Simple SummaryDespite substantial progress and effort, the clinical efficacy of chemotherapy remains suboptimal due to chemoresistance, which is partly attributed to adipocytes in the tumor microenvironment. Adipocytes, major stromal cells in the bone marrow, are known to uptake lipophilic molecules. As many chemotherapeutics are hydrophobic, bone marrow adipocytes may contribute to the development of chemoresistance. However, detailed investigation has been challenging due to the limited accessibility and heterogeneity of the bone marrow microenvironment. To address these barriers, we have established 2D and 3D in vitro culture models of human bone marrow adipocytes. Our models provide compelling evidence that bone marrow adipocytes sequester doxorubicin in lipid droplets, resulting in a reduction in its cytotoxicity. These findings reveal an orthogonal mechanism for bone marrow tumor microenvironment-driven chemoresistance.AbstractChemoresistance is a significant problem in the effective treatment of bone metastasis. Adipocytes are a major stromal cell type in the bone marrow and may play a crucial role in developing microenvironment-driven chemoresistance. However, detailed investigation remains challenging due to the anatomical inaccessibility and intrinsic tissue complexity of the bone marrow microenvironment. In this study, we developed 2D and 3D in vitro models of bone marrow adipocytes to examine the mechanisms underlying adipocyte-induced chemoresistance. We first established a protocol for the rapid and robust differentiation of human bone marrow stromal cells (hBMSCs) into mature adipocytes in 2D tissue culture plastic using rosiglitazone (10 μM), a PPARγ agonist. Next, we created a 3D adipocyte culture model by inducing aggregation of hBMSCs and adipogenesis to create adipocyte spheroids in porous hydrogel scaffolds that mimic bone marrow sinusoids. Simulated chemotherapy treatment with doxorubicin (2.5 μM) demonstrated that mature adipocytes sequester doxorubicin in lipid droplets, resulting in reduced cytotoxicity. Lastly, we performed direct coculture of human multiple myeloma cells (MM1.S) with the established 3D adipocyte model in the presence of doxorubicin. This resulted in significantly accelerated multiple myeloma proliferation following doxorubicin treatment. Our findings suggest that the sequestration of hydrophobic chemotherapeutics by mature adipocytes represents a potent mechanism of bone marrow microenvironment-driven chemoresistance.