Breast cancer is the most common cancer in women and is second most common cancer worldwide. This represents about 12% of all new cancer cases and 25% of all cancers in women. Recently, researchers and oncologists are paying attention to the interaction intercurring among the cellular and acellular actors that play a role in the remodeling of the tumor microenvironment. The investigations of the cross-talk among cancer cells and stroma may enhance the knowledge of the mechanism of tumor resistance to the chemotherapeutic or promote the development of targeted therapy. A faster and more efficacy anti-cancer drug testing will be also possible using of three dimensional tumor models in preclinical studies in vitro. This will take the place of two dimensional flat cell culture and animal models. Developing a cancer preclinical model copycatting the tumor microenvironment is still challenging and is highly demanded since pharmaceutical companies lose huge amount of money in drug screening. Naturally-derived biomaterials are used to support the growth of cells due to their biocompatibility and mechanical properties. In this work, freeze-dried scaffold made up of silk protein fibroin from mulberry silkworm Bombyx mori are used to develop 3D breast cancer model. Healthy mammary fibroblasts are cultured alone or in combination with two cancer cell lines (MCF-7 and MDAMB-231) and are used to mimic non-invasive and metastatic phenotypes of the solid tumors. The proliferation and metabolic analyses are performed as well as gene expression. In particular, ECM marker analyses (Collagen I, MMP-2, MMP-3, MMP-9 and α-SMA) give information on how cancer cells and fibroblasts interact. The 3D breast cancer model developed could be a promising tools for the improvement of preclinical study workflow, since they are cheap, easy-to-handle and they are able to copycat the tumor microenvironment.