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

Area of Science:

• Biomedical Engineering
• Oncology
• Biomaterials Science

Background:

• Osteosarcoma (OS) is an aggressive bone cancer with limited treatment advancements.
• Traditional 2D cell cultures do not accurately mimic the complex tumor microenvironment.
• Existing 3D models often involve low-throughput and destructive analysis methods.

Purpose of the Study:

• To develop a scalable, fluorescence-enabled 3D osteosarcoma model (FOS3D) for enhanced tumor microenvironment analysis.
• To enable non-destructive, high-content characterization and drug response profiling of OS.
• To facilitate the study of OS adaptations within a physiologically relevant 3D context.

Main Methods:

• Fabrication of gelatin methacryloyl hydrogels with tunable stiffness (5-50 kPa) encapsulating GFP-expressing OS cells.
• Utilized whole-well fluorescence scanning for non-destructive proliferation assessment.
• Integrated light-sheet microscopy for volumetric imaging of tumor organization.
• Performed transcriptional profiling and immunohistochemistry to analyze microenvironment-driven adaptations.
• Conducted longitudinal monitoring for chemotherapeutic screening.

Main Results:

• FOS3D models accurately reflect OS stromal stiffness ranges.
• Non-destructive fluorescence scanning correlated well with traditional proliferation assays.
• Volumetric imaging revealed dynamic changes in tumor spatial distribution.
• Identified microenvironment-driven adaptations in ECM remodeling, stemness, and drug resistance.
• Demonstrated successful chemotherapeutic screening of OS cell lines, capturing dose-dependent responses and increased 3D treatment tolerance.

Conclusions:

• FOS3D offers a scalable and versatile platform for osteosarcoma research.
• The model enables non-destructive, longitudinal monitoring and high-content analysis.
• FOS3D facilitates robust drug screening in a physiologically relevant 3D tumor microenvironment.
• This toolkit advances the characterization and therapeutic development for osteosarcoma.