FOS3D: A Fluorescence‐Enabled Toolkit for Characterizing a Three‐dimensional Osteosarcoma Model

Osteosarcoma (OS) remains a rare, aggressive primary bone malignancy for which therapeutic progress has been limited for several decades. Three-dimensional (3D) tissue-engineered models can better reproduce the tumor microenvironment than monolayer cultures; however, their routine use is often constrained by low-throughput workflows and reliance on destructive analytical endpoints. Here, we present FOS3D, a fluorescence-enabled OS model based on gelatin methacryloyl hydrogels incorporating stable green fluorescent protein (GFP)-expressing tumor cells. By varying polymer concentration and photo-crosslinking duration, hydrogels spanning compressive moduli of approximately 5-50 kPa were generated, covering stiffness ranges reported for OS stromal environments. Whole-well fluorescence scanning enabled rapid, non-destructive quantification of proliferation in intact constructs and showed correlation with DNA and metabolic assays. Integration with light-sheet microscopy further enabled volumetric imaging of tumor organization, revealing time-dependent changes in spatial distribution. Transcriptional profiling and immunohistochemistry confirmed microenvironment-driven adaptations associated with extracellular matrix remodeling, stemness, and drug-resistance pathways. Finally, GFP-based longitudinal monitoring enabled chemotherapeutic screening of cisplatin and doxorubicin across two OS cell lines, capturing dose-dependent responses and the increased treatment tolerance characteristic of 3D cultures. Collectively, FOS3D provides a scalable fluorescence-enabled toolkit for high-content characterization and drug-response profiling of OS in physiologically relevant 3D environments.