Elevated DLL3,the Ligand Of Notch Signaling Pathway,in Stomach Cancer By Tumor Associated Macrophages Enhances Cancer Cell Proliferation

Introduction and AimNotch signaling pathway,a highly conserved signaling pathway,involves in the development and progression of tumors.DLL3,aligand in Notch signaling pathway,functions oncogene or tumor suppressor gene in different tumors.However,little is known about the role of DLL3 in gastric cancer.Tumor-associated macrophages(TAMs).macrophages infiltrating into tumor tissues,are the main infiltrating cells in the tumor microenvironment(TME).TAMs regulate the growth of tumor cells by various pathways.It's known that Notch signaling pathway participates in regulating the differentiation and function of TAMs.In this study,a co-culture model of gastric cancer cells and macrophages was constructed in vitro to investigate the direct or indirect interactions of gastric cancer cells,macrophages and tumor microenvironment mediated by DLL3.Methods1?Construct a co-culture model of MKN45 and macrophages(M?).M? in the upper chamber while MKN45 cultured in the lower chamber.The proliferation of MKN45 was detected by MTT assay.And the secretion of IL-10,IL-1? and IL-12 in supernates was detected by ELISA.Indeed,the expression of Notch signaling was detected by Western blot.2?DLL3-overexpression MKN45 stable cell line(MKN45-DLL3)was constructed.Also DLL3-siRNA was transiently transfected into MKN45(MKN45-siDLL3).The role of up-regulation and down-regulation of DLL3 on MKN45 proliferation was detected by MTT.3?MKN45-DLL3 was co-cultured with M?.And the secretion of IL-10,IL-1? and IL-12 in supernates was detected by ELISA.4?Whole transcriptome sequenced MKN45-DLL3 analyzing the gene regulated by DLL3.Got the gene IL-33,which was associated with M?,then identified by qPCR and ELISA.And stimulated M?with rIL-33.The secretion of IL-10,IL-1?and IL-12 in M? was detected by ELISA.5?Co-immunoprecipitation and mass spectrometry(IP-MS)were performed to detect the proteins that interacted with DLLS in MKN45.Results1?MKN45 co-culturing with M? promoted MKN45 cell proliferation and the secretion of IL-10,IL-1? and IL-12 in TME.And up-regulated the expression of DLL3 in MKN45.2?DLL3 was successfully up-regulated and down-regulated in MKN45 cells,and promoted and inhibited the proliferation of MKN45 cells.The up-regulation of DLLS in MKN45 induced more secretion of IL-10 and IL-12 in TME.3?Up-regulation of DLL3 in MKN45 induced IL-33 expression.However,IL-33 did not contribute to the secretion of IL-10,IL-1? and IL-12 by M?.4?DLL3 in MKN45 interact with HSPA8(heat shock cognate 71 kDa protein)and LG3BP(galectin-3-binding protein).DLL3 might target HSPA8 and LG3BP to endosomal/lysosomal degradation.ConclusionCo-culture of M? and MKN45 cells up-regulated the expression of DLLS in MKN45,promoted the proliferation of MKN45,and increased the secretion of IL-10 and IL-12 in the TME.This is a new insight of the interaction between gastric cancer and macrophages via the Notch signaling pathway.