Osteosarcoma (OS) is an aggressive primary bone cancer that mainly affects children and young adults, and is characterized by high genomic complexity. Current treatment relies on chemotherapy, yet many patients exhibit resistance or develop metastatic disease. Current experimental models for OS research rely primarily on 2D monolayer culture or xenograft models. However, 2D cultures culture generally fail to retain tumor phenotypes and drug response in vivo, whereas mouse models are costly and impractical for high-throughput drug screening. Recently tissue engineered 3D cancer models have emerged as new cancer research tools, which better recapitulate in vivo tumor signaling and drug responses than 2D cultures. However, most tissue engineered cancer models to date are limited to soft tissues. Unlike soft tissues, bone is characterized by a highly- mineralized extracellular matrix (ECM) comprised of 70% minerals such as hydroxyapatite (HA) crystals. However, the role of bone mineral in driving OS progression and drug response remains largely unknown. Furthermore, previous OS studies rely on a narrow set of cell lines that have been in culture for decades, which may no longer reflect the biology and drug response in vivo The overall goal of this proposal is to integrate a scalable and physiologically relevant 3D OS model with high-dimensional sequencing tools to elucidate OS genomic heterogeneity and drug resistance, as well as screening novel combination therapies using multiple patient-derived OS cell lines. Our 3D models is specifically designed with high-throughput screening in mind, and leverages on a patented microribbon (µRB)-based scaffold invented by the Yang (PI) lab. This multi-PI application will bring together expertise in biomaterials design and 3D tumor models (Yang lab/Stanford) with expertise in patient-derived xenograft (PDX) cell lines, genomics and preclinical therapeutics of OS (Sweet- Cordero lab/UCSF). We hypothesize that OS signaling and drug responses in 3D culture can be modulated by tuning the type and size of mineral cues 3D gelatin µRB scaffolds to better mimic the in vivo phenotype, and combinational therapies that target identified signaling using 3D OS model will lead to better treatment outcomes for OS in vivo. To test these hypotheses, we will carry out the following aims. Aim 1: Develop 3D OS models with optimized niche cues for deep characterization of OS signaling and heterogeneity using multiple OS PDX cell lines and compare results to mouse orthotopic OS models. AIM 2: To harness 3D OS models to determine the regulatory pathways involved in mediating receptor tyrosine kinase expression in OS and identify lead drug candidates by screening a panel of targeted drug therapies. Aim 3: To identify novel combination therapies for PDX OS cell lines and elucidate potential drug resistance mechanisms using 3D OS models. This study will pioneer integrating 3D OS model with PDX cell lines and high-dimensional sequencing e...