Abstract Background: The processes regulating cellular dormancy in tumor cells remain poorly characterized. We investigated the microenvironmental cues responsible for the induction of cellular dormancy in breast cancer cells in different culture scenarios. Methods: We characterized an in vitro model in distinct conditions defined hierarchically by the following: a) normoxia or hypoxia (1% O2 and 5% CO2), b) dishes coated with fibronectin or laminin or none, and c) presence or absence of 10 ng/ml basic fibroblast growth factor (FGF-2) in the culture medium, added on day 0 of culture. Two cell lines (MCF-7 and MDA-MB-231) were cultured at clonogenic densities for 9 days (day-1, day0, up to day+7). The day +7 cells were characterized using crystal violet staining for colony growth (</=12 cells=dormant), p-p38/p-ERK ratio (>1=dormant), Ki67 expression (no expression=dormant) by immunofluorescence, and β-galactosidase staining as a senescence marker. Results: For induction of dormancy in MCF-7 cells, under hypoxic conditions, fibronectin or laminin were sufficient, whereas under normoxic conditions, FGF-2 with fibronectin (but not laminin) was required. For induction of dormancy in MDA-MB-231 cells, under hypoxic conditions, FGF-2 with laminin (but not fibronectin) was sufficient, whereas under normoxic conditions, laminin or fibronectin alone were sufficient, but the addition of FGF-2 led to a proliferative state. The detailed results are shown in Table 1. The dormant cells showed lack of β-galactosidase staining and were viable after 7-day doxorubicin treatment (2 μM), whereas there was complete cell death in proliferating cells. Conclusion: Specific combinations of FGF-2, laminin and fibronectin in microenvironment induce a state of dormancy in MCF-7 and MDA-MB-231 cells under normoxic and hypoxic conditions. The dormant cells show resistance to doxorubicin and lack senescence markers, suggesting their possible role in drug resistance and disease relapse. Table 1. Dormancy induction under specific conditions. Breast cancer cell type Microenvironmental cues p-p38/p-ERK signaling ratio Phenotypic states MDA-MB-231 Normoxia, none (Control) 0.39 Proliferative MDA-MB-231 Normoxia, Fibronectin 1.36 Dormant MDA-MB-231 Normoxia, Laminin 1.69 Dormant MDA-MB-231 Normoxia, FGF-2, Fibronectin 0.21 Proliferative MDA-MB-231 Normoxia, FGF-2, Laminin 0.72 Proliferative MDA-MB-231 Hypoxia, FGF-2, Laminin 1.05 Dormant MDA-MB-231 Hypoxia, FGF-2, Fibronectin 0.26 Proliferative MDA-MB-231 Hypoxia, Laminin 0.27 Proliferative MDA-MB-231 Hypoxia, Fibronectin 0.33 Proliferative MCF-7 Normoxia, none (Control) 1.00 Proliferative MCF-7 Normoxia, FGF-2, Fibronectin 1.44 Dormant MCF-7 Normoxia, FGF-2, Laminin 1.00 Proliferative MCF-7 Normoxia, Fibronectin 0.86 Proliferative MCF-7 Hypoxia, none (Control) 0.93 Proliferative MCF-7 Hypoxia, Fibronectin 2.45 Dormant MCF-7 Hypoxia, Laminin 1.3 Dormant Citation Format: Pulkit Datt, Jinesh Maniar, Prerana P. Dange, Vaishali Kailaje, Mithlesh K. Lakhera, Mansi Samarth, Tanuja Durve, Sudeep Gupta. Microenvironmental cues that regulate breast cancer cellular dormancy in the bone marrow niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 78.