Abstract 5493: 3D bone marrow niche: Scalable and physiologically relevant <i>ex vivo</i> drug screening platform for acute myeloid leukemia and multiple myeloma

Introduction: Preclinical screening of drugs targeting hematological cancers, like Acute Myeloid Leukemia (AML) or Multiple Myeloma (MM), is challenging due to limited ex vivo growth and short viability of cells from bone marrow and blood cancers. Consequently, most of the preclinical studies rely on short-term suspension cultures of cell lines and/or primary cells, which undergo spontaneous apoptosis and do not predict the in vivo response. Physiologically relevant in vitro screening of cells from hematological malignancies requires the recapitulation of the human bone marrow niche (BMN) to provide cell-cell interactions and paracrine factors that are critical for the ex vivo proliferation of malignant hematopoietic cells. In our 3D BMN platform, homogeneous and reproducible 3D endothelial-mesenchymal networks that resemble pre-vascularization are used to integrate primary cells isolated from malignant bone marrow disorders and assess Standard of Care (SoC) drug responses Methods: Bone marrow and whole blood from newly diagnosed and relapsed-refractory (RR) patients with AML and MM were processed to isolate, respectively, CD34+ myeloid blasts and CD138+ myeloma cells. Heterologous mesenchymal stromal cells (MSC) and endothelial cells were embedded in a protein-rich hydrogel in a 384-well format until network formation was microscopically observed. Cryopreserved primary cancer cells were integrated into the 3D BMN and exposed to SoC drug panels for 6-7 days. Quantitative assessment of the SoC drug effects was performed using our proprietary automated high content imaging (HCI) analysis platform. Immunofluorescence (IF) staining and flow cytometry were used for in-depth characterization of BMN-malignant cell co-cultures. Results: 3D BMN networks were homogeneously distributed throughout the hydrogel and supported the integration of AML and MM cells. Colony-like clusters of cancer cells were observed within the BMN networks, suggesting outgrowth of the cells, which was validated by Ki67 staining. IF staining was also used to characterize and observe the maturation of the BMN over time (CD31, fibronectin). FACS analysis confirmed MSCs immunophenotype and revealed the proportion of myeloid blasts and myeloma cells in the bone marrow of each donor. Dose-dependent responses to Cytarabine and Daunorubicin were observed in primary AML and MM cells integrated in 3D BMN. Conclusion: The complex bone marrow microenvironment is critical for the maintenance, proliferation, and drug responses of malignant cells from bone marrow disorders. Here, we present a robust and reproducible 3D BMN platform that supports medium-throughput HCI drug screening in primary patient cells from different hematologic malignancies. In addition, our HCI analysis offers the possibility to study off-target effects to the MSCs and endothelial cells within the niche. Citation Format: Talita Stessuk, Dorrith Verstegen, Sophia Ajala, Hanna Vermeer, Jolie Flach, Marrit Putker, Nataliia Beztsinna, Ludovic Bourre. 3D bone marrow niche: Scalable and physiologically relevant ex vivo drug screening platform for acute myeloid leukemia and multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5493.