Hepatic stellate cell (HSC) line LX-2 is activated by liver cancer stem-like cells (LCSLCs) and produces various cytokines that make up most of the hepatocellular carcinoma (HCC) microenvironment.The new genistein derivative,7-difluoromethoxyl-5,4’-di-n-octylgenistein (DFOG),shows anticancer effects in multiple malignancies by controlling forkhead box M1 (FOXM1).In this study,we aimed to assess whether DFOG disrupts the crosstalk between human HSC LX-2 cells and LCSLCs.Distinct generations of MHCC97H-derived spheres were obtained with the second generation considered as LCSLCs which displayed enhanced self-renewal ability and elevated expression levels of CD133,CD44,and EpCAM proteins,as well as tumorigenicity,as revealed by colony formation assay in vitro and tumorigenicity assay in vivo.LX-2 and MHCC97H cells were co-cultured with/without DFOG (1,5,and 10 μM,respectively) using the transwell system.FOXM1 overexpression and/or knockdown were employed for mechanistic investigations.Our results suggested that Co-CM promoted LX-2 cell transformation into liver cancer-associated HSCs.Meanwhile,FOXM1 was up-regulated and the level of hepatocyte growth factor (HGF)was increased in LX-2 cells and in the supatant after Co-CM stimulation.Sphere and colony formation abilities in MHCC97H cells,and protein levels of CD133,CD44,and EpCAM,were also markedly elevated.DFOG dose-dependently inhibited the above effects,similar to FOXM1 knockdown in LX-2 cells.FOXM1 overexpression reversed the inhibitory effects of DFOG or FOXM1 knockdown or both on LX-2 cell activation and LCSLC feature induction in MHCC97H cells by LCSLC/LX-2 co-culture.This study demonstrated that DFOG disrupts the crosstalk between HSCs and LCSLCs to suppress LCSLC features via down-regulating FOXM1 expression and reducing HGF secretion in HSCs.