Nutrient Stress Drives Fibroblast PD-L1 Secretion Via ACSS2/IL-6 Activation In Esophageal Squamous Cell Carcinoma

【Objective】To investigate the molecular mechanism by which ACSS2 regulates the expression and secretion of IL-6 in esophageal squamous cell carcinoma cells under nutrient stress,and to reveal the mechanism by which IL-6 from esophageal squamous cell carcinoma cells promotes the secretion of sPD-L1 in tumor-related fibroblasts.【Methods】(1)Immunohistochemical staining was used to anaiysis the distribution festures of ACSS2 in 50 cases esophageal squamous cell carcinoma;ACSS2 and CD31co-expression were detected by immunohistochemistry and immunofluorescence;RT-PCR and Western blotting were used to detect the level of ACSS2 in ESCC and Het-1A with nutrient stress for different time.(2)RT-PCR and ELISA were used to detect IL-6,TGF-β,TNF-αmRNA and secretion level in ESCC with nutrient stress;RT-PCR was used to measure the correlation between ACSS2 and IL-6 mRNA in the wake of nutrient stress for different time in ESCC;combining with siRNA-ACSS2,IL-6 expression and secretion were deteceted by RT-PCR,Western blotting and ELISA in ESCC with nutrient stress;immunohistochemical staining was used to anaiyze IL-6characteristics of distribution and the correlation to ACSS2 in ESCC tissues.(3)The changes of TLR4,MyD88,p-p65 and p65 were detected by Western blotting in ESCC with nutrient stress;combining with NF-κB inhibitor and siRNA-ACSS2,ACSS2 and NF-κB pathway activation were detected by Western blotting and the secretion of IL-6 was determined by ELISA in ESCC with nutrient stress.(4)Flow cytometry and immunoblotting were used to analyze PD-L1 expression in ESCC with nutrient stress;immunohistochemical staining was used to anaiysis the the expression of PD-L1 in ESCC tissues;PD-L1~+cell types and the relationship to ACSS2 furtherly were explored by immunohistochemistry and immunofluorescence;the expression of PD-L1 in CAFs treated witn upernatant from ESCC with nutrient stress or not was determined by flow cytometry;immunoblotting and ELISA were used to analyse the relationship between PD-L1 in CAFs and IL-6 in ESCC;CCK8was used to determine the effect of sPD-L1 from CAFs on CD8~+T cell viability.【Results】(1)The expression of ACSS2 in ESCC tissues was higher in infiltration,breaking through basement membrance and forming cancer embolus than other regions.The intensity of ACSS2 was negative correlation with CD31.ACSS2transcription and protein expression were increased in ESCC rather than in normal esophageal squamous epithelial cells with nutrient stress for different time(2)The mRNA and secretion levels of IL-6 were significantly up-regulated in ESCC after nutrient stress treatments,while TGF-βmRNA and secretion levels showed an opposite trend and TNF-αshowed no statistical significance changes.The mRNA and secretion levels of IL-6 in ESCC were affected by ACSS2.IL-6 was mainly expressed in tumor cells and was positively correlated with the expression of ACSS2 in ESCC tissues.(3)ACSS2 induced by nutrient stress in ESCC upregulated p-p65 but had no effect on TLR4,MyD88 and p65.Combined with siRNA-ACSS2 and NF-κB inhibitors,ACSS2 regulated the activation of NF-κB and the increase of IL-6secretion in ESCC with nutrient stress.(4)Nutrient stress treatment had no significant effect on the expression of PD-L1in ESCC.PD-L1~+cells were CAFs in ESCC tissues.IL-6 from ESCC stimulated PD-L1 secretion from CAFs.The increase of PD-L1 secretion in CAFs inhibited the activity of CD8~+T cells.【Conclusions】Nutrient stress can drives the immune escape through the secretion of IL-6 via ACSS2/NF-κB in ESCC and further resulting soluble PD-L1 upregulation from CAFs.