Acetylcholine Impairs The Recruitment Of CD8+ T Cells In Perineural Invasive Pancreatic Ductal Adenocarcinoma Via Regulating CCL5

OBJECTIVES:To determind the different expression of perineural invasion(PNI)associated protein among PDAC;To investigate the biological process of perineural invasive PDAC using gene expression profile microarray;To definite the specific type of immune cell infiltrated within perineural invasive PDAC;To explore the effect and probable mechanism of acetylcholine impairs the recruitment of CD8+ T cells in perineural invasive PDAC.METHODS:We exploited the public database to analyse gene expression with different perineural invasion capacity and validated CD74 as a significantly different target.We utilized tissue microarray of Ren Ji cohort to valid the expression of CD74 and its downstream neurotrophic factor GDNF during the course of perineural invasion.Furthermore,we used the gene expression profile microarray to determind the aberrant expression gene and biological process betweeen PNI group and non-PNI group in Ren Ji cohort of 50 PDAC cases.We found that progress pathway including immunoreaction and immune cell chemotaxis wasremarkably down-regulated.Subsequently,we validated the CD8+ T cells distribution infiltrated within perineural invasive PDAC.Ultimately,we introduced a series of laboratory technique such as HPLC,protein microarray,cell chemotaxis assay and RNA-scope to explore the effect and probable mechanism of acetylcholine impairs the recruitment of CD8+ T cells in perineural invasive PDAC via regulating CCL5.RESULTS:We sorted out several probably candidate perineural invasion associated gene by analysing the GEO database and validated CD74 high expression in perineural invasive group.CD74 was positive expressed in PDAC tissue and prominently correlated with the incidence of PNI of PDAC patients(p=0.021).We investigated and validated CD74down-stream potential neurotrophic factor GDNF by knocking down CD74 or using exogenous agonist rMIF,respectively.Then we assessed the different process pathway between PNI and non-PNI group from gene expression profile microarray and detected that progress pathway including immunoreaction and immune cell chemotaxis was remarkably down-regulated.As well,distribution of CD8+ T cells infiltrated within perineural invasive PDAC was suppressed.Next,results from HPLC indicated that the concentration of acetylcholine from parasympathetic nerve system increased in PNI group.CD8+ T cells in PBMC showed a strong chemotactic response toward the supernatant of pancreatic cancercells and this response can be restricted by the pre-treatment of acetylcholine while mecamylamine(MEC),the antagonist of nicotinic acetylcholine receptors,could rescue the acetylcholine induced reduction of CD8+ T cell chemotaxis.Finally,with the aid of protein microarray and RNA-scope,we explored and validated the probable mechanism of acetylcholine impairs the recruitment of CD8+ T cells in perineural invasive PDAC via regulating CCL5.CONCLUSION:Perineural invasion is one of characterastic biological processes among PDAC and there is complicated and vital interaction between peripheral nerve and tumor.Tumor cells promotes perineural plasticity and incidence of perineural invasion while neurotransmitters,neurotrophic factors and chemokines support survival and invasion of tumor cells.Tumor-specific expressed membrane protein CD74 may induce perineural invasion of PDAC through GDNF.In perineural invasive PDAC,progress pathway including immunoreaction and immune cell chemotaxis was remarkably suppressed,especially aberrant secreted parasympathetic nerve system neurotransmitter acetylcholine significantly attenuated the recruitment capacity of CD8+ T cells by tumor.Finally,we validated aberrant increasing acetylcholine impaired the recruitment of CD8+ T cells in perineural invasive pancreatic ductal adenocarcinoma thus via regulating the expression of chemokine CCL5.