In vitro breast cancer tissue models play key roles in studying cancer biology and drug discovery. In efforts to recreate native breast cancer spatial organization, 3D in vitro models have received increased attention. These models can be categorized into two major classes: scaffold-based and scaffold-free systems. Scaffold based systems, such as pre-fabricated scaffolds and assembled hydrogels composed of natural and/or synthetic materials, promote cell-extracellular matrix (ECM) interactions. In the case of scaffolds, cells are seeded on the surface of a matrix, whereas in hydrogels, cells are surrounded by a matrix in all dimensions. In both cases, the cells can receive important physical and biochemical cues from the scaffolds that impact their function. In contrast, scaffold-free systems, such as cancer cell spheroids, promote extensive cell-cell interactions, as cells are densely packed in aggregate forms via cell-cell adhesion ligands. These cell-cell interactions via direct contact, in addition to secreted paracrine factors, are also important signals that regulate cell behavior. Despite substantial progress in developing 3D breast cancer models, significant challenges still remain. The breast cancer microenvironment typically possesses both strong cell-cell and cell-ECM interactions, and recapitulating them in in vitro 3D models is essential. However, in most in vitro systems, enhancing one of these interactions often results in decreasing the other. Collagen fibers are one of the major ECM molecules in breast tumors, and analyses of patient biopsies indicate that breast cancer cells often reside in collagen fiber-rich ECMs. Collagen fibers are highly ordered and hierarchical. In nature, collagen molecules assemble into fibrils with diameters on the order of a hundred nanometers. These fibrils bundle to form fibers with diameters of ~1-20 microns. Collagen fibers provide structural, mechanical and biochemical signaling to resident cells, which influences their behavior. However, few biomaterial systems have been developed based on natural collagen fibers for 3D cell growth and tissue formation. Here, we developed a strategy for 3D breast tissue model construction in vitro using extracted collagen fibers from decellularized natural tissues. In this platform, breast cancer cells and supporting cells are cultured within the gaps between individual collagen fibers, which resembles natural conditions. This system maintains strong cell-ECM and cell-cell interactions for resident breast cancer cells and the surrounding stromal fibroblasts or mesenchymal cells. Using this platform, a number of in vitro breast cancer models have been established, including inflammatory breast carcinoma, ductal carcinoma, and pleomorphic breast carcinoma. Importantly, implanting the model tissue onto the chicken chorioallantoic membrane for 9 days resulted in tissue histologically resembling ECM-rich patient breast cancer biopsy tissues. In summary, the extracted native collagen fibers enable the construction of breast cancer models in vitro through maintained cell-cell and cell-ECM interactions. These models histologically resemble in vivo tumor models and patient biopsies. With simple preparation, this platform can be easily scaled up for rapid deployment for downstream applications, such as drug discovery and mechanistic studies of tumor cell interactions as well as cancer progression. With the flexibility to change the cancer cell and surrounding cell types, this system is expected to have great utility for the study of other cancers as well. Citation Format: Rui Tang, Aixiang Ding, Marvin Rivera, Eben Alsberg. Modeling breast cancer tissue in vitro using extracted native collagen fibers [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-53.