The objective of this study is to design a cancer invasion model by making use of cancer-associated fibroblasts (CAF) or tumor-associated macrophages (TAM) and gelatin hydrogel microspheres (GM) for the sustained release of drugs. The GM containing adenosine (A) (GM-A) were prepared and cultured with TAM to obtain three-dimensional (3D) TAM aggregates incorporating GM-A (3D TAM-GM-A). The GM-A incorporation enabled TAM to enhance the secretion level of vascular endothelial growth factor. When co-cultured with HepG2 liver cancer cells in an invasion assay, the 3D TAM-GM-A promoted the invasion rate of cancer cells. In addition, the E-cadherin expression level decreased to a significantly greater extent compared with that co-cultured with TAM aggregates incorporating GM, whereas the significantly higher expression of N-cadherin and Vimentin was observed. This indicates that the epithelial-mesenchymal transition event was induced. The GM containing transforming growth factor-β1 (TGF-β1) were prepared to incorporate into 3D CAF (3D CAF-GM-TGF-β1). Following a co-culture of mixed 3D CAF-GM-TGF-β1 and 3D TAM-GM-A and every HepG2, MCF-7 breast cancer cell, or WA-hT lung cancer cell, the invasion rate of every cancer cell enhanced depending on the mixing ratio of 3D TAM-GM-A and 3D CAF-GM-TGF-β1. The amount of matrix metalloproteinase-2 (MMP-2) secreted also enhanced, and the enhancement was well corresponded with that of cancer cell invasion rate. The higher MMP secretion assists the breakdown of basement membrane, leading to the higher rate of cancer cell invasion. This model is a promising 3D culture system to evaluate the invasion ability of various cancer cells in vitro. Impact statement This study proposes a cell culture system to enhance the tumor-associated macrophage function based on the combination of three-dimensional (3D) cell aggregates and gelatin hydrogel microspheres (GM) for adenosine delivery. An additional combination of 3D cancer-associated fibroblasts incorporating GM containing transforming growth factor-β1 allowed cancer cells to enhance their invasion rate. This co-culture system is promising to evaluate the ability of cancer cell invasion for anticancer drug screening.