Ewing sarcoma (ES) is a pediatric malignancy, which carries adverse prognosis in its metastatic form. Bone metastases, although less common, associate with the worst clinical outcome. Yet, this form of the disease remains understudied due to challenges in its modeling. Recently, we have shown that ES bone metastases are triggered by severe hypoxia in primary tumors, which leads to the formation of polyploid cells, the progeny of which, hereafter named HYP-4n cells, have a high propensity for bone metastasis. However, the mechanisms underlying the ability of HYP-4n cells to colonize the bone remain unknown. This type of scientific question can be addressed by functional genomics approaches, such as CRISPR screens. Yet again, such assays are challenging in the setting of bone metastasis in vivo, due to the low number of osseous lesions developing in animal models and their clonal nature. On the other hand, most of the existing in vitro models fail to faithfully recapitulate heterocellular interactions occurring in the bone environment. Thus, we propose the 3D bicellular microfluidic platform (3D-BMP), which mimics the conditions ES cell encounter during osseous dissemination, as a research tool that will enable high-throughput genomic screens on bone metastasis. 3D-BMP consists of a bioengineered microvessel covered with endothelial cells and embedded in the bone-mimicking matrix with osteoblasts and osteoclasts. It allows for control of important parameters of the bone environment, i.e. matrix stiffness, hydrostatic pressure and oxygen tension. As such, it enables testing for rapid structural and functional changes occurring in the bone during the metastatic process and simultaneous analysis of multiple invading colonies, both of which are not achievable in animal models. The goal of our project is to test the utility of 3D- BMP in studies on the biology of ES bone metastasis, including large scale genomic assays. To this end, we will use HYP-4n cells, with high propensity for bone metastasis, and their diploid counterparts derived from the same ES cell lines cultured in normoxia (NOR-2n), which do not metastasize to bone, to optimize the experimental conditions in the 3D-BMP to best reflect their differences in metastatic properties and test their interactions with the bone environment (Aim 1). This model will be used to perform a focused CRISPRi drop-out assay targeting genes the most highly upregulated in HYP-4n cells to select candidate molecules crucial for dissemination to the bone tissues and survival in this environment (Aim 2). The selected targets will be first validated via 3D-BMP and then tested in vivo, using an orthotopic xenograft model, to provide evidence for the biological relevance of our platform. The proposed study will: 1) validate 3D-BMP as a model recapitulating key steps of osseous spread; 2) test the experimental pipeline involving CRISPR screen in 3D-BMP followed by in vivo validation as a research tool for the identific...