYN regulate the expression of CXCL8 in esophageal cancer cells,we performed the transcription factor(TF)PCR array and verified it was the TF NFATC2 that affects CXCL8 in part ?.Then we confirmed that IDO1 activates NFATC2 through KYN to promote the transcription and secretion of CXCL8.Finally,in part IV,we demonstrate that IDO1 in esophageal cancer can also increase neutrophil recruitment in vivo through activating the NFATC2/CXCL8 pathway using both animal model and tumor tissues from esophageal cancer patients.In summary,the current research mainly studies the expression and significance of IDO1 in esophageal cancer and explores the role and mechanism of IDO1 in esophageal cancer microenvironment,providing the theoretical basis for the application of treatment strategy targeting IDO1 in esophageal cancer patients.Part ? The expression and clinical significance of IDOlin Esophageal cancerMethods:1)Q-PCR was performed to detect the expression of IDO1 in tumor tissues and matched normal tissues in EC patients.2)IHC was used to detect the expression of IDO1 in EC tissues.Results:1)Higher level of IDO1 expression in cancer tissues is observed compared with matched normal tissues in EC patients.2)IDO1 expression is significantly associated with TNM stage and tumor grade in EC patients.3)Higher expression of IDO1 predicts significantly poorer outcome in EC patients.Part ? IDO1 mediates recruitment of neutrophils through upregulating CXCL8 in esophageal cancer cellsMethods:1)LEGENDplexTM Human Proinflammatory Chemokine Panel and Human Th Cytokine Panel Kit was used to screen for cytokines and chemokines associated with IDO1 function.2)LC/MS was conducted to analyze tryptophan and kynurenin concentration in supernatant of esophageal cancer EC1 after IDO1 knockdown.3)Magnetic Separation Methods was used to isolate and purify neutrophils from peripheral blood.4)Transwell assay was performed to detect neutrophil recruitment by esophageal cancer cells after IDO1 knockdown and KYN treatment.5)Elisa was used to detect CXCL8 secretion in EC 1 supernatant after different treatment.6)Flow cytometry was used to evaluate the purity of neutrophil isolation and the expression of CXCR1 and CXCR2.Results:1)IDO1 has no effect on esophageal cancer cell proliferation and apoptosis.2)IDO1 promotes the CXCL8 secretion in esophageal cancer cells.3)The secretion of CXCL8 in esophageal cancer cells is promoted by IDO1 through KYN production.4)IDO1 mediates recruitment of neutrophils through KYN/CXCL8/CXCR1 in esophageal cancer cells.Part ? IDO1 promotes CXCL8 production via activating NFATC2Methods:1)Transcription factor PCR array was performed to screen for the downstream transcription factors closely related to IDO1 and KYN function.2)IF was conducted to observe NFATC2 nuclear translocation after KYN treatment.3)Western Blot was used to prove that KYN can mediate the transfer of NFATC2 from the cytoplasm to the nucleus.4)Dual-luciferase reporter assay was conducted to observe the activation of CXCL8 promoter.5)CHIP assay was used to validate the direct binding of the transcription factor NFATC2 to the CXCL8 promoter region.Results:1)IDO 1 promotes CXCL8 production via NFATC2.2)KYN induces NFATC2 activation.3)NFATC2 promotes CXCL8 transcription by directly binding to the promoter region of CXCL8.Part IV IDO1 increases neutrophil infiltration in EC tissues via NFATC2/CXCL8 activationMethods:1)Animal experiments were performed to explore whether IDO1 can activate NFATC2/CXCL8 pathway and increase the neutrophil chemotaxis to the tumor site in vivo.2)IHC was used to detect the expression of CXCL8 and NE in EC tissues.Results:1)In animal models,IDO1 increases neutrophil recruitment by activating the NFATC2/CXCL8 signaling.2)CXCL8 overexpression in esophageal cancer tissue predicts a poor prognosis in EC patients.3)More neutrophils infiltrate in IDO1 high CXCL8 high EC tissues.Conclusion1)IDO1 overexpression in EC tissues predicts a poorer prognosis in EC patients.2)IDO1 in EC cells increases neutrophil recruitment through CXCL8 upregulation.3)IDO1 upregulates CXCL8 expression by activating KYN/NFATC2 pathway.4)IDO1 increases neutrophil infiltration by activating KYN/NFATC2/CXCL8 pathway in EC tissues.