Abstract Breast cancer recurs in approximately 20% of disease-free patients 5+ years after successful treatment of the original tumor. These late recurrences often occur at metastatic sites, including the bone marrow and lungs, and are hypothesized to arise from disseminated tumor cells (DTCs) that reactivate after a long period dormancy. Interactions between DTCs and the microenvironment at these sites are thought to be important regulators of both breast cancer cell dormancy and reactivation. New approaches are needed to study this complex process and test hypotheses about key cell-microenvironment interactions in dormancy toward improved treatment strategies. In this work, we investigated the role of cell-matrix and cell-cell interactions on dormancy and activation of breast cancer cells utilizing well-defined materials to mimic the extracellular matrix (ECM) of metastatic sites and selectively introducing metastatic niche cells via dynamic co-culture. We established synthetic ECMs with well-defined properties that mimicked key aspects of the mechanical properties and biochemical content of bone marrow and lung metastatic sites (Young's modulus E˜0.5-5 kPa, respectively, and rich in collagen). These synthetic ECMs were constructed with a bioinert, multifunctional poly(ethylene glycol) crosslinked with a cell-degradable peptide and decorated with integrin-binding peptides derived from collagen and laminin, creating three-dimensional (3D) environments for the culture of breast cancer cells and niche cells. We cultured breast cancer cells of different metastatic potential (estrogen receptor positive [ER+, T47Ds] and triple negative [ER-, MDA-MB-231s]) within matrices, with or without co-culture of niche cells, and examined differences in markers of dormancy for up to 6 weeks. Both breast cancer cell types exhibited good viability in 3D culture (> 90%). Different degrees of cell proliferation (metabolic activity, EdU assay, cell/cluster number and volume) were observed over the first 2 weeks in culture, dependent upon matrix composition. Interestingly, ER- MDA-MB-231s were more responsive to differences in the biochemical content of the matrix, with increased elongation and proliferation observed within collagen mimic environments, whereas fewer differences were observed in the responses of the ER+ T47Ds to these same compositions. Further, both cell types exhibited significant decreases in proliferation in response to increased matrix density, mimicking the moduli of bone marrow to lung tissues. Dormant single cells or small cell clusters, reminiscent of micrometastases, were observed over weeks 2-6. Immunostaining and bioinformatics analyses revealed increased expression of autophagy markers for these dormant cells, suggesting a potential mechanism of survival. Additionally, co-culture with specific niche cells, including human mesenchymal stem cells and osteoblasts, was observed to enhance or suppress breast cancer cell proliferation within these well-defined environments. These studies have established a new controlled 3D breast cancer dormancy culture model, with on-going investigations examining cell re-activation for the identification of therapeutics to maintain dormancy and prevent recurrence. Citation Format: Kloxin AM, Ovadia EM, Pradhan L, Sawicki LA. Investigating breast cancer dormancy in response to microenvironment cues with well-defined synthetic extracellular matrices [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-06.