Geometry is a key factor in defining the transcriptional background of bone marrow metastatic neuroblastoma cells in a multicellular 3D in vitro model

Abstract text (incl. figure legends and references)

Introduction: Metastasis remains one of the leading causes of cancer death. Metastases are frequent in pediatric neuroblastoma (NB), a developing tumor of the sympathetic nervous system. Of particular concern are invasions of neoplastic cells to the bone marrow and bone, which are strongly correlated with tumor aggressiveness.

Objectives: In this study, we investigated how the geometry, chemistry, and cellular complexity of a 3D in vitro NB bone metastasis model made of β-tricalcium phosphate (β-TCP) scaffolds with interconnected microchannels affect tumor cell behavior.

Material and Methods: The interaction between mesenchymal stromal cells (MSC), endothelial cells (EC), and NB cells and the extracellular matrix was investigated using two-photon microscopy. The release of cytokines was determined by ELISA. Extensive computational analyses were performed to determine how geometry affects the transcriptional background of NB cells. The in-silico data were validated using an immunofluorescence approach.

Results: We confirmed that the tumor microenvironment is dynamically shaped by stromal cells, promoting the growth of NB cells in the metastatic niche. Compared with their 2D counterparts, 3D β-TCP structures produce proinflammatory and immunomodulatory cytokines and define the intracellular localization of Connexin 43. Moreover, our data confirmed that 3D growth conditions were crucial for the type of tumor cell response to conventional chemotherapeutic agents. Finally, we verified the crucial contribution of geometry in defining the in vivo resembling transcriptional landscape of tumor cells by activating a subset of genes critical for tumor progression.

Conclusions: Our results demonstrate that geometry is an important modulator of cell-cell interaction and of molecular signaling pathways active in metastatic NB cells. The 3D conditions of the metastatic niche support the maintenance of stem-like properties in the tumor cell compartment.