Decoding novel therapeutic targets for the bone metastatic neuroblastoma using a tailored 3D in vitro engineered model recapitulating bone-like microenvironment

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Metastatic cancers remain a therapeutic challenge with a significant number of patients failing to respond sufficiently to multimodal therapies. In Neuroblastoma, most frequent extracranial solid tumors developing in preschool children, single targeted therapies show only limited tumor specificity causing severe side effects and frequently fail due to heterogeneous expression of target molecules and tumor cell plasticity. Specificity of the disease that reflects embryonal origin and onset in children within their first years of life often impedes a possibility for collecting enough tumor material for more comprehensive molecular and cellular biology analyses. Better molecular understanding of the metastatic disease is required for finding new druggable targets and propose more effective therapeutic strategies. In order to address this issue, 3D in vitro models that mimic native structure of bone marrow infiltrated by tumor cells are required. We recently introduced such a model based on a simplified scaffold system with tailored interconnected micro-channels and confirmed in a proof-of-principle study its biocompatibility with neuroblastoma cell growth. In here proposed research project, a more complex composition of the bone and metastatic bone marrow environment will be established by leveraging advances of biomedical engineering technologies, biomaterials, and 3D printing technique. A multi-cellular 3D co-culture model made of stromal, endothelial, hematopoietic stem and neuroblastoma cells will be then characterized using confocal, two-photon and electron microscopy to evaluate its functional resemblance with native bone marrow. Next, molecular and cellular biology approaches will be applied to scrutinize more in details how metastatic microenvironment sustain neuroblastoma cells survival and shape their response to drugs. In particular, transcriptomics and epigenetic analyses will be performed to reveal main genes that are influenced by 3D growth conditions. Validations of their protein products will be done in following using immunocytochemistry approach to assess expression and distribution inside the scaffolds. As well, immunologic profiling will be deciphered to investigate which molecules are favored in the artificial bone marrow niche and their biological role will be determined in vitro. Based on the available literature, the potential for targeting pro-tumorigenic effectors will be evaluated, and selected blockers will be tested in vitro by measuring cell viability and toxicity. The results of this project will improve knowledge of how metastatic neuroblastoma interact with non-tumor cells and indicate putative new druggable targets. This could open new strategies for more effective and tailored treatments of tumor cells disseminated into the bone marrow and define applicability of proposed in vitro model for studying more sophisticated tumor-related processes.