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

ABSTRACT 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.