Hepatocellular carcinoma (HCC) progression is closely related to pathological fibrosis, which involves heterotypic intercellular interactions (HIIs) between liver cancer cells and fibroblasts. Here, we studied them in a direct coculture model, and identified fibronectin from fibroblasts and integrin-α5β1 from liver cancer cells as the primary responsible molecules utilizing CRISPR/Cas9 gene-editing technology. Coculture led to the formation of 3D multilayer microstructures, and obvious fibronectin remodeling was caused by upregulated integrin-α5β1, which greatly promoted cell growth in 3D microstructures. Integrin-α5 was more sensitive and specific than integrin-β1 in this process. Subsequent mechanistic exploration revealed the activation of integrin-Src-FAK, AKT and ERK signaling pathways. Importantly, the growth-promoting effect of HIIs was verified in a xenograft tumor model, in which more blood vessels were observed in bigger tumors derived from the coculture group than that derived from monocultured groups. Hence, we conducted triculture by introducing human umbilical vein endothelial cells, which aligned to and differentiated along multilayer microstructures in an integrin-α5β1 dependent manner. Furthermore, fibronectin, integrin-α5, and integrin-β1 were upregulated in 52 HCC tumors, and fibronectin was related to microvascular invasion. Our findings identify fibronectin, integrin-α5, and integrin-β1 as tumor microenvironment-related targets and provide a basis for combination targeted therapeutic strategies for future HCC treatment.