Tumor growth and metastasis entangle the alteration and recruitment of non-malignant cells to the primary tumor, among them immune cells, constituting the tumor microenvironment (TME). Communication between tumor cells and their stroma has been shown as a fundamental driving force of the tumoral process. A great deal of effort has been focused on depicting their specific interactions and crosstalk. However, most research has been carried out in 2D conventional cultures that alter cell morphology and intracellular signaling processes. Considering these premises, we have developed a 3D cell co-culture model to mimic T cell infiltration into the tumor mass and explore tumor-immune cells interactions in the TME. Expression of specific cell markers and assessment of cell proliferation were carried out to characterize the proposed 3D co-culture model. Additionally, the study and profiling of the secretome revealed a subset of particular cancer-related inflammation proteins prompted upon 3D cultivation of tumor cells in presence of lymphocytes, pointing out an intercellular communication. Altogether, these results suggest that our 3D cell co-culture model can be a useful tool to identify and study critical factors mediating the crosstalk between tumor and immune cells in the TME. Finally, the potential of this model as a drug-screening platform has been explored using docetaxel as a model antitumoral compound.