Engineering a multicellular metastatic bone marrow niche using foamed beta tricalcium phosphate scaffolds for advanced neuroblastoma in vitro modeling and drug testing

Bone marrow metastasis is a hallmark of high-risk neuroblastoma (NB) and is associated with poor prognosis, therapy resistance, and disease recurrence. Despite significant advances in cancer biology, current in vitro models inadequately reproduce the complexity of the metastatic bone marrow microenvironment, limiting the predictive value of preclinical studies. Here, we developed versatile three-dimensional (3D) foamed beta tricalcium phosphate (βTCP) scaffolds to engineer an in vitro multicellular NB metastatic bone marrow niche. We fabricated spongy βTCP scaffolds using a polyurethane mold and an organic slurry, and then sintered them to obtain mechanically stable constructs with interconnected porosity. We used thermal and spectroscopic analyses to assess scaffold structure, and advanced microscopy for morphological and functional evaluations of the metastatic in vitro model. Human mesenchymal stromal cells efficiently colonized the scaffolds, maintained viability for 28 days, and remodeled the acellular microenvironment through extracellular matrix protein deposition. Moreover, modulation of RUNX2 and Collagen I gene levels confirmed their pro-osteogenic commitment. Sequential introduction of endothelial cells, hematopoietic stem cells, and NB cells enabled generation of a multicellular metastatic niche that recapitulates key cellular components of the bone marrow. Chemokine profiling revealed modulation of inflammatory, angiogenic, and stromal-remodeling pathways, including NF-κB, STAT3, IL-2 signaling, epithelial-to-mesenchymal transition, and pro-angiogenic signatures. Functional validation demonstrated that therapeutic responses differed substantially between non-malignant and metastatic multicellular systems, highlighting the critical contribution of the complex cell-to-cell interactions to treatment outcomes. Collectively, these findings establish a biomimetic, versatile 3D in vitro model that reproduces essential structural and biological features of the metastatic bone marrow niche, providing a valuable tool for further NB research and preclinical drug screening.